BULE HORA UNIVERSITY

COLLEGE OF NATURAL AND COMPUATIONAL SCIENCE

DEPARTMENT OF BIOTECHNOLOGY

A PROPOSAL SUBMITTED TO DEPARTMENT OF BIOTECHNOLOGY

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF BACHELOR OF SCIENCE IN BIOTECHNOLOGY.

TITLE: ANTIMICROBIAL ACTIVITIES OF SANICULA ELATA LEAF

AGAINST DENTAL CARIES BACTERIA
BY:- ID NO

BERHANU BERIHUN ………………………………………………RU4574/13

ADVISOR:-Mr. NIGUSSE HOTESSA

BULE HORA, ETHIOPIA

JANUARY, 2016 E.C

 Table of Contents

List OF Table...........................................................................................................III

Least of Abbreviation...............................................................................................III

List of Figure............................................................................................................III

CHAPTER-ONE........................................................................................................1

1. INTRODUCTION..................................................................................................1

1.1.BACK GROUND OF STUDY........................................................................1

1.2 Statement of the Problem.................................................................................2

1.3. Objectives of the Study...................................................................................3

1.3.1. General objectives.......................................................................................3

1.3.2. Specific Objectives.......................................................................................3

1.6. Significance of the Study................................................................................3

Chapter-Two..............................................................................................................5

2. Literature Review...................................................................................................5

2.1.Description and Distribution Sanicula Elata....................................................7

CHAPTER-THREE...................................................................................................8

3. MATERIALS AND METHODS...........................................................................8

3.1.Study design and setting...................................................................................8

3.2.Sample collection and culturing dental specimen............................................8

3.3.Bacterial Isolation and Characterization..........................................................9

3.3.1.Bacterial isolation.........................................................................................9

3.3.2. Dextran Production Test..............................................................................9

3.3.3 The Fermentation of Different Carbohydrates Sources...............................9

3.4. Preparation of crude extract..........................................................................10

3.5. Antimicrobial Assay......................................................................................11

4. Work Plan and Budget Breakdown......................................................................12

 4.1. Work plan......................................................................................................12

4.2. Budget Breakdown........................................................................................13

References................................................................................................................14
List OF Tables

Table 1. Work Plan......................................................................................................20

Table 2. Budget Breakdown.........................................................................................21

List of Abbreviation
CAM: Complementary and Alternative Medicine
IZ: Inhibition zone
MBC: Minimum bactericidal concentration
MIC: Minimum inhibition concentration
 CHAPTER-ONE

1. INTRODUCTION
1.1.BACK GROUND OF STUDY
The mouth is colonized by 200 to 300 bacterial species, but only a limited number of
these species participate in dental decay (caries) or periodontal disease. Dental decay
is due to the irreversible solubilization of tooth mineral by acid produced by certain
bacteria that adhere to the tooth surface in bacterial communities known as dental
plaque. The tooth surface normally loses some tooth mineral from the action of the
acid formed by plaque bacteria after ingestion of foods containing fermentable
carbohydrates. This mineral is normally replenished by the saliva between meals
(Philip et al., 2000). However, when fermentable foods are eaten frequently, the low
pH in the plaque is sustained and a net loss of mineral from the tooth occurs. The
major pathway is concerned with energy metabolism; in this process, the enzyme
invertase splits sucrose into its component glucose and fructose molecules, which
are then converted to lactic acid by the glycolytic pathway. Streptococcus mutans is
the main cause of dental decay. Various lactobacilli are associated with progression
of the lesion. This low pH selects for aciduric organisms, such as S mutans and
lactobacilli, which (especially S mutans) store polysaccharide and continue to
secrete acid that demineralize teeth long after the food has been swallowed
(Chandrabhan et al., 2012).

