UNIVERSITAS INDONESIA

PERIONDONTITIS DISEASE TREATMENT

Final Exam

HOME GROUP B

GROUP PERSONNEL:

Chandra Dwi Prakoso (1206212445)

Farisa Imansari (1206212426)

Ramadhan Iskandar (1206212400)

Sri Dwi Aryani (1206212395)

DEPARTMENT OF CHEMICAL ENGINEERING

FACULTY OF ENGINEERING

UNIVERSITY OF INDONESIA

DEPOK 2015

LIST OF CONTENT

CHAPTER I INTRODUCTION ..................................................................................... 1

1.1. Background ........................................................................................................... 1

1.2. Problem Formulation ............................................................................................ 1

1.3. Objectives ............................................................................................................. 1

CHAPTER II DISCUSSION .......................................................................................... 2

2.1. Determining Bioactive Compound and Polymer ................................................. 3

2.2. Injection for Periodontal Disease Treatment ........................................................ 5

2.3. Drug Release Strategy .......................................................................................... 6

2.4. Preparation and Pre-Treatment of Each Component Used .................................. 6

2.5. Spray Drying of Gelatin and Propolis .................................................................. 7

2.6. Microparticle Testing ........................................................................................... 7

2.7. In Vitro Release of Propolis from the System ...................................................... 8

2.8. Antibacterial and Toxicity Testing ....................................................................... 8

CHAPTER III CONCLUSION ...................................................................................... 9

REFERENCE .................................................................................................................. 10

1

CHAPTER I

INTRODUCTION

1.1. Background

Periodontal disease is a world-wide prevalent chronic infection which is caused by accumulation of specific

microorganisms or group of microorganisms in the dental plaque, resulting in progressive loss of the

alveolar bone around the teeth with increased probing depth and recession or both. If left untreated it can

lead to loosening and subsequent loss of teeth. It prevails in all groups, ethnicities, races and both the gender.

The conventional plaque removal treatment known is scaling and root planning. This treatment removes

the plaque and biofilm mechanically by using hand-scaler. The disadvantage of this treatment is that it

causes inflammation few days after doing the treatment.

In order to avoid the inflammation, drug delivery system can be introduced to cure this disease. Injectable

drug delivery systems can be rapidly delivered using syringe without causing pain. It is used for the delivery

of antimicrobial agents directly into the periodontal pocket easily with therapeutic agents and reaches to

large population of pathogens inside the pocket.

1.2. Problem Formulation

What is the bioactive compound for treating periodontitis?

What is the suitable polymer for the drug carrier?

What method that can be applied for the encapsulation?

What is the most suitable preparations for the drug?

What is the strategy for the drug release?

What are the testing needed for the microsphere?

1.3. Objectives

To determine suitable active pharmaceutical ingredients for periodontitis disease

To determine most suitable method for drugs usage

To determine most suitable method for microencapsulation production

To determine practical strategy for drugs development research

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CHAPTER II

DISCUSSION

Step June July August September October November

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

Problem Identification

Literature Review

Determining Bioactive

Compound, polymer, and

method

Preparation and Pre-

treatment of each

component used

Spray-drying Encapsulation

Microparticle Testing

In vitro release profile

analysis

Antibacterial activity and

toxicity testing

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2.1. Determining Bioactive Compound, Polymer and Method

The health industry has always used natural products as an alternative, to the conventional

allopathic formulations available for the treatment of various afflictions. Propolis, a natural antibiotic is a

resinous yellow brown to dark brown substance that honey bees collect from tree buds, sapflows, shrubs or

other botanical sources to seal unwanted open spaces in the hive, protecting it from outside contaminants.

The main chemical classes present in propolis are flavonoids, phenolics and other various aromatic

compounds. Flavonoids are well known plant compounds that have antibacterial, antifungal, antiviral,

antioxidant and anti-inflammatory proprieties. Propolis has been used in general for various purposes and

has a promising role in future dentistry. The development propolis in global dentistry industry caused by it

potential to cope with several dentistry problem such as anticaries, antiplaque, chronic periodontitis, and

oral candiadiasis,

Anticaries

Few microorganisms found in the oral cavity are able to adhere to the teeth and, among these, a

limited group is cariogenic. The specific cariogenic microbiota consists of Streptococcus mutans,

Lactobacillus and some Actinomyces species. Based on literature reports showing that propolis resin is a

product with antiinflammatory and bactericidal activity, several in vitro and some in vivo studies have

demonstrated its potential use in the treatment of bacterial diseases. It was concluded in one study that the

propolis extract used as mouthrinse possesses antimicrobial activity against S. mutans present in the oral

cavity. The extract might be used as an alternative measure to prevent dental caries (De Carvalho DSA, et

al, 2007).

