08 - Chapter 2 PDF

CHAPTER 2 LITERATURE REVIEW 25
The literature related to the novel drug delivery systems of eugenol against inflammation
and periodontal infections topic have been extensively reviewed and compiled as follows.
2.1. Pharmacological actions of eugenol
A review on the research done on the pharmacological actions of eugenol revealed that
exhibits pharmacological effects on almost all systems.
2.1.1. Analgesic activity
Kurian and coworkers studied the antinociceptive ability of eugenol (100 mg kg-1) in
several mouse models of pain and found that the effect was more pronounced in the
inflammatory phase than the neurogenic phase (Kurian et al., 2006). Eugenol can
alleviate neuropathic pain (Guenette et al., 2006). Guenette et al. (2007) in their study in
male Sprague-Dawley rats, showed that eugenol, at a dose of 40 mg kg-1, is capable of
prolonging reaction time to thermal stimuli. All these results suggested the possible use
of eugenol as an analgesic agent.
2.1.2. Antioxidant effect
The potential use of eugenol as a therapeutic antioxidant has been demonstrated by the
studies of Nagababu and coworker (Nagababu and Lakshmaiah, 1994). In their study, the
formation of thiobarbituric acid reactive substances (TBARS) was decreased by eugenol
at concentrations of 4-15 µM. Tests with compounds that are reactive with the hydroxyl
radical in the presence of eugenol showed that it competes with these reactive compounds
for this radical and thus could protect skin damage by UV-light (Taira et al., 1992). The
antioxidant effect of eugenol-grafted chitosan hydrogel has also been reported (Jung et
al., 2006). Eugenol can cause significant suppression of lipid peroxidation and low
density lipoprotein oxidation. Eugenol-like compounds significantly reduce copper-
dependent oxidation of low density lipoprotein, and lipid peroxidation and autooxidation
of Fe2+ (Masae et al., 2005). Electron spin resonance spectroscopic studies by Fujisawa
and coworkers showed that eugenol (18.50 mU per 100 µM sol) significantly scavenged
reactive oxygen species (Fujisawa et al., 2002). Ogata et al. found that the antioxidant
action of eugenol (62.5 – 250 µM) is mediated through the inhibition of lipid
peroxidation at the level of initiation (Ogata et al. 2000). Vidhya and coworkers
investigated the short and long term antioxidant activities of eugenol (1000 mg kg-1) on
rat intestine and observed that the compound helps in the removal of toxic materials from
DEPARTMENT OF PHARMACEUTICS JAMIA HAMDARD

the intestine as it caused the induction of glutathione-S-transferases (Vidhya and Devaraj,

1999). Antioxidants and free radical scavengers like eugenol have also been monitored as
agents for preventing the progress of Parkinson disease. A study demonstrated that
eugenol (0.1 - 1.0 µM kg-1) inhibits lipid peroxidation and thereby suppresses 6-
hydroxydopamine-induced dopamine depression thereby suggesting its potential use in
Parkinson disease (Kabuto et al., 2007). The antioxidant activity of eugenol is
comparable with that of butylated hydroxyanisole and butylated hydroxytoluene (Jirovetz
et al., 2006). Although many in vitro studies have been carried out on the antioxidant
activity of eugenol, there have been insufficient in vivo studies.
2.1.3. Protective effect
Eugenol modulates both the NMDA receptor and superoxide radical and thus seems to
provide protection against ischemic injury. Eugenol can attenuate NMDA induced acute
neurotoxicity and decrease xanthine/xanthine oxidase produced oxidative neuronal injury
(Wie et al., 1997). Eugenol (10.7 mg kg-1day-1) has been found to show an in vivo
antioxidant property and a better inducing effect on phase II enzymes which counteract
the metabolizing enzyme reduction caused by carbon tetrachloride (Kumaravelu et al.,
1995). In vitro studies on liver microsomal monooxygenase activities and carbon
tetrachloride (CCl4)-induced lipid peroxidation have shown that eugenol inhibits both
(Nagababu et al., 1995). The in vivo studies by the same researchers also showed a
protective effect of eugenol at doses of 5 and 25 mg kg-1 against CCI4 induced
hepatotoxicity. The methanolic extract of leaves of Ocimum suave (Lamiaceae), which
contains eugenol as a major constituent (Ntezurubanza et al., 2008), has shown gastric
cytoprotective and anti-ulcer effects in experimental Wistar rats (Tan et al., 2002).
Eugenol pre-treatment (10-100 mg kg-1) reduces both the number of gastric ulcers and the
gravity of lesions induced by ulcerogenic agents like platelet activating factor and ethanol
(Capasso et al., 2000). The mechanism of the gastroprotective action of eugenol has been
established by the studies of Morsy and Fouad (Morsy and Fouad, 2008). Gastric
mucosal lesions, gastric acid outputs and pepsin activity in male Sprague-Dawley rats
were markedly reduced by a single oral dose of eugenol (100 mg kg-1), used as a
pretreatment one hour before indomethacin injection. This pretreatment resulted in a
marked increase in mucin concentration, suppression of the rise in gastric mucosal

malondialdehyde and total nitrite, and attenuation of the decrease in reduced glutathione.
It was further confirmed that the protective action of eugenol is through opening of these
channels and not through the transient receptor potential vanilloid 1. The DNA-protective
activity of eugenol has been observed by Yogalakshmi and coworkers in thioacetamide-
induced liver injury in rats at a dose of 10.7 mg kg-1 day-1 (Yogalakshmi et al., 2010).
Eugenol imparts a dose dependent protection against nicotine-induced superoxide
mediated oxidative damage at a concentration range of 1-20 µg mL-1 (Kar Mahapatra et
al., 2009). Recently eugenol has been found to ameliorate gentamicin-induced
nephrotoxicity and oxidative damage by scavenging oxygen free radicals, decreasing
lipid peroxidation and improving intracellular antioxidant defense (Said, 2011).
2.1.4. Anesthetic action
A randomized, single-blind study was carried out in 73 subjects to evaluate the topical
anesthetic action of clove oil in comparison with benzocaine (Rapp, 2007). The pain
scores between the clove gel and the benzocaine groups were not significantly different,
but were significantly lower than those of the placebo groups. The study showed that
clove gel is a cheap and easily available topical anesthetic which could be used as an
alternative to benzocaine gel in dentistry. A reversible, dose dependent anesthesia has
also been reported after eugenol administration (5 – 60 mg kg-1, iv) in male Sprague–
Dawley rats (Guenette et al., 2006). A clinical study was conducted in 100 adult patients
to study the effect of eugenol as an intravenous anesthetic. However, they concluded that
it is not advisable to use eugenol except in special circumstances as the incidence of
development of venous thrombosis around the site of injection was high (Right and
Payne, 1962). The possible use of eugenol as a local anesthetic has also been
demonstrated by the studies of Park and coworkers, who suggested its suitability for non-
dental anesthesia as well (Park et al., 2009).
2.1.5. Antibacterial, antifungal and antiviral activities
The activity has been studied of Eugenia aromaticum on several microorganisms and
parasites, including pathogenic bacteria, and herpes simplex and hepatitis C viruses (El-
Zemity et al., 2008). Burt and coworker studied the antibacterial activity of clove bud oil
and found it to be effective (Burt and Reinders, 2003). Eugenol is active against Neisseria
gonorrhoeae, with a minimum inhibitory concentration of 85-256 mg L-1 (Shokeen et al.,

2008). Moreover, eugenol shows synergistic effects with antibiotics against Gram-
negative bacteria (Hemaiswarya and Doble, 2009). Mytle and coworkers have
demonstrated the ability of clove oil to inhibit the growth Listeria monocytogenes. A 1%
v/w concentration was sufficient for the inhibition of growth at 5oC and 15oC (Mytle et
al., 2006). Eugenol was found to be active against sessile cells in Candida albicans
biofilms (He et al., 2007). The compound showed fungicidal activity in vitro for
exponentially growing Candida albicans. The mechanism of this action was found to be
through envelope damage (Chami et al., 2005). In the in vivo study, treatment with
eugenol caused a significant reduction in the colony count number in an
immunosuppressed rat model of oral candidiasis in comparison with the untreated control
group (Chami et al., 2004a). Chami and coworkers also evaluated eugenol in an
immunosuppressed rat model of vaginal candidiasis for its efficacy in the prophylaxis and
treatment of this condition (Chami et al., 2004b). Eugenol has been demonstrated to have
activity against Penicillium (100 mg L-1), Aspergillus (100-140 mg L-1) and Fusarium
species (140-140 mg L-1) also (Campaniello et al., 2010).
Eugenol can cause damage to viral envelopes of freshly formed virons and can cause
inhibition of viral replication at the initial stage (Tragoolpua and Jatisatienr, 2007). The
anti-viral activity of eugenol, clove flower bud extract and clove essential oil against
Herpes simplex virus has been carried out (Tragoolpua and Jatisatienr, 2007). Direct
inactivation of viruses and inhibition of intracellular and extracellular viruses after
replication were observed with eugenol. In another study for antiviral activity against
HSV-1 and HSV-2 viruses (Benencia and Courrèges, 2000), 50% inhibitory
concentration values were 25.6 µg mL-1 and 16.2 µg mL-1 for HSV-1 and HSV-2,
respectively. Eugenol demonstrated a synergistic action with acyclovir against in vitro
replication of herpes virus. Topical eugenol therapy was observed to suppress herpes
virus induced keratitis in mouse.
2.1.6. Anticonvulsant activity
Zelger and coworkers investigated the anticonvulsant activity of phenyleugenol,
benzyleugenol and phenylethyleugenol, which are synthetic derivatives of eugenol
(Zelger et al., 1983). All three compounds showed significant anticonvulsant activity in
the maximal electroshock seizure test. Phenyleugenol and benzyleugenol were found to

have a high therapeutic index. All these studies indicated that eugenol could be taken as a
lead molecule in the development of novel anticonvulsant drugs.
2.1.7. Anti-inflammatory action
Eugenol inhibits cycloxygenase and thus inhibits prostaglandin H synthase (Thompson
and Eling, 1989). This can be the result of its competition with arachidonic acid. Eugenol
is able to resist the release of proinflammatory mediators like interleukin-1β, tumor
necrosis factor-α and prostaglandin E2 from macrophages and is thus useful for acute
inflamed dental pulps and apical periodontitis as an anti-inflammatory agent (Lee et al.,
2007). Eugenol can also inhibit cycloxygenase-2 in macrophages. Eugenol caused
reduction of both paw and joint swelling in arthritis induced male Sprague-Dawley rats at
a dose of 33 mg kg-1 (Sharma et al., 1994). An in vivo study by Rodrigues and coworkers
on the effect of an hydroalcoholic extract of clove on pro-inflammatory cytokines
production by macrophages of mice showed that clove oil (200 mg kg-1) caused cytokine
inhibition due to the presence of eugenol, which imparts an anti-inflammatory activity
(Rodrigues et al., 2009). The in vivo anti-inflammatory activity of eugenol in
lipopolysaccharide induced lung injury has been demonstrated on administration of 160
mg kg-1, ip (Magalhães et al., 2010). Recent reports suggest that an anti-inflammatory
property of eugenol enhances the chemotherapeutic potential of gemcitabine and induces
anticarcinogenic and anti-inflammatory activity in human cervical cancer cells (Hussain
et al., 2011).
2.1.8. Penetration enhancement
The utility of clove oil (82.6% eugenol) as a penetration enhancer has been demonstrated
by Shen and coworkers (Shen et al., 2007). Permeation enhancement was found to be
significant both in vivo and in vitro. The in vivo effect observed was comparatively
weaker than that observed in vitro. A marked increase in drug flux was observed with
clove oil in in vivo percutaneous absorption studies in rabbit. All the results suggested the
potential ability of clove oil, of which eugenol is the major active component, as a
penetration enhancer in transdermal drug delivery systems (Shen et al., 2007). In vitro
evaluation of penetration enhancers, including eugenol, has been conducted by Mutalik
and coworkers using mouse skin (Mutalik and Udupa, 2003). In their study, eugenol was
also found to increase skin retention and solubility of glibenclamide and glipizide. Zhao

and coworkers studied the in vitro penetration enhancement effect of eugenol on

percutaneous absorption of a drug through porcine epidermis (Zhao and Singh, 1998).
The study showed that eugenol was able to enhance markedly the permeability
coefficient of tamoxifen with respect to the control group. The study also found that lipid
extraction and increased partitioning of the tamoxifen to the stratum corneum is
responsible for the observed permeability enhancement action of eugenol.
2.1.9. Cardiovascular actions
Eugenol (0.2 – 20 µmol) produces dose-dependent, reversible vasodilator responses that
are partially dependent on the endothelium (Criddle et al., 2003). Sensch and coworkers
showed that eugenol (60 – 600 µmol L-1) exerted negative inotropic effects in heart
muscle of Guinea-pig (Sensch et al., 2000). The effect was found to be comparable with
that of nifedipine, a calcium channel blocker. Eugenol (0.006 – 6 mM) causes
hypotensive effects in hypertensive rats due to vascular relaxation (Interaminense et al.,
2007). The blocking of voltage-sensitive and receptor-operated channels by eugenol is
responsible for its smooth muscle relaxant effect and these actions are mediated through
endothelial-generated nitric oxide (Damiani et al., 2003). Choudhary and coworkers
observed eugenol to counteract isoproterenol-induced cardiac hypertrophy (Choudhary et
al., 2006). They conducted their study in male Wistar rats using a dose of 1 mg kg-1 twice
daily. They showed that serum calcineurin activity in vitro was suppressed by eugenol,
and isoproterenol-induced oxidative stress and apoptosis were reduced. Eugenol was also
found to increase cardiac calcineurin and protein kinase C activity in ventricular tissue to
normal values.
2.1.10. Anticancer activity
Significant induction of activity of glutathione S-transferase by eugenol was observed in
liver and small intestine (Zheng et al., 1992). Miyazawa and coworkers showed that due
to the presence of a C-4 hydroxy group in their structure, trans-isoeugenol and eugenol
had significant suppressive effects on the SOS-inducing activity of chemical mutagens
such as furylfuramide (Miyazawa and Hisama, 2003). The antimutagenic effects of a
eugenol derivative, dehydroeugenol, have also been demonstrated by the same authors.
Studies have demonstrated that eugenol provides protection from chemical induced skin
cancer (Kaur et al., 2010; Pal et al., 2010).