Herbs have been a valuable source of medication in virtually all cultures and
societies worldwide due to their important antimicrobial principles and phyto-
constituents and wider therapeutic potentials. As various extracts of herbs and
medicinal plants are being reported with antibacterial activities, much effort should
be made in their identification, studying biologically active ingredients, efficacy and
potency testing and scientific validation for their significant and practical multi-
beneficial uses (Mogammad et al., 2011). The natural products obtained from plants
contain very rich biologically active constituents which have great potential against
bacterial species and they act as main ingredient in various pharmaceutical products.
Extracts or phyto-constituents derived from various parts of medicinal plants for
prevention and cure of several diseases provide therapeutic modalities with broad
spectrum antimicrobial activities against various pathogenic microorganisms
1
(Dhama et al., 2014)
Traditionally people use Sanicula elata plant and others for treatment dental disease
due to burning properties. This plant will have antimicrobial substances that inhibit
dental caries bacteria. Dental caries can be treated with effective antibiotics like
chlorohexidine, digluconate, penicillin, methicillin, ampicillin, erythromycin and
cephalotin. However, excessive use of these chemicals has been reported to change
the oral and intestinal flora and can cause other problem such as vomiting, tooth
staining or oral cancer. Beside this antibacterial agents can also promote the
development of resistant bacterial strains. Due to adverse effects of chemical based
remedies the use of plants and plant based products are organic and emerged out as a
best alternative. The herbal medicines have broad spectrum antibiotic effect, less cost
and easily available, less side effects and it difficult to develop resistance by pathogen
(Chaiya et al., 2013). So this research will study about antimicrobial activities
sanicula elata plant from their crude extracts on dental care bacteria Streptococcus
mutants).
1.2 Statement of the Problem
One of the main problems in exploring the potential of Sanicula elata leaf extract in
preventing dental caries is the limited research available on this specific plant. While
there have been some studies investigating its antimicrobial and anti-biofilm
properties, more comprehensive research is needed to understand the full range of
bioactive compounds present in the extract and their mechanisms of action. Without
a thorough understanding of these factors, it is challenging to develop effective oral
care products or therapies based on Sanicula elata.
Another problem is the lack of standardization and quality control measures for
Sanicula elata leaf extract. In order to ensure consistent and reliable results, it is
essential to establish standardized extraction methods and quality control protocols
to guarantee the presence and concentration of bioactive compounds responsible for
the anti-caries properties. Without proper standardization, there may be variations in
the efficacy of different Sanicula elata leaf extracts, hindering their widespread use
in dental care.
Even if the research on Sanicula elata leaf extract proves its efficacy in preventing
dental caries, there may be challenges in its practical application and acceptance by
the dental community and consumers. Incorporating a new ingredient into oral care

2
products or developing natural therapies based on Sanicula elata requires significant
investment and regulatory approvals. Additionally, there may be skepticism or
resistance from dental professionals and consumers who are accustomed to
conventional dental care methods. Educating and raising awareness about the
potential benefits of Sanicula elata leaf extract in preventing dental caries will be
crucial to overcome these challenges and promote its acceptance and use in oral care
practices.
while Sanicula elata leaf extract shows promise in preventing dental caries, there are
several challenges that need to be addressed, including limited research,
standardization and quality control issues, and practical application and acceptance.

1.3. Objectives of the Study

1.3.1. General objectives

 This study aims to isolate and characterize dental decaying

bacteria and to check antimicrobial activity of leaf sanicula elata
plant against selected dental care.

1.3.2. Specific Objectives

• To identify and characterize pathogenic bacteria (S. mutans) that

affect teeth

• To extract antimicrobial crudes from Sanicula elata plant leaf

• To assess antimicrobial effect of sanicula elata plant against

Streptococcus mutans

1.6. Significance of the Study

Dental caries is a common oral health issue caused by the demineralization of tooth
enamel due to the acidic by-products produced by bacteria in the mouth. The
prevention and management of dental caries are crucial for maintaining oral health.
Several studies have investigated the antimicrobial and anti-caries properties of
Sanicula elata leaf extracts, highlighting its potential significance in dental care

Sanicula elata leaf extracts have been found to exhibit strong antimicrobial activity
against various oral pathogens. The presence of bioactive compounds such as
flavonoids, tannins, and phenolic acids in the leaf extract contributes to its
3
antimicrobial properties. These compounds have been shown to inhibit the growth and
activity of bacteria responsible for dental caries, including Streptococcus mutans,
Streptococcus sobrinus, and Lactobacillus acidophilus.
Sanicula elata leaf extracts have demonstrated the ability to inhibit biofilm formation
on tooth surfaces. Dental plaque, a biofilm formed by bacteria, plays a crucial role in
the initiation and progression of dental caries. The anti-biofilm properties of Sanicula
elata leaf extracts can help prevent the attachment and colonization of cariogenic
bacteria on tooth surfaces, reducing the risk of dental caries development.

