“FORMULATION AND EVALUATION OF ANTIMICROBIAL
ACTIVITY OF HERBAL EXTRACTS OF
ANARCARDIUM OCCIDENTALE DENTAL IMPLANTS”
MASTER OF PHARMACY DISSERTATION PROTOCOL
SUBMITTED TO THE
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES KARNATAKA,
BANGALORE.
BY
MR.ARUN NAIRB.Pharma
Under The Guidance of
Dr. A R SHABARAYA.M.Pharm, Ph.D.
DEPARTMENT OF PHARMACEUTICS.
SRINIVAS COLLEGE OF PHARMACY, VALACHIL, MANGALORE – 574143
2013-2015
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BANGALORE, KARNATAKA
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
MR.ARUN NAIR
1stYEAR M.PHARM,
DEPT. OF PHARMACEUTICS,
SRINIVAS COLLEGE OF PHARMACY,
VALACHIL, MANGALORE-574143.
1.
Name of the Candidate and
Address:
2.
Name of the Institution:
3.
Course of Study and Subject:
MASTER OF PHARMACY
(PHARMACEUTICS)
4.
Date of Admission:
25-07-2013
5.
Title of the Project:
SRINIVAS COLLEGE OF
PHARMACY,
VALACHIL, FARANGIPETE POST,
MANGALORE-574143.
‟FORMULATION AND EVALUATION OF ANTIMICROBIAL ACTIVITY
OF HERBAL EXTRACTS OF
ANARCARDIUM OCCIDENTALE DENTAL IMPLANTS”
6. Brief Resume of the intended work:
6.1 − Need of the study:
Two major dental diseases in the world are dental caries and periodontal disease,
both of which are caused by various bacteria in the oral cavity. Dental carries is a
common oral disease that usually develops secondary to the formation of plaque
biofilms on the tooth surfaces. The causative agents are Gram-positive bacteria
such as Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., and
some non-mutans streptococci while Periodontitis is an inflammatory response to
the over growth of anaerobic organisms in the subgingiva and if unchecked,
results in the destruction of the bone and soft tissues supporting the tooth, which
results in tooth loss.1Based on the prevalence of pathogens observed in
periodontitis patients, it was observed that Porphyromonas gingivalis and
Prevotella intermedia were most frequently recovered in patients showing angular
pattern of bone loss.1,2
To prevent tooth decay and periodontal diseases, fluoride compounds has been
used to prevent the corrosion of enamel and antibiotics such as penicillin,
erythromycin, tetracycline or antibacterial agents such as chlorhexidine has also
been used. But the excess use of fluorine causes the hardening of cartilage and
stomach-ache and the use of synthetic bactericides or antibacterial agents cause
various side-effects and adverse drug reactions. Moreover in conventional mode
of drug administration, many do not reach target areas in the body in sufficient
concentration because of premature inactivation and excretion. The systemic drug
administration has been useful in treating periodontitis but having disadvantages
that, drug is diluted several thousand folds before it reaches the site and exposes
the rest of the body to potential side effects. This problem can be overcome by
administering the drug directly in to the intended site of action with lesser dose.
Sustained drug delivery systems are able to provide very precise control over drug
release for prolonged period of time eliminating the need for frequent dosing and
minimizing side effects, there by increasing the patient compliance and comfort.
A site-specific system aims at delivering the therapeutic agent at sufficient levels
inside the pocket and at the same time minimizing the side effects associated with
systemic drug administration.3
The global need for alternative prevention and treatment options and products for
oral diseases that are safe, effective and economical comes from the rise in
disease incidence (particularly in developing countries), increased resistance by
pathogenic bacteria to currently used antibiotics and chemotherapeutics,
opportunistic infections in immune compromised individuals and financial
considerations in developing countries.4,5 Medicinal plants have been used for
thousands of years in folk medicine for maintaining oral hygiene. Most of these
herbs are alkaline with high antibacterial activity. Hence these herbs help to
maintain acid-alkaline balance of the saliva, decrease plaque/calculus formation
and are less prone to periodontal diseases. It is also observed that the
microorganisms found in inflamed gums are resistant to antibiotics but not to
antibacterial plant extracts like neem.6
Here we will be formulating our dental implants using natural antibiotic obtained
from the dried leaves extract of Anarcardium Occidentale of family
Anarcardiaceace. A lot of studies have been carried out on the antimicrobial
potentials of crude extract of different leaves, bark, bulbs, stems and roots.7
It is a multipurpose tree whose leaves, stems and bark extracts are used
extensively for the treatment of diarrhoea, dysentery and colonic pain.8 The
cashew tree produces many resources and products. Various parts of the plant
possess curative qualities like antidiabetic9,antiinflammatory9,antibacterial10and
antifungal properties11.
In this present work we will formulate Antibiotic dental implants of Anarcardium
Occidentale for the treatment of periodontal disease which is mainly caused by
anaerobic bacteria. Antibiotic cones formulations provide a long term treatment
and reduce the cost and frequency of dosing compare to conventional therapy.
6.2 – Review of literature:
A great deal of work has been done by scientists about the current application and
future possibilities for altering the drug activities and stability by formulating
them in the form of film or implant films. The black pigmented P. gingivalis, in
particular, possess virulence factors that are relevant to the pathogenesis of oral
infections3,14. Human periodontal disease has been associated with a complex
microbiota while the development of destructive periodontitis seems to be the result
of a specific infection. Gram positive coccoid bacteria have been related to
periodontal health, while periodontal disease was associated with Gram negative
rods. Human periodontitis is initiated and perpetuated by a small group of bacteria
that colonize the subgingival region, mainly Gram negative, anaerobic or
microaerophilic bacteria.15

