TJPS Vol.

42 (Supplement Issue) 2018

Thai Journal of Pharmaceutical Sciences (TJPS)

34th International Annual Meeting in Pharmaceutical Sciences and
2nd CU FPhS - RIKEN CDB Symposium
(IAMPS34 and 2nd CU FPhS - RIKEN CDB)
Formulation development of oral deodorant spray containing
Pinus merkusii heartwood extract
1 1 2 1,3
Alisa Yeesamun , Fueangfah Watthana , Worawan Saingam and Apirak Sakunpak *
1
Faculty of Pharmacy, Rangsit University, Pathum Thani, Thailand, 12000
2
The Herbal Medicinal Products Research and Development Center (Cooperation between Rangsit University and Harbin
Institute of Technology and Heilongjiang University of Chinese Medicine), Faculty of Pharmacy, Rangsit University,Pathum
Thani, Thailand, 12000
3
Department of Pharmacognosy, Faculty of Pharmacy, Rangsit University, Pathum Thani, Thailand, 12000

* Corresponding author: E-mail: apirak.s@rsu.ac.th

Keywords: formulation development; oral deodorant spray; Pinus merkusii

Introduction
1
Dental caries is considered to be the most frequent bacterial infectious diseases in humans .
2,3
Streptococcus mutans is an importance pathogen and is a common cause of dental caries . Currently, a
number of antibiotics are used for treating these infections. However, because these antibiotics are associated
4,5
with significant side effects . Plants have proven to be a valuable source for biologically active compounds.
Indeed, from ancient history some plant extracts have been used to treat various diseases. Our previous study
of antibacterial activity against S. mutans (S. mutans ATCC 12175, S. mutans NRPC 801 and S. mutans NRPC
804) of 22 ethanolic plant extracts revealed that three ethanolic extracts of Senna garrettiana, Pinus merkusii
and Dracaena lourieri showed the inhibitory activity against all bacteria strains. However, highest inhibitory
6
activity was observed with ethanol extract of P. merkusii heartwood . The purpose of this study was to develop
oral deodorant spray by mixing the extract of P. merkusii heartwood in order to produce anti-S. mutans effects.
In addition, the formulation was characterized for their physicochemical properties were also conducted.

Material and method

Chemicals
Ethanol, ethyl acetate and hexane (analytical grade) were obtained from Merck (Darnstadt, Germany).
Methanol (HPLC grade) was purchased from B&J (USA) Gentamicin, sodium chloride and dehydroabietic were
purchased from Sigma (St. Louis, MO, USA).

Microorganisms and media

S. mutans DMST18777 was obtained from Faculty of Pharmacy, Srinakharinwirot University, Thailand,
26120. Brain-Heart infusion (BHI) and agar were purchased from Becton, Dickinson and Company (MD, USA).

Preparation of extract
Heartwood of P. merkusii was collected from Loei Province, Thailand, in October 2016 and reference
voucher specimens were deposited at Faculty of Pharmacy, Rangsit University, Pathumthani, Thailand. The
plant was dried at 50 ºC for 24 h in a hot air oven and reduced to coarse powders. Dried plant 10 g was
extracted with ethanol, ethyl acetate and hexane (50 ml) by sonication method at room temperature for 30 min,
and then the extract passed through filter paper and concentrated with a rotary evaporator.

149
 TJPS Vol.42 (Supplement Issue) 2018
Preparation of inoculum
S. mutans was grown on Brain Heart Infusion agar plate (BHI) at 37 °C for 48 h in an anaerobic jar.

Minimum inhibition concentration (MIC)

Broth dilution method was used for evaluation of MIC of the extract. The extract was diluted in
10%ethanol with the concentration of 100 mg/ml and diluted with BHI to concentration of 1000-2 μg/ml. The
8 6
inoculum was adjusted with 0.85% NaCl to contain 10 CFU/ml, and then diluted 1:100 in media to contain 10
CFU/ml. Fifty-microliter of adjusted inoculum was added to each well plate then, ten-microliter of extract
solution was added and incubated at 37 °C for 48 h in an anaerobic jar. Gentamycin and dehydroabietic acid
were use as positive control.