4
 Chapter-Two

2. Literature Review

Human mouth is one of the major habitats in which diversity of microorganism live
together. It is similar to other environmentally-exposed sites in the body in having a
characteristic (autochthonous) and diverse micro-flora in health. Far from having a
passive relationship with the host, recent research has confirmed earlier (and largely
forgotten) studies that demonstrated that the resident micro-flora of animals and
humans plays a positive role in the normal development of the host (Flip et al 2000).
This resident microflora also plays an active role in the maintenance of the healthy
state by contributing to the host defenses and preventing colonization by exogenous
microorganisms. Implicit in this statement is that disease can be a consequence of
disruption of this resident microflora. Curiously, this microflora consists of
organisms with apparently contradictory requirements; for example, facultative,
micro-aerophilic, capnophilic and obligately anaerobic species (with either
saccharolytic or asaccharolytic metabolic lifestyles) are able to co-exist. This
contradictory requirement will cause dental decay and other problems (Marsh et al.,
2000).Dental decay is due to the dissolution of tooth mineral by acids derived from
bacterial fermentation of sucrose and other dietary carbohydrates. These bacteria
live in bacterial communities known as dental plaque which accumulates on the
tooth surface. Dental caries is one of the most common chronic infectious diseases
in the world. Bacterial plaque accumulated on dental surfaces and composed of
native oral flora is the primary etiologic agent of dental caries. Cariogenic bacteria
interact by various recognized ways including co-aggregation, metabolic exchange,
cell- cell communication, and exchange of genetic material. These mechanisms
benefit bacterial survival and can make dental biofilms difficult therapeutic targets
in dental diseases (Yadav et al., 2012). For almost a century it was believed that any
bacterial community on the tooth surface could cause decay, and treatment was
almost exclusively the mechanical cleaning of these surfaces by tooth brushing,
using some type of mild abrasive. Such treatments based upon debridement and, in
extreme cases, upon dietary carbohydrate restriction, were singularly unsuccessful in
reducing dental decay. Dental caries cause destruction of enamel, dentin or
cementum of teeth due to bacterial activities. The burden of dental caries is still a
5
major health problem in most industrialized countries as it affect 60% - 90% of
school-aged children and the vast majority of adult. This is largely due to the
increasing consumption of sugar and inadequate exposure to fluorides (Chandrabhan
et al., 2012)
Streptococcus mutans is the main cause of dental decay. Lactobacillus casei and
Streptococcus faecalis are also associated with progression of the lesion.
Streptococcus mutansis acidogenic bacteria that ferment sucrose produce acids, which
in vitro lower the pH value to below 5.0. The major pathway is concerned with energy
metabolism; in this process, the enzyme invertase splits sucrose into its component
glucose and fructose molecules, which are then converted to lactic acid by the
glycolytic pathway. When S mutans, lactobacilli, and other plaque species were
compared in vitro for their ability to ferment sucrose at different pH values, S mutans
was found to be more active than the other bacteria at pH 5.0, and thus, it is probably
most active in vivo at the very pH at which the teeth begin to demineralize.
Eventually, enough mineral is lost so that a cavitation occurs in the enamel, and if this
enlarges so that it extends into the dentin, a semi closed system is formed in which the
pH value drops below 5.0. Under these acidic conditions, growth of lactobacilli is
favored, and these organisms succeed as the predominant flora in the carious lesion
(Pathak et al., 2011).

Medicinal plants have been used as traditional treatments for numerous human
diseases for thousands of years and in many parts of the world. Even today, due to
high cost of effective antibiotics and the predicament of antibiotic resistance
microbial strains worldwide, about 60- 85% of the population of developing world
relies either on herbal or on indigenous forms of Complementary and Alternative
Medicine (CAM) medicines for their various general health related issues and
countering several diseases/disorders. The natural products derived from medicinal
plants have proven to be an abundant source of biologically active compounds, many
of which have been the basis for the development of new lead chemicals for
pharmaceuticals (Yadav et al., 2013).

