Formulation of Anti-plaque Toothpaste Ekstrak Gambir

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Jurnal Farmasi Indonesia Vol. 5 No. 2 Juli 2010: 70 -77

70

FORMULATION OF ANTI-PLAQUE TOOTHPASTEFROM STANDARDIZED GAMBIR EXTRACTAND ITS ANTIMICROBIAL ACTIVITY

Henny Lucida, Rustini, Dian Saufitri, Dachriyanus

Faculty of Pharmacy Andalas University, Padang

Correspondence: Henny Lucida, PhD, AptFakultas Farmasi Universitas Andalas, Kampus UNAND Limau Manih Padang,

Indonesia, email: [email protected]

ABSTRAK

Telah dilakukan formulasi pasta gigi ekstrak gambir terstandarisasi serta uji dayaantimikrobanya terhadap pertumbuhan bakteri penyebab plak Streptococcus mutans. Ujipendahuluan menunjukkan bahwa ekstrak gambir terstandarisasi pada kadar 7% memilikidiameter hambat optimal terhadap pertumbuhan mikroba uji. Berdasarkan ini dibuat 3formula pasta gigi dengan kadar ekstrak gambir terstandarisasi sebesar 7% dengankosentrasi kalsium karbonat (abrasif) yang bervariasi, berturut-turut 37, 42 dan 47%. Semuaformula diuji sifat-sifat farmasetik dan aktivitas antimikrobanya, sebagai pembandingdigunakan pasta gigi “F”. Hasil evaluasi sifat farmasetika sediaan menunjukkan semuaformula memenuhi persyaratan pastagigi yang baik. Formula 3 (F3) memberikan diameter

hambat paling baik (19,25 + 0,50 mm), sedangkan pembanding “F” memberikan diameterhambat (20,0 + 0,00 mm). Terdapat perbedaan bermakna diameter hambat antar perlakuan(basis versus pasta gigi ekstrak gambir) pada taraf kepercayaan 0,05.

Kata kunci: ekstrak gambir, katekin, pasta gigi, antiplak, Streptococcus mutans

ABSTRACT

A toothpaste containing standardized gambir extract had been formulated and anantimicrobial assay of the toothpaste against Streptococcus mutans had been done.Preliminary study showed that the concentration of gambir extract of 7 % obtained anoptimal range of inhibition of S. mutans growth. Three formulas were then prepared, whichcontained 7 % extract and various concentrations of calcium carbonate (37, 42 and 47 %respectively). The formulas were subjected to evaluation procedures including thepharmaceutical properties and the antimicrobial activity in comparison to commercial formula“F”. Results indicated that all formulas had good pharmaceutical properties. F3 showed thebest inhibition against the growth of S mutans (diameter of inhibition = 19,25 + 0,50 mm)while that of the commercial toothpaste “F” was (20,0 + 0,00 mm) , which was significantlydifferent from that of the toothpaste base at probability 0.05.

Keywords: gambir extract, catechin, anti-plaque, toothpaste, Streptococcus mutans

INTRODUCTION

Gambir, the dried extract of theleaves of Uncaria gambir (Hunter)

Roxb., has been the prime commodityof West Sumatera province. The

province has supplied 80% of the totalgambir produced in Indonesia andexported to other countries through

India and Singapore. Gambir has alsobeen used for long time by local people

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Formulation of anti-plaque toothpaste from gambir extract and its antimicrobial activity

(Henny Lucida, Rustini, Dian Saufitri, Dachriyanus)

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to be chewed with piper bettle leaveswhich was believed to strengthen the

teeth.Dental caries has been a problem

found in people of developing countriessuch as Indonesia. This was initiated bydental plaque formation and may leadto gingivitis. The incidence isassociated with component of diet, lifestyle and dental hygiene which maycreate serious problems if not treatedproperly (1,2). There have been severalpublications on the effectiveness ofsome natural product compounds forthe prevention of dental caries.

