Areca Catechu

Areca catechu palm is a stem tall slender, single-trunked palm that has the ability to grow up to 30 meters, the common practice of the plant is by the use of the husk of Areca nut as herbal “chewing sticks” instead of tooth brushes to maintain oral health and hygiene, the seeds are well known for masticatory seed and are used in medicine as well (Jose et al., 2011; Joseph and Singh, 2008). The Areca nut is the seed of the Areca catechu that grows mostly in the tropical Pacific, Asia and parts of east Africa. The habit of chewing the Areca nut for its stimulating properties has been used in about one tenth of the world’s population making it one of the most consumed psycho active substance (Lingappa et al., 2011). Dried form of the Areca nut was claimed to strengthen gums, sweeten the breath, eliminate bad taste and act as dentifrices (Shwetha HR et al., 2017). It has been reported that the main components of Areca are polyphenols, fat polysaccharides and protein. Also the nuts contain alkaloids arecoline, tannic acid which was suggested that it can suppress bacteria in the mouth (Al-Bayati, 2016). It has been reported that Areca nut exerts a direct antimicrobial effect against oral bacteria including Streptococcus mutans, Streptococcus salivarius, Candida Albicans and Fusiform nucleatum (Bobby Cyriac et al., 2012; Shwetha HR et al., 2017)

Presence of Staphylococcus aureus and Escherichia coli in the oral cavity

The oral cavity is considered as a home to microbial communities with important implications for human health and disease, it contains more than 300 known species of bacteria(Bik et al., 2010; Wilson Melanie J et al., 1997). Some oral infections are caused by at least in part by Staphylococcus aureus e.g., angular cheilitis, periodontitis, and staphylococcus mucositis(G McCormack et al., 2015; Smith et al., 2003; SMITH et al., 2001). 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(Kenneth Todar, 2012; Ryan Mac, 2017).

Antibacterial activity of Areca Catechu

Many researches have reviewed the antimicrobial activity of the Areca nut, as in aqueous/methanolic or as in hot/cold extracts of the leaves, seeds and green husks from different regions of the world, and they showed a broad spectrum antimicrobial activity against Gram positive  and Gram –negative bacteria (Bhat Sarpangala et al., 2017; Negi and Dave, 2010; Pahadia et al., 2013; Rahman et al., 2014; Reena R Nelson Anthikat, 2009)

Materials and Methods

Bacterial Isolates

The bacterial isolates of Escherichia coli and Staphylococcus aureus were obtained from King Khalid UniversityHospital (KKUH), King Saud University. Riyadh, Saudi Arabia.

Plant material and preparation of extracts (aqueous and methanolic):

Fifty grams of Areca catechu seeds 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 (0.22 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 (0.22 µm) and kept at -20^oC for later use. The supernatant is referred to as methanolic extract (Janakat et al., 2004).

Agar well-diffusion test

The antibacterial activity was tested by 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. Then the plates were 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 (Perez C et al., 1990).

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 (Viksø Nielsen et al., 1997).

The broth media dilution method was used to determine the MIC of the extract of Areca catechu seeds; 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 in this study were tested for their sensitivity to aqueous and methanolic extracts of Areca catechu seeds.

Results and Discussion

In this study, the antibacterial properties of the Areca nut 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 possibility forming of the diameter of the inhibition zones resulting from exposure of the bacteria to the aqueous and methanolic extracts of Areca nut. 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) (Bobby Cyriac et al., 2012; Joseph and Singh, 2008; Pahadia et al., 2013; Rahman et al., 2014; Reena R Nelson Anthikat, 2009). Staphylococcus aureus was the most readily inhibited bacterium(Joseph and Singh, 2008; Rahman et al., 2014) followed by Escherichia coli (Bik et al., 2010) unlike the results of 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 (Bobby Cyriac et al., 2012),  while the inhibition of bacteria growth reported here for Areca catechu extracts are greater than those reported by (Chatragadda et al., 2017) using  Staphylococcus aureus and Escherichia coli.

The MICs for the aqueous and methanolic extracts of Areca nut 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 (Chatragadda et al., 2017).

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 Areca catechu seeds by agar-diffusion method at different concentrations.

* Inhibition zones in mm (Means of triplicate)

Table 2: Antibacterial activity of methanolic extract of Areca catechu seeds by agar-diffusion method at different concentrations.

*Inhibition zones in mm (Means of triplicate)

Table 3: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of aqueous extract of Areca catechu seeds.

MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration

Table 4: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of methanolic extract of Areca catechu seeds.

MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration

Conclusion and Recommendations

This study concluded thatthe methanolic extract of the Areca nut contains a potent antibacterial agent that is protein in nature and may be used in the treatment of some oral infections.

Although that this 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, we have some reservations towards the side effects that needs to be studied and evaluated more thoroughly. The results highlight the traditional use of “Areca nut” and some scientific validation of the claimed biological antimicrobial activity in vivo.

Further clinical trials are required to determine the efficacy of Areca catechu seeds extract on Escherichia coli or/and Staphylococcus aureus.

Acknowledgment

The author is thankful to King Khalid University Hospital for providing the bacterial isolate and to Prof. Suliman Ali Alharbi, College of Science for providing laboratory facilities to perform the research and to his guidance and expertise.

References

1. Al-Bayati N.J.M.  In-vitro antibacterial and antifungal effect of areca nut extract. Res. J. Pharm. Biol. Chem. Sci. 2016;7:282–286.
2. Amudhan M, Begum H, Hebbar K. A review on phytochemical and pharmacological potential of Areca catechu L. Seed. Int .J. Pharm Sci Res.  2012;3:4151–4157.
3. Bhat S.K, Sarpangala M, Devasya A.   Antimicrobial properties of areca NUT, Areca catechu, l: a review. Int. J. Res. Ayurveda Pharm. 2017;8:8–12. https://doi.org/10.7897/2277-4343.083134.
   CrossRef
4. Bik E.M, Long C.D, Armitage G.C, Loomer P, Emerson J, Mongodin E.F, Nelson K.E, Gill S.R, Fraser-Liggett C.M, Relman D.A.  Bacterial diversity in the oral cavity of ten healthy individuals. ISME J. 2010;4:962–974. https://doi.org/10.1038/ismej.2010.30
   CrossRef
5. Cyriac B.M, Vidya P, Varghese I, Shantaram M, Jose M.  Antimicrobial properties of Areca catechu (Areca nut) husk extracts against common oral pathogens. Int. J. Res. Ayurveda Pharm. 2012;3:81–84.
6. Chatragadda R, Mohanraju R, Karthick P, Kada N.M.  Efficacy and effect of Areca catechu nuts. J. Terr. Mar. Res. 2017;1:50–53.
7. McCormack G.M, Smith A.J, Akram A, Jackson M, Robertson D, Edwards G.  Staphylococcus aureus and the oral cavity: An overlooked source of carriage and infection? Am. J. Infect. Control. 2015;43:35–7. https://doi.org/10.1016/j.ajic.2014.09.015.
   CrossRef
8. Hernandez B.Y, Zhu X, Goodman M.T, Gatewood R, Mendiola P, Quinata K, Paulino Y.C. Betel nut chewing, oral premalignant lesions, and the oral microbiome. PloS One. 2017;12:e0172196. https://doi.org/10.1371/journal.pone.0172196.
   CrossRef
9. Hossain M.F, Anwar M, Akhtar S, Numan S.M. Adverse effects on health posed by consumption of Areca nut (Areca catechu L., family: Palmaceae). Int. J. Community Med. Public Health. 2017;2:357–360. https://doi.org/10.18203/2394-6040.ijcmph20151028.
   CrossRef
10. IARC, 2004. IARC Monographs [WWW Document]. Betel-Quid Areca-Nut Chewing Some Areca-Nut-Deriv. Nitrosamines. URL http://monographs.iarc.fr/ENG/Monographs/vol85/index.php (accessed 6.11.18).
11. Janakat S, Al-Fakhiri S, Sallal A.K.  A promising peptide antibiotic from Terfeziaclaveryi aqueous extract against Staphylococcus aureus in vitro. Phytother. Res. PTR. 2004;18:810–813. https://doi.org/10.1002/ptr.1563.
    CrossRef
12. Jose M, Varghese I, Shantaram M.   In Vitro Phytochemical analysis and Antioxidant activity in five selected herbal materials used for oral health and hygiene. Int. J. Appl. Biol. Pharm. Technol. 2011;2;95–103.
