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THE JOURNAL OF ALTERNATIVE AND COMPLEMENTARY MEDICINEVolume 7, Number 5, 2001, pp. 523–527Mary Ann Liebert, Inc.

Potential Use of Tea Extract as a Complementary

Mouthwash: Comparative Evaluation of TwoCommercial Samples

CHARLES O. ESIMONE, M.Pharm.,1 MICHAEL U. ADIKWU, Ph.D.,1

SUNDAY V. NWAFOR, M.Pharm.,2 and CHUKWUMEZIE O. OKOLO, B.Pharm.2

ABSTRACT

Objective: To evaluate the potential of using tea extracts as complementary mouthwash andto test the comparative efficacy of two commercial samples.

Design: A randomized controlled trial with 30 healthy human volunteers was carried out. Thsubjects were randomly assigned to 5 groups of 6 subjects per group. The ability of Ndu tea ®

(from Cameroon) and Lipton® tea (from Nigeria) to reduce colony forming units (CFU) in thliquid expectorated after 60 seconds of gargling from the mouth of the volunteers at 5 and 6minutes were evaluated. These were compared to the values obtained from bank water andMinty Brett® (thymol 0.047%), a standard antiseptic.

Setting: University of Nigeria, Nsukka, Enugu State, Nigeria.

Subjects: Thirty healthy human volunteers (18 males and 12 females, between 22–30 years oage) who met the eligibility requirement of being nonsmokers and not taking any other antimicrobial agent were selected for the study.

Result: Relative to the bank water, the results indicated that the hot water extract of both teassignificantly ( p , 0.05) reduced CFU per milliliter in the liquid expectorated after gargling a both 5 and 60 minutes. Minty Brett showed higher activity than both tea extracts; however, unlike Minty Brett both extracts still reduced the CFU per milliliter at time 60 minutes (an indication of longer duration of activity). The combination of the tea extracts with sodium lauryl sulfate (1.2% w/v), a surfactant and emulsifier, significantly increased the antimicrobial activityrelative to each tea alone. Comparatively, the activity of Ndu tea was found to be slightly highe

than that of Lipton tea but this was not significant ( p , 0.05).Conclusion: Lipton and Ndu tea extracts potently reduced the CFU per milliliter. This activ

ity was potentiated by sodium lauryl sulfate. Although Minty Brett had more potent antimicrobial activity, both tea extracts have longer duration of activity. The results indicate the potential usefulness of tea extracts as a complementary mouthwash.

INTRODUCTION

The tea plant (Camellia sinensis; Fam.,

Theacea) originated in the southwest partof China and has been cultivated for more than

3000 years. It is a perennial evergreen plant thais a semitree or shrub depending on the environment. Nearly one half of the world popula

tion consumes tea (Chen and Yu, 1994). Thfresh leaves picked from the tea plant ar

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processed into various kinds of tea including black tea, green tea, oolong tea, scented tea, etc., by means of different manufacturing methods(Chen and Yu, 1994).

The components of tea include caffeine,polyphenols, trace elements and vitamins (Mark,

1992). The mild stimulant effect and sense of well-being produced by tea have been attributedto its caffeine content (Mark, 1992). Tea polyphe-nols (one of the most important components of tea) mainly include catechins, anthocyanins, andphenolic acids (Mark, 1992). Many of the physi-ologic activities reported for tea extract have been found to be because of the polyphenol moi-ety (Tagg and Millin, 1975; Ikeda et al., 1992;Sakagami et al., 1992; Xu et al., 1992). The amount

of catechin in tea is approximately 12–24% andrepresents more than 50% of the total amount of tea polyphenols. Cadmium, cobalt, and fluoridehave been identified among the elements in tea(Horwitz and Vander Linden, 1974).

