“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 SCIENCESBANGALORE, KARNATAKA

ANNEXURE-II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate and Address:

MR.ARUN NAIR1stYEAR M.PHARM,DEPT. OF PHARMACEUTICS,SRINIVAS COLLEGE OF PHARMACY,VALACHIL, MANGALORE-574143.

2. Name of the Institution:SRINIVAS COLLEGE OF PHARMACY,VALACHIL, FARANGIPETE POST, MANGALORE-574143.

3. Course of Study and Subject: MASTER OF PHARMACY(PHARMACEUTICS)

4. Date of Admission: 25-07-2013

5. Title of the Project:

‟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 B22 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 AR23 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.

Materials and Methods:

Materials:

1. Drug                              : Leaves extract of Anarcardium occidentale.

7. 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 GuideThe work, which is assigned toMr.ARUN NAIR is under my guidance.

11. 11.1 Name and Designation of the Guide

Dr. A R SHABARAYA M.Pharm, Ph.D.Assistant Professor,Srinivas College of PharmacyValachil, Mangalore- 574143

11.2 Signature

11.3 Name and Designation of the Co-Guide

Prof.KRISHNANANDA KAMATH K.M.PharmSrinivasCollege 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. SHABARAYAPrincipal and Director,Srinivas College of Pharmacy,Valachil, Mangalore- 574143