Efficacy of Bark (Juglans regia L.) Extracts Against Periodontitis Bacteria: an In Vitro Study

Full text

5492 Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3
Efcacy of Bark (Juglans regia L.) Extracts Against
Periodontitis Bacteria: an In Vitro Study
Khawlah A. Salman1, Sahar M. Jawad2, Najla Naji Chafat3, Alaa S.J. Al-Bdery4
1Lecturer, Department of Food Sciences, Faculty of Agriculture, University of Kufa, Najaf, Iraq,
2Lecturer, Department of Soil Science and Water Resources, Faculty of Agriculture, University of Kufa, Najaf, Iraq,
3Asst. Prof., Department of Pathological Analyses, Faculty of Sciences, University of Thi-Qar, Thi-Qar, Iraq,
4Asst. Lecturer, Al-Najaf General Directorate of Education, Najaf, Iraq
Abstract
Juglans regia is one of the medicinal plants widely applied in many applications due to its pharmacological
value and desirable characteristics of its parts. Hence, there is a motivation of medicinal and cosmetics
applications. In this paper, the crude aqueous extracts from Juglan regia bark were screened for in vitro
antibacterial properties against clinical isolates of Periodontitis bacterial causative agents (Granulicatella
adiacens, Staphylococcus sciuri, and Kocuria spp. The antibacterial test was carried out using the Kirby
Bauer method. The tested extract from this medicinal plant with the different concentrations (100 mg/ml,
250 mg/ml, 500 mg/ml) were screened. The standard antibiotics Ciprooxacin (5 μg/ml) and Cefotaxime
(30µg/ml) were used as controls. The extract of 250 mg/ml being more effective in action as compared to the
others. Furthermore, Kocuria spp showed the most isolate affected by the extract. This research has revealed
the active inhibitory effect of bark extract against all the tested isolates. This extract contains active chemical
components that contribute to biological activity thereby assisting to combat bacterial infections. However,
many studies need to be carried out to identify the responsible constituents for growth inhibition.
Keywords: Juglan regia bark, aqueous extract, antibacterial activity, Periodontitis.
Introduction
The demand is driven recently to use alternatives
natural compounds instead of chemical ones such as
plant extracts from medicinal and aromatic plants in
addition to wild plants in defending diseases caused
by different microbes fungal, bacterial, viral, nematode
and insect 1,2. The inclination towards the use of natural
materials as a substitute for the articial ones has
increased dramatically due to some consequences of
the latter such as undesirable side effects, complexity,
the emergence of the medicine resistance trait and
cost benets, competition by manufacturers 3-7. Many
Corresponding author:
Khawlah A. Salman
Email: khawla.alzur@uokufa.edu.iq
traditional medicines are made by grounded the leaves
or bark, and the derivative mixture may contain
hundreds of potentially active molecules. Identifying
such molecules and examined their effectiveness is
well established as antioxidant materials8, anti-tumor9,
anti-inammatory10,11, in addition to the antimicrobial
activity12. Juglan regia tree normally known as walnut is
considered as one of the important medicinal plants that
have proven to be effective in therapeutic and beauty
elds 13). Related parts of juglan regia such as shells,
kernel, and seed, bark have been examined by many
researchers in various studies14. The bark of Juglan
regia locally known as Deirum in Asia and middleeast
countries. This medicinal part can be utilized as a tooth
cleaner, cosmetic product for coloring the lips by women
and the cosmetic industry15. Juglan regia bark is available
in different sizes and shapes, brous and characterized

Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3 5493
with resinous, scented features16. Furthermore, Walnut
(Juglans regia L.) bark has been determined as a
valuable medicinal source to treat a range of diseases
and its activity as anti-inammatory, antioxidant, blood
purify, anticancer, depurative, diuretic, and laxative
activities has been revealed17. The chemical composition
of juglan regia bark mainly compromise from various
therapeutic components such as 𝛽-sitosterol, ascorbic
acid5, juglone, folic acid, gallic acid, regiolone, and
quercetin-3-𝛽-L-arabinoside, avonoids, pectic
substances, sterols and phynols18. Juglans regia L. bark
is well recognized as a bioactive compound against fungi
Aspergillus niger, Alternaria alternate, Trichoderma
vierns and Fusarim solani Candida alibicans 19). The
inhibition activity has been established not only as
antifungal but also, its application as an antibacterial
agent has been conrmed20. The extract of Juglans regia
bark exhibited a broad-spectrum antimicrobial activity
in a dose-dependent manner against some pathogenic
bacteria such as Gram-positive bacteria (Staphylococcus
aureus and Streptococcus mutans, Streptococcus
salivarius), Gram-negative bacteria (Escherichia coli
and Pseudomonas aeruginosa 21. A wide range of oral
pathogenic bacteria is applied in many antimicrobial
studies using natural products from medicinal plants
for the promise to discover the potential replacement
remedies to the synthetic antibiotics. Hence, this study
was carried out to evaluate the antibacterial activity
of aqueous extract of Juglans regia bark against three
species of periodontitis bacteria.