Jothi V, Vijay KT, Vasudev B, Giliyar SB2 investigated the
antimicrobial effect of Anacardium occidentale(cashew) leaf extract on
Porphyromonas gingivalis and Prevotella intermedia. Methanol and
aqueous extracts of cashew leaves were prepared. Its antimicrobial activity
against P. gingivalis and P. intermedia was tested using the agar diffusion
method at various dilutions of 75μl,50μl,25μl,10μl and 5μl respectively.
The agar plates were then incubated at 37°C for 72 hours after which the
zone of inhibition was measured and the mean was recorded for each test
solution and compared with the standard. This preliminary study suggests
that the antimicrobial action of A.occidentale leaf could be utilized for the
preparation of chemical plaque control formulations.

Sastravaha G, Yotnuengnit P, Booncong P, Sangtherapitikul P12 have
done research on Centella asiatica and Punica granatum medicinal herbs
that have been reported to promote tissue healing and modulate host
responses. An innovative herbal medicament was formulated in the form
of biodegradable chips for sub-gingival application. Twenty patients with
initial pocket depth 5-8 mm were enrolled into the study. After baseline
examination, scaling and root planning of non-target teeth, the target teeth
received scaling and root planning followed by sub-gingival delivery of
medicated chips in the test group and unmediated chips in the placebo
group. Probing pocket depth, attachment level, bleeding on probing,
gingival index, and plaque index were recorded at baseline, 3 and 6
months. The results showed significant improvements of pocket depth and
attachment level in the test sites when compared with the placebo sites at 3
months and with the placebo and control sites at 6 months. All treatment
sites exhibited a similar trend of decreasing plaque score. However, the
test sites seemed to show slightly better percentage of bleeding on
probing. The results indicate that local delivery with C. asiatica and P.
granatum extracts plus scaling and root planning significantly reduced the
clinical signs of chronic periodontitis.

Gaetti Jardim Jr. JE, Landucci LF, Arafat OKK, Ranieri RV, Ramos
MMB, Ciesielski FIN et.al13 have studied on the extracts of plants from
Brazilian savanna, which are used in popular medicine. Study evaluated
the inhibitory activity of the alcoholic and aqueous extracts from savanna
plants on periodontal bacteria. The minimal inhibitory concentrations were
evaluated by the agar dilution method, using Wilkins-Chalgren agar.
Antimicrobial activity of plants extracts on microbial biofilms was
determined in microplates. Psidium cattleianum and Myracrodruon
urundeuva extracts demonstrated significant inhibitory activity on all
bacterial strains tested while alcoholic and aqueous extracts showed
similar results. The extracts from these two species were able to inhibit
both planktonic cells and microbial biofilm.