Tea extracts have been shown to have severaluseful antimicrobial effects. Extracts have beenshown to inhibit the growth of both gram-posi-tive and gram-negative bacteria including me-thicillin-resistant Staphylococcus aureus and phy-

topathogens such as Erwinia species (Toda et al.,1989; Hamilton-Miller, 1995). Tea extracts havealso been shown to inhibit the growth of clini-cal isolates of Helicobacter pylori in a manner sug-gesting bactericidal action (Diker and Hasceite,1994). Ethanol extract of Japanese green tea ef-fectively inhibits the growth of canine oral bac-teria and thus has been suggested to be usefulas an antiperiodentitis agent and/or flora con-trol agent in dogs (Isoghai et al., 1992). Work in

humans and animals suggests that tea reducesthe incidence of caries (Onishi et al., 1981, 1989;Otake et al., 1991). Trichophyton mentagrophytes,

Mycoplasma pneumoniae, Mycoplasma ovale, Tri-chophyton rubrum, and phytogenetic fungi butnot Candida albicans, Crytococcus neoformans, or

Mycoplasma salivarium were inhibited by tea ex-tracts (Okubo et al., 1991). Tea extracts have beenshown to prevent rotavirus and enterovirusfrom infecting monkey kidney cells in tissue cul-

ture (Mukoyama et al., 1991).There are suggestions that tea catechins may

have some commercial usefulness in the general

cost, availability, and widespread use, we wereprompted to evaluate two commercial tea extracts (Lipton,® Lever Bros., Nsukka, Nigeria, andNdu® tea, Camaroon Development Co., SouthWest Province, Camaroon) as mouthwashes.

MATERIALS AND METHODS

Culture medium

The medium used was nutrient agar (OxiodBasingstoke, Hampshire, England). This waprepared in accordance with the manufacturer’s specifications, divided into 10-mL quantities in Bijou bottles, and autoclaved at 121°Cfor 15 minutes. Sterile distilled water was usedfor all dilutions.

Tea sample

Two commercial tea samples were used: Lipton and Ndu tea (bought from Cameroon).

Antiseptic

Minty Brett® (Pharma-Deko PLC, LagosNigeria) containing thymol B.P 0.047% waused as a standard mouthwash.

Sterilization of materials

The petri dishes, wide-mouthed bottles, flasks25-mL beakers (wrapped with foil), test tubesand pipettes plugged with cotton wool were sterilized in a hot-air oven at 160°C for 1 hour.

Extraction of tea

Infusion of each of the tea leaves containing8% w/v of the dry powdered leaf was made

using distilled water boiled to 100°C. This wassoaked for 15 minutes. The infusion flask washaken intermittently to aid extraction. At theend of the 15 minutes, the infusion was filteredand the residue discarded. The extract was sterilized by autoclaving at 121°C for 15 minuteand allowed to cool before use. Prior to sterilization, each aqueous extract was divided intotwo portions. Sodium lauryl sulfate (MerckDarmstadt, Germany) was added to one por

tion at a concentration of 1.2% w/v.Evaluation of the antiseptic properties of

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were randomly assigned to 5 groups of 6 subjects per group. Cigarette smokers and those us-ing any antibiotics or any other mouthwashwere excluded from the study. The volunteerswere not allowed to eat during the test period.

For the blank test, 25 mL of sterile distilled

water was given to subjects to rinse theirmouths thoroughly for 60 seconds and the wa-ter was expectorated into sterile wide-mouth bottles. They were labeled “blank” and used asa negative control. Two hours after the blankgargling, the subjects in each group were given20 mL of the following to rinse their mouth for60 seconds: group 1: Lipton extract alone;group 2: Lipton extract plus sodium lauryl sul-fate (1.2% w/v); group 3: Ndu tea extract alone;

group 4: Ndu tea extract plus sodium laurylsulfate (1.2% w/v); and group 5: Minty Brett(20 mL).

The liquid was discarded after gargling forthe 60-second interval. Five and 60 minutes af-ter gargling, the mouth was rinsed again with25 mL of sterile distilled water and the liquidseparately expectorated into prelabeled wide-mouth bottles.

Estimation of microbial load

Tenfold serial dilutions of the different liq-uids were done and the dilutions that gave agood countable number of colonies were ob-tained. Exactly 0.1 mL of the sterile distilledwater dilutions of each sample was inoculatedinto a petri dish into which aseptic sterilemolten nutrient agar was added and the twosolutions mixed gently but thoroughly. The

seeded agar was allowed to set and then theplates were incubated at 37°C for 24 hours andthe colonies counted. The viable number of aer-obic colony forming units per milliliter (CFU)of each washing was determined. Mean CFUper milliliter for the three subjects in eachgroup at each stage of the washing (blank, 5,and 60 minutes) were calculated and the resultswere expressed as mean 6 standard error of the mean (SEM).