Gambir was reported as potentialinhibitor of dental plaque formation(3,4). This was suggested to be due tothe polyphenolic groups of tannins andflavonoids in gambir. The activity oftannins and flavonoids in the inhibitionof dental plaque formation is associatedwith inhibition of the synthesis ofinsoluble glucans catalyzed byglucosyltransferase (GTF) enzyme

from Streptococcus mutans (3, 5, 6).Hattori et al (1990) reported thatpolyphenols in cocoa, coffee and teashowed an inhibition on GTF activity atconcentration 1 – 10 mM. In addition,Badria dan Zidan (2004) reported thattannins, alkaloids dan flavonoids innatural products showed a weak tomoderate inhibition (< 25 – 50 %)against S. mutans strain atconcentration range of 6,25 – 125

g/Ml (7). Moreover, polyphenols from

green tea inhibited the growth of S.mutans at concentration 250 – 1000

g/mL (5).Gambir contains catechin as a major

component which gives its specifictaste and odor. Being a polyphenoliccompound, catechin shows antioxidantand antibacterial activities. Therefore itis potential to be formulated as cancerchemopreventive agents (8),antibactericidal agents (7), and as adrug for diseases associated with free

radical formation such as prematureaging, alzheimer, cardivascular etc.

Figure 1. Chemical structure ofcathecin

The Laboratory of Natural ProductChemistry of Faculty of Pharmacy Andalas University has been able topurify catechin from gambir extract (9).The product was a standardized gambirextract because the catechin contentwas more than 80 % compared tocatechin standard. This article reports astudy on the formulation of a toothpasteof the standardized gambir extract forthe prevention of dental plaque. Apreliminary study to determine the

effective inhibition concentration of thestandardized extract against S. mutanswas done, followed by formulation oftoothpastes and antimicrobial assay ofthe toothpastes against S. mutans incomparison to commercial ones.

MATERIALS AND METHODS

Gambir extract was obtained fromthe Laboratory of Natural ProductChemistry, Faculty of Pharmacy

Andalas University, catechin standardwas from PT. Nusantara Beta FarmaPadang. A spectrophotometric UV-Visible method (Shimadzu 1601) wasused to determine catechinconcentration. Validation of theanalytical method showed a linearregression between concentration ofcatechin / standardized gambir extractversus absorbance ( y = 0.00604 +13.16 x ; r = 0.999 ).

The gambir extract was subjected to

standardization procedures (10) suchas:Morganoleptics, determination of

OOH

OH

OH

OH

OH

A B

C




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water loss on drying, determination ofash content, determination of the

solubility in water and ethanol 96%, anddetermination of catechinconcentration.

Bacterials used were obtained fromBalai Laboratorium KesehatanYogyakarta which was identified andthen confirmed by using chemical andbiochemical methods. A series ofsolution containing standardized gambirextract at concentrations of 1, 2, 3, 4, 5,6, 7, and 8 % respectively were put into

paper disks and then inoculated on thenutrient agar medium containing S.

mutans. The petridishes were thenincubated at 37 oC for 24 hours. Theinhibition diameter of the standardizedgambir extract of each solution wasmeasured.

Three formulas of toothpaste weremade (Table 1) which contained 7% thestandardized gambir extract andvarious concentrations of calciumcarbonate (37; 42 and 47 %respectively) as abrasive.

Table 1. Composition of the standardized gambir extract toothpastesIngredient (%) Phase

(Function)Formulae

F1 F2 F3

Glycerin A(humectant)

23 23 23

Sodium Carboxy MethylCellulose (NaCMC)

A (gellingagent)

0.5 0.5 0.5

Aqua destillata B (solvent) 20.02 15.02 10.02Sodium Metabisulphite B

(antioxidant)1 1 1

Sodium Saccharine B(sweetener)

0.5 0.5 0.5

Standardized Gambir Extract Activesubstance

7 7 7

Nipagin C(preservative)

0.18 0.18 0.18

Calcium Carbonate C (abrasive) 37 42 47Colloidal Silica Dioxide C (gelling

agent)3.5 3.5 3.5

Glycerine D(humectant)

5 5 5

Sodium Lauryl Sulphate D (surfactant) 2 2 2Menthol D (corrigen

odoris)

0.3 0.3 0.3

To make the toothpaste, NaCMCwas mixed with glycerine untilcompletely wet (phase A), then watercontaining sodium metabisulphite andsodium saccharine (phase B) wasadded, stirred to make a gel mass.Standardized gambir extract (7 %) wasadded to the gel mass. Separately,nipagin was mixed with colloidal silicadioxide and calcium carbonate

homogenously (phase C). Phase C wasthen added to phase A and B which

were stirred to form a paste. Nipaginsolution in ethanol and sodium laurylsulphate in glycerin (phase D) wereadded to the paste and stirredhomogenously.