Antiplaque

Koo H et al studied the effect of a mouth rinse containing selected propolis on 3-day dental plaque

accumulation and polysaccharide formation. Six volunteers took part in a double-blind crossover study

performed in two phases of 3 days. During each phase the volunteers refrained from all oral hygiene and

rinsed with 20% sucrose solution 5 times a day to enhance dental plaque formation and wit mouth rinse

(placebo or experimental) twice a day. On the 4th day, the plaque index (PI) of the volunteers was scored

and the supragingival dental plaque was analyzed for insoluble polysaccharide (IP). The PI for the

experimental group was significantly less than for the placebo group. The experimental mouth rinse reduced

the IP concentration in dental plaque by 61.7% compared to placebo (p < 0.05). An experimental mouth

rinse containing propolis was thus efficient in reducing supragingiva plaque formation and IP formation

under conditions of high plaque accumulation.

Chronic Periodontitis

Researchers in Brazil have found that periodontal diseases are amendable to treatment by gree

propolis. A study was conducted on four patients at a periodontics clinic in southeastern Brazil who had

varyin degrees of dental problems: calculus, gingivitis, bleeding, fluid accumulation, gingival recession,

toot mobility, pus formation, and bone loss. Treatment consisted of daily tooth brushing with propolis and

washing the mouth with a propolis solution. The propolis was applied in certain periodontal pockets once

a week for five weeks. All the periodontal pockets irrigated with propolis showed a 95% decline in gingivitis

and pus. Because propolis is cheap and accessible to the population, its effectiveness I treating periodontal

disease is extremely relevant to public health. The authors therefore recommend that 10% Brazilian green

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propolis be used in conjunction with treatment of chronic periodontitis (Cairo de Maral). A study was done

in KLE‘s VK institiute of dental sciences, Belgaum, Karnataka, India, to explore the efficacy of propolis

extract as a subgingival irrigant in periodontal treatment. And it was conclude tha subgingival irrigation

with propolis extract as an adjuvant to periodontal treatment was more effective than scaling and root

planning both by clinical and microbiological parameters (Coutino AO, et al, 2009).

Oral Candiadiasis

According to Brazilian researchers, green propolis is effective against oral candidiasis. Green

propolis is collected from honeybees in southeastern Brazil. In one study, twelve patients were treated with

propolis. After cleaning their prosthesis and their oral cavity, they dried the infected area and applied the

propolis extract topically in candidiasis oral mucosa lesions with a swab, four times daily for a week. A

control group of six patients performed the same treatment with Nystatin, a standard antifungal product.

All 18 patients—whether treated with propolis extract or Nystatin—showed a remission of the candidiasis

lesion in less than three weeks: 11 patients after 7 days, and 6 patients after 15 days ( G.P. Rezende, et al,

2008 ). According to Estrela et al., ‘oily vehicles‘ become an issue if a calcium hydroxide paste is used as

an intracanal medicament because oily substances have low solubility in water and do not allow immediate

availability of the hydroxyl ions released from calcium hydroxide. Thereby, a less effective antimicrobial

action is expected. Otherwise, oily vehicles could be an option when calcium hydroxide is used as an

obturation agent. One could consider associating calcium hydroxide with propolis in order to add all

beneficial biological properties of propolis, particularly its antiinflammatory, immunomodulating,

antibacterial, antifungal and antiviral properties to those of calcium hydroxide Moreover, as an oily

substance, propolis may promote low-speed dissociation and diffusion when used as a component in an

endodontic paste for primary teeth. It is also important for endodontic compounds to accompany the

physiological resorption of primary teeth. The association of propolis with calcium hydroxide could

aggregate the benefits of each material. However, propolis would not jeopardize the antimicrobial activity

of calcium hydroxide. By testing a mixture of propolis and calcium hydroxide against a polymicrobial

culture extracted from primary tooth root canals, better results were observed than those obtained with

calcium hydroxide plus propylene glycol. Calcium hydroxide was chosen because it is well established as

an endodontic dressing, but its use is still controversial in primary teeth. However it was confirmed in this

study that association of calcium hydroxide with propolis can be beneficial in terms of antimicrobial activity

(G.P. Rezende., et al, 2008).