Statistical analysis

The significance of the difference for the CFU

RESULTS

The result from the blank trials indicated thathe expectorated liquid from the subjects in thevarious groups had different mean viable counexpressed as CFU per milliliter. Ndu and Lip

ton tea, respectively, decreased in a significan( p , 0.05) manner, the mean viable count. Thisactivity was considerably potentiated in eachcase by sodium lauryl sulfate (Table 1, Fig. 1)The ability of the two commercial tea productto reduce viable cell count was found to betime-dependent; hence, at the 5-minute interval, the order of activity was thus: MintyBrett . Lipton tea 1 SLS 5 Ndu tea 1 SLS .Ndu tea alone . Lipton tea alone; while at 60

minutes, the order was as follows: Lipton 1SLS .Ndu tea 1 SLS .Minty Brett . Liptontea alone . Ndu tea alone (Table 1, Fig. 1)These changes in CFU per milliliter at times 5and 60 minutes were statistically significantUnlike Minty Brett, for which the activity decreased at the 60-minutes interval, Ndu andLipton teas (alone and in combination withsodium lauryl sulfate) maintained a consistenincrease in activity. However, the antimicrobia

activity of Minty Brett was significantly highethan that of Lipton and Ndu teas (and theicombination with sodium lauryl sulfate) at 5minute times. In terms of comparative activityNdu tea was found to be slightly higher than(not significant) that of Lipton tea.

DISCUSSION

Mouthwashes are used to cleanse and deodorize the oral cavity. Generally, they containantibacterial agents and are most often used fotheir deodorizing, refreshing, and antiseptic effects (Nairn, 1980). The results of this study indicate that both types of tea used for the research had significant antimicrobial activity because they were able to reduce the viable celcount profoundly. Such antimicrobial propertyis a desired property of mouthwashes. Our re

sults further support the earlier suggestionthat components of tea may be useful in orahygiene (Tsunoda et al 1991) The finding tha

TEA EXTRACT AS MOUTHWASH 52

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remembered that a number of alkyl sulfates aravailable as surfactants but by far the most popular member of this group is sodium lauryl sulfate, which is used as an emulsifier and solubilizer in pharmaceutical systems (George et al1990) and in toothpastes. Therefore, it is possi ble that the synergistic antimicrobial action osodium lauryl sulfate with the tea extractmight have been the result of a reduction in sur

face tension on the microorganism, thereby enhancing the ease of attachment of tea polyphenols (the major antimicrobial component) onthe microbial cell walls and cell membranesThe ability of sodium lauryl sulfate-tea combination to exhibit antimicrobial activity highethan that of Minty Brett (a standard mouthwash) at 60 minutes is indicative of a veritablesubstitute with longer duration of action.

The slight difference in the antimicrobial po

tency (not significant) of the two commercial teextracts (Lipton tea, Ndu tea) could be becausof the differences in soil content geographica

ESIMONE ET AL526

TABLE 1. VIABLE CELL COUNT OBTAINED WITH THE DIFFERENT AGENTS AFTER

5 MINUTES AND 60 MINUTES

Time Mean viable count (CFU/mL)Group Agent (min) 3 106

6 SEM

1 Lipton alone

Blank 9.866

0.1205 6.27 6 1.4460 1.76 6 0.51

2 Lipton 1 SLSBlank 1.20 6 0.14

05 0.19 6 0.0260 0.06 6 0.02

3 Ndu tea aloneBlank 08.67 6 20.51

05 5.07 6 2.6660 2.44 6 2.23

4 Ndu tea 1 SLSBlank 3.37 6 0.36

05 0.54 6 0.3160 0.29 6 0.10

5 Minty BrettBlank 3.47 6 0.18

05 0.16 6 0.0860 0.56 6 0.54

n5 6CFU/mL, colony forming units per milliliter.

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ferences in the polyphenol content of the tea,which determines the antimicrobial properties.