Minimum bactericidal concentration (MBC)

The MBC was defined as the lowest concentration of the extract to kill microorganism. The incubation
mixtures of S. mutans and extract that showed positive result of inhibitory effect were streaked on each media
plate and incubated at 37 °C for 24 hours in anaerobic condition. The lowest concentration that did not show
any growth was taken as the MBC.

Formulation development
Optimization of excipients for oral spray
Optimization of sprays included selection and investigation of type and concentrations of co-solvent
system. The co-solvent system focuses on dielectric constant and solubility of P. merkusii heartwood extract.
The solubility study was presented. P. merkusii heartwood extract is freely soluble in ethanol thus set a
dielectric constant at semi-polar. The various co-solvents were water, ethanol, propylene glycol (PG) and
polyethylene glycol 400 (PEG 400).

Preparation of oral deodorant spray

The spray was prepared by simple solution method. First the co-solvent system was made by
dissolving P. merkusii heartwood extract in ethanol and stirring until a homogeneous solution. Then, the other
solvents such as water, PG and PEG 400 were added and mixed well. Oral spray from P. merkusii heartwood
extract was prepared by added other additives for good taste, good physical characteristics and stable. The
ingredients of oral spray are shown in Table 1.

Table 1. Preparation of oral spray from P. merkusii heartwood extract

Ingredients Amount (g) Function

P. merkusii heartwood extract 0.2 Active ingredient
Purified water 35.0 Co-solvent
Ethanol 10.0 Co-solvent
PG 15.0 Co-solvent
PEG400 40.0 Co-solvent
Menthol 0.3 Soothing agent
®
Xylitab 4.0 Sweetening agent
Peppermint oil 0.1 Flavoring agent
Paraben conc. 1.0 Preservative

Evaluation of oral spray

Physical appearance

P. merkusii heartwood extract oral spray was set aside at room temperature (30  2 °C) for 0, 7, 14 and
28 days, clarity of solution were observed.

150
 TJPS Vol.42 (Supplement Issue) 2018
pH
About 20 mL of oral spray solution was taken in a 30 ml glass beaker. The pH was determined using
pH meter (SevenCompact Mettler Toledo). The measurement of pH of each formulation was done in triplicate
and mean values were calculated. pH was determined for 0, 7, 14 and 28 days.

Qualitative analysis of dehydroabietic acid by high performance liquid chromatography

Qualitative analysis of dehydroabietic acid was analysed using a HPLC method, coupled with UV
detector set at 210 nm. The HPLC system consisted of a quaternary pump system (Agilent, 1260 VL),
autosampler (Agilent, 1260 TCC) and UV/VIS with diode array detector (Agilent, 1260 DAD VL). A reverse-
phase Zorbax C-18 column 4.6 mm i.d. x 100 mm was eluted by using a mixture (90:10 v/v) of methanol and
water as the mobile phase with a flow rate 1.0 ml/min. The injection volume was 10 µl. Validation parameters of
linearity, accuracy, and precision were confirmed for this method.

Results

Anti-S. mutans activities of ethanol, ethyl acetate and hexane extracts of P. merkusii heartwood are
shown in Table 2. All extracts showed the inhibitory activity against S. mutans. However, highest activity was
observed with hexane extract with the MIC value of 20.8 μg/ml and MBC value of 41.7 μg/ml. The result
suggested that hexane extract was capable of extraction anti-S. mutans compounds from P. merkusii
heartwood. Thus, the hexane extract was further to develop oral deodorant spray.

Table 2. MIC and MBC of P. merkusii heartwood extract

Solvent MIC (µg/ml) MBC (µg/ml)

Hexane extract 20.83 41.67
Ethyl acetate extract 83.33 166.67
Ethanol extract 666.67 666.67
Dehydroabietic acid 41.67 41.67
Gentamycin 0.52 16.67

The physical appearance of oral spray formulation was clear. P. merkusii heartwood extract was
dissolved in ethanol as presents a clear solution. pH of oral spray is 6.76  0.12. An evaluation of oral spray
was presented in Table 3. The chromatogram of P. merkusii heartwood extract oral spray was compared with
chromatogram of standard dehydroabietic acid (Figure. 1) and the content of dehydroabietic acid in oral spray
are shown in Table 4.