The toothpastes were thensubjected to evaluation proceduresincluding organoleptics, homogeneity,physical stability at room temperature,pH, spreading ability, skin irritation,

particle size determination,concentration of catechin and





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antimicrobial assay of the toothpastesin comparison to commercial (“F”)

toothpaste. A good formula was chosenbased on the pharmaceutical propertiesand the antimicrobial activity of thetoothpastes.

RESULTS AND DISCUSSION

Gambir extract was obtained bymaceration with ethyl acetate andpurified by using hot water.Standardization procedures showed

various physicochemical characteristicsof the extract (Table 2) with the

average catechin concentration of(88.89 % + 1.088). There were onlyquality requirements for gambir (11) butnone for gambir extract. The catechinlevel of the extract used in this studysupported a good quality ofstandardized gambir extract. Thus, thisstandardized gambir extract was usedas an active ingredient for antibacterialtoothpaste.

Table 2. Physicochemical characteristic of gambir extract obtained

No. Physicochemicalcharacteristics

Requirements for 1st quality gambir *)

Observed

1. Organoleptics Powder, yellow – brownish yellow,specific smell

Powder, brownishyellow, specificsmell

2. Solubility- Water

-Aethanol- Ethyl acetate

-

--

Sparingly soluble (1: 92)Soluble (1 : 1.8)Soluble (1 : 4.2)

3. Water loss on drying(% w/w)

< 14 13

4. Ash content (% w/w) < 5 0.15. Catechin content (%

w/w)- 88.89 + 1.088

*) SNI for gambir, 2000

Identification procedures on thebacterials used in this study showedthat the they were gram positive, did

not produce catalase enzyme, -hemolysis positive, oxidation testnegative, grew well in a bacitracincontaining medium but did not grow in6.5% NaCl broth medium. Theseresults supported its identity as Smutans (12).

An antimicrobial assay ofstandardized extract gambir against Smutans showed that the inhibitiondiameters of the extract over theconcentration of 1 % - 8 % were in therange of 6.00-12.67 mm (Table 3).

Concentration of 7 % was chosen asoptimum because the toothpaste

formula of F1 with concentration ofextract from 1- 8% showed the bestinhibition (17.69 mm) (Table 3).

Formulation of toothpastes was thendone with the concentration ofstandardized extract gambir 7% atvarious abrasive (CaCO3)concentrations (Figure 2). Beside theantibacterial active agent, the presenceof an abrasive determined the efficacyof a toothpaste in cleaning teeth. Abrasive was usually used at the rangeof 30-50%. The use of abrasive atvarious concentrations will result inviscosity differences and maybe theeffectiveness of the toothpaste. These

might correlate with the diameter




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inhibition of each formula againstplaque causing bacterials.

Figure 2. The antiplaque toothpastesat various abrasive

concentrations (F1=37%,F2=42% and F3=47%)

All formulas (F1, F2 and F3) showedviscous semisolid form with brown color

and specific smell (Figure 2). Thephysicochemical and pharmaceuticalproperties of the toothpaste before andafter 6 week storage were presented inTable 4. Spreading ability of thetoothpastes was determined usingmanual extensometer which value wasinversely proportional to viscosity. Thespreading value decreased with anincrease in abrasive concentrationbecause the viscosity increased. Thespreading value of F1, F2 and F3 was

1.085; 0.840 and 0.505 cm2

respectivelyand that of commercial toothpaste “F”was 0.695 cm2.

Table 3. Antimicrobial activity against the growth of S mutans

Concentration ofstandardized gambir

extract (%)

Diameter of inhibition (mm)

Standardizedgambirextract

ToothpasteF1

1 6.00 13.50

2 6.00 14.003 7.50 14.254 7.60 14.005 9.83 15.306 9.67 16.087 11.16 17.608 12.67 17.60

* Diameter of inhibition of the toothpaste base = 13.40 mm

There was a slight increase in thepH of toothpaste after storage, althoughit remained in the range of salivary pH

(5.6 – 7.6) (13). The change in pH maycorrelate with a decrease in catechinconcentration during storage. Catechin,a weak acid, degraded by oxidation(14). There might be H+ consumedduring the degradation resulted in anincrease in pH. Besides, S. mutans grew optimally at acidic pH values byproducing an acid through the activityof glucosyltransferase enzyme onsucrose. This would decrease the pH ofsaliva and resulted demineralization of

enamel at the surface of the teeth.Therefore, a toothpaste with a neutral

to weak basic pH value will beadvantageous.