Our drug consist of two materials, propolis as an Active Pharmaceutical Ingredients (API) and

gelatin as a polymer or non-API. The chemical composition and biological properties of propolis

preparations may vary according to geographical location and different plant sources, as well as the

extraction procedure used (Sforcin, 2007). The references provided reported on differences in biological

activity between propolis preparations from different sources. Concentrations of the purported biologically

active constituents in some propolis preparations are low or undetectable in other propolis preparations

(Kumazawa et al., 2004; Bankova, 2005a, 2005b). The fact that propolis composition is very variable

creates a problem for standardization (Marcucci, 1995). A universal chemical characterisation of propolis

would be impossible (Bankova, 2005a). Because the term “propolis” is not characterised with respect to

the chemical composition, the botanical source as well as the chemical composition of the specific propolis

preparation should be clarified (Bankova, 2005a; Sforcin, 2007).

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In spite of propolis being commonly used in cosmetic and medicinal preparations owing to its

antiseptic, anti-inflammatory, and anesthesic properties, it is not completely innocuous because 1.2 to 6.6

patients who were patch-tested for dermatitis were sensitive to propolis. The main allergens were 3-methyl-

2-butenyl caffeate and phenylethyl caffeate, that is, components present in the poplar-type propolis. Clinical

allergy in humans is presented as contact dermatitis or oral mucositis, beekeepers being the most affected.

Nevertheless there has been a recent rise in this incidence among biocosmetic users, on account of the

increasing popularity of natural products such as propolis. According to these authors, patients with an

allergy to propolis may be at risk of cross-sensitization with balsam of Peru, a common allergen found in

flavoring agents, perfumed products, certain spices, and products that contain the peel of citrus fruit.

Therefore, propolis is a complex natural product with a great diversity of chemical structures and

subsequent biological activities, nevertheless, it is not completely innocuous and care must been taken,

mainly when such a product has a great diversity of origins. An absence of quality control may be pernicious

to human health

For non-API substance which is gelatin, the Food and Drug Administration (FDA) has affirmed

the safety of gelatin. However in December 2003, the first American case of bovine spongiform

encephalopathy (BSE) was confirmed in a dairy herd in the state of Washington. The discovery led to

measures to keep BSE from entering the food supply, measures that may soon impact softgel capsules for

dietary supplements. After this FDA issued guidance that charged gelatin importers, manufacturers, and

suppliers with determining the tissue, species, and country source of all materials. The guidance also banned

specific risk materials (SRMs), such as brains, spines, and spinal cords from use in gelatin. Bone and hides

from cattle showing any sign of neurological disease were prohibited regardless of origin.

Lastly for the price propolis and gelatin are quite expensive than some chemical drugs agent. For

the propolis the price is Rp. 120,000 per 6 mL, while for the gelatin the price is Rp. 91,000 per kg. However

by the efficence process the needs of raw materials can be reduced to get economical product price at the

end.

2.2. Injection for Periodontal Disease Treatment

The therapy of periodontal disease is mainly based on extensive oral rinses with solutions and

systemic administration of drugs. However, to obtain an effective concentration of therapeutic agents in the

pocket after systemic administration, repeated high doses over a prolonged period of time is required.

Hence, this type of treatment contains the risk of systemic side effects and the emergence of resistant strains

and superimposed infections. These problems have evoked an interest in the development of localized drug

delivery systems to offer prolonged delivery of therapeutic agents into the periodontal pocket.

Treatment now must focus on etiology, potential squeal, and longer-term ramifications of untreated

disease. Objectives of conservative periodontal treatment should include altering or eliminating microbial

etiology and the contributing factors for periodontal disease, halting disease progression and returning the

dentition to a state of health and comfort, obtaining host healing, and achieving gains in clinical attachment

levels.

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For these reasons, we used injectable drug delivery systems because can be rapidly delivered using

syringe without causing pain. It is used for the delivery of antibiotic agents directly into the periodontal

pocket easily with therapeutic agents and reaches to large population of pathogens inside the pocket. The

cost of the therapy is considerably reduced compared to the need of time for the placement. The effective

drug for periodontal disease by injection is microparticle which powder form and antibiotics used are

propolis and gelatin. There are 2 steps for periodontal disease treatment using injection. The steps are pre-

treatment and treatment. Usually, pre-treatment for periodontal disease is needed to cleaning the tooth

before the drug is injected. This method is commonly called “The Scaling and Root Planning”.

After pre-treatment was done, the next

step is inject the drug especially antibiotic using

Injector. By injection is a locally administered

antibiotic treatment for gum disease. It's a

powder composed of 100,000 tiny

microspheres that release antibiotics over time.