Materials and methods
Collecting plant samples and preparing them for
extraction
The stem bark was obtained from the local market, to
be prepared by grinder and the grounded parts were kept
at room temperature. The stock extracts solution was
prepared from Derum (J regia L.) by dissolving (1gm)
in 1mL to obtain dilution 500,250,100 mg / mL aqueous
extract and then ltered by Whatman lter paper.
Specimens Collection
In this study, 20 samples were collected from the
Collage of Dentistry, University of Kufa, from patients
with periodontitis. Samples were collected from patients
with cotton rolls and carefully cleaned with sterile cotton.
For each site, 2 paper points (30-40) were inserted into
the periodontal pocket for 30 seconds, the pocket depth
was equal to or exceeding (3.5-7) mm. It was then placed
in a sterile container with saline (2 ml) and inoculated on
the agar plates.
Antimicrobial assay
The isolated bacterial strains of periodontitis were
diagnosed by the Vitek 2 system for identity and they
were: Granulictaella adiacens, Staphylococcus sciuri
and Kocuria spp. One colony of each isolate was
inoculated in a brain heart infusion broth (BHI) and
incubated for 3 hours at 37 °C to obtain culture turbidity
with standard turbidity (0.5 McFarland standard). On
Muller Hinton agar spread 0.1ml of the culture with
a sterile swab, dry at room temperature for (10-15)
minutes. Inhibitory activity was detected by the agar
well diffusion method, after sterilizing the cork borer,
four wells were made on the surface of the culture media
with a diameter of 10 mm then add (100 μl) to each well
in different concentrations of derum extract. antibiotic
discs were applied at the center such as ciprooxacin
(5 μg) and Cefotaxime (30µg) for comparison with the
plant extract, after which the plate was incubated for
18-24 hours at a temperature of 37 °C. The diameter of
the inhibition zones was recorded. The experiment was
repeated twice and the average of four replicates was
calculated.
Statistical Analysis
The data gathered and exported to a Microsoft Excel
spreadsheet where descriptive statistics were performed.
The data was analyzed processed using SAS version
9.1. Two-way ANOVA was also carried to determine if
there was any interaction be tween the effect of extracts
concentration and the pathogenic bacteria. P ≤ 0.05 is
considered signicant in both tests (Tukey test). Further-

5494 Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3
more, the analysis was done to nd the difference between
the means (4 replicates). One-way ANOVA (Analysis
of variance) was car ried out to demonstrate statistical
difference using the vary ing zones of inhibition when
the extracts from Juglans regia L. bark was used against
the isolates included in this study.
Results
The results of an in vitro antibacterial assay of the
aqueous extract of bark (Juglans regia ) against a group
of a clinically signicant panel of prederontitis causative
bacteria at different concentrations of 100 mg/ml, 250
mg/ml, and 500 mg/ml are demonstrated in this study.
Figure 1 demonstrated that isolate Granulicatella
adiacens exhibited signicant inhibitory response (P<
0.05) at the average concentration level (250 mg/ml),
however, this isolate did not show signicant effect (P>
0.05) when the highest concentration (500 mg/ml) was
used. Regarding Staphylococcus sciuri isolate, there is
a high response (P< 0.05) at the average concentration
level (250 mg/ml), which reveals that the two isolates
were in parallel response. However, a concentration of
100 mg/ml of the bark extract is established to inhibit
the growth of Staphylococcus sciuri isolate signicantly
P< 0.05).