Jintakorn K, Surat L and Pirasut R14 investigated antimicrobial activity
against periodontopathic bacteria using agar diffusion technique. These
plant extracts demonstrated comparable antimicrobial efficacy against
black-pigmented bacteria strains to standard drug chlorhexidine. The
CD50 of C. formosum and C. lansiumon HGF cells were 0.37 and 0.16
mg/ml, respectively, demonstrated using the MTT assay.

Mastiholimath VS, Dandagi PM , Gadad AP, Patil MB, Manvi FV,
Chandur VK17 has formulated and evaluated Ornidazole implants for
periodontitis have been reported. Ornidazole has an excellent activity
against anaerobic micro-organism was prepared by solvent casting
technique. The physicochemical parameters were evaluated. In-vitro
antibacterial activity was carried out on streptococcus mutans.

Nagaraja R, Udupa N18 has formulated Doxycycline HCl and Tinidazole
in biodegradable carrier’s dental implants have been reported. The in-vitro
drug release patterns and stability of these devices were studied. The
formulation showed an initial burst release followed by more sustained of
the drugs throughout the period of study (42 days).

Prabushankar GL, Gopalkrishna B, Manjunatha KM, Girisha CH19
has formulated and evaluated Levofloxacin dental films for Periodontitis.
Films were prepared by solvent casting technique. Kinetic models were
studied for zero order, first order equation, Hixson- Crowell and Higuchi
models.

Manoj K, Prabhushankar GL, Satheshbabu PR20 has formulated and
In vitro evaluated of periodontal films containing Metronidazole. Films
were prepared by solvent casting technique. Metronidazole is a nitro
imidazole used to treat protozoal
infection. The films Evaluated for
thickness uniformity, folding endurance, weight uniformity, content
uniformity, tensile strength and surface pH.

Karunakar B, Sabu K, Udupa N, Varma AR21 has designed and
evaluated tinidazole dental implants for the controlled release therapy.

Udupa N, Karunakar B 22 has designed and evaluated norfloxacin
dental implants using solvent casting method. Attempt was made to
formulate Norfloxacin as targeted sustained release dental implant which
can be directly placed near the site of action in periodontitis and also to
study various pharmaceutical parameters like physicochemical, stability
and drug release characteristics.

Agarwal RK, Dennis HR, Glenn LM, Richard AR 23 developed and
characterized Tetracycline poly(lactide/glycolide) films for the treatment
of
periodontitis.
Film
was
capable
of
delivering
therapeutic
concentrations of tetracycline HCl for duration of two weeks into the
intra-cervicular fluid within the inflamed periodontal pocket.

Mohammed GA, Narayana CR, Kanthraj K, Harish NM, Prabhakar
P24 has formulated chitosan-based ciprofloxacin and diclofenac film for
periodontitis. Chitosan films containing ciprofloxacin alone and in
combination with diclofenac sodium were prepared by solvent casting
method. Some of the drug-loaded films were cross linked with 2%
gluteraldehyde for 2 and 4 hours, respectively. The films were then
evaluated for their physicochemical properties including weight variation,
tensile strength, thickness, in-vitro release, stability and antibacterial
activity.

Deasy P B, Collins AEM, Maccarthy DJ, Russell RJ25 has formulated
strips containing tetracycline HCl or Metronidazole for the treatment of
periodontal disease.

Rafael PR, Jose VC, Jose MSB, Enrique CP26 worked on Antibiotic use
in dental practice. The use of antibiotics in dental practice is characterized
by empirical prescription based on clinical, bacteriological and
epidemiological factors, with the use of broad spectrum antibiotics for
short period of time, and the application of a very narrow range of
antibiotics.
The
simultaneous
prescription
of
nonsteroidal
anti-
inflammatory drugs can be modified with the bioavailability of the
antibiotics.

Ifesan BOT, Fashakin JF , Ebosele F and Oyerinde AS28 prepared the
ethanolic , hexane and water extracts from leaves of Anacardium
occidentale(cashew), Cocos nucifera(coconut), Citrus sinesis(sweet
orange), Citrus limon(lemon) and Carica papaya (pawpaw) and screened
for there antimicrobial and antioxidant activities. The antimicrobial
activity of the extract against microorganisms (Acinetobacter spp.,
Bacillus cereus, Escherichiacoli, Shigella dysenteriae, Staphylococcus
aureus, Salmonella typhi, Aspergillus niger and Aspergillus flavus) was
determined using modified agar-well diffusion method.