The microspheres are filled with the antibiotic

propolis and gelatin. Injection of antibiotic will

add to the pocket between your gum and tooth.

It's a painless procedure, and antibiotic

injection with SRP (Scaling and Root Planning)

can make your gum disease treatment more

effective by killing bacteria left behind after

SRP.

2.3. Drug Release Strategy

In order to get good release profile and suitable dosage of propolis extract, the gelatin and propolis

ratio is 6:1. We assume that every treatment taken by patient, about 0.5 gram of the drug injected into the

pocket. In every gram of the drug contains 4% extracted propolis, as we can calculate the propolis content

is 20 mg. This amount of propolis extract is considered allowable to be absorbed as drug for human body.

2.4. Preparation and Pre-Treatment of Each Component Used

Preparation of Extracted Propolis

Place chopped propolis 30% and ethanol 70% into a turbo extraction machine. Filter the liquid

through a filter paper.

Preparation of Propolis Microparticles

Propolis extractive solution was dispersed in a series of gelatin solutions, using dripping technique,

at 25◦C and with magnetic agitation by 30 min (Bruschi and Gremião, 2001). The final dispersions were

spray-dried in a BÜCHI Mini Spray Dryer model B-191 (Büchi, Switzerland) through the nozzle using a

peristaltic pump. The resultant dried products were collected and kept away from rehydration until further

tests. An experiment was developed using mannitol with the aim to reduce the microparticles aggregation.

The utilized amount of mannitol was 20% (w/w) in relation to the total amount to be obtained.

Figure 2.1 Injecting drug to the periodontal pocket

(sumber: google.com)

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2.5. Spray Drying of Gelatin and Propolis

Gelatin microparticles containing propolis (PM) were obtained by spray-drying.

Figure 2.2 Block flow diagram of production of propolis microparticle

Propolis extract (PE) was prepared with a propolis/ethanol ratio of 30/70 (w/w) by turbo extraction,

filtered through filter paper, and made up to the initial weight with the ethanol. PE was dispersed in a gelatin

solution containing 20% (w/w) of mannitol, using dripping technique, at 25ºC and with magnetic agitation

by 30 minutes. The amount of gelatin utilized was a function of the dryness residue of PE (6/1). The final

dispersion was spray-dried in a BÜCHI Mini Spray Dryer model B-191 (Büchi, Switzerland) the following

spray-drying conditions: inlet temperature (160ºC), feed rate (6%), aspiration (80%), and pressure (3%).

PPG-5- Ceteth-20 (PPG; 85% w/w) was warmed (50–60C) with slow stirring. Isopropyl myristate (IM;

10%w/w) and water (5%) were added to the PPG and the mixture was stirred until homogenization. Finally,

PM (particle size 2.48 ±0.81 µm) were mixed thoroughly into these preparations to form systems

containing 6.41% (w/w) microparticles, equivalent to 4% (w/w) of propolis extract, the concentration

normally utilized.

2.6. Microparticle Testing

Evaluation of Sedimentation Characteristics

Formulation was decanted into a 7.5-ml cylinder with a diameter of 1.0 cm. After 1, 2, 4, 8, 12, 24,

and 168 hours, the sedimentation volume F was determined. Formulations were evaluated according to the

number of movements necessary to convert the sediment to a homogeneous dispersion (Patel, Kennon, &

Levinson, 2001). To obtain an acceptable dispersion, the sedimentation volume F should remain at or above

0.9 for 1 hour, but a longer period is preferred for our purpose (Gabriëls & Plaizier- Vercammen, 2004).

Oscillatory Rheological Analysis

Oscillatory analysis of formulations was performed using an AR 2000 controlled stress/controlled

rate rheometer (T.A. Instruments, Surrey, England) in oscillation mode at 25 and 37 •} 0.1°C. Flow

properties of the periodontal formulations determine the ease of product administration into the periodontal

pocket and the (time-dependent) recovery of the products following administration (Jones et al., 2001).

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As the PPG concentration was increased, the consistency index of the systems increased significantly. The

steady shear behavior of the systems was also influenced by the temperature and the presence of propolis

microparticles. There was a significant decrease in the consistency index as the temperature was increased,

while the flow behavior index increased significantly. The presence of propolis microparticles led to a

significant increase in the consistency index and a significant decrease in the flow behavior index. The flow

properties of the systems, at temperatures of 25 and 37°C, are displayed graphically in Figures 3 and 4 in

appendix.