Respecting the third isolate Kocuria spp, the manner
of action was slightly different, as thereby with an
increase in the concentration of the bark extract, there
was a high inhibition activity (P< 0.05) between 100
mg/ml, 250 mg/ml, whereas, there were no statistically
signicant differences accompanied (P> 0.05) between
both concentrations (250 mg/ml and 500 mg/ml) Figure
3.
Fig. 1 The inhibitory effect of three different concentrations of bark Juglans regia against three treated isolates.
The error bars represent ± standard deviation (SD). Bars with different letters demonstrate signicant differences
(ANOVA, two-way, Tukey, P<0.05, n=4).

Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3 5495
Fig. 2 The response of the average treated isolates (Granulicatella adiacens, Staphylococcus sciuri and Kocuria
spp.) towards the average concentrations of aqueous bark extract of Juglans regia L.. The error bars represent
± standard deviation (SD). Bars with different letters demonstrate the signicant differences (ANOVA, one way,
Tukey, P<0.05, n=7).
The nding also revealed that the concentration of 100 mg/ml and 250 mg/ml had a signicant antibacterial
effect against the tested bacteria (P<0.05) as shown in Figure 3. However, the inhibition effect was approximately
similar continued to be in the same manner in the case of 500 mg/ml and there were no signicant differences were
recorded (P>0.05).
Fig. 3 Inhibition rate for three different concentrations of the bark extract of Juglans regia L. as average
isolates. The error bars represent ± standard deviation (SD). Bars with different letters demonstrate the
signicant differences (ANOVA, one way, Tukey, P<0.05, n=7).

5496 Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3
The inhibition zones caused by the standard
antibiotics were also observed in different degrees of
inhibition based on the sensitivity of the tested bacteria.
As can be noticed from Table 1 there was a signicant
antigrowth activity of both antibiotics used 30mm
of Ciprooxacin and 38mm of Cefotaxime against
Granulicatella adiacens isolate, whereas the lowest
inhibition zones were detected on Kocuria spp. The
data suggested that this bacteria was not highly sensitive
neither to the bark extracts nor to the typical antibiotics.
Table 1 Average of inhibition zones of bark extract on tested isolates. The presented values are the average
of 4 replicates of inhibition zones (mm). Average of inhibition zones of the standard antibiotics are shown.
Isolates
Control Concentration (mg/ml)
Ciprooxacin
(5 μg/ml)
Cefotaxime
(30µg/ml) 100 250 500
Granulicatella adiacens 30 38 23 28 25
Average of inhibition zones
(mm)
Staphylococcus sciuri 25 20 11.67 17.67 17.3
Kocuria spp 10 8 3 7.67 11
Discussion
As an adaption of bacteria that cause infection to
withstand antibiotics, the possibility for antimicrobial
resistance to cause a global health crisis emerges
large3,7. This issue has led the need for the development
of new medicines. Scientists and policymakers have
transformed their efforts and working together to
conceive new antibacterial agents naturally to overcome
such a threat8.
Periodontitis one of the bacterial infections mostly
associated with bacterial plaque 21. Some people avoid
using the commercial available mouth rinses products
due to the harmful chemicals and colors; alcohol 20.
Therefore, the current research was carried out to
investigate the antibacterial activity of bark (Juglans regia
L.) Aqueous extracts against clinical bacterial isolated
from patients with periodontitis. From the nding, the
bark of Jugaln regia extract showed a considerable
level of antibacterial activity against examined clinical
isolates. Generally speaking the extract from Juglans
regia L. bark proved to be signicantly active against
Granulicatella adiacens at (250mg/ml) concentration.