2.1.11. Other pharmacological actions

An antigenotoxic effect of eugenol was studied in humans by Rompelberg and coworkers
(Rompelberg et al., 1996), but no evidence was observed for these effects. Tajuddin and
coworkers investigated the effect of a 50% ethanolic extract of clove on sexual function
improvement in male Swiss mice (500 mg kg-1) and observed an enhanced sexual
behavior of the mice (Tajuddin et al., 2003).
2.1.12. Molecular mechanisms and biochemical changes
Eugenol showed inhibition of high-voltage-activated calcium channel currents in both
capsaicin-sensitive and capsaicin-insensitive dental primary afferent neurons (Lee et al.,
2005). However, the action of eugenol was unaffected by capsazepine. This suggested
that the action is not mediated by transient receptor potential vanilloid 1 activation. N-
type calcium currents were inhibited by eugenol in the cell line C2D7. The study showed
that high-voltage-activated calcium channel current inhibition in dental primary afferent
neurons by eugenol is responsible for its dental analgesic action. The mechanism of
anesthetic action of eugenol by inhibition of sodium channel currents has been
established by the whole-cell patch-clamp method in rat dental primary afferent neurons
(Park et al., 2006). Capsaicin-sensitive and capsaicin-insensitive neurons are both
inhibited by eugenol and its irritable action is possibly due to the inhibition of voltage-
gated K+ currents (Li et al., 2007). Yang and coworkers showed that eugenol produces its
effects in the sensory nerve endings in the teeth through vanilloid receptor 1, at least
partially (Yang et al., 2003).
The analgesic effect of eugenol is probably due to the inhibition of Ca(V)2.3 calcium
channels. A study by Chung and coworkers showed that the mechanism of inhibition of
Ca(V)2.3 calcium channels by eugenol is not involved through transient receptor
potential vanilloid 1 and thus appeared to be distinctly different from that of capsaicin
(Chung et al., 2008). Both capsaicin receptor mediated and capsaicin receptor
independent pathways are involved in the activation of Ca2+ channels by eugenol
(Ohkubo and Kitamura, 1997). Eugenol has the ability to inhibit pro-inflammatory
mediators like nitric oxide synthase, lipoxygenase and cyclooxygenase. Eugenol exerts its
analgesia by inhibition of Na+ currents and the inhibition is independent of the stimulus
frequency (Cho et al., 2008). Inotropic effects of eugenol have been investigated in rat

left ventricular papillary muscles by Damiani and coworkers (Damiani et al., 2004). The
contraction force was depressed without affecting the contractile machinery. Complete
blockade of inward Ca2+ current was observed with 0.5 mM of eugenol. Xu and
coworkers explained that the transient receptor potential vanilloid 3 is expressed by
eugenol. It is a warm-sensitive Ca2+-permeable cation channel in the skin, tongue and
nose (Xu et al., 2006).
Eugenol has been reported to potentiate the GABA response. The compound’s ability to
potentiate GABAA receptors can modulate neural transmission in the brain just like, for
example, benzodiazepine and barbiturate (Aoshima and Hamamoto, 1999). Salah and
coworkers showed that eugenol causes effects such as relaxation of rat ileal strip, reduced
intestinal transit in rats, and the potentiation of the diarrhea inducing effect of castor oil
(Salah et al., 2002). They suggested that all these effects are mediated through Ca2+
channel modulation. Eugenol accelerates inactivation of the Ca2+ current in isolated
canine and human ventricular cardiomyocytes (Magyar et al., 2004).
2.1.13. Metabolism and pharmacokinetics
Fischer and coworkers investigated the metabolism of eugenol in male and female
healthy volunteers (Fischer et al., 1990). The study revealed that eugenol is rapidly
absorbed and metabolized after oral administration. It is almost completely excreted in
the urine within 24 h. Only less than 0.1% of the administered dose was excreted
unmetabolized in urine. Eugenol was found in the urine in the form of conjugates and
metabolites. Among the metabolite conjugates, 55% consisted of eugenol-glucuronide
and sulfate. The epoxide-diol pathway, allylic oxidation, synthesis of a thiophenol and a
substituted propionic acid, and migration of the double bond were also found to be other
routes of eugenol metabolism in humans. A study was made by Guénette and coworkers
of the pharmacokinetic parameters of eugenol using non-compartmental analysis after
gavage administration in male Sprague–Dawley rats in a dose of 40 mg kg-1 (Guenette et
al., 2007). Plasma T1/2 of eugenol was found to be 14.0 h and blood T1/2 was 18.3 h.
Glucuronide and sulfate conjugates of eugenol in urine after eugenol administration were
also identified in male Sprague–Dawley rats (Guenette et al., 2006).
A summary of the research work carried out to demonstrate the different pharmacological
activities of eugenol and clove oil is given in Table 2.1. Further research is required to

collect more information about the pharmacological effects of eugenol, and also to find
new areas of therapeutic applications. A detailed knowledge of eugenol pharmacology
could be utilized to consider eugenol as a lead molecule for the development of new
drugs (Awasthi et al., 2008) with enhanced therapeutic efficacy.
2.2. Novel eugenol delivery systems
Kriegel et al. (2010) prepared antimicrobial nanofibers by electrospinning
microemulsions composed of eugenol solubilized in an aqueous nonionic micellar
surfactant solution (Surfynol V R 465) with poly (vinyl alcohol) (PVA). Nanofibers
contained microemulsions composed of 0.75–1.5 % w/w eugenol and 5–10 % w/w
Surfynol. Scanning electron microscopy revealed substantial difference in fiber
morphology depending on microemulsion composition with fiber diameters increasing as
the concentration of either surfactant or essential oil component in the fibers increased.
Release studies suggested a burst release of the encapsulated eugenol, potentially due to
the hydrophilicity of the polymeric carrier resulting in rapid dissolution of the carrier
matrix and high-fiber porosity. The eugenol release rate depended on the amount of
eugenol and surfactant incorporated within the fibers. The anti-microbial activity of
nanofibers carrying eugenol was evaluated against two strains of Salmonella typhimurium
(2476 and 2576) and Listeria monocytogenes (Scott A and 101) using a macrobroth
dilution assay. Presence of nanofibers in bacterial suspensions successfully suppressed
growth of food-borne pathogens and in some cases decreased initial cell numbers.
Nanofibers were more efficient against Gram-negative than Gram-positive bacterial
strains.
Gel formulation of eugenol loaded nanostructured lipid carriers was developed for
periodontal delivery such that it would effectively deliver drug in a sustained manner. A
systematic study was performed for obtaining the nanostructured lipid carriers by using
32 factorial design approach with percentage of lipid and surfactant as independent
variables at three different levels. Hot homogenization method was adopted for
preparation of nanostructured lipid carrier dispersion. Nanostructured lipid carrier
dispersion was characterized for particle size and entrapment efficiency. Results of
regeression analysis revealed that entrapment efficiency was dependent on lipid
concentration. Optimized nanostructured lipid carrier dispersion was formulated into gel

Table 2.1 Pharmacological studies of eugenol and clove oil (Pramod et al., 2010)
Activity studied Experimental Effect (Dose/Concentration) Ref.
EUGENOL
Analgesic Mice Significant antinociceptive effect (100 mg kg-1) Kurian et al., 2006
Male Sprague-Dawley rats Prolonged reaction time (40 mg kg-1) Guenette et al., 2007
Antioxidant Microsomal mixed function oxidase mediated Decreased TBARS, inhibition of oxygen uptake, monooxygenase Nagababu and Lakshmaiah,
peroxidation activity inhibition (4-15 µM) 1994
LDL oxidation Suppressed oxidation (1.5 µM) Masae et al., 2005
Lipid peroxidation Significant suppression (0.05 – 0.15 mM) Masae et al., 2005
Reactive oxygen scavenging activity Significant activity (18.50 mU/100 µM sol) Fujisawa et al., 2002
Free radical reaction Inhibited lipid peroxidation at propagation step (62.5 – 250 µM) Ogata et al., 2000
Rat intestine Induced glutathione-S-transferases (1000 mg kg-1) Vidhya and Devaraj, 1999
Lipid peroxidation Inhibited (0.1 -1.0 µM kg-1) Kabuto et al., 2007
DPPH scavenging activity Activity similar to BHA & BHT (20 µg mL-1) Jirovetz et al., 2006
Hydroxyl radical scavenging Activity greater than quercetin (0.6 µg mL-1) Jirovetz et al., 2006
Protective NMDA neurotoxicity Prevented acute neuronal swelling and reduced neuronal death Wie et al., 1997
(100- 300 µM)
Carbon tetrachloride intoxicated rat liver Counteracted metabolic enzyme reduction (10.7 mg kg-1 day-1) Kumaravelu et al., 1995
Liver monoxygenase activity & Carbon Inhibition (5 & 25 mg kg-1) Nagababu et al., 1995
tetrachloride induced lipid peroxidation
Induced gastric lesions in rat Reduced number and severity of ulcers observed (10-100 mg kg-1) Capasso et al., 2000
Induced ulcer in male Sprague-Dawley rats Gastroprotective action (100 mg kg-1) Morsy and Fouad, 2008
Adult male Wistar rats Curtailed thioacetamide (TA)-induced hepatic injury (10.7 mg kg-1 Yogalakshmi et al., 2010
day-1)
Gentamicin-induced nephrotoxicity and oxidative Ameliorated GM-induced nephrotoxicity and oxidative damage Said, 2011
damage in rat kidney
Nicotine-induced oxidative damage Dose dependent protection (1-20 µg mL-1) Kar Mahapatra et al., 2009
Anesthetic Randomized, single-blind study Activity similar to benzocaine Rapp, 2007
Male Sprague-Dawley rats Reversible & dose dependent activity (5- 60 mg kg-1) Guenette et al., 2006
Antibacterial, Candida albicans biofilms Active against sessile cells (20-2000 mg L-1) He et al., 2007
Antifungal & Candida albicans Antifungal activity by envelope damage (0.2 %) Chami et al., 2005
Antiviral Oral candidiasis in immunosuppressed rats Significant reduction in the colony counts (0.5 mL, 24 mM) Chami et al., 2004a
Vaginal candidiasis in immunosuppressed rats Reduction in the colony counts of Candida albicans (20 mg kg-1 Chami et al., 2004b
day-1)
Penicillium species Growth inhibition (100 mg L-1) Campaniello et al., 2010
Aspergillus species Growth inhibition (100-140 mg L-1) Campaniello et al., 2010
Fusarium species Growth inhibition (140-150 mg L-1) Campaniello et al., 2010
Anti-herpes simplex virus Active against HSV-1 and HSV-2 viruses (16.2 and 25.6 µg mL-1) Benencia and Courrèges, 2000

Activity studied Experimental Effect (Dose/Concentration) Ref.

