Introduction

Natural products are used as traditional remedies for cosmetic and therapeutic needs in many parts of the world. Recently, the use of such products has been emphasized in treating various infections as some chemically synthesized drugs have undesirable effects ^(1)(2)(3). Antimicrobial activities of Juglans regia bark, leaves and seeds of the plants against gram positive bacteria (Staphylococcus aureus) and gram negative bacteria (Escherichia coli) strains are reported in the literature ^(1)(2)(4).  In some areas of Saudi Arabia women used the Juglans regia plant bark (Derum) for teeth cleaning and sparkling and as a dye for coloring the lip, they also use the boiled plant bark to extract deep brown dye to dye their hair ^(4)(5)(6).

Juglans regia

Walnut (Juglans regia) is one of the most widespread tree nuts worldwide. The walnut tree’s formal botanical name “Juglans regia” is roman in origin which means “Jupiter’s acorn”. The tree is native to the old world and it is spreading from the Balkans eastward to the western Himalayan chain and was cultivated in Europe as early as 1000 BC. Nowadays, it is commercially throughout Southern Europe, northern Africa, eastern Asia, the USA and western South America.  China is considered the leading world producer followed by US, Iran, Ukrania, Romania, France and India although the production in other countries has increased vastly in recent years such as Chile and Argentina .In recent decades an increasing tendency towards the use of natural substances instead of the synthetic ones has been observed. As the synthetic materials and products are more complex in comparison to natural substances, it will take a long time for them to complete their natural cycles and return to nature; thus causing a lot of environmental pollution. Also with the increase in the price of raw materials, the problem of cost benefits for chemical production is becoming more considerable. Natural antioxidants, such as phenolic compounds, used as natural antioxidants, are gaining importance, due to their benefits for human health, decreasing the risk of degenerative diseases by reduction of oxidative stress and inhibition of macromolecular oxidation ^(3)(7)(8).

Presence of Staphylococcus aureus and Escherichia coli in the oral cavity

The oral cavity is well known as a reservoir for many microorganisms, it contains more than 300 known species of bacteria ⁽⁹⁾. Some oral infections are caused by at least in part by Staphylococcus aureus e.g., angular cheilitis, parotitis, and staphylococcus mucositis ^(10)(11)(12). Recent studies have suggested that S. aureus can be isolated from the oral cavity of particular patient groups such as children and individuals with systemic diseases⁽¹¹⁾. Furthermore, the presence of E. coli is well documented to be located in the intestines, although a small amount of the bacteria is found in the mouth ^(12)(13).

Antibacterial activity of Juglans regia

The antimicrobial activity of Juglans regia was reviewed in many literatures, including hot/cold solvent and aqueous/methanolic extracts of leaves, barks fruits and green husks from different regions of the world, which showed broad spectrum antibacterial activity against gram positive bacteria and gram negative bacteria ^{(8)(1)(7)(3)(14)(15)(2)(16)}

Materials and Methods

Bacterial Isolates

Escherichia coli and Staphylococcus aureus were obtained from King Khalid University Hospital (KKUH), Riyadh, Saudi Arabia.

Plant material and preparation of extracts (aqueous and methanolic)

Fifty grams of Juglans regia bark (Derum) were cut into small pieces and soaked in sterilized distilled water 1:3 (w/v) for 24 hours. Then, the mixture was homogenized in a house hold blender for one minute at full speed. The homogenate was then filtered through double layer of cheese cloth and centrifuged at 3000xg for 10 minutes at 4^oC. The supernatant was then filtered through sterile filter (0.45 µm) and kept at -20^oC for later use. The supernatant is referred to as aqueous extract. The same protocol was used for methanolic extraction but the sample was soaked in 95% methanol. After centrifugation, methanol was evaporated using a steam of air at room temperature. The sample was then re-suspended to its original volume with distilled water. This was filtered through sterile filter (0.45 µm) and kept at -20^oC for later use. The supernatant is referred to as methanolic extract ^(17).

Agar well-diffusion test

Relative antimicrobial activity was determined using agar-well diffusion method. Escherichia coli or/and Staphylococcus aureus were spread onto the surface of LB agar with sterile swab (0.1 ml containg106 cell/ml). Six mm diameter wells were punched into the agar and filled with 0.1ml of the aqueous extract or protein fractions that showed antibacterial activity. The plates were then incubated overnight at 37^oC. All experiments were carried out in triplicates. After 24 hours of incubation inhibition zones were measured. Control wells were filled with sterilized distilled water ⁽¹⁸⁾.