There was a decrease in catechin

concentration after 6 weeks storage ( 2-10%) which might be due todegradation of catechin in gambirextract by oxidation, even thoughsodium metabisulphite as antioxidanthas beed added. This was supportedby the change in color from pale brownto darker. The antioxidant atconcentration 1% might not be effectiveto protect catechin from degradation.

Determination of antimicrobialactivity against S mutans by diffusion

agar method (Table 5 & Figure 3)showed that the toothpaste base itself





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had a diameter inhibition (13.38 – 13.75mm) against S mutans, this might be

due to the presence of sodium laurylsulphate and menthol in the base. Theinhibition diameter of the toothpasteswere in the range of 17.75-19.25 mm(Table 5 & Figure 3)) which considered

as strong activity antibacterial (morethan 17 mm) (15). Toothpaste F3

showed the best inhibition (19.25 +0.50 mm) which correlate with thehighest catechin concentration found inF3 (Table 4).

Table 4. The physicochemical and pharmaceutical properties of the standardizedgambir extract (7%) toothpastes

No.

Parametersevaluated

The physicochemical and pharmaceutical properties of

Tootpaste F1 Tootpaste F2 Tootpaste F3

A B* A B* A B*

1. Organoleptics Palebrown Brown Palebrown Brown Palebrown Brown

2. pH 6.83 7.57 6.90 7.53 6.90 7.503. Spreading

ability (cm2)**1.085 - 0.840 - 0.505 -

4. Mean particle

size (m)

8.32 - 7.20 - 7.40 -

5. Irritation test No - No - No -6. Catechin

concentration(%)

80.20 +0.78

78.37 +1.22

79.10 +0.79

76.18 +1.33

82.22+ 1.15

72.04 +0.63

7. Antimicrobial

activity(diameter ofinhibition(mm))***

17.75 +

0.50

- 18.30 +

0.37

- 19.25

+ 0.50

-

*storage condition: room temperature, protected from light for 6 weeks**the spreading area of 0.5 g toothpaste at 20 g loading for 1 minute***diameter of inhibition of commercial toothpaste “F” was (20.00 + 0.00)

-: not determined

Table 5. The diameter of inhibition of 7% gambir extract toothpastes (F1, F2and F3), toothpaste base (B1, B2 and B3), the gambir extract (E)

and commercial toothpaste “F” (K+) against the growth of S. mutans No. Treatments Inhibition diameter (mm)

1 2 3 4 Average+ SE

1. B1 13.0 13.5 13.0 14.0 13.38 + 0.472. F1 17.0 18.0 18.0 18.0 17.75 + 0.503. B2 14.0 13.0 14.0 14.0 13.75 + 0.504. F2 18.5 18.7 18.0 18.0 18.30 + 0.375. B3 13.0 13.0 14.0 14.0 13.50 + 0.576. F3 19.0 19.0 19.0 20.0 19.25 + 0.507. K (+) 20.0 20.0 20.0 20.0 20.00 + 0.008. 7% Gambir Extract 13.0 14.0 13.0 13.0 13.25 + 0.50

9. K (-) / DMSO 0.0 0.0 0.0 0.0 0.00 + 0.00




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Figure 3. The diameter of inhibition of 7% gambir extract toothpastes (F1, F2and F3), toothpaste base (B1, B2 and B3), the gambir extract (E)and commercial toothpaste “F” (K+) against the growth of S. mutans

Data in Table 5 (n = 4) showed theinhibition diameter of all treatments inthis study (the toothpaste’s base, the

gambir extract toothpaste, positive andnegative controls, total = 9). Statisticalanalysis of the data showed that therewas significant different betweentreatments (B1 vs F1; B2 vs F2 and B3vs F3) at p = 0.05 (Fcalc = 15.64; Ftab= 2.34), but no significant differentamong repetition (Fcalc = 0.08).

CONCLUSION

The standardized gambir extract

toothpaste (7%) containing abrasive atvarious concentrations (37, 42 and 47%respectively) showed an inhibitionagainst the growth of S mutans withdiameter inhibition of 17.75 – 19.25mm. These results supported thepotential use of this Indonesianoriginally natural product as an antiplaque agent. Further studies wereneeded to find an antioxidant to preventdegradation of catechin in thestandardized gambir extract.

REFERENCES

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5. Hattori M, Kusumoto IT, Namba T,Ishigami T, Hara Y. Effect of tea

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Formulation of Anti-plaque Toothpaste Ekstrak Gambir

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