Anticonvulsant Maximal electroshock seizure test Synthetic eugenol derivatives showed significant activity Zelger et al., 1983
Anti-inflammatory Prostaglandin H synthase inhibition Inhibited cycloxygenase (100-200 µM) Thompson and Eling, 1989
Human macrophages Resisted the release of proinflammatory mediators & inhibited Lee et al., 2007
cycloxygenase-2
Male Sprague-Dawley rats Reduced paw and joint swellings in induced arthritis (33 mg kg-1) Sharma et al., 1994
Mice Reduced lipopolysaccharide induced lung inflammation (160 mg Magalhães et al., 2010
kg-1, ip)
Cytotoxicity studies in HeLa and normal cells Enhanced the chemotherapeutic potential of gemcitabine Hussain et al., 2011
Penetration In vitro evaluation in mouse skin Increased drug flux (5 %) Mutalik and Udupa, 2003
enhancer In vitro penetration through porcine epidermis Significant enhancement of permeability coefficient of drug (5 %) Zhao and Singh, 1998
Cardiovascular Rat mesenteric vascular bed Dose-dependent, reversible vasodilatation (0.2 – 20 µmol) Criddle et al., 2003
Guinea-pig heart muscle Potassium current inhibition (60 – 600 µmol L-1) Sensch et al., 2000
Hypertensive rats Vascular relaxation (0.006 – 6 mM) Interaminense et al., 2007
Smooth muscle relaxant effect Action mediated through endothelial-generated nitric oxide (300 Damiani et al., 2003
Isoproterenol-induced cardiac hypertrophy in rats µM)
Oxidative stress and apoptosis were reduced (1 mg kg-1 twice daily) Choudhary et al., 2006
Anticancer Mice Protection against chemically induced skin cancer (30 µL) Kaur et al., 2010
Swiss mice Restriction of skin carcinogenesis at dysplastic stage (1.25 mg kg-1) Pal et al., 2010
CLOVE OIL
Antioxidant DPPH scavenging activity Activity similar to BHA & BHT (0.5 µg mL-1) Jirovetz et al., 2006
Hydroxyl radical scavenging Active more than quercetin (0.2 µg mL-1) Jirovetz et al., 2006
Lipid peroxidation Active more than BHT Jirovetz et al., 2006
Anti-inflammatory Mice Caused cytokine inhibition (200 mg kg-1) Rodrigues et al., 2009
Penetration Excised rabbit abdominal skin Significant increase in drug flux (1-3 %) Shen et al., 2007
enhancer In vivo percutaneous absorption in rabbit Increase in drug flux (1-3 %) Shen et al., 2007
Anticancer Antimutagenic activity against MNNG, 4NQO, Suppressive effect on mutagens (ethyl acetate extract) Miyazawa and Hisama, 2003
AfB1 and Trp-P-1.
Aphrodisiac Male Swiss mice Improved sexual function (500 mg kg-1 of 50 % ethanolic extract) Tajuddin et al., 2003

CHAPTER 2 REVIEW OF LITERATURE 36
using Pluronic F127 and characterized for rheological behavior and in vitro release. In
vitro release from the gel showed its sustained release potential as compared to plain drug
loaded gel. Stability study of nanostructured lipid carrier dispersion and its gel
formulation as per ICH guidelines for six months revealed its stability over the entire
period. The factorial design was found to be well suited to identify the key variables
affecting the entrapment efficiency (Pokharkar et al., 2011).
Gomes et al. (2011) synthesized spherical poly (DL-lactide-co-glycolide) (PLGA)
nanoparticles with entrapped eugenol for future antimicrobial delivery applications. The
emulsion evaporation method was used to form the nanoparticles in the presence of PVA
as a surfactant. The inclusion of eugenol into the PLGA nanoparticles was accomplished
in the organic phase. Synthesis was followed by ultrafiltration (performed to eliminate the
excess of PVA and antimicrobial compound) and freeze-drying. The nanoparticles were
characterized by their shape, size, entrapment efficiency, and antimicrobial efficiency.
The entrapment efficiency for eugenol was approximately 98%. Controlled release
experiments conducted in vitro at 37 °C and 100 rpm for 72 h showed an initial burst
followed by a slower rate of release of the antimicrobial entrapped inside the PLGA
matrix. Eugenol loaded nanoparticulate formulations proved to be efficient in inhibiting
growth of Salmonella spp. (Gram-negative bacterium) and Listeria spp. (Gram-positive
bacterium) with concentrations ranging from 20 to 10 mg mL-1.
Shu-Ya (2010) carried out studies on encapsulation of several active substances including
eugenol by β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD),
and by an emulsion–diffusion method with polycaprolactone (PCL). The size distribution
of the β-CD and 2-HP-β-CD inclusion complex was polydisperse as compared with
eugenol encapsulated with PCL. Thermo gravimetric analysis (TGA) analysis revealed
that the encapsulation efficiency of PCL was higher than β-CD eugenol and 2-HP-β-CD
eugenol inclusion complexes. Thus, the emulsion–diffusion method was found to be the
most effective for eugenol encapsulation due to their complete wrapping of eugenol by
PCL layer as revealed by TEM analysis.
Shah et al. (2010) prepared and evaluated the pharmacological activity of nano-
encapsulated eugenol in irritable bowel syndrome (IBS). The eugenol loaded stearic acid
solid lipid nanoparticles (Eu-SLN) were prepared by hot-homogenization-ultrasonicaton

method and characterized for particle size, polydispersity index and encapsulation
efficiency. Pharmacological activity of the prepared Eu-SLN was studied using restraint
stress induced increase in defecation as an animal model. Rats were divided in 7 groups
namely, control, stress control, eugenol (25 and 50 mg kg-1), Eu-SLN (25 and 50 mg kg-
1
) and placebo nanoparticles. Particle size, polydispersity index and encapsulation
efficiency of nanoparticles was found to be 478 nm, 0.385 and 65% w/w, respectively.
Restraint stress significantly increased the fecal counts compared to control group.
Eugenol at both the doses (25 and 50 mg kg-1) significantly decreased the stress induced
increase in fecal pellet counts. Among the two doses of eugenol, only the dose of 50 mg
kg-1 was able to reduce fecal pellet counts near to normal. Eu-SLN (25 and 50 mg kg-1)
mitigated the effect of stress on defecation, similar to eugenol at 50 mg kg-1. Placebo
SLN was not able to decrease effect of stress. In contrast to eugenol at 25 mg kg-1, Eu-
SLN at the dose of 25 mg kg-1 significantly decreased fecal pellet counts to normal levels
and suggested that SLN increased the pharmacological activity of eugenol in IBS. The
study concluded that nanoparticulate formulation could be beneficial to improve the
therapeutic efficacy of eugenol by reducing the dose.
Nanoparticles with both antioxidant and antibacterial properties have been prepared by
grafting eugenol on chitosan nanoparticles. Aldehyde groups were first introduced in
eugenol, and the grafting of this oil to chitosan nanoparticles was carried out via the
Schiff base reaction. The surface concentration of the grafted essential oil component was
determined by X-ray photoelectron spectroscopy (XPS). The antioxidant activity of the
eugenol-grafted chitosan nanoparticles (CHEU NPs) were assayed with
diphenylpicrylhydrazyl (DPPH). Antibacterial assays were carried out with a
representative Gram-negative bacterium, Escherichia coli and a Gram-positive
bacterium, Staphylococcus aureus. The grafted eugenol conferred antioxidant activity to
the chitosan nanoparticles, and the essential oil component-grafted chitosan nanoparticles
achieved an antibacterial activity equivalent to or better than that of the unmodified
chitosan nanoparticles. Cytotoxicity assays using 3T3 mouse fibroblast showed that the
cytotoxicity of CHEU NPs was significant lower than those of the pure essential oils
(Chen et al., 2009).

Jadhav et al. (2004) reported the development and evaluation of controlled-release

mucoadhesive tablets for gingival application, containing eugenol, which were prepared
by taking carbopol 934 P and hydroxypropyl methylcellulose (HPMC) K4M in the ratio
of 1:2, 1:1, and 2:1. Incorporation of eugenol (10 mg) in a mucoadhesive formulation
provided controlled release for a period of 8 h, which is advantageous over conventional
use. In vitro mucoadhesion was measured as detachment force in grams and the
formulations showed good correlation in vivo. The release study indicated that increase in
Carbopol increased the release rate of eugenol from the formulation whereas HPMC
retarded it. Increased in vitro bioadhesion was related to HPMC content of the
formulation. The release kinetics of eugenol in vitro correlated with the in vivo results.
This indicated the increased potential of eugenol as antibacterial, local analgesic, and
anaesthetic treatment.
2.3. Herbal remedies against inflammation
The in vivo and in vitro mechanistic anti-inflammatory actions of cucurbitacin E
(Citrullus lanatus var. citroides) were examined by Abdelwahab and coworkers. The
results showed that lipopolysaccharide/interferon gamma increased nitric oxide
production in RAW264.7 macrophages, whereas L-nitro-arginine methyl ester and
cucurbitacin E curtailed it. Cucurbitacin E did not reveal any cytotoxicity on RAW264.7
and WRL-68 cells. Cucurbitacin E inhibited both cyclooxygenase enzymes with more
selectivity toward COX-2. Intraperitoneal injection of cucurbitacin E significantly
suppressed carrageenan-induced rat's paw edema. Oxygen radical absorbance capacity
and ferric reducing antioxidant power assays showed that cucurbitacin E is not a potent
reactive oxygen species scavenger. It could be concluded that cucurbitacin E is
potentially useful in treating inflammation through the inhibition of cyclooxygenase and
reactive nitrogen species but not reactive oxygen species (Abdelwahab et al., 2011).
Rafael and coworkers studied the bioactivity of fruits of three topical anti-inflammatory
plant species: Bryonia dioica, Tamus communis and Lonicera periclymenum. Different
analyses and assays were performed in order to characterize their phytochemical
composition and to find biologically active compounds for pharmaceutical application.
Black-bryony ripened fruits revealed the highest antioxidant properties which are in
agreement to its highest concentration in phenolics, flavonoids, ascorbic acid, tocopherols

and lycopene. The studied fruits revealed interesting antioxidant properties and bioactive
phytochemicals that could provide scientific evidence for their folk uses as anti-
inflammatory species (Rafael et al., 2011).
The petroleum ether fraction (PEF) of the ethanol extract of the hip of Rosa multiflora
has been identified to be the active fraction in inflammation animal models (i.e., oral
administration of PEF (168.48, 42.12 and 10.53 mg kg-1) evoked a significantly (P <
0.001) dose-dependent inhibition of the xylene-induced mice ear edema). Down-
regulating COX-2 expression (P < 0.001) and reducing nitric oxide production (P < 0.05)
through inhibiting inducible NO synthase (iNOS) activity (P < 0.001) was proposed to be
the partial mechanism of action of PEF. GC–MS analysis indicated that unsaturated fatty
acids are enriched in PEF and may be responsible for the anti-inflammatory activity of
PEF and this herb. The results of their study provided pharmacological and chemical
basis for the application of the hip of Rosa multiflora Thunb in inflammatory disorders
(Guo et al., 2011).
The anti-inflammatory effect of kirenol, isolated from ethanolic extract of Siegesbeckia
orientalis, 0.4–0.5% w/w has been found to have similar effect to that of piroxicam gel at
4 h after carrageenan injection. The analgesic activity of topical preparation with more
than 0.4% w/w was observed in the late phase. These effects were proposed to be due, at
least in part, to the pro-inflammatory cytokine production of IL-1β and TNF-α. The
administration of kirenol cream at the dose of 0.3, 0.4 and 0.5% (w/w) significantly
inhibited the development of joint swelling induced by CFA, which was auxiliary
supported by histopathological studies (Wang et al., 2011) .
The immunomodulatory activity of Phaseolus angularis ethanol extract (Pa-EE) in toll
like receptor (TLR)-activated macrophages induced by ligands such as
lipopolysaccharide (LPS), Poly (I:C), and pam3CSK has been investigated by assessing
nitric oxide (NO) and prostaglandin (PG)E2 levels. To identify which transcription
factors such as nuclear factor (NF)-κB and their signaling enzymes can be targeted to Pa-
EE, biochemical approaches including reporter gene assays, immunoprecipitation, kinase
assays, and immunoblot analyses were also employed. Finally, whether Pa-EE was orally
available, ethanol (EtOH)/hydrochloric acid (HCl)-induced gastritis model in mice was
used. Pa-EE dose-dependently suppressed the release of PGE2 and NO in LPS-,

Poly(I:C)-, and pam3CSK-activated macrophages. Pa-EE strongly down-regulated LPS-

induced mRNA expression of inducible NO synthase (iNOS) and cyclooxygenase
(COX)-2. Interestingly, Pa-EE markedly inhibited NF-κB, activator protein (AP)-1, and
cAMP response element binding protein (CREB) activation; further, according to direct
kinase assays and immunoblot analyses, Pa-EE blocked the activation of the upstream
signaling molecules spleen tyrosine kinase (Syk), p38, and transforming growth factor β–
activated kinase 1 (TAK1). Finally, orally administered Pa-EE clearly ameliorated
EtOH/HCl-induced gastritis in mice. The study results suggested that Pa-EE can be
further developed as a promising anti-inflammatory previous remedy because it targets
multiple inflammatory signaling enzymes and transcription factors (Yu et al., 2011).
Various preparations of Tripterygium wilfordii Hook F (TwHF) have been used in the
treatment of a number of autoimmune and inflammatory diseases since the 1960s (Tao
and Lipsky, 2000). Triptolide is a diterpenoid triepoxide purified from this Chinese herb.
TwHF has been used in traditional Chinese medicine for more than two thousand years.
However, its potential value was recognized by the western medicine only after
investigators observed the effectiveness of TWHF in the treatment of leprosy and
rheumatoid arthritis. Triptolide has been identified as the major component responsible
for the immunosuppressive and anti-inflammatory effects of TWHF. Triptolide inhibits
both Ca2+-dependent and Ca2+-independent pathways and affects T cell activation
through inhibition of interleukin-2 transcription at a site different from the target of
cyclosporin A. Triptolide also has inhibitory effects on a variety of proinflammatory
cytokines and mediators and on the expression of adhesion molecules by endothelial
cells. Triptolide is effective for the treatment of a variety of autoimmune diseases and in
prevention of allograft rejection and graft-versus-host disease in both animals and
humans (Chen, 2001). In another study triptolide inhibited nuclear factor-kappaB
transcriptional activation at a unique step in the nucleus after binding to DNA (Qiu and
Kao, 2003).
Chauhan and coworkers described a novel herbal composition comprising an extract of
flowering and fruiting heads of the plant, Sphaeranthus indicus. The said extract of
Sphaeranthus indicus contains a compound, 3a-hydroxy-5a,9-dimethyl-3- methylene-
3a,4,5,5a,6,7,8,9b-octahydro-3H-naphtho[1 ,2-b]furan-2-one (7- Hydroxy-4,1 1 (13)-