The second susceptible strain was Staphylococcus sciuri
that being affected by the extract with a diameter of
inhibition (17.67 mm) at (250mg/ml) concentration,
whereas the inhibition zones were increased gradually
by increasing the concentrations from 100 mg/ml to
500 mg/ml in the case Kocuria spp. Interestingly, the
concentrations used showed variances in antibacterial
growth, particularly, at the concentration ranged from
250 mg/ml -500 mg/ml, they were slightly comparable
in activity against all the examined bacteria. Therefore,
it is worth noting that it would have been better to select
concentrations within these two ranges to demonstrate
the accurate efcacy of the extract because the overlap
between concentrations has led to indenite the ideal
concentration of inhibition. Comparatively, the zones of
inhibition formed by standard antibiotics utilized were
varied, the data suggested that the sensitivity of the
tested isolates was incompatible. The aqueous extract
had signicantly antibacterial action against some oral

Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3 5497
bacteria such as Staphylococcus aureus, S. salivarius,
and S. sanguis compared to control. However, there was
no observed activity on S.mutans when compared with
the antibacterial agent Erythromycin 20. The activity of
Juglan regia bark extract improved by the synthesis
of nanoparticles materials, this synthesized particles
showed signicant inhibition against Streptococcus
mutans, which is the main causative agent for dental
cavities. The nanoparticles also showed promising
antibiolm action by inhibiting the glucosyltransferase
enzyme16. Study indicated that the ethanolic extracts of
Juglan regia bark exhibited more efcient antimicrobial
activity against oral microbes included yeast and bacteria
in comparison to the aqueous extract21. The ndings of
this study obviously showed that the aqueous extracts
of Juglans regia bark considerably inhibited the growth
of the examined oral bacteria, and available reports are
agreeable with the current outcomes. The antibacterial
characteristic of the medicinal plant might be as sociated
with metabolites such as phenolic compounds, terpenoid,
alkaloids, avonoids, and steroids. The bark of juglone,
regiolane, contains ketones, sterol, and avonoid 8,29.
Conclusion
Plants of pharmacologically active constituents
have been used by tradi tional therapists to treat a lot
of infectious diseases. In compliance with the ndings;
the extract has exhibited desirable antibacterial activity
against all the tested isolates. This study consolidates the
use of natural products as medicines and conrms the
antibacterial potentials of the bark of Juglan regia (Walnut
plant). The results strongly support the traditional use of
this plant (Derum) as a preventive remedy for various
microbial diseases of soft and hard tissues in the oral
cavity. And the possibility of using it as an oral rinse as
a prevention or as a treatment against oral pathogenic
bacteria. Undoubtedly, the extract contains chemical
substances that take part in metabolic activities, thereby
helping to ght against bacterial infections. Howev-
er, further biochemical, chemical and pharmacologi cal
investigations must be carried to identify and examine
the puried bioactive components that are responsible
for the antimicrobial activity and their mechanism of
action.
Conict of Interest: None
Funding: Self
Ethical Clearance: Not required
References
1) Carrol D, Chassagne F, Dettweiler M, Quave C.
Antibacterial activity of plant species used for oral
health against Porphyromonas gingivalis. PLOS
ONE, 2020. 15(10), p.0239316.
2) Farooqui A, Khan A, Borghetto I, Kazmi S, Rubino
S, Paglietti B. Synergistic Antimicrobial Activity
of Camellia sinensis and Juglans regia against
Multidrug-Resistant Bacteria. PLOS ONE, 2015,
10(2), p.e0118431.
3) Khalid M, Bilal M, Munir H, Shah S, Khurshid M,
El-Shazly M, Iqbal H. In-vitro Evaluation of Anti-
Bacterial, Anti-biolm and Cytotoxic Activity
of Naturally Inspired Juglans regia, Tamarix
aphylla L., and Acacia modesta with Medicinal
Potentialities. Journal of Pure and Applied
Microbiology, 2020. 14(2), pp.1133-1142.
4) Aritonang H, Koleangan H, Wuntu A. Synthesis
of Silver Nanoparticles Using Aqueous Extract
of Medicinal Plants’ (Impatiens balsamina and
Lantana camara) Fresh Leaves and Analysis of
Antimicrobial Activity. International Journal of
Microbiology, 2019, pp.1-8.
5) Boula M, Lamchouri F, Touk H. Chemical
Analysis, Phenolic Content, and Antioxidant
Activities of Aqueous and Organic Moroccan
Juglans regia L. Bark Extracts. Current Bioactive
Compounds. 2020 Dec 1;16(9):1328-39.
6) Ala M, Pirmohammadi R, Khalilvandi-Behroozyar
H, Anassori E. Potential of walnut (Juglans
regia) leave ethanolic extract to modify ruminal
fermentation, microbial populations and mitigate
methane emission. Animal Production Science,
2020. 60(9), p.1189 .