CONCLUSION

Our study revealed that the tea extracts sig-nificantly reduced the microbial load in theoral cavity. This is an indication of the po-tential usefulness of tea extract as a comple-mentary mouthwash. The results also indi-cate that mixtures of aqueous tea extract andsodium lauryl sulfate have more reliable andefficient antimicrobial property than MintyBrett at prolonged duration (60 minutes)when used as a mouthwash or gargle. No sig-

nificant difference was noted in the efficacyof the two commercial tea extracts to decreaseviable cell count from liquid expectoratedfrom the mouth.

REFERENCES

Chen Z, Yu Y. Tea. In: Encyclopedia of Agricultural Sci-ence. Volume 4 S.W. Index, 1994;4:281–288.

Diker KS, Hasceife G. The bactericidal activity of tea

against Helicobacter pylori. Lett Appl Microbiol 1994;19:299–300.

George Z, Hans S, James S. Disperse systems. 18th edi-tion. In: Remington’s Pharmaceutical Sciences.1990:257–270.

Hamilton-Miller JMT. Antimicrobial properties of tea(Camellia sinensis L.). Antimicrob Agents Chemother1995;39(11):2375–2377.

Horwitz C, Vander Linden SE. Cadium and cobalt in teaand coffee and their relationship to cardiovascular dis-ease. South African Med J 1974;48:230–233.

Ikeda I, Imasato Y, Sasaki E, Nakayama M, Nagao H,

Takeo T, Yayaba F, Sugano M. Tea catechins decreasemicellar solubility and intestinal adsorption of choles-terol in rats. Biochem Biophys Acta 1992;1127:141–146.

Isoghai E, et al. Inhibitory effects of Japanese green teaextracts of canine oral bacteria. Bifid Bacteria Microflora1992;11:53–59.

Mark V. Physiological and clinical effects of tea. In: TeaCultivation and Consumption. London: Chapman andHall, 1992;707.

Mukoyama A, Ushijima H, Nishimura S, Koiki I, Toda MHara Y, Shimamura T. Inhibition of rotavirus infection by tea extracts. Jpn J Med 1991;44:181–186.

Nairn JG. Solutions, emulsions, suspensions and extracts

In: Remington’s Pharmaceutical Sciences. 16th edition1980:1445.

Okubo S, Toda M, Hara Y, Shimamura T. The antifungaand fungicidal activities of tea extract and catechin. Jpn J Bacteriol 1991;46:509–514.

Onishi M, Ozaki F, Yoshino F, Murakami Y. Experimental evidence of caries preventive activity of nonfluoridcomponent of tea. J Dent Health 1989;31:158–161.

Onishi M, Shimura N, Nakamura C, Sato C. A field teson the caries preventive effect of tea drinking. J DenHealth 1981;31:13–19.

Otake S, Makimura M, Kuroki T, Nihishara Y, Hirasaw

M. Anticaries effects of polyphenolic compounds from Japanese green tea. Caries Res 1991;25:438–443.

Sakagami H, Asano K, Hara Y, Shinamura T. Stimulationof human monocytes and polymorphonuclear cell iodination nd interleukin-1 production by epigallocatechingallate. J Leukocyte Biol 1992;51:478–483.

Tagg GV, Millin DJ. The nutritional and therapeutic valuof tea—A review. J Sci Food Agric 1975;26:1439.

Toda MS, Okubo R, Shinamura T. The bactericidal activity of tea and coffee. Lett Appl Microbiol 1989;8123–125.

Tsunoda T, Yamazaki T, Mukai M. Oral preparations con

taining catechins for periodontitis and halitosis contro Japanese patent JP 03218320. September 1991.

Xu Y, Ho CT, Amin SG, Han C, Ching FL. Inhibition otobacco-specific nitrosamine-induced lung tumorigenesis in A/J mice by green tea and its major polyphenoand antioxidants. Cancer Res 1992;52:3875–3879.

Address reprint requests toSunday V. Nwafor, M.Pharm

Department of Pharmacology and ToxicologyUniversity of Nigeria, Nsukk

Faculty of Pharmaceutical ScienceEngu Stat

Nigeria

E-mail: [email protected]

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