eudesmadien-12,6-olide) (compound 1 ), as a bioactive marker. The patent also covered

the methods of manufacture and administration of the said compositions to a subject in
need of treatment for an inflammatory disorder. The invention also related tumor necrosis
factor-& agr; (TNF- & agr;) and interleukin (IL-1 , IL-6, IL-8) inhibitory activity of the
said compositions. The invention related inhibition of the expression of intercellular
adhesion molecule 1 (ICAM-1 ), vascular-cell adhesion molecule 1 (VCAM-1 ), and E-
Selectin by the compositions. The compositions optionally contain at least one anti-
inflammatory agent or can be used in combination with at least one anti- inflammatory
agent (Chauhan et al., 2007).
2.4. Novel drug delivery systems for the treatment of inflammation
Poly(amidoamine) dendrimer-erythromycin conjugates have been developed for drug
delivery to macrophages involved in periprosthetic inflammation. In their study, a
specific dendrimer-erythromycin conjugate nanodevice was investigated for the treatment
of periprosthetic inflammation. To sustain pharmacological activity, erythromycin was
conjugated to poly(amidoamine) dendrimer (PAMAM) through an ester bond. A
bifunctional PAMAM dendrimer was prepared having neutral hydroxy and reactive
amine groups on the surface and was reacted with erythromycin prodrug (erythromycin-
2'-glutarate). The cytotoxicity, efficacy and antibacterial properties were evaluated on
macrophages associated with periprosthetic inflammation. The conjugate was
noncytotoxic and showed significant reduction of nitrite level (by 42% as compared with
untreated cells and free erythromycin). The zone of inhibition of the conjugate on
bacterial growth at different concentrations showed similar activity compared to free
erythromycin. The anti-inflammatory properties of erythromycin combined with the
targeting potential of the dendrimer could lead to sustained and targeted intracellular
delivery. Thus the anti-inflammatory properties of erythromycin combined with the
targeting potential of the dendrimer can lead to sustained and targeted intracellular
delivery (Bosnjakovic et al., 2011).
A biodegradable drug delivery system has been developed by Eperon and coworkers for
the treatment of postoperative inflammation in cataract surgery. They developed drug
delivery system using poly(D,L-lactide-co-glycolide) loaded with triamcinolone
acetonide. The developed drug delivery system significantly reduced postoperative ocular

inflammation and was proved to be very promising since it could replace oral treatment
and reduce the number of intraocular injections (Eperon et al., 2008). The ability of
methylprednisolone-polyamidoamine dendrimer conjugate to improve the airway
delivery has been evaluated by Inapagolla and coworkers in a pulmonary inflammatory
murine model that was based on an 11-fold enhancement of eosinophil lung accumulation
following five daily inhalation exposures of sensitized mice to the experimental allergen,
ovalbumin. These results demonstrated that conjugation of MP with a dendrimer
enhances the ability of methylprednisolone to decrease allergen-induced inflammation,
perhaps by improving drug residence time in the lung. This was supported by the fact that
only 24% of a single dose of dendrimer delivered to the peripheral lung is lost over a 3-
day period. Therefore, conjugation of drugs to a dendrimer could provide an improved
method for retaining drugs within the lung when treating such inflammatory disorders as
asthma (Inapagolla et al., 2010).
Carmichael and coworkers demonstrated that peptide-mediated transdermal delivery of
botulinum neurotoxin type A reduces neurogenic inflammation in the skin. They found
that a short synthetic peptide (TD-1) can facilitate effective transdermal delivery of
botulinum neurotoxin type A through intact skin. The findings showed that botulinum
neurotoxin type A could be administered subcutaneously or topically with a novel
transdermal delivery peptide to reduce inflammation produced by activating nociceptors
in the skin. Their study concluded that peptide-mediated delivery is an easy and non-
invasive way of administering the toxin that might prove to be useful in clinical practice
(Carmichael et al., 2010).
Chakravarthy and coworkers developed doxorubicin-conjugated quantum dots to target
alveolar macrophages and inflammation. Pulmonary inflammatory diseases still often
remain challenging to treat, despite decades of advances and several available agents.
Their study results demonstrated that nanoparticulate platform could provide targeted
macrophage-selective therapy for the treatment of pulmonary disease (Chakravarthy et
al., 2011). Vascular inflammation is a common, complex mechanism involved in
pathogenesis of a plethora of disease conditions including ischemia-reperfusion,
atherosclerosis, restenosis and stroke. Chacko and coworkers have reviewed targeted
nanocarriers for imaging and therapy of vascular inflammation and expressed that

specific targeting of imaging probes and drugs to endothelial cells in inflammation sites
holds promise to improve management of these conditions. They found that nanocarriers
of diverse compositions and geometries, targeted with ligands to endothelial adhesion
molecules exposed in inflammation foci have been devised for this goal (Chacko et al.,
2011).
Singka and coworkers examined the effect of sodium carbonate (on the topical delivery
of methotrexate from a loaded nanogel in vitro and the modulation of prostaglandin E(2)
(PGE(2)) production in skin ex vivo. A nanogel based on co-polymerised N-
isopropylacrylamide and butylacrylate was synthesized, characterized and loaded with
methotrexate. The added sodium carbonate led to solubilisation and methotrexate release,
hence increasing the concentration gradient, flux and reducing PGE(2) production
(Singka et al., 2010).
2.5. Herbal remedies for the treatment of periodontal diseases
There are a number of herbs that can help eliminate inflammation and infection
associated with periodontal diseases. Proper oral hygiene, of course, goes a long way in
treating and preventing periodontal disease. Coenzyme Q-10, a natural supplement helps
to increase tissue oxygenation in the body that helps to provide healthy blood flow to the
gums. A reported clinical trial demonstrated improvement in plaque and calculus levels
by twice daily intake of 25 mg of Coenzyme Q-10 in patients suffering from periodontal
disease (Lockwood, 2007). A number of neutraceuticals are currently being used in the
prevention and treatment of periodontal disease. Evidences have been reported for the
relation between Vitamin C and periodontal disease. Significant gum bleeding can occur
in vitamin C deficiency (Leggott et al., 1986; Mason, 2007). Vitamin C along with
bioflavonoids helps to speed up the healing process. Clinical studies have also been
reported to lower plague, gingival and bleeding indices by use of formulations containing
extracts of bloodroot (Barnes et al., 2007). Aloe vera gel has been reported to sooth gum
tissue and relieves pain and discomfort when applied on gums. Clove oil reduces
infection and relieves pain. Echinacea and goldenseal have also been reported to relieve
infection and inflammation (Lisa, 2009).
Clove oil will reduce infection and relieves pain. Clove oil is commonly used for the
relief of toothache (Barnes et al., 2007). In dentistry, clove oil is applied in an undiluted

form using a plug of cottonwool soaked in the oil and applied to the cavity of the tooth
(Barnes et al., 2007). A study by Cai et al. (1996) reported preferential activity of crude
methanolic extract of clove against Gram-negative anaerobic oral pathogens which cause
periodontal diseases (Cai and Wu, 1996). This study included isolation of eight active
constituents and the antibacterial effect of these isolated compounds were studied. The
authors reported kaempferol and myricetin to have significant growth-inhibitory effect
against periodontal pathogens.
Chan et al. (2003) evaluated the effects of herbal medicines in treating periodontal
diseases. Three Chinese herbal compositions widely used for periodontal diseases were
tested for their ability to retard progression of experimental periodontitis in hamsters,
inhibit bacterial growth, and induce mutations. The study also included evaluation of
major components of these compositions. The results showed that between the animal
groups there were no significant differences in terms of the degree of inflammation,
alveolar bone resorption, and rate of repair with or without the use of Chinese herbal
medicine but hamsters treated with one composition (Conth Su) showed early
regeneration of epithelium. Among the evaluated herbs, Chi Tong Ning demonstrated
superior bacterial inhibition ability of MIC 0.025 g mL-1. While both of the above
mentioned compositions were capable of inhibiting bacterial growth, none of the
individual herb components showed comparable bacterial inhibition abilities. Also no
signs of induction of cell mutations were observed for the tested herbal composites or
their components using the Ames test. The results of the study demonstrated that
traditional Chinese herbal medicines, which have been used for hundreds of years by
Chinese people to treat periodontal diseases, could effectively inhibit bacterial growth
without causing cell mutation.
Cao et al. (1998) reviewed articles published on the effects and mechanisms of herbal
medicine on periodontal disease. Particularly, two modifications of an ancient compound
prescription, Guchiwan (Tooth-firming pills) and Guchigao (Tooth-firming extract), were
serially studied. It was pointed out in the review that various herbal extracts had shown
suppressive effects on bone resorption by isolated osteoclasts during in vitro studies. The
author emphasized an indepth study on traditional Chinese medicine for their possible use
in periodontal diseases.

Cai et al. (2000) demonstrated the growth inhibitory effect of methanolic extract of
Diospyros lycioides on oral pathogens that could cause periodontal diseases. The twigs of
this plant were used to clean teeth by people in Namibia as chewing sticks. The active
compounds were isolated and structures were elucidated. The compounds showed growth
inhibitory effect on oral cariogenic baceria and periodontal pathogens.
A phase II clinical trial (NCT00731432) was initiated for the evaluation of efficacy of a
transmucosal herbal periodontal patch in reducing gingival inflammation in diabetic
patients. The study was sponsored by Izun Pharma Ltd., New York, USA (Caine, 2008).
But the study could not be completed as it has been suspended.
Auh et al. (2011) described a composition for the prevention or treatment of periodontal
disease, comprising an extract of Coptidis rhizoma or a combined extract of Coptidis
rhizoma and Pharbitidis semen as active ingredients. They claimed their composition to
stimulate anti-inflammation, osteoblast differentiation and alveolar bone regeneration,
and to prevent the alveolar bone destruction, thereby being effective for the prevention
and/or treatment of periodontal disease.
Kim et al. (2010) patented a herbal mixture extract of Pleurotus eryngii, Acanthopanacis
cortex and Notoginseng radix and a composition for prevention and treatment of
periodontal disease containing the herbal mixture extract as an active ingredient. They
invented a herbal mixture extract having activities of inhibiting the generation and
activation of osteoclasts by enhancing the expression of osteoprotegerin (OPG) in
osteoblasts, preventing alveolar bone from destruction by inhibiting the proliferation of
osteoclasts and maintaining the growth of periodontal ligament cells and gingival
fibroblasts. They also claimed a composition for prevention and treatment of periodontal
disease containing the above mixture as an active ingredient.
Piramal et al. (2008) described a composition for treatment and prevention of periodontal
diseases using a bioadhesive formulation comprising curcuminoids as an active agent.
The composition included curcumin, tetrahydrocurcumin, bishydrocurcumin, crude drug
and solvent extracts of Curcuma longa, one or more bioadhesive polymers such as
hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium
carboxymethylcellulose, hydroxyethyl cellulose and carbomers and sodium chloride,

sodium bicarbonate or mixtures and one or more excipients. This orally applicable
composition can be used for the treatment of gingivitis and other periodontal diseases.
Greve and Greve (2005) described the use of vervain (ironweed, Verbena officinalis,
Verbenaceae) and/or ribwort (Plantago lanceolata / P. angustifolia, Plantaginaceae)
either alone or as a mixture of these two herbs or of their constituents or extracts. Aerial
parts of the plants were preferred for the composition though all parts could be used. A
combination of these two herbs or of the constituents or extracts of the two herbs were
claimed to be very effective. Pharmaceutical additives and/or adjuvants such as fillers,
extenders, binders, wetting agents, stabilizers, colorants, buffers, aroma chemicals,
flavouring agents and/or preservatives could be included in addition to active ingredients.
Use in the form of extracts or constituents of the two plants was preferred The extracts
had herb/extract ratio from 50:1 to 1:1. The extracts could be prepared in such a way that
0.2 to 100 parts of liquid extract, or 0.1 part of dry extract was obtained from dried or
fresh plant material with the aid of suitable liquid extractants such as water, alcohols, in
particular ethanol or ethanol-water mixtures. The composition could be liquid, gel-like or
pasty and could contain diluents compatible with vervain and/or ribwort. Twenty drops of
2.5% of an extract which contained nearly 60% v/v ethanol preparation per half a glass of
water (50 ml) was sufficient for achieving adequate inhibition of microorganisms. The
semi-solid forms such as toothpaste or oral gels contained approximately 2.5% of an
extract which contained almost 80%v/v ethanol. In the case of lozenges, chewable tablets
and (chewable) pastilles, it was preferred that such preparations contain approximately
250 mg of dry extract per unit. A dry extract prepared by concentrating the liquid extract
which originally had an herb/extract ratio of approximately 1:1 and an alcohol component
of approximately 80% v/v was claimed to be advantageous.
Kim et al. (2006) provided a herbal mixture extract of Pleurotus eryngii, Acanthopanacis
Cortex and Notoginseng Radix and a composition for prevention and treatment of
periodontal disease. The herbal mixture extract had potential of inhibiting the generation
and activation of osteoclasts and inhibiting the proliferation of osteoclasts and
maintaining the growth of cells of periodontal ligament and gingival fibroblasts.
Inhibition of generation and activation of osteoclasts was by enhancing the expression of