2.7. In Vitro Release of Propolis from the Systems

In vitro release of propolis from the

formulations was determined using a periodontal

pocket simulator apparatus (Figure 1 Appendix),

through a flow system. The release medium was

artificial saliva composed of potassium phosphate

dibasic (0.082%), potassium phosphate monobasic

(0.036%), sorbitol solution 70% (4.27%), potassium

chloride (0.063%), sodium chloride (0.087%),

magnesium chloride hexahydrate (0.013%), calcium

chloride dihydrate (0.007%), metilparaben (0.180%),

hidroxyethylcellulose 4400 (0.025%), and water

(95.24%) (Nakamoto, 1979). A periodontal pocket

simulator apparatus, based on a flow system, with

artificial saliva, was used to investigate the release of

propolis from the formulations. The results from literature are presented in Figure 2.3.

2.8. Antibacterial Activity and Toxicity Testing

Susceptibility tests were performed using ATCC strains Prevotella intermedia (33563), Prevotella

melaninogenica (25845), Porphyromonas gingivalis (33277), Actinobacillus actinomycetemcomitans

(29523 and 29522), Capnocytophaga gingivalis (33624), Fusobacterium nucleatum (10953) which were

cultured in enriched Brain Heart Infusion agar, for 5 days. Minimum Inhibitory Concentration (MIC) was

determined as the lowest concentration of the propolis extract, which inhibited the growth of the tested

microorganisms.

Multiple dose toxicity studies.

Suspensions prepared from ethanolic extracts of propolis were fed to 5-wk-old mice at doses of 2230 to

4000 mg/kg. After 2 wk of treatment, no deaths were noted, body weights had increased normally and no

abnormalities were found on necropsy (Kaneeda and Nishina, 1994).

Figure 2.3 The effects of PPG and isopropyl

myristate on the release of propolis

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CHAPTER III

CONCLUSION

Bioactive compound selected is propolis because it has antibacterial activity toward periodontal

microorganism

Suitable preparations for periodontal disease drug is injection, because can be rapidly delivered

using syringe without causing pain. It is used for the delivery of antibiotic agents directly into the

periodontal pocket easily with therapeutic agents and reaches to large population of pathogens

inside the pocket.

Propolis-gelatin microsphere obtained by spray drying method

Testing for the microparticle are sedimentation evaluation and rheological analysis, besides release

profile also analysed by using pocket simulator apparatus and using artificial saliva. Antibacterial

and toxicity testing also needed.

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REFERENCE

Bankova V, 2005a. Recent trends and important developments in propolis research. Evidence-based

Complementary and Alternative Medicine, 2, 29-32.

Bankova V, 2005b. Chemical diversity of propolis and the problem of standardisation. Journal of

Ethnopharmacology, 100, 114-117.

Coutino A O, Sanikop S. Propolis (Honeybee) extract as a subgingival irrigant in periodontal treatment.

KSDJ 2009; 28(2): 31-35.

De Carvalho Duailibe S A, Gonçalves A G, Ahid F J M. Effect of a propolis extract on streptococcus

Mutans counts in vivo. J Appl Oral Sci. 2007;15(5):42

G P Rezende, L R Costa, F C Pimenta, D A Baroni In vitro Antimicrobial Activity of Endodontic Pastes

with Propolis Extracts and Calcium Hydroxide:A Preliminary Study. Braz Dent J 2008;19(4): 301-305

Koo H, Cury JA, Rosalen PL, Ambrosano GM, Ikegaki M, Park YK. Effect of a mouthrinse containing

selected propolis on 3 day dental plaque accumulation and polysaccharide formation. Caries Res. 2002

Nov-Dec;36(6):445-8

Kumazawa S, Hamasaka T and Nakayama T, 2004. Antioxidant activity of propolis of various geographic

origins. Food Chemistry 84, 329-339.

Marcucci MC, 1995. Propolis: chemical composition, biological properties and therapeutic activity.

Apidologie, 26, 83-99.

Popova MP, Graikou K, Chinou I, Bankova VS. GC-MS profiling of diterpene compounds in

Mediterranean propolis from Greece. J Agric Food Chem. 2010;58:3167–76

Sforcin JM, 2007. Propolis and the immune system: a review. Journal of Ethnopharmacology, 113, 114

Tandon S. Textbook of pedodontics, 2 ed, Hyderabad (India): paras medical publisher

2008. P 404.

V.R. Santos. Oral candidiasis treatment with Brazilian ethanol propolis extract. Phytotherapy Research

2005; 19:652-654.