Herbal plants have been used as a source of valuable medication in virtually all
cultures worldwide due to presence of important antimicrobial principles, immune-
modulatory activities, and maintenance of general health, precious therapeutic
properties and healing potentials; thus ensure prevention and cure for several diseases
6
and disorders of humans and animals. Extracts or phyto-constituents derived from
various parts of medicinal plants for prevention and cure of several diseases provide
therapeutic modalities with broad spectrum antimicrobial activities against various
pathogenic microorganisms. The emergence of drug resistance as well as modern
developments in therapeutic field have revived the use of traditional medicines and
the plant- based remedies as potential source of therapeutic aids in health system in
treating and combating important bacterial pathogens and various types of bacterial
diseases (Bairwa et al., 2012).

2.1.Description and Distribution Sanicula Elata

Sanicula elata is species of flowering plant derived from family of Apiaceae and
have different name from place to place. It is known by the name ‘’gororsa’’ in
oromia region. It grows at moisture place that have relatively high water containing
and hot place both at high land and low land. It medicinal activities differ based on
highland and low land has somewhat high burning effects. The plant has fibrous
root, lobed and glossy leaf shape, dark green color, lobed palmate leaf margin,
yellow flower are borne in tight spherical umbels, weak stem and propagated by
seed.

Figure 1. Picture of Sanicula elata plant

Traditionally people use them for treatment of dental disease by chewing and
holding it on their teeth. It deeply burn the place of gingiva and tongue. These
people use it without knowing its antimicrobial by only its burning ability. Many
studies and investigating the activity of traditional medicinal plants against oral
pathogens indicate they have high effect against these pathogens (Mogammad et al.,
2011)

7
 CHAPTER-THREE

3. MATERIALS AND METHODS

3.1.Study design and setting

This prospective cross sectional study will conduct at Biology laboratory at Bule
Hora University. Ten dental samples (n=10) will be collected for the study in clear
open mouth disposable containers from patients who will clinically diagnosed as
suffering from dental disease. All the samples will be take immediately to the BHU
(Bule Hora University) biology laboratory for isolation and identification of dental
carries bacteria species, specifically the entire study will be divided into four major
steps. The first step included collection of sample to the laboratory. In the second
step, isolation and identification of dental decay or disease causing bacteria
(particularly in our study streptococcus mutans bacteria based on their colony
morphology and biochemical tests. Third step will be collection of sanicula elata
plants and extracting of antimicrobial crudes from its leaf by chloroform, ethanol
and its powder. Lastly, I Will conduct antimicrobial assay using disc diffusion
method to assess inhibition activities of sanicula elata against streptococcus mutans
bacteria comparing its inhibition zone within control antibiotic. Antimicrobial
susceptibility of streptococcus mutans strains will determine the minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC) after culturing
bacterial species on Muller Hinton agar.
The control antibiotics used is Amoxicillin to compare antibacterial
activities of sanicula elata extracts within standard one.
3.2.Sample collection and culturing dental specimen

I will prepare three different medium used for optimal isolation. The dental samples
will primarily inoculate on nutrient broth and nutrient agar and will be incubate
anaerobically at 37oc for 24 hours. Subculture will be done in the Blood agar and
Brian heart infusion until I will get pure colony. Further incubation will be done
anaerobically at 35-37oc for 24-36 hours. Sanicula elata plant sample will be
collected from different places of Bule Hora. The plants leaves selected for study
and its antimicrobial crudes will be extracted from its leaf by chloroform, ethanol,
distilled water and its leaf powder.
8
3.3.Bacterial Isolation and Characterization

3.3.1.Bacterial isolation

Point of each collected sample will be inoculated on nutrient broth and nutrient agar
using sterile spreading glass. Cultures will be incubated anaerobically at 37 oc for 24
hours. Colonies grown on nutrient-agar medium will spread on the blood agar plates
and incubated anaerobically for two days. Subcultures will be repeated several times
in order to obtain pure cultures.