osteoprotegerin (OPG) in osteoblasts, preventing alveolar bone from destruction by

inhibiting the proliferation of osteoclasts.
Thame (1989) described an oral hygiene method for treatment of periodontal diseases of
bacterial etiology and for reducing plaque by use of a dried methanolic extract of Vinca
rosea in the oral cavity. The composition contained 0.03 - 50% w/w of methanolic
extract. The oral rinse and toothpaste formulations of the compositions contained suitable
excipients.
Zhu (2006) described a Chinese herbal preparation for treating periodontitis, consisting of
fleece-flower (18-25%), dried rehmanria (11-18%), liquorice (3-7%), fresh-water turtle
shell (11-17%), dogwood fruit (7-15%), dodder seed (4-11%), Derla andrographis (4-
12%) and Tokyo violet herb (8-15%). The formulation could be administered in dosage
forms such as hard capsule, soft capsule, tablet, pill, granule, oral liquid etc. The active
ingredients of the above herbs prepared by supercritical carbon dioxide fluid extraction
could also be used.
Pawar (2005) explained a dentifrice herbal tooth powder which removed plaque, stain or
patches and cleaned and polished tooth surfaces without any abrasive action. The
composition comprised of the powder of Acacia catechu, Menthol and camphor in the
proportion of 91%, 2.7% and 6.3% respectively. The powder of Acacia catechu was used
to remove tarter, plaque and stain and in cleansing and polishing tooth surface without
any abrasion action. The powders of menthol and camphor were used as a flavouring
agent. A clinical study on this dentifrice herbal tooth powder reported 87-95%, 70-72%
and 80-95% reductions in plaque, gingivitis and dental calculus respectively, in merely
15 days.
Behl et al. (2004) developed a synergistic herbal formulation comprising of active
fractions from Azadirachta indica, Citrullus colocynthis and Cucumis sativus extract and
a carrier or additive. They also explained a process for preparation of formulation
comprising of extraction of neem bark and leaves in aqueous solution or polar solvents,
Citrullus colocynthis, preferably underground parts using a homogenizer in an aqueous
polar solvent and the anti-oxidant portion from Cucumis sativus by cold extraction using
aqueous polar solvents at temperature. They claimed the composition to be useful for
teeth and gums as mouth-wash or mouth rinse. This herbal formulation was described to

be useful for preventing dental plaque and gingivitis in humans and also as an anti-
microbial agent for preventing periodontal diseases. A clinical study conducted on 60
volunteer subjects to evaluate the efficacy of fraction from neem, fraction from rullms
colocynthis and a mixture of them on the reduction of dental plaque led to significant
reductions of dental plaque.
Dulin (2006) explained the use of a natural plant herb, herbal extract or essential oils like
eucalyptus oil and clove oil either alone or in combination with an anti-microbial
compound, a fluoride ion-providing compound, analgesic, enzyme etc. The composition
was formulated in the form of a liquid or a gel which moistened a single-use disposable
sterile cotton roll to be received in a buccal vestibule. The system was therapeutically
effective in treatment of periodontal diseases on topical administration.
Shi et al. (2003) developed compositions of herbs and methods of using these
compositions for the treatment and prevention of microbial infection, in particular for
dental caries or periodontal disease. The composition consisted of mixture of any two or
more herbs such as Phellodendron amurense, Paris polyphylla, Prunus mume,
Glycyrrhiza uralensis, Aframomum villosum, Sanguisorba officinalis, Elsholtzia
splendens, Eugenia caryophyllata, Rhus chinensis, Atractylodes chinensis koidz, perilla
frutescens, Coptis chinensis, Sophora flavescens, Bletilla striata, Aframomum
cardamomum, Sophora tonkinensis, Melia toosendan, and Medicinal rhubarb root. The
study was conducted for evaluation of the herbal formula using human cell lines and
Ame’s DNA mutagenesis tests and found the formulation to be safe.
Musick (2006) detailed the use of the herb, mastic gum (Isle of Chios) in alone or
combination with antimicrobials like metronidazole, Keflex, amoxicillin, tetracycline,
clathrimycin, bismuth in a method for treating the cause of a diagnosed non-gastric
disease such as periodontal disease and gingivitis. Mastic gum might be helpful in
maintenance therapy. The therapy could extend up to 120 days. Optimal success was
claimed by a 90-day therapy for preventing recurrence and was found to be effective in
treating Helicobacter sp. infections outside or involving the upper GI tract.
Levine et al. (2002) described compositions containing extracts of Echinacea purpurea
and Sambucus nigra and the extract(s) of one or more plants such as Hypericum
perforatum, Commiphora molmol and Centella asiatica. In the management of

inflammatory mucosal diseases of both viral and non-viral origin these compositions
were described to have significant application. The composition was found to be useful in
many mucosal diseases including gingivitis and periodontal diseases. Tissue samples
were taken during a double-blind matched-sample clinical trial of a transmucosal
adhesive patch of this herbal composition in sixteen patients for the treatment of gingival
inflammation showed no protease activity. The result suggested significant inhibitory
effect of the herbal composition.
Romanowski et al. (2005) described compositions that were safe and effective for
preventing and treating oral disease and for maintaining good oral health for both humans
as well as animals. They could be used regularly. They claimed compositions which
contained xylitol, a preservative and an antimicrobial agent in a carrier. The composition
might further contain a herbal ingredient such as olive leaf extract, black walnut green
hulls, clove leaf, thyme herb, grapefruit seed extract, Aloe vera, calendula flower,
Echinacea pupurea, gota kola extract, chamomile flower, green tea leaf, oregano leaf,
peppermint oil, cinnamon bark, eucalyptus leaf, lavender oil etc. This invention provided
long-term improvement in oral care and provided permanent solution to oral disorders.
The composition might be formulated as a solution which could be diluted before use, as
powders that may be added to beverages, candies, dental chew, oral gels toothpaste,
lozenges, whitening molds etc. Use of pleasant flavors was said to make the formulation
more appealing, especially for children and elderly. The composition was particularly
useful for treating and preventing oral problems such as gingivitis, periodontal disease,
stomatitis and halitosis. It also prevented build-up of tartar on teeth and also suppressed
oral and throat cancer. The composition may optionally contain antimicrobials,
immunomodulators and/or anticancer agents. The composition could exert both local and
systemic effects. For animals, food and water could be used for incorporating these
compositions.
Chen (2005) described the use of an extract of a herb from the family Scutellaria which
served as a phytoestrogen and a method of treating estrogen-related disorder such as
periodontal disease, tooth loss etc. The extract could be of Glycyrrhiza uralensis,
Glycyrrhiza glabra, Taraxacum mongolicum, Medicago sativa, Brassica oleracea or
Eclipta prostrata.

Reddy et al. (2000) explained that the effect of herbal remedies for periodontal diseases
can be enhanced by the use of probiotics using genera such as Lactobacillus and
Propionibacterium. They conducted clinical trials on herbal tooth powder and tooth paste
formulations intended for bad breath, bleeding gums and periodontal disorders. Khadira,
shilajit, guggulu, arka, cinchona, eucalyptus, vidonga karkata, shringi beejarasa,
ardhraka, amlaki and scariva were the herbal ingredients whereas Lactobacillus
acidophilus and Propionibacterium shermanii were the probiotics used. The
formulations containing probiotics were found to enhance the activity of the herbal
remedy than those without probiotics. Similar results were observed when probiotics
were added to a marketed herbal toothpaste.
Domb and Wolnerman (2003) described the usefulness of a solid, self-bioadhesive
composition for reducing the depth of periodontal pockets in a patient by topical
application that adheres to the oral mucosal tissue. The composition consisted of atleast
one herbal or homeopathic active agent. The herbal active may be herbal extracts,
tinctures, essential oils either alone or in mixtures. Essential oils such as lemon oil,
citronella oil, citron oil, cedarwood oil, juniper berries oil, pomela peel oil, lemon basil
oil, cinnamon oil, cajeput oil, eucalyptus oil, fennel oil, girofle oil, lavender oil, geranium
oil, clove oil, myrte oil, origano oil, pine oil, rosemary oil, sarriette oil, thyme oil,
spearmint oil and tea-tree oil could be used. Gotu Kola, Salvia offincinalis, Echinacea,
Hypericum, Myrrah, Elder, Plantago, Baptisia, Camphoria, Uncaria, Calendula,
Phytolaca, Catechu black, Krameria, Tsuga, Coneflower, grape fruit seed extract,
Rosmarinus, Crataegus, Glycerrhiza, Angelica, Styrax, Krameria, Matricaria, Mallow,
Propolis, Sage, Barberine from Hydrastis canadensis L. and other plant from the family
Berberidaccae, gentian from the Gentianaceae family of plants for the treatment of fungal
infections, Lonicera flower extract, Taraxacum extract, Scutellaria root extract, Gardenia
fruit extract, Pueraria root extract, Radix gentianae, Pulsatilla root extract, Longdancao
antifungal agent and combinations thereof can be used for herbal tincture actives. A solid
bioadhesive carrier in 40-99% w/w was used in the formulation for bioadhesivity.
Basu and Hoffman (2002) described a composition useful for the treatment of disorders
such as periodontal disease and any periodontal disorder caused as a result of smoking.
The composition consisted of either or both extract of Citrus reticulata and Prunus mume

with one or more of the extracts of Magnoliae officinalis, Coptis chinensis, Agastaches
rugosa, Phellodendron chinense, Zingiberis officinalis, Fraxinus rynchophylla and
Glycyrhizae inflate. Citrus reticulata and Prunus mume were reported to show significant
inhibition of LPS-induced TNF-alpha secretion in human peripheral blood mononuclear
leukocyte cells. The extracts of other seven herbs mentioned earlier exhibited significant
inhibition of TNF-alpha induced release of PGE sub 2.
Compositions of a mixture of two or more ingredients from a list of myrrh extract or oil,
mulberry bark extract, Cimicifuga heraleifolia extract and green tea extract have been
patented by Suh et al. (1993). Each of the above ingredients was in quantities of 0.001-10
%w/w of the mixture. The extraction was done with water, alcohol or acetone.
Toothpastes, breath fresheners, mouthwashes, chewing gums or sweets were the claimed
formulations of these compositions. Antibacterial activity was claimed against cariogenic
and plaque-forming bacteria. They also claimed their use in inhibiting bacterial
glycosyltransferase. Use of these compositions was proposed for prophylaxis of caries
and periodontal disease and also for treating bad breath.
Moon et al. (2003) described method for preparation of plant extract powder and use of
this extract in oral formulations for prevention and treatment of periodontal diseases and
tooth decay. The plant extract was loaded into a porous powder carrier, which was further
coated by a water insoluble coating material. The extracts was used in quantities of 0.05 –
5.0%w/w. Pine, liquorice, cassia seed, cinnamon, nothosmyrnium root, sophora, lonicera
flower, platycodon, green tea, dayflower, Korean angelica root, liriope rhizome, moutan,
Arabian myrrh, seseleos radix, Angelicae Dahuricae Radix, Lagerstroemia indica,
morusk, ginger, sanguinaria, asarum, cimicifuga, Chinese galls, Grapefruit seed, lycium
root, cnidium, Alpinia katsumadai Hayata, gardenia, Lythrum salicaria L., dandelion,
propolis, flavonoid, nepta herb, Reynoutria japonica Houtt, scutellaria, machilia, black
adzuki bean, camomile, ratanhia or Sage oil as single or in combination. The carriers may
be inorganic abrasives, high molecular weight substances, colloidal humed silica etc.
Colloidal humed silica having submicron particle size was advantageous in liquid
formulations. The water insoluble coating agent was claimed to be easily disintegrated in
the mouth and the active is released when applied in the mouth. The composition may be
formulated as toothpaste, oral cleaner, oral purifier etc.