Agedah CE, Bawo DDS, Nyananyo BL29 has carried out work on the
antimicrobial capabilities of plant extract derived from the leaves of the
cashew plant, Anacardium occidentale L. (Family Anacardiaceae), on two
common human pathogens of clinical importance, Escherichia coli and
Staphylococcus aureus were investigated. The antimicrobial properties of
A. occidentale are derived from the presence of a polyphenol, anacardic
acid and other compounds, tatrols and tanins.
6.3 – Objectives of the study:
The objectives of the study are to prepare polymeric cones containing the
antibacterial agent for periodontal pockets.
Specific objectives of the present investigation are as follows:
1. To formulate dental implants containing herbal extracts of
Anarcardium Occidentale.
2. To evaluate the various pharmaceutical and physiochemical
parameters of drugs , estimate the drug content and carry out
stability studies.
3. To evaluate the antimicrobial activity of drug containing implants
against bacteria which cause periodontitis.
4. To achieve localized action.
5. To avoid intermittent doses and prevent dose dumping.
6. To bypass first pass metabolism of biodegradable drugs.
7. To achieve better patient compliance.
8. To enhance the bioavailability of the drug.
7. Materials and Methods:
Materials:
1. Drug
: Leaves extract of Anarcardium occidentale.
2. Polymers
: Poly (ε-caprolactone) , Polyvinyl pyrollidone ,
Hydroxypropyl methyl cellulose etc.
3. Plasticizers
: Dibutyl phthalate
4. Solvent
: Aqueous or Organic
Method:
Extraction27
The aqueous or ethanolic extraction of the plant material were prepared. The
aqueous or ethanolic extraction of the plant material was carried out by
suspending 25g of the finely ground leaves of Anarcardium Occidentale in 125ml
of distilled water or 95% ethanol. The extracts were decanted and filtered through
Whatman filter paper No1. The residues obtained were reconstituted in 95%
ethanol at stock concenteration of 0.2g/ml. The extract solutions were stored in
the refrigerator at 4±2°C until used.
Phytochemical screening30
Solvent Casting Technique.17
1. Drug extract and Polymer are dissolved in suitable solvent at room
temperature using magnetic stirrer.
2. Addition of a suitable plasticizer.
3. The polymeric solution is then poured on a Petri dish and dried.
4. Films were removed.
5. Films of specific size are cut, packed in aluminium foil and stored in
desiccators for evaluation.
7.1 Source of data:
Review of literature from
Journal such as

Drug Development and Industrial Pharmacy.

European Journal of Medicinal Plants.

Indian Journal of Pharmaceutical Sciences.

International Journal of Biology.

International Journal of Pharma and Bio sciences.

International Journal of Pharmacy and Pharmaceutical Sciences.

International Journal of Pharmaceutical Technology and research.

International Journal of Applied Biology and Pharmaceutical Technology.

Journal of Pharmacy and Pharmacology.

Journal of Controlled Release.

Internet browsing.