The identification of S. mutans is based on (according to the information in Bergeys

Manual of Determinative Bacteriology 9th ed., 1994). Furthermore biochemical tests
conducted to confirm the bacterial species including growth on high sucrose
concentration, salt and acid media, dextran production and catalase tests. The
identification also will depend on the dextran production test which will be done
according the method of Guth of (1970).
Typical colony will be selected after sufficient growth and sub culture

3.3.2. Dextran Production Test

A 2ml of Nutrient broth medium will be inoculated with loop full of bacterial culture
then incubate anaerobically at 37°C for two days. Centrifuge the culture at 3000xg
for 10 minutes, add 0.1ml of supernatant of each culture, add to each three tubes and
stir with 0.3 ml of 10% sodium acetate. Add 0.8-fold volume of acetone to the tube
1; a 1.2 fold volume of ethanol to tube 2 and 1.5-fold volume of methanol will be to
the tube 3. Shack each tube for 3 minutes and observe. The flocculation in each tube
indicates dextran production.

3.3.3 The Fermentation of Different Carbohydrates Sources

The bacterial isolates will be fermented different carbohydrate sources: determine

following the methods of Fngold and Baron (1986). Brain heart infusion broth
supplemented with 10% of sucrose. Brain heart infusion broth medium used as
negative control, while Sucrose as positive control and add aseptically to the
autoclaved brain heart infusion broth and incubated anaerobically at 37°C for 72 hrs.
The PH of media has lowered to 4.5 as compared with the controls indicated the
ability of these bacteria to ferment and convert it to lactic acid these carbohydrates
9
sources.

3.3.4 Tolerance to High Concentration of Sodium Chloride

Test the ability of bacterial isolates to growth in 4% NaCl using nutrient broth
medium and incubate anaerobically at 37°C for 48 hrs. Turbidity will indicate for
the ability of the bacterial isolates to tolerate this concentration of NaCl as compared
with control which did not contain this concentration of NaCl.
3.3.5 Catalase test

Place nutrient agar in slant, inoculate bacterial species and spread on slant tube and
incubate at optimum temperature for 24‐48 hours. dropp 3% of H2O2 in the slant
culture. will not bubble formed in test tube

3.3.6. Gram test

Morphological characterization was done using a differential staining method after
incubation. The colonies were smeared onto three microscopic slides then stained
with gram stain and observed under light microscope because gram staining not only
morphology but also we could determine gram reaction of specific bacteria. Primary
stain was done by crystal violet and it stains all cells purple. Gram's iodine reagent
serves as a mordant, a substance that forms an insoluble complex by binding to the
primary stain. The resultant crystal violet-iodine (CV-I) complex serves to intensify
the color of the stain, and all the cells will appear purple-black at this point. In gram-
positive cells only, this CV-I complex binds to the cell wall. Decolorizing Agent
Ethyl Alcohol 95% was added and this reagent serves a dual function as a lipid
solvent and as a protein-dehydrating agent. In gram-positive cells, the low lipid
concentration is important to retention of the Mg-RNA-CV-I complex. Counterstain
Safranin was the final reagent, used to stain red those cells that have been previously
decolorized. Since only gram-negative cells undergo decolorization, they may now
absorb the counterstain. Gram-positive cells retain the purple color of the primary
stain.

3.4. Preparation of crude extract

The leaves of Sanicula elata should be clean and sterilize with 70% alcohol and dry.
After drying the plant parts will powder using mortal and pistil. 3g of powder will
extract exhaustively at room temperature by 20 ml of 95% ethanol, chloroform and
distilled water for 24hrs with shaker and by using filter paper and extracts will
10
concentrate. After that the suspension will filter and the extracts will be deliver in
sterile clean tube with suitable labeling and will kept at 4°c until used.

3.5. Antimicrobial Assay

Perform antibacterial assay using agar well disc diffusion method. Prepare Muller
Hinton agar by adding 20g in 500ml distilled water and dispense on 18 sterile petri
dish and add 0.1ml of culture broth and spread with sterile spreader. A five pore
will be made in the plate with the help of cork borer (0.65cm). 100µl of plant
extract (that extracted by distilled water, ethanol, chloroform and 500mg of solid
plants powder) and control antibiotic (Amoxicillin) compound will introduce into
the pore and the plates will keep in the refrigerator for diffusion for 30min and then
incubate over night at 37oc. interprete the antimicrobial activity measuring diameter
zone of inhibition in cm. Sensitivity of clinical isolates to commonly used standard
antibiotic Ampicillin 20µl, per disk will also evaluate and compare within plant
extract.