Determination of minimal inhibitory concentration (MIC) minimal bactericidal concentration (MBC)

The MIC is defined as the lowest concentration (mg/ml) of the extract resulting in clear broth media, while minimum bactericidal concentration (MBC) is defined as the lowest concentration (mg/ml) of the extract resulting in no growth of bacteria after culture on agar media. In this study the (MIC) and (MBC) were determined using broth dilution method ⁽¹⁹⁾.

The broth media dilution method was used to determine the MIC of the extract of Juglans regia bark; the final concentration of the extract was 0.5–40 mg/ml in brain heart infusion broth; all of different concentrations were inoculated with 100 µl of strains and incubated at 37° C for 24 hour. All tests were performed in triplicate.

The bacteria used here were tested for their sensitivity to aqueous and methanolic extracts of Juglans regia bark (Derum).

Results and Discussion

The antibacterial properties of the Derum extracts were firstly determined by agar-diffusion method and was screened for its antimicrobial properties against Escherichia coli and Staphylococcus aureus. Tables 1and 2 show the diameter of the inhibition zones resulting from exposure of the bacteria to the aqueous and methanolic extracts of Derum. The inhibition of bacteria in this study was directly related to the concentration of extracts, where inhibition zones increasing with increasing extract concentration (0.5-4mg) ^(2)(1)(4). Staphylococcus aureus was the most readily inhibited bacterium ⁽⁴⁾⁽¹⁾ followed by Escherichia coli ⁽⁸⁾ unlike the results of Pereira ⁽²⁰⁾ where E. coli showed a complete resistance to the extract.

The methanolic extracts in this study were hardly to inhibit the bacteria. The results are similar to those presented by Alkhawajah ⁽⁴⁾,  while the inhibition of bacteria growth reported here for Juglans regia extracts are greater than those reported by ⁽²¹⁾ using  Staphylococcus aureus and Escherichia coli.

The MICs for the aqueous and methanolic extracts of Derum varied with the bacteria used (Table 3-4). The lowest observed MICs were for both Escherichia coli and Staphylococcus aureus (1mg/ml) in case of aqueous extract while Escherichia coli and Staphylococcus aureus showed MICs of 6 and 5 mg/ml respectively in case of methanolic extract, which is much higher than 0.1 mg/ml as in Al-Khawajah ⁽⁴⁾ .

The minimum bactericidal concentration (MBC) of methanol extracts of Escherichia coli was 36 mg/ml compared with values of 29 mg/ml for the other bacteria.

Table 1: Antibacterial activity of aqueous extract of Juglans regia bark by agar-diffusion method at different concentrations.

Table 2: Antibacterial activity of methanolic extract of Juglans regia bark by agar-diffusion method at different concentrations.

Table 3: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of aqueous extract of Juglans regia bark.

MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration

Table 4: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of methanolic extract of Juglans regia bark.

MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration

Conclusion and Recommendations

In conclusion; aqueous extract of the Derum contains a potent antibacterial agent that is protein in nature and may be used in the treatment of some oral infections.

The present study supports the use of natural products for medication as antibacterial agents found in plant extract which exhibits antibacterial activity to some opportunistic oral bacteria. Also, it highlights the traditional use of “Derum” and some scientific validation of the claimed biological antimicrobial activity in vivo.

Further clinical trials are required to determine the efficacy of Juglans regia bark extract on Escherichia coli or/and Staphylococcus aureus.

Acknowledgment

The author is thankful to King Khalid University hospital for providing the bacterial isolates and to Dr Suliman AlHarbi / College of Science for providing laboratory facilities to perform the research and to his guidance and expertise.