CHAPTER 2 REVIEW OF LITERATURE 52
Table 2.2 Summary of patents on herbal remedies for periodontal infections (Pramod et al., 2009)
Plant/Herb Dosage Form References
Coptidis rhizoma and Pharbitidis semen - Auh et al., 2011
Pleurotus eryngii, Acanthopanacis cortex and Notoginseng radix. Herbal mixture Kim et al., 2010
Curcuma longa Bioadhesive Piramal et al., 2008
Verbena officinalis, Plantago lanceolata and P.angustifolia. Toothpaste, Oral gel, Greve and Greve, 2005
Lozenges, chewable tablets
and (chewable) pastilles
Pleurotus eryngii, Acanthopanacis and Notoginseng. - Kim et al., 2006
Vinca rosea Oral rinse, Toothpaste Thame, 1990, 1989
Fleece-flower, Rehmanria, Liquorice, Dogwood fruit, Dodder Hard capsule, Soft capsule, Zhu, 2006
seed, Derla andrographis and Tokyo violet herb. Tablet, Pill, Granule, Oral
liquid
Acacia catechu Willd, Menthol and camphor. Dentifrice Pawar, 2005
Azadirachta indica, Citrullus colocynthis and Cucumis sativus. Mouthwash or mouth rinse Behl et al., 2004
Eucalyptus oil and clove oil. Gel Dulin, 2006
Phellodendron amurense, Paris polyphylla , Prunus mume, - Shi et al., 2003
Glycyrrhiza uralensis, Aframomum villosum, Sanguisorba
officinalis, Elsholtzia splendens, Eugenia caryophyllata, Rhus
chinensis, Atractylodes chinensis koidz, perilla frutescens , Coptis
chinensis, Sophora flavescens, Bletilla striata , Aframomum
cardamomum, Sophora tonkinensis, Melia toosendan and
Rhubarb.
Isle of Chios - Musick, 2006
Echinacea purpurea, Sambucus nigra, Hypericum perforatum, - Levine et al., 2002
Commiphora molmol and Centella asiatica.
Olive leaf extract, black walnut, clove leaf, thyme herb, grapefruit Beverages, candies, dental Romanowski et al., 2005
seed extract, Aloe vera, calendula flower, Echinacea pupurea, chew, oral gels toothpaste,
gota kola extract, chamomile flower, green tea leaf, oregano leaf, lozenges, whitening molds
peppermint oil, cinnamon bark, eucalyptus leaf and lavender oil.

Plant/Herb Dosage Form References

Glycyrrhiza uralensis, Glycyrrhiza glabra, Taraxacum - Chen, 2005
mongolicum, Medicago sativa, Brassica oleracea and Eclipta
prostrata.
lemon oil, citronella oil, citron oil, cedarwood oil, juniper berries Topical bioadhesive Domb and Wolnerman,
oil, pomela peel oil, lemon basil oil, cinnamon oil, cajeput oil, 2003
eucalyptus oil, fennel oil, girofle oil, lavender oil, geranium oil,
clove oil, myrte oil, origano oil, pine oil, rosemary oil, sarriette
oil, thyme oil, spearmint oil, tea-tree oil, Gotu Kola, Salvia offinc,
Echinacea, Hypericum, Myrrah, Elder, Plantago, Baptisia,
Camphoria, Uncaria, Calendula, Phytolaca, Catechu black,
Krameria, Tsuga, Coneflower, grape fruit seed extract,
Rosmarinus, Crataegus, Glycerrhiza, Angelica, Styrax, Krameria,
Matricaria, Mallow, Propolis, Sage, Hydrastis canadensis,
Taraxacum extract, Scutellaria root extract, Gardenia fruit
extract, Pueraria root extract, Radix gentianae and Pulsatilla root
extract.
Citrus reticulate, Prunus mume, Magnoliae officinalis, Coptis - Basu and Hoffman, 2002
chinensis, Agastaches rugosa, Phellodendron chinense,
Zingiberis officinalis, Fraxinus rynchophylla and Glycyrhizae
inflate. Citrus reticulate and Prunus mume.
Pine, liquorice, cassia seed, cinnamon, nothosmyrnium root, Powder, toothpaste, oral Moon et al., 2003
sophora, lonicera flower, platycodon, green tea, dayflower, cleaner, oral purifier
Korean angelica root, liriope rhizome, moutan, Arabian myrrh,
seseleos radix, Angelicae Dahuricae Radix, Lagerstroemia indica,
morus bark, ginger, sanguinaria, asarum, cimicifuga, Chinese
galls, Grapefruit seed, lycium root, cnidium, Alpinia katsumadai
Hayata, gardenia, Lythrum salicaria L., dandelion, propolis,
flavonoid, nepta herb, Reynoutria japonica Houtt., scutellaria,
machilia, black adzuki bean, camomile, ratanhia and Sage oil.

Use of dried ethanolic extract of periwinkle (Vinca rosea) in the oral cavity as a method
for treatment of periodontal diseases has been described by Thame (1990). The
formulation contained 0.03 - 50% w/w of the extract. A carrier such as water, glycerin
and lower alcohols could be used for the extracts. The composition could be oral rinse or
toothpaste and may include anionic surface active agents such as sodium lauryl sulfate.
The ethanolic extract was shown to possess enhanced antimicrobial action with sodium
lauryl sulphate.
Table 2.2 summarizes the patents granted on herbal remedies for periodontal infections
2.6. Novel drug delivery systems for periodontal diseases
To date, a significant number of approaches for the management of periodontal disease
have been proposed. Table 2.3 gives a summary of some investigated novel localized
drug delivery system for the treatment of periodontal infections.
Unfortunately, the difficulty in accessing the periodontal pocket due to the complexity in
anatomy of the root and the contours of the lesion which leads to poor penetration of
junctional epithelium by some of these systems has rendered most of them only partially
successful (Medlicott et al., 1994; Schwach-Abdellaoui et al., 2000). Unreliability of the
conventional delivery system on periodontal disease treatment has significantly increased
the interest of researchers to focus on nanotechnological based approaches as it has
proved to be promising. Better penetration of active moiety into the junctional epithelium
combined with optimal drug release profiles are among the important benefits of this
approach (Piñón-Segundo et al., 2005). The local tissue concentration of a drug can be
enhanced by incorporating the active agent into controlled release delivery systems to be
placed directly in the periodontal pocket for local delivery (Schwach-Abdellaoui et al.,
2000). The local delivery of antimicrobial therapy to periodontal pockets has the benefit
of drug reaching at the target site at low dose thus minimizing exposure of the total body
from the drug (Rams and Slots, 1996; Schwach-Abdellaoui et al., 2000). Sustained local
delivery systems might also be recommended for sites considered as difficult to
instrument because of depth or anatomical complexity, for example in the case of
furcation defects (Kornman, 1993; Needleman, 1991). Every targeted drug delivery
system should has the advantage of delivering pharmacologically active moiety to the
specific target sites at a rate and concentration that will yield maximum therapeutic

efficacy while reducing side effects to minimum level (Vyas and Khar, 2002). Several
studies have shown that an effective approach to optimize the targeting and sustaining or
enhancing the action of drugs is to associate the active moiety with a carrier system.
Several varieties of drug carrier systems are available like liposomes, niosomes,
dendrimers, nanoparticles, micelles and microemulsions. Numerous nanoparticle-based
drug delivery systems have currently been developed or under development. Their use
aim to minimize drug degradation upon administration, prevent undesirable side effects,
and increase drug bioavailability and the fraction of the drug accumulated in the
pathological area. Among particulate drug carriers, polymeric nanoparticles,
microparticles, micelles, and liposomes are the most extensively studied and possess the
most suitable characteristics for encapsulation of many drugs (Vladimir, 2006). The
selection of a particular nanocarrier system depends on its advantages and limitations
combined with the drug properties and the therapeutic effect sought (Kreuter, 1994).
Table 2.4 indicates some of the advantages of different colloidal carrier systems.
2.6.1. Particulate and colloidal drug delivery systems
2.6.1.1. Microparticles
This approach facilitates accurate delivery of small quantities of potent drugs; reduced
drug concentrations at sites other than the target organ or tissue; and protection of labile
compounds before and after administration and prior to appearance at the site of action.
The characteristics of microspheres containing drug should be correlated with the
required therapeutic action and are dictated by the materials and methods employed in the
manufacture of the delivery system (Burgess and Hickey, 2007). A number of
biodegradable and non-biodegradable polymers had been used to prepare microspheres
for the delivery of drugs into the periodontal pocket. In one study, tetracycline was
loaded into the poly (lactide) (PLA)/poly(lactide-co-glycolide) (PLGA) microparticles
and was used for periodontal infection treatment. It was observed that, the rate of in vitro
release of tetracycline from the carrier particle was influenced by the polymer choice
(lactide/glycolide ratio, polymer molecular weight and crystallinity) and by the pH of the
medium (the release rate is increased as the pH increases) (Esposito et al., 1997). In
another study, microcapsules prepared with Pluronic F127 and loaded with tetracycline

Table 2.3 Summary of investigated localized drug delivery system for the treatment of periodontal infections
Type of delivery
Drug(s) Polymer(s) References
system
Microspheres Doxycycline Chitosan Shanmuganathan et al., 2008
Doxycycline PLGA + PCL Mundargi et al., 2007
Tetracycline Pluronic F 127 Baker et al., 1988
Tetracycline PLGA Esposito et al., 1997
Histatin peptides PLGA Jeyanthi et al., 1997
Bone morphogenetic proteins Glycidyl methacrylated dextran/gelatin Chen et al., 2007
scaffolds
Chlorhexidine PLGA Yue et al., 2004
Nanoparticles Metronidazole benzoate Thiolated chitosan -poly(methacrylic acid) Saboktakin et al., 2011
Triclosan PLGA Piñón-Segundo et al., 2005
Triclosan Cellulose acetate phthalate Piñón-Segundo et al., 2005
Harungana madagascariensis PLGA Moulari et al., 2006
leaf extract
Antisense oligonucleotide Chitosan Dung et al., 2007
Films Ciprofloxacin & Diclofenac Chitosan Ahmed et al., 2009
Chlorhexidine Ethyl cellulose Soskolne et al., 1983
Chlorhexidine diacetate Bycoprotein & glycerol Steinberg et al., 1990
Tetracycline Atelocollagen powder Minabe et al., 1989
Tetracycline PLGA Agarwal et al., 1993
Metronidazole Ethyl cellulose Golomb et al., 1984
Metronidazole Chitosan & PCL El-Kamel et al., 2007
Metronidazole PLGA Ahuja et al., 2006a
Metronidazole Poly(ortho ester) Vasavada and Junnarkar,
1997
Ornidazole Polyvinyl alcohol & carboxymethyl Wang et al., 2007
chitosan

Type of delivery
system
Amoxycillin & Eudragit L® and Eudragit S® Higashi at al., 1991
Clindamycin
Minocycline PCL Kyun et al., 1990
Minocycline Ethyl cellulose Elkayam et al., 1988
Taurine Chitosan Ozmeric et al., 2000
Ipriflavone Chitosan & PLGA Perugini et al., 2003
Chips (Periochip®) Chlorhexidine gluconate Cross-linked hydrolyzed gelatin & glycerin Goffin, 1998
Strips Chlorhexidine Hydroxypropyl cellulose Ozcan et al., 1994
Chlorhexidine Acrylic polymer Addy et al., 1985
Tetracycline HCl Poly(hydroxybutyric acid) Collins et al., 1989
Tetracycline HCl Polyethylmetha acrylate (acrylic) Addy and Langeroudi, 1984
Metronidazole Hydroxypropyl cellulose Wade et al., 1992
Metronidazole Poly(2-hydroxyethyl)-methacrylate Deasy et al., 1989
Tetracycline Hydroxypropyl cellulose Noguchi et al., 1984
Doxycycline Hydroxypropyl cellulose & methacrylic Taner et al., 1994
acid
Ofloxacin Polyhydroxybutyric acid Higashi et al., 1990
Tetracycline HCl PLGA Maze et al., 1995
Chlorhexidine Ethyl cellulose Paolantonio et al., 2008
Chlorhexidine Poly(hydroxybutyric acid) Friedman and Golomb, 1982
Doxycycline Acrylic polymer Larsen, 1990
Gels Doxycycline Carbopol 940 Botelho et al., 2010
Metronidazole Chitosan Akncbay et al., 2007
Tetracycline Hydroxyethyl cellulose & Jones et al., 1996
polyvinylpyrrolidone
Metronidazole Hydroxyethyl cellulose & polycarbophil Jones et al., 1997
Propolis Poloxamer 407 & Carbopol 934P Bruschi et al., 2007
Saguinarium PLA & N-methyl 2-pyrrolidone Polson et al., 1996