Laboratory based experiments and evaluation.
7.2Method of approaches :
1. Formulation of Dental Cones.
Solvent casting technique.
2. Evaluation of Dental Cones such as follows :
a. Physicochemical evaluation :
Drug Content.3,17
Film (size of 7x2 mm2) was taken from different areas of the film and placed into
a 10 ml volumetric flask, into which 10 ml of solvent was added and kept aside
till the film is completely dissolved. Withdraw 1 ml of solution and diluted to
10ml with suitable buffer. The absorbance of the solution was measured at
suitable wavelength.
Tensile strength. 17
Tensile strength of the films were determined by Universal strength testing
machine. It consists of two load cell grips, the lower one is fixed and upper one is
movable. The test film of specific size (4 × 1 cm2) was fixed between these cell
grips and force was gradually applied till the film breaks. The tensile strength of
the film was taken directly from the dial reading in kilograms.
Thickness. 17
Thickness of the film was measured using digital screw gauge (Mitutoyo) at
different areas of the film and the average was then calculated.
Folding endurance.19
The folding endurance of the films were determined by repeatedly folding the
film at the same place up to 300 times till it broke or folded, which is considered
satisfactory to reveal good film properties. This test was carried out on all the
films.
Weight variation.19,20
Film (size of 7x2 mm2) was taken from different areas of film. The weight
variation of each film was calculated.
c. In- vitro drug release study21:
USP II Static dissolution method.
7.3 Does the study require any investigations or interventions to be
conducted on patients or other humans or animals? If so, please
describe briefly.
Not applicable
o
Has ethical clearance been obtained from your institution in
case of 7.3?
Not applicable
8.
List of references:
1. Monetti M ,Usin MM , Tabares S , Gonzalez A , Cabral HR and Sembaj
A. The presence of periodonto pathogens associated with the tumour
necrosis factor alpha expression in patients with different periodontal
status. Acta Odontologica Latinoamericana 2012;25(1):82-8.
2. Jothi V, Vijay KT, Vasudev B, Giliyar SB. Antimicrobial effect of
Anacardium
occidentale
leaf
extract
against
pathogens
causing
periodontal disease. Adv Biosci Biotechnol 2013;(4):15-18.
3. Mohammed GA, Narayana CR, Kanthraj K, Harish N M , Prabhakar P.
Preparation and evaluation of periodontal strips of Gatifloxacin for
periodontal Diseases. Int J Pharma Bio Sci 2010;1(3):1-8.
4. Tichy J, Novak J. Extraction, assay and analysis of antimicrobials from
plants with activity against dental pathogens (Streptococcus sp.).
J.Alternative and Complementary Med 1998;4(1):39–45.
5.
Bhardwaj A, Bhardwaj SV . Role of medicinal herbs in prevention and
treatment of dental diseases. AAM. (2012), [cited December 31, 2013];
1(3): 95-101.
6. Jose M, Bhagya B, Shantaram M. Ethnomedicinal herbs used in oral
health and hygiene in coastal Dakshina Kannada. J Oral Health comm dent
2011; 5(3):119-23.
7. Akinpelu DA.Antimicrobial activity of Anarcardium occidentale bark.
Fitoterapia 2001;72(3):286-7.
8. Omojasola, PF and Awe S. The antibacterial activity of the leaf extracts of
Anacardium occidentale and Gossypium hirsutum against some selected
microorganisms. Biosci Res Comm 2004;16(1):25-58.
9. Srisawat, S ,Teanpaisan R, Wattanapiromsakul C and Worapamorn W,
Antibacterial activity of some thai plants against Porphymonas gingivalis ;
2005 ; Sep 1-4 ; Malacca. Malacca :International Association for Dental
Research, 20th Southeast asia Division & Southeast Asia Association for
Dental Education, 16th Annual Scientific Meeting.
10. Doss VA and Thangavel KP. Antioxidant and antimicrobial activity using
different extracts of Anacardium occidentale L. Int J Appl Biol Pharm
Technol 2011;(2):436-43.
11. Dahake AP, Joshi VD and Joshi AB. Antimicrobial screening of different
extract of Anacardium occidentale Linn.leaves .Interdisciplinary journal
of contemporary research in business 2009;(1);856-8.
12. Sastravaha G, Yotnuengnit P, Booncong P, Sangtherapitikul P. Adjunctive