7) Panth N, Paudel K, Karki R. Phytochemical prole
and biological activity of Juglans regia. Journal of
Integrative Medicine, 2016. 14(5), pp.359-373.
8) Fernández-Agulló A, Castro-Iglesias A, Freire M,
González-Álvarez J. Optimization of the Extraction
of Bioactive Compounds from Walnut (Juglans
major 209 x Juglans regia) Leaves: Antioxidant

5498 Indian Journal of Forensic Medicine & Toxicology, July-September 2021, Vol. 15, No. 3
Capacity and Phenolic Prole. Antioxidants, 2019.
9(1), p.18.
9) Nicu A. Antibacterial activity of ethanolic
extracts from fagus sylvatica l. and juglans regia l.
leaves. farmacia, 2018. 66(3), pp.483-486.
10) Muzaffer U, Paul V, Prasad N, Karthikeyan
R, Agilan B. Protective effect of Juglans regia
L. against ultraviolet B radiation induced
inammatory responses in human epidermal
eratinocytes. Phytomedicine, 2018.42, pp.100-111.
11) Jafer F, Naser L. The Biological Activity of
Aqueous and Methanolic Extracts of Juglans regia
on Yeasts and Pathologic Bacteria. Archives of
Clinical Microbiology, 2020;20(1):2405-10..
12) Beiki T, Najafpour G, Hosseini M. Evaluation
of antimicrobial and dyeing properties of
walnut (Juglans regia L.) green husk extract for
cosmetics. Coloration Technology, 2017. 134(1),
pp.71-81.
13) Oğuz H, Gökdoğan O, Baran M, Oğuz I. Potential
of walnut (Juglans regia L.) nursery production
and its economic importance in Turkey. Acta
Horticulturae, 2019. (1258), pp.149-154.
14) Ribeiro A, Estanqueiro M, Oliveira M, Sousa Lobo
J. Main Benets and Applicability of Plant Extracts
in Skin Care Products. Cosmetics, 2015. 2(2),
pp.48-65.
15) Strelyaeva AVS, Lezhava DIL, Luferov ANL,
Kuznetsov RMK, Bobkova B, Lazareva YuB L,
Kostikova ENK. Study of Quality Medicinal Plants
Bark Walnuts and Extract from it. Pharmacognosy
Journal, 2020. 12(2), pp.282-286.
16) Croitoru A, Ficai D, Craciun L, Ficai A, Andronescu
E. Evaluation and Exploitation of Bioactive
Compounds of Walnut, Juglans regia. Current
Pharmaceutical Design, 2019. 25(2), pp.119-131.
17) Jahanban Esfahlan, Ostadrahimi Tabibiazar,
Amarowicz A. Comprehensive Review on the
Chemical Constituents and Functional Uses of
Walnut (Juglans spp.) Husk. International Journal
of Molecular Sciences, 2019. 20(16), p.3920.
18) Gaytán-Sánchez N, Rodríguez-García R,
Hernández-Castillo F, Díaz-Jiménez L, Villarreal-
Quintanilla J, Flores-López M, Carrillo-Lomelí
D, Peña-Ramos F. Antifungal activity of Juglans
spp. and Carya sp. ethanol extracts against
Fusarium oxysporum on tomato under greenhouse
conditions. Industrial Crops and Products, 2019.
138, p.111442.
19) Aldawood T, Alyousef A, Alyousef S.
Antibacterial effect of Juglans regia l bark extract
at different concentrations against human salivary
microora. Journal of Oral Medicine, Oral Surgery,
Oral Pathology and Oral Radiology, 2017. 3(4),
pp.214-217.
20) Alkhawajah A. Studies on the Antimicrobial
Activity of Juglans regia. The American Journal of
Chinese Medicine, 1997. 25(02), pp.175-180.
21) Zakavi F, Golpasand Hagh L, Daraeighadikolaei A,
Farajzadeh Sheikh A, Daraeighadikolaei A, Leilavi
Shooshtari Z. Antibacterial effect of Juglans regia
bark against oral pathologic bacteria. International
journal of dentistry. 2013 Jan 1.