Type of delivery
system
Doxycycline hyclate PLA & N-methyl 2-pyrrolidone Polson et al., 1997
Metronidazole Glycerol monooleate & sesame oil Noyan et al., 1997
Tetracycline PLGA Maze et al., 1996
Tetracycline Pluronic L 101&glycerol monooleate Esposito et al., 1996
Minocycline Hydroxyethyl cellulose & aminoalkyl Hayashi et al., 1998
methacrylate copolymer
Fibers Amoxycillin trihydrate Polyvinyl acetate Ahuja et al., 2006b
Tetracycline HCl Cellulose acetate Goodson et al., 1979
Chlorhexidine Cellulose acetate Coventry and Newman, 1982
Tetracycline HCl Ethylene vinyl acetate Tonetti et al., 1990
Tetracycline HCl PCL Tonetti et al., 1990
Wafer Silver nitrate PLGA & Ethyl cellulose Bromberg et al., 2000
Benzylpenicillin PLGA & Ethyl cellulose Bromberg et al., 2001
Tetracycline PLGA & Ethyl cellulose Bromberg et al., 2000
Implant Secnidazole and/or PLA and PLGA Gad et al., 2008
Doxycycline
Micelles Triclosan Micelles of poloxamine T1107 Chiappetta et al., 2008
Lidocaine & Prilocaine Lutrol F127 & Lutrol F68 Scherlund et al., 2000
Dendrimers Triclosan Polyamidoamine Gardiner et al., 2008
Poly (dl-lactic-co-glycolic acid) (PLGA); Poly lactic Acid (PLA); Poly-ε-caprolactone (PCL)

were reported. This formed a gel at body temperature and the microcapsules were held in
the periodontal pocket throughout the duration of treatment (Baker et al., 1988).
Table 2.4 Advantages of some colloidal carrier systems
Carrier system Size (µm) Site Long Reference
specificity term
stability
Liposomes 0.05 to ≥ 1 +++ - Vyas and Khar, 2002
Niosomes 0.5 to > 1 +++ + Vyas and Khar, 2002
Dendrimers ≤ 0.01 +++ ++ Svenson and Tomalia, 2006
Nanoparticles 0.01 to 1 +++ +++ Williams III and Vaughn,
2007
Microemulsions 0.1 to 1 - + Park and Yeo, 2007
Microspheres 1 to 200 ++ +++ Burgess and Hickey, 2007
Micelles 0.01 to 0.5 ++ ++ Elena et al., 2006
Good (+++), Moderate (++), Low (+), Poor (-)
PLGA microspheres have also been investigated for the delivery of histatins. Non-ionic
surfactant was used to maintain the stability of the histatins during the release. The in
vitro release of total peptide lasted for about one month (Jeyanthi et al., 1997). Despite
the fact that these in vitro studies seem promising, some questions related to the retention
of such formulations in the periodontal pocket need clarification (Schwach-Abdellaoui et
al., 2000).
PLGA microspheres containing minocycline were developed and then evaluated alone or
as an adjunct to scaling and root planing for subgingival treatment in adult periodontitis.
This study showed that, on treatment with minocycline microspheres as an adjunct to
scaling and root planing, a significant reduction in probing depth was achieved during the
period of one month. The data showed that, the group treated by this strategy gave better
response than all other groups. However, there were no differences in probing depth
reduction among the groups at 6 months. It has been reported that minocycline local
delivery system, both with or without scaling and root planing, achieved a significant
reduction in Porphyromonas gingivalis within one month period of therapy (Jones et al.,
1994). Mundargi and associates developed novel biodegradable microspheres using the
blends of PLGA and poly(ε-caprolactone) (PCL) in different ratios for controlled delivery
of doxycycline in the treatment of human periodontal pocket. In vitro release studies
showed that, up to the period of 11 days, doxycycline concentrations in the gingival

crevicular fluid were higher than the minimum inhibitory concentration of doxycycline
against most of the periodontal pathogens. Significant results were obtained with respect
to both microbiological and clinical parameters for up to 3 months even as compared to
commercial doxycycline gel. In vivo testing suggested that these novel blend
microparticles prepared from PLGA and PCL were effective in controlled delivery of
doxycycline to the periodontal pockets (Mundargi et al., 2007). Chen and coworkers
evaluated the biological activity enhancement of a novel glycidyl methacrylated dextran
(Dex-GMA)/gelatin hybrid hydrogel containing microspheres loaded with bone
morphogenetic proteins (BMP) as periodontal cell/tissue scaffold (Chen et al., 2009).
Larger number of human periodontal ligament cells (PDLCs) attached was observed in
scaffolds containing microspheres loaded with BMP when compared to those without
microspheres. When osteogenic differentiation of PDLCs in such scaffolds was
evaluated, the alkaline phosphatase (ALP) activity, osteocalcin content, and calcium
deposition became maximum for the scaffold containing microspheres loaded with BMP,
as compared with those without microspheres. Adsorption occured with the same amount
of BMP aqueous solution, although both values were significantly higher than those in
BMP-free scaffold. In addition, they also studied osteoinduction activity following the
implantation of these scaffolds into the periodontal defects in dogs. In terms of
histological examinations, significantly more new bone formation and periodontal
ligaments regeneration were observed throughout scaffold containing microspheres. They
concluded that the attachment, proliferation, and osteogenic differentiation of PDLCs
could be enhanced by Dex-GMA/gelatin hydrogel scaffold containing microspheres
loaded with BMP, and such scaffold was promising to enhance periodontal tissue
regeneration in periodontal therapy (Chen et al., 2007).
In another investigation, an implantable, anti-microbial delivery device for the treatment
of periodontal disease was developed. In this polymer-based delivery system, the
encapsulation efficiency, release characteristics, and bioactivity of anti-microbial agent
were controlled by the complexation of the drug with cyclodextrins of differing
lipophilicity. The microparticles of PLGA containing chlorhexidine free base,
chlorhexidine digluconate and their association or inclusion complex with methylated-
beta-cyclodextrin and hydroxypropyl-beta-cyclodextrin were prepared by single emulsion

and solvent evaporation techniques. It was observed that encapsulation efficiency and
release of the chlorhexidine derivatives from the microparticles was a function of the
lipophilicity of the cyclodextrin. Complexation of the poorly water soluble chlorhexidine
with the more hydrophilic hydroxypropyl-beta-cyclodextrin resulted in higher
encapsulation efficiency and longer duration of sustained release over a 2-weeks period
than complexation with the more lipophilic methylated-beta-cyclodextrin. Preliminary
studies showed that the chlorhexidine release from PLGA microparticles was biologically
active against bacterial population that is relevant in periodontitis (P. gingivalis and B.
forsythus) and a healthy inhibition zone was maintained in agar plate assay over a period
of at least a 1-week. Therefore, this PLGA/cyclodextrin delivery system may prove
useful for the localized delivery of chlorhexidine salts and other anti-microbial agents in
the treatment of periodontal disease where prolonged-controlled delivery is desired (Yue
et al., 2004). Recently, Shanmuganathan and coworkers developed doxycycline-loaded
chitosan microspheres using water-in-oil emulsion technique. In vitro release studies
showed that a burst release of 42% in 6 h was achieved and maintained an equilibrium
concentration of 72% in 24 h. Assessment of antibacterial activity showed that
doxycycline-loaded chitosan microspheres were able to inhibit a significant number of
periodontal pathogens (Shanmuganathan et al., 2008). Although these results may
suggest some interesting potential for this therapy, one must always be cautious in
extrapolating findings from studies with a small sample size.
2.6.1.2. Nanoparticulate drug delivery systems
In recent years, formulation scientists and researchers are involved deeply in
investigating novel delivery systems which would optimize the pharmacological action of
drugs coupled with the reduction of their toxic side effects in vivo. One response is the
use of colloidal drug carriers that can provide site specific or targeted drug delivery
combined with optimal drug release profiles. Among these carriers, nanoparticles have
been the most extensively investigated (Jaeghere et al., 1999). The exploitation of the
unique attributes of nanoparticulate drug delivery systems to combat oral biofilm
formation and infection has increased markedly over the past decade (Allaker, 2010). The
advantages of nanoparticles which include small size, ability of site specificity, high
dispersibility in an aqueous medium, better stability during storage as compared with

liposomes, controlled release rate and improved dissolution and bioavailability make
them suitable candidate for delivering drugs into the periodontal pocket. In addition,
these systems reduce the frequency of administration which would improve patient’s
compliance and also can provide a uniform distribution of the active agent over an
extended period of time thus resulting into high benefit to low risk ratio as compared to
conventional formulations (Kong et al., 2006). Bako and associates demostrated that
biocompatible nanoparticles composed of 2-hydroxyethyl methacrylate and
polyethyleneglycol dimethacrylate could be used as a drug delivery system for dental
applications. The polymer-based nanoparticles were prepared via micellar
polymerisation, which resulted in a well dispersible white powder material with particle
size in the range of 50–180 nm. These nanoparticles are suitable for incorporation into a
hydrogel matrix and to design new drug delivery devices for dental applications (Bakó et
al., 2007).
Piñón-Segundo and coworkers prepared triclosan-loaded nanoparticles by the
emulsification–diffusion process, in an attempt to obtain a novel delivery system
adequate for the treatment of periodontal disease. The polymers used were PLGA, PLA
and cellulose acetate phthalate, while poly (vinyl alcohol) was used as stabilizer and up to
89.4% entrapment efficiencies was obtained. The in vitro release studies were carried out
under sink condition. In all release studies, the maximum amount of triclosan released
was higher than 97% of the drug loaded into nanoparticles, according to the drug loading.
A preliminary in vivo study in dogs with induced periodontal defects suggested that
triclosan-loaded nanoparticles penetrates through the junctional epithelium and helped
decrease gingival inflammation (Piñón-Segundo et al., 2005). The in vitro bactericidal
activity of the Harungana madagascariensis leaf extract (HLE) was studied on the oral
bacterial strains largely implicated in dental caries and gingivitis infections. In this
investigation, HLE-loaded PLGA nanoparticles were prepared using interfacial polymer
deposition following the solvent diffusion method. The study showed that, the
encapsulation of HLE into a colloidal carrier enhanced its antibacterial activity as
compared with the free drug (Moulari et al., 2006). In an attempt to develop antibiotic-
free therapy for microbial infections, nanoparticles attached with antimicrobial enzymes
was designed and studied. Satishkumar and associates developed a system in which hen-

egg lysozyme (antimicrobial enzyme) was covalently attached to two types of

polystyrene latex nanoparticles: positively charged, containing aliphatic amines surface
group; and negatively charged, containing sulphate and chloromethyl surface group. The
particles showed lower activity as compared to free enzyme and it was also found that it
could be used for targeted antimicrobial activity which makes it applicable in periodontal
treatment (Satishkumar and Vertegel, 2008).
A recent study has revealed that the retention time of metronidazole benzoate at its
absorption site could be increased by formulating it into nanoparticles using thiolated
chitosan (TCS)-poly(methacrylic acid) as a local, oral mucoadhesive delivery system that
can be applied and removed by the patient for the treatment of periodontal diseases
(Saboktakin et al., 2011).
Shefer and coworker patented a nanoparticulate controlled release system useful for site
specific delivery of biologically active ingredients over an extended period of time and
targeting biological surfaces comprising the oral cavity. The nanoparticles possessed an
average particle diameter of about 0.01 to 10 µm and comprised of biodegradable solid
hydrophobic core and a bioadhesive/mucoadhesive positively charged surface. The
bioadhesive properties of the nanoparticles were attributed to the positively charged
surfactant entrapped on the particle surface. These nanoparticles can be incorporated into
any suitable oral hygiene product including gels, chewing gums, toothpaste and
mouthwash for the treatment and prevention of periodontal disease. The nanoparticles are
particularly effective for targeted controlled delivery of biological active ingredients into
the periodontal pocket (Shefer and Shefer, 2003). Antisense oligonucleotide-loaded
chitosan-tripolyphosphate (TPP) nanoparticles were prepared and evaluated.
Chitosan/oligonucleotide-TPP nanoparticles, which were prepared by adding TPP after
the formation of chitosan/oligonucleotide complex, showed the sustained release of
oligonucleotides and are suitable for the local therapeutic application in periodontal
diseases (Dung et al., 2007). Although these studies showed promising results, it is
necessary to conduct several more studies in order to evaluate the capacity of these
carriers as drug delivery systems for periodontal infection treatment. Olgun and
coworkers prepared antimicrobial polycaprolactone composite films containing 12.5%
silica and 0.15% silver nanorods using the roll-milling method. They evaluated the

destruction of E. coli and S. aureus on the surface of the composite films after 6 h of
incubation at 37°C. For the E. coli, no bacterial contamination was detected after 6 h and
the film surface was completely disinfected with 100% reduction of the microbial
contamination. For the S. aureus, 94% reduction of the bacterial contamination was
observed after 6 h. They then compared the results with the composite films containing
triclosan. The antimicrobial activity tests with 0.25% triclosan incorporated
polycaprolactone-silica composite films showed 70% reduction of E. coli and 95%
reduction of S. aureus after 6 h (Olgun et al., 2009).
The localized delivery of proteins like growth factors has significant therapeutic potential
in regenerative medicine (Kolahi, 2010). Nanoparticles are the most suited carriers for
this localized delivery. By providing the regenerating cells a natural microenvironment
and an artificial extracellular matrix they will proliferate. The delivery of bioactive
molecules like fibroblast growth factors to these extracellular matrices is the key factor in
proliferation. The in vivo stability and efficacy of these bioactive molecules can be
maintained for a prolonged time with the help of nanoparticulate drug delivery systems.
Natural polymers like carbohydrates, gelatin and biodegradable polymers like PLGA
have been widely exploited for the controlled delivery of proteins. Chen and coworkers
developed novel composite nanoparticles based on glycidyl methacrylate(GMA) and
natural polymers like gelatin/dextran using a simple method without any organic solvents
(Chen et al., 2009). Bone morphogenetic protein (BMP) was immobilized on to
nanoparticle composites containing dextran-GMA/gelatin by swelling the polymers in
aqueous solution of BMP. They also prepared BMP encapsulated nanoparticles by using
spray-freeze dried BMP. The maximum drug loading was obtained for BMP encapsulated
nanoparticles compared to that of BMP immobilized particles. The in vitro drug release
from immobilized nanoparticles was found to be sustained. The study concluded that in
terms of bioactivity of the proteins surface immobilization was satisfactory.
Nanoparticles are also effective in endodontic treatment. Endodontic treatment is
associated with elimination of endodontic infection and healing of apical periodontitis.
Pagonis and coworkers investigated the application of photodynamic therapy based on
PLGA nanoparticles as an adjunct in antimicrobial endodontic treatment. They used
methylene blue as the photosensitizer and loaded this photosensitizer in to PLGA