periodontal treatment with Centella asiatica and Punica granatum
extracts. A preliminary study. J Int Acad Periodontol 2003;5(4):106-15.
13. Gaetti-JardimJr E , Landucci L , Arafat OKK , Ranieri RV, Ramos
MMB, Ciesielski FINS et al. Antimicrobial activity of six plant extracts
from the Brazilian savanna on periodontal pathogens. Int J Odontostomat
2011; 5(3):249-56.
14. Jintakorn K, Surat L, Pirasut R. Antimicrobial activity against
periodontopathic bacteria and cytotoxic study of Cratoxylum formosum
and Clausena lansium.J Med Plants Res 2011;5(25); 5988-92.
15. Pandit JK. Targeted devices for periodontal disease, Edited by Jain NK.
Controlled and novel drug delivery. New Delhi: CBS publisher and
distributors; 2004.
16. Sweetman SC. Martindale. The Complete Drug Reference. Great Britain:
Pharmaceutical press; 2002.
17. Mastiholimath VS, Dandagi PM, Gadad AP, Patil MB, Manvi FV,
Chandur VK. Formulation and evaluation of ornidazole dental implant for
periodontitis. Indian J Pharma Sci 2006; 68(1):68-71.
18. Nagaraja R, Udupa N. Preparation and evaluation of dental implants
containing doxycycline hydrochloride and tinidazole in biodegradable
carrier. Indian J Pharma Sci 1998;60(6):405-6.
19. Prabushankar GL, Gopalkrishna B, Manjunatha KM, Girisha CH.
Formulation and evaluation of Levofloxacin dental films for Periodontitis.
Int J Pharmacy Pharma Sci 2010;2(1):162-8.
20. Manoj K, Prabhushankar GL, Satheshbabu PR. Formulation and In-Vitro
evaluation of periodontal films containing Metronidazole. Int J Pharm
Tech Res 2010;2(4):2188-93.
21. Karunakar B, Sabu K, Udapa N, Varma BRR. Design and evaluation of
tinidazole dental implants. Drug Dev Ind Pharm 1994;20(3):409-16.
22. Udupa N, Karunakar B. Design and evaluation of norfloxacin in dental
implants. Indian J Pharma Sci 1993;55(2):68-9.
23. Agarwal RK, Dennis HR, Glenn IM, Richard AR. Development and
Characterization of tetracycline-poly (lactide/ glycolide) films for the
treatment of periodontitis. J.Controlled Release 1993;23(2):137-46.
24. Mohammed GA, Narayana CR, Kanthraj K, Harish N M , Prabhakar P.
Formulation of chitosan based ciprofloxacin and diclofenac film for
periodontitis therapy. Trop J Pharm Res 2009; 8(1):33-41.
25. Deasy PB, Collins AEM, Maccarthy DJ, Russell RJ. Use of strips
containing tetracycline hydrochloride or metronidazole for the treatment
of advanced periodontal disease. J Pharm Pharmacol 1989;41(10):694-9.
26. Rafael PR, Jose VC, Jose MSB, Enrique CP. Antibiotic use in dental
practice .Med Oral Patol Oral Cir Bucal 2007;12(3):186-92.
27. Oygade JO, Awotoye JT , Adewunmi A , Thorpe HT. Antimicrobial
activity of some Nigerian medicinal plants , screening of antibacterial
activity .Biosci Res Commun 1999;11(3):193-7.
28. Ifesan BOT , Fashakin JF , Ebosele F , Oyerinde AS. Antioxidant and
antimicrobial Properties of Selected Plant Leaves. Eur J Med Plants
2013;3(3):465-473.
29. Agedah CE, Bawo DDS, Nyananyo BL . Identification of antimicrobial
properties of cashew, Anacardium occidentale L. (Family Anacardiaceae)
.J Appl Sci Environ Manage 2010;14 (3):25 –7.
30. Abulude FO, Ogunkoya MO and Adebote VT. Phytochemical and
antibacterial investigations of crude extracts of leaves and stem barks of
Anacardium occidentale. Cont J Biol Sci 2009;(2):12-6.
9.
Signature of the candidate
(ARUN NAIR)
10.
Remarks of the Guide
11.
11.1 Name and Designation of the
Guide
The work, which is assigned to
Mr.ARUN NAIR is under my guidance.
Dr. A R SHABARAYA M.Pharm, Ph.D.
Assistant Professor,
Srinivas College of Pharmacy
Valachil, Mangalore- 574143
11.2 Signature
11.3 Name and Designation of the
Co-Guide
Prof.KRISHNANANDA KAMATH K.
M.Pharm
SrinivasCollege of Pharmacy,
Valachil, Mangalore- 574143
11.4 Signature
-----------
11.5 Head of the Department
Dr. A R SHABARAYA M.Pharm.,Ph.D.
Principal and Director,
Srinivas College of Pharmacy,
Valachil, Mangalore- 574143
11.6 Signature
12.
12.1 Remarks of the Principal
Recommended and forwarded for
favourable consideration.
12.2 Signature
Dr. A. R. SHABARAYA
Principal and Director,
Srinivas College of Pharmacy,
Valachil, Mangalore- 574143