Table 3. Evaluation of oral spray

Days
Evaluation
0 7 14 28
physical appearances clear clear clear clear
pH 6.78 6.63 7.05 6.80

151
 TJPS Vol.42 (Supplement Issue) 2018
Dehydroabietic acid

Figure 1. Chromatogram of dehydroabietic acid (A) and P. merkusii heartwood extract oral spray (B)

Table 4. Content of dehydroabietic acid in oral spray

Test (n=3) Content of dehydroabietic acid (%w/w)

1 0000.0
2 0000.0
3 0000.0
Average  SD 0000.0  00000.0

Discussion
7
The extraction of the antimicrobial compounds depends on the polarity of the compounds . The result
show that the major anti-S. mutans compound in P. merkusii heartwood is a non polar compound which can be
extracted in n-hexane. Like in the present study the best result was obtained with n-hexane extract followed by
ethyl acetate and ethanol extract. Solubility study of P. merkusii heartwood extract was presented freely soluble
in ethanol, soluble in PG 400, sparingly soluble in PG, slightly soluble in glycerin, sorbitol and water. P.
merkusii heartwood extract, aqueous solubility of a chemical compound is not enough to be applicable in oral
spray formulations. Thus, different kinds of soluble techniques including co-solvency that applied in this study
to increase the solubility of extract. The co-solvent system were water, ethanol, PG and PEG 400. All solvents
were safe, available, less expensive and common solvents used in solution preparations. The system was
successes to solute extract. The physical appearance of oral spray formulation was clear. Menthol and
®
peppermint oil were created cooling sensation after spray in month. Xylitab made a sweet. All additive
promoted a good formulation. After 28 days, Oral spray was curtained in physical and chemical (pH).

Conclusion

These results suggest that P. merkusii heartwood extract may be a new potential source as natural
anti-S. mutans applied in pharmaceutical such as; oral deodorant spray.

152
 TJPS Vol.42 (Supplement Issue) 2018
Acknowledgments

The authors wish to thank the Faculty of Pharmacy and Sino-Thai Traditional Medicine Research
Center (Cooperation between Rangsit University, Harbin Institute of Technology, and Heilongjiang University
of Chinese Medicine), Rangsit University, Pathum Thani, Thailand, for all chemicals and instruments. This
research was partly funded by the Research Institute of Rangsit University, Pathum Thani, Thailand (Grant
No. 12/2560).

References

1. Venditti M, Baiocchi P, Santini C, Brandimarte C, Serr P,Gentile G, Girmenia C, Martino P. Antimicrobial

susceptibilities of Streptococcus species that cause septicemia in neutropenic patients. Antimicrob Agents
Chemother. 1989; (33): 580-2.
2. Isturiz R. Global resistance trends and the potential impact on empirical therapy. J Antimicrob Agents.
2008; (32) supple 4: 201-206.
3. Linder L, Andersson C, Marie-Louise S. Protoplast Formation and Localization of Enzymes in
Streptococcus mitis. Infect Immun.1983; 40(3): 1146-1154.
4. Katherine A, Bowden WGH, Richmond DA. Antibody binding to Streptococcus mitis and Streptococcus
oralis cell fractions. Arch Oral Biol. 2008; 53(2): 141-149.
5. Venditti M, Baiocchi P, Santini C, Brandimarte C, Serra P, Gentile G. Antimicrobial susceptibilities of
streptococcus species that cause septicemia in neutropenic patients. Antimicrob Agents Chemother.
1989; 33: 580-582.
6. Sakunpak A, Sueree L. Antimicrobial activity of 22 plant extracts against oral bacteria (Streptococcus
mutans). Thai J Pharm Sci. 2017; 41 (Suppl.): 153-156.
7. Bacon K, Boyer R, Denbow C, O'Keefe S, Neilson A, Williams R. Evaluation of different solvents to extract
antibacterial compounds from jalapeño peppers. Food Sci Nutr. 2017; 5(3): 497-503.

153