nanoparticles (Pagonis et al., 2010). The in vitro studies showed that PLGA nanoparticles
were targeted to the biofilms of E. faecalis in the root canals of extracted teeth. PLGA
nanoparticles are being widely used in drug delivery applications especially due to their
high biocomaptability, biodegradability, narrow particle size distribution and modulation
of hydrophilic/lipophilic properties (Muthu, 2009). The mechanical strength of PLGA
based systems can be improved by synthesizing its copolymers. The biolological
properties of these copolymers are dependent upon the crystalline behaviour.
Polyglycolic acid is highly crystalline since it lacks the methyl side group. A wide variety
of combinations of these two monomers can be prepared with distinct physicochemical
properties suiting various applications. PLGA copolymers are less hydrophilic when it
contains more lactide content and they absorb less water thereby promoting slow
degradation. The glass transition temperature of PLGA copolymers is above
physiological temperature and they behave as glassy materials. PLGA and its copolymer
based nanoparticles have been widely utilized for the delivery of various antibacterial
agents like triclosan, streptomycin, gentamycin etc.
2.6.1.3. Micelles
Polymeric micelles are created from amphiphilic polymers that spontaneously form
nanosized aggregates when the individual polymer chains ("unimers") are directly
dissolved in aqueous solution. Hydrophobic drugs may be solubilized within the core of
the micelle or alternatively, conjugated to the micelle-forming polymer (Elena et al.,
2006). This drug carrier system is also found to be suitable for periodontal treatment.
Diego and associates investigated the effect of the incorporation of the antibacterial agent
triclosan into polymeric micelles of poloxamine T1107. They investigated the
aggregation phenomenon primarily by means of critical micellar concentration in a broad
range of pH. Triclosan-loaded aggregates proved to be active in vitro against a broad
spectrum of bacteria but more importantly, also against two representative clinical
pathogens: methicilin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus faecalis (VREF) which makes it suitable for the treatment of periodontal
diseases. While the MRSA was sensitive to even very low triclosan levels attainable in
poloxamine-free aqueous media, the VREF was inhibited only when exposed to higher
drug levels affordable exclusively using an inclusion system. These findings indicated the

release of the drug from the reservoir and also showed a significant decrease in the
number of colony-formation units (CFUs) when the biofilm was exposed to the triclosan/
poloxamine as compared to the limited activity of the polymer-free triclosan control
(Chiappetta et al., 2008). In another study, thermosetting microemulsions and mixed
micellar solutions were investigated as drug delivery systems for anesthetizing the
periodontal pocket. The structure of the systems consists of the active ingredients
lidocaine and prilocaine, as well as two block copolymers, Lutrol F127 and Lutrol F68.
The results obtained for dilute solutions showed discrete micelles with a diameter of 20-
30 nm and a critical micellization temperature of 25-35°C. The distribution study of the
active ingredients indicates a preferential solubilization of the active components in
micelles over unimers. Several advantages were observed by micellar-based systems over
emulsion-based formulations including good stability, ease of preparation, increased drug
release rate, and improved handling due to the transparency of the formulations
(Scherlund et al., 2000).
2.6.1.4. Liposomes
Liposome-delivered photosensitizer has been observed to show enhanced activity against
pathogens (Maisch, 2007) and is likely to be exploited much in coming years for the
treatment of periodontal diseases. Triclosan loaded lectinized and non lectinized
liposomes proved to be more effective in eliminating periodontal pocket bacteria than
free triclosan in targeting oral and skin biofilmforming pathogens (Drulis-Kawa
and Dorotkiewicz-Jach, 2010; Jones et al., 1994, 1993). Another study by Jones and
coworkers has reported that it is possible to target triclosan and chlorhexidine loaded
liposomes, both cationic and anionic, to bacterial biofilms (Jones et al., 1997). Robinson
and associates investigated the potentials of immunoliposome to reduce dental plaque.
The study results revealed that liposomes can be targeted to oral bacteria with the help of
monoclonal antibodies. They also observed that the affinity of the immunoliposomes for
the biofilm is affected by the presence of the liposome attached to the antibody. It is the
hydrophobicity that helps liposome in targeting bacteria by non-specific adsorption. The
electrostatic interactions are likely to predominate in case of cationic liposomes as most
bacteria have a net negative charge on the cell surface (Robinson et al., 1998). Vyas and
coworkers carried out studies with metronidazole loaded liposomes and the results

suggested that principle mechanism for drug delivery to plaque forming bacteria is
lectin–carbohydrate interactions (Vyas et al., 2001). Jin and associates investigated the
therapeutic effects of doxycycline loaded nano-liposomes in rat model. They prepared
gels in to which nano-liposomes were incorporated and compared the efficacy with
control group and minocycline treated group. Amelioration of periodontitis was higher in
the case of nano-liposomes compared with control and restoration was higher than that of
minocycline treated group (Jin et al., 2010).
2.6.1.5. Dendrimers
Dendrimers are highly branched carriers with multiple end-groups for drug attachment. It
consists of series of chemical shells built on a small core molecule and its surface may
consist of acids or amines groups which are mainly for functional groups attachment. A
number of drugs can be entrapped or encapsulated in dendrimers and thus could be of use
for delivering active moiety into the periodontal pocket (Svenson and Tomalia, 2006).
Gardiner and associates developed triclosan-loaded polyamidoamine dendrimers which
was aimed to increase the delivery efficiency of the drug into the periodontal pocket.
Solubilisation studies over the pH range 5-12 demonstrated an increase in the level of
triclosan solubility with increase in dendrimer concentration. The studies showed that
conjugating the antibacterial triclosan with polyamidoamine dendrimers could enhance
its delivery efficiency, and thus could be used for the treatment of periodontal diseases
(Gardiner et al., 2008).
2.6.2. Advantages of nanotechnology-based drug delivery systems in periodontal
diseases
An ideal targeting system should have long circulating time, it should be present at
appropriate concentrations at the target site i.e., above minimum inhibitory concentration
in case of antibiotics, and it should not lose its activity or therapeutic efficacy while in
circulation (Vyas and Khar, 2002). Nanotechnology-based delivery systems can be
designed in such away to possess all of the above characteristics and hence, drug
products have an advantage over other normal drug products (Sahoo et al., 2007).
Recently, researchers have involved in the design and development of novel drug
delivery system with more emphasis on nanotechnology-based delivery systems. Ideally,
all these systems can improve the stability, absorption, and therapeutic concentration of

the drug within the targeted tissue, as well as permit reproducible and long-term release
of the drug at the target site (Kayser et al., 2005; Kubik et al., 2005). Nanoparticulate
delivery systems provide protection for agents susceptible to degradation or denaturation
in regions of variable pH, improving patient comfort and also prolong the duration of
exposure of a drug by increasing retention of the formulation through bioadhesion
(Arangoa et al., 2001). Nanotechnology is relatively new, and although the full scope of
contributions of these technological advances in the field of human health care remains
unexplored, recent advances suggest that nanotechnology will have a profound impact on
periodontal disease prevention, diagnosis, and treatment (Burgess and Hickey, 2007). In
the near future, nanodentistry will make it possible to maintain near perfect oral health
through the use of nanomaterials, biotechnology, and nanorobotics. Through this, it will
be possible to provide high quality dental care to the millions of the world’s population
who currently receive no significant dental care (Burgess and Hickey, 2007; Freitas Jr,
2000; Ure and Harris, 2003). Some studies showed that nanoparticles can penetrate into
the junctional epithelium and therefore, can provide a potential intrapocket carrier system
for the delivery of active substances to the periodontal pocket. Ideally, nanoparticles may
establish some physical bonding with the tissue, avoiding their being flushed out by the
crevicular fluid that fills the periodontal pockets. Therefore, nanoparticles could be a
drug-delivery system used to maintain an effective drug release rate in the periodontal
pocket (Piñón-Segundo et al., 2005).
2.6.3. Applications of micro and nanotechnologies for gene delivery in periodontal
diseases
Gene therapy is a novel approach which relies on genetic engineering (Gorav et al.,
2010). Molecular techniques can be employed to introduce, suppress or manipulate
specific genes, thereby promoting an individual’s own cells to generate a therapeutic
agent (Chen et al., 2009). The delivery of growth factors in periodontal infection can be
achieved by various means such as direct infusion of the gene using viral or non viral
vectors and incorporating gene into delivery cells ex vivo and then transferring these cells
into body (Aruna and Hemalata, 2010). The usefulness of gene therapy for periodontitis
has been studied in rats by Jin and colleagues (Jin et al., 2003). Gelatin sponges were
prepared and seeded with rat sygenic fibroblast cells transduced with adenovirus

encoding BMP-7 (Ad-BMP-7) and placed into periodontal bone defects. The adenoviral
gene transfer approach showed progression in periodontal bone regeneration and
periodontal formation. But these viral delivery systems can cause carcinogenesis,
mutagenesis or can evoke immune response against viral proteins. The third –generation
periodontal regenerative therapies involve nanoscale technology which can overcome
most of the drawbacks (Gorav et al., 2010). A novel gene delivery approach by Chen and
workers involved the utilization of ultrasound for transferring nano/micro bubbles
through the periodontal soft tissues. The nano and microbubbles were prepared form
distearoyl phosphatidylcholine and a mixture of lipid bubbles/vector plasmid DNA
carrying firefly luciferase was injected to the periodontal tissues of male Wistar rats. The
experimental results demonstrated that lipid bubbles in combination with ultrasound
technique enhanced the transfection of gene to rat periodontal tissues. Bioluminescence
imaging and confocal microscopy showed the localization of gene expression in the
muscle cells of gingival tissues. Highly porous nanostructured films can also be designed
for sustained delivery of bioactive agents into the periodontal site. Rachelson and
coworkers prepared highly porous nanostructured films of Poly-DL-lactide-co-glycolide
using freeze drying of inverted emulsion (Rachelson and Zilberman, 2009). The inverted
emulsion, being able to preserve liquid structure in solid phase, led to the formation of
highly porous films. They incorporated water soluble antibiotics like ceftazidime hydrate
and mafenide hydrate. These porous films can be employed as coatings in fracture
fixation devices, periodontal treatment as well as in wound dressings. Thus highly porous
nano and microstructured films of biocompatible polymers like PDLGA can be employed
for multiple purposes.
2.6.4. Delivery of bone density regulators
Bone protective agents like alendronate sodium and residronate sodium are generally
given systemically for modulating the proliferation of osteoclasts. But they have also
been proved to be effective, when given locally, in various dental bone applications like
periodontal bone loss (Nafea et al., 2007). Conversion of osteoblasts into osteoclasts is
the crucial phenomena in bone remodelling (Aruna and Hemalata, 2010). Nafea and
associates prepared alendronate sodium loaded PLGA nanoparticles with high loading
efficiency (Nafea et al., 2007). They employed multiple emulsion technique for the

preparation of drug loaded polymer particles and characterized the obtained nanoparticles
for surface morphology, in vitro drug release, entrapment efficiency and degradation
pattern of polymer matrix. The drug loading was high and microspheres exhibited a
triphasic release over 13 days. The controlled delivery of these bone density regulators
will be therapeutically advantageous in the advanced stages of periodontitis where
progressive bone loss occurs (Pragati et al., 2009). Nano and microparticulate carrier
systems are the specific alternative to meet this purpose.
2.6.5. Metallic nanoparticles in periodontal diseases
Carbon nanofibres are a promising group of metallic nanoparticles in the periodontal drug
delivery. Their main characteristic features which helps in rendering them suitable for
drug delivery include good mechanical properties, chemical stability, high aspect ratio
and surface properties that allow easy functionalization with more biocompatible
hydrophilic groups. Liu and coworkers prepared carbon fibres conjugated with tricalcium
phosphate nanoparticles using polyacrylonitrile fibres for sintering (Liu et al., 2010). The
prepared nanocomposite was characterised for their biological properties and observed
that they exhibited good biocompatibility and pronounced effect on cell growth. Due to
solubility of tricalcium phosphate in acidic solution the nanofibre composite can be cut in
to short organism eliminable fragments. Silver nanoparticles are also being widely used
for therapeutic applications due to their antibacterial activity (Raybachuk et al., 2009;
Saravana and Vijayalakshmi, 2006). Advantage of silver nanoparticles is that they are
biocompatible and effective even at lower concentration. Hydroxy apatite is another
material of interest which constitute a major component of bone. Hydroxyapatite
nanoparticles are often advantageous in the later phases of periodontal diseases where
bone degeneration takes place. They can be delivered by incorporating into various
composites, films or membranes. Shin and associates developed chitosan–hydroxyapatite
complex for periodontal therapy. Hydroxyapatite was incorporated as nanoparticles into
chitosan film. They experimentally established that the rapid healing property of
chitosan and bone remodelling tendency of hydroxyapatite could be achieved by this
complex. Thus synergy of both the materials was accomplished. Ostim® and VITOSS®
are two marketed products based on hydroxyapatite nanoparticles which are used in
dentistry. These discrete nanoparticles can be homogenously distributed into resins or

coatings to form nanocomposites which offer the following advantages like hardness,
high flexural strength, good handling properties and reduction in filler shrinkage. Fillers
like aluminosilicate powder are also incorporated for mechanical strength.

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CHAPTER 2 LITERATURE REVIEW 25

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the intestine as it caused the induction of glutathione-S-transferases (Vidhya and Devaraj,