This research project was supported by a grant from the “Research Center of the Center for Female Scientific and Medical Colleges”, Deanship of Scientific Research, King Saud University

References

1. Bushra Abbas and Ganan Mohammad. Study on the effect of Juglans regia extract on some Periodontal bacteria and compare it with Imipenem. thiqaruni.org/bio/8/(28).doc‏. 2009;5(1):164–9.
2. Nirmala R. Deshpande a, Jyoti P. Salvekara AAK, Asha A. Kale*a AB. Antimicrobial Activity Study of Juglans regia L. JPR [Internet]. 2011 Feb 21;4(2). Available from: http://jpronline.info/index.php/jpr/article/view/5867
3. Sharafti-Chaleshtori R et al. Biological characterization of Iranian walnut (Juglans regia) leaves. Turk J Biol. 2011;35:635–9.
4. Alkhawajah AM. Studies on the antimicrobial activity of juglans regia. Am. J. Chin. Med. 1997;25(2):175–80.
5. I Muhammad et al. Ethanobotanical studies on plant resources of RESOURCES OF Ranyal Hills, District Shangla, Pakistan. Pak.J Bot. 2007;39(2):329–37.
6. Darmani H, Nusayr T, Al-Hiyasat AS. Effects of extracts of miswak and derum on proliferation of Balb/C 3T3 fibroblasts and viability of cariogenic bacteria. Int J Dent Hyg. 2006 May;4(2):62–6.
7. Milind P and Deepa K. Walnut: Not A Hard Nut to crack. IRJP. 2011;2(5):8–17.
8. Nael Abu Taha. Utility and importance of walnut, Juglans regia Linn: A review. African Journal of Microbiology Research [Internet]. 2011 Dec 30 [cited 2012 Sep 9];5(32). Available from: http://www.academicjournals.org/AJMR/abstracts/abstracts/abstract%202011/30Dec/Taha%20and%20Al-wadaan.htm
9. Wilson, Melanie J, Weightman, A.J.; Wade, W.G. Applications of molecular ecology in the characterization of uncultured microorganisms associated with human disease. Reviews in Medical Microbiolog. 1997 Apr;8(2):91–102.
10. Smith AJ, Jackson MS, Bagg J. The ecology of Staphylococcus species in the oral cavity. J. Med. Microbiol. 2001 Nov;50(11):940–6.
11. Smith AJ, Robertson D, Tang MK, Jackson MS, MacKenzie D, Bagg J. Staphylococcus aureus in the oral cavity: a three-year retrospective analysis of clinical laboratory data. British Dental Journal. 2003;195(12):701–3.
12. Ryan Mac. What are the different types of bacteria found in the human mouth? [Internet]. 2011 Mar. Available from: http://www.livestrong.com/article/161300-what-are-the-different-types-of-bacteria-found-in-the-human-mouth/
13. Kenneth Todar. The Normal Bacteria Flora of Humans. Todar’s Online TextBook of Bacteriology [Internet]. Available from: http://textbookofbacteriology.net/index.html
14. Ndukwe KC, Okeke IN, Lamikanra A, Adesina SK, Aboderin O. Antibacterial activity of aqueous extracts of selected chewing sticks. J Contemp Dent Pract. 2005 Aug 15;6(3):86–94.
15. Perumal Samy R, Ignacimuthu S. Antibacterial activity of some folklore medicinal plants used by tribals in Western Ghats of India. Journal of Ethnopharmacology. 2000 Jan;69(1):63–71.
16. Cruz-Vega DE, Verde-Star MJ, Salinas-González N, Rosales-Hernández B, Estrada-García I, Mendez-Aragón P, et al. Antimycobacterial activity of Juglans regia, Juglans mollis, Carya illinoensis and Bocconia frutescens. Phytotherapy Research. 2008;22(4):557–9.
17. Janakat S, Al-Fakhiri S, Sallal A-K. A promising peptide antibiotic from Terfezia claveryi aqueous extract against Staphylococcus aureus in vitro. Phytother Res. 2004 Oct;18(10):810–3.
18. Perez C, Pauli M, Bazerque P. An Antibiotic assay by the well agar method. Acta Biol Med Exp. 1990;15:113–5.
19. Viksø-Nielsen A, Christensen TMIE, Bojko M, Marcussen J. Purification and characterization of β-amylase from leaves of potato (Solanum tuberosum). Physiologia Plantarum. 1997;99(1):190–6.
20. Pereira JA, Oliveira I, Sousa A, Valentão P, Andrade PB, Ferreira ICFR, et al. Walnut (Juglans regia L.) leaves: Phenolic compounds, antibacterial activity and antioxidant potential of different cultivars. Food and Chemical Toxicology. 2007 Nov;45(11):2287–95.
21. Clark AM, Jurgens TM, Hufford CD. Antimicrobial activity of juglone. Phytotherapy Research. 1990;4(1):11–4.