13. Joseph I, Singh A.J.A. 2008. Antimicrobial Activity of Selected Medicinal Plants, <em>Craetva magna</em> (Linn.), <em>Pongamiaglabra</em> (Linn.) and <em>Areca catechu</em> (Linn.). Ethnobot. Leafl. 2008.
14. Todar K.  2012. The Normal Bacterial Flora of Humans [WWW Document]. URL http://textbookofbacteriology.net/normalflora.html (accessed 6.12.18).
15. Lalitha P, Arathi K.A, Shubashini K, Sripathi, Hemalatha S, Ponnusamy J.  Antimicrobial activity and phytochemical screening of an ornamental foliage plant, Pothosaurea (Linden ex Andre). Int. J. Chem. 2010;1: 63–71.
16. Lingappa A, Nappali D, Sujatha G, Shiva. 2011. Areca nut: to chew or not to chew? [WWW Document]. URL https://web.b.ebscohost.com/abstract?direct=true & profile=ehost& scope=site& authtype=crawler& jrnl=09762663&AN=78583133&h=kJwjE4WGayPHy1cqflpRQC6n0 LNn6ygf428J xMQP84TRWYNR% 2fMfwfTe% 2bAJDY3pf9rLkeMIcxuO6LVjHVDkLgeg% 3d%3d&crl=f& result Ns=Admin Web Auth & result Local=Err Crl Not Auth & crl hashurl=login. aspx% 3 f direct % 3 dtrue % 26 profile % 3 dehost%26scope %3 dsite % 26 authtype %3 dcrawler % 26 jrnl % 3 d09762663% 26AN% 3d78583133 (accessed 6.12.18).
17. Negi B.S, Dave B.P. In Vitro Antimicrobial Activity of Acacia catechu and Its Phytochemical Analysis. Indian J. Microbiol. 2010;50:369–374. https://doi.org/10.1007/s12088-011-0061-1.
    CrossRef
18. Pahadia A, Gawde R, Agrawal S.  Antimicrobial activity of hydro alcoholic extract of Areca catechu. 2013;8.
19. Perez C, Pauli M, Bazerque P.  An Antibiotic Assay by Agar Well Diffusion Method. 1990;15:113–5.
20. Pithayanukul P, Nithitanakool S, Bavovada R. Hepatoprotective potential of extracts from seeds of Areca catechu and nutgalls of Quercusinfectoria. Mol. Basel Switz. 2009;14:4987–5000. https://doi.org/10.3390/molecules14124987.
    CrossRef
21. Rahman M, Sultana P, Islam S.M, Mahmud M, Rashid M.M, Hossen F. Comparative Antimicrobial Activity of Areca catechu Nut Extracts using Different Extracting Solvents. Bangladesh J. Microbiol. 2014;31:19–23. https://doi.org/10.3329/bjm.v31i1.28460.
    CrossRef
22. Reena R, Anthikat N. Study on the Areca Nut for its Antimicrobial Properties. J. Young Pharm. 2009;1:42–45. https://doi.org/10.4103/0975-1483.51874.
    CrossRef
23. Mac R. 2017. What Are the Different Types of Bacteria Found in the Human Mouth? [WWW Document]. LIVESTRONG.COM. URL https://www.livestrong.com/article/161300-what-are-the-different-types-of-bacteria-found-in-the-human-mouth/ (accessed 6.12.18).
24. says M.S. 2017. جوزة الفوفل (Areca nut).
25. Shwetha H.R,Babu C.P.  ArecanutAs An Elixir For Dental Caries?  IOSR J. Dent. Med. Sci. 2017;16:36-40.
26. Smith A.J, Jackson M.S, BAGG J.  The ecology of Staphylococcus species in the oral cavity. J. Med. Microbiol. 2001;50:940–946. https://doi.org/10.1099/0022-1317-50-11-940.
    CrossRef
27. Smith A.J, Robertson D, Tang M.K, Jackson M.S, MacKenzie D, Bagg J. Staphylococcus aureus in the oral cavity: a three-year retrospective analysis of clinical laboratory data. Br. Dent. J. 2003;195:701–703. https://doi.org/10.1038/sj.bdj.4810832.
    CrossRef
28. Viksø‐Nielsen A, Christensen T.M.I, Bojko E.M, Marcussen J.  Purification and characterization of β-amylase from leaves of potato (Solanum tuberosum). Physiol. Plant. 1997;99:190–196. https://doi.org/10.1111/j.1399-3054.1997.tb03448.x.
    CrossRef
29. Melanie W.J, Weightman A .J, Wade W.G. 1997. Applications of molecular ecology in the characterization of… : Reviews in Medical Microbiology [WWW Document]. URL https://journals.lww.com/revmedmicrobiol/abstract/1997/04000/applications_of_molecular_ecology_in_the.5.aspx (accessed 6.12.18).