11
 4. Work Plan and Budget Breakdown
4.1. Work plan

Table 1 Work Plan and Activities

Activity December January February March April May June

15- 21-30 1-15 16-30 1-30 day 1-15 day 1-20 day 1-5
20 days days days day
days

Title selection 

Write up of proposal   

Proposal submission 

Proposal 
presentation

Field of material   
preparation

Field of data 
collection

Media preparation

literature review 

12
13
Research submission   

Research approval   
and review

Research defense 

4.2. Budget Breakdown

Table 2 Budget breakdown

14
 References

Bairwa, R., Gupta, P. and Srivastava, B. (2012).Traditional Medicinal Plants: Use in

Oral hygiene. International Journal of Pharmaceutical and Chemical
Sciences 1(4): 2277- 5005.

Chaiya1, A., Saraya, S., Chuakul, W., and Temsiririrkku, R. (2013). Screening for
Dental Caries: Preventive Activities of Medicinal Plants against
Unit Quantity Cost in ETB Total cost

media Kg 1 1500 1500

chloroform Kg 1 1000 1000

Ethanol Kg 1 500 500

distilled water Kg 1 400 400

Agar Kg 1 1800 1800

NaCl Kg 0.25 800 800

Ampicillin Kg 4 500 500

Pen Number 6 20 120

Flash In GB 1 500 500

A4 Paper Package 1 500 500

Print Per Paper 50 3 300

Mobil Card Number 10 15 150

Transport 3 1000 3000

Total 11070

Streptococcus mutans. Mahidol University Journal of pharmaceutical science

40(1): 9-17.

Chandrabhan, D., Hemlata, R., Renu, B. and Pradeep, V. (2012). Isolation of dental
caries bacteria from dental plaque and effect of tooth pastes on acidogenic
15
 bacteria. Open Journal of Medical Microbiology 2: 65-69.

Dhama, K., Tiwari, R., Chakraborty, S., Saminathan, M., Kumar, A., Karthik, K.,
Wani, M.Y., Amarpal, Singh, S.V. and Rahal, A. (2014). Evidence based
antibacterial potentials of medicinal plants and herbs countering bacterial
pathogens especially in the era of emerging drug resistance. International
Journal of Pharmacology, 10: 1-43.

Essam F. A., Hashim M. Z. and Faris A. (2014). Isolation and Identification of

Streptococcus mutans (H5) produced glucosyltransferase and cell-associated
glucosyltransferase isolated from dental caries. Int.J.Curr.Microbiol.App.Sci
3(6): 850-864.

Philip D. Marsh (2000). Role of the oral microflora in health. Microbial Ecology in
Health and Disease12: 130–137

Mogammad T.P., Charline W.J., Lewrens X.G., Johan M. and Abdul M. (2011). An
in-vitro analysis of antimicrobial efficiency herbal toothpastes on selected
plaque colonizers. Int. Journal of clinical dental Sceince.2(3).

Nada H.A. Al-Mudallal, Essam F.A. Al-Jumaily, Nidhal.A.A.Muhimed and Abd Al-
Wahid Al- Shaibany (2008).Isolation and identification of mutan's
streptococci bacteria from human dental plaque samples.Journal of Al-
Nahrain University. 11(3):98-105.

Pathak, A., Sardar, A., Kadam, V., Pekadwad, B. and Karuppayil, M. (2012).
Efficiency of some medicinal plants against human dental pathogens. Indian
Journal of Natural products and Resources 3(1): 123-127.

Yadav, R. and Yadav, S.K. (2012). Dental disease and its cure. Asian Journal of
Pharmaceutical and Clinical Research 6: 0974-2441.

David Beighton, Roy R. B. Russell and Hazel Hayday (1980). The

isolation and characterization of Streptococcus mutans
Serotype A from dental plaque of monkeys (Macaca
fascicularis). Journal of General Microbiology, 124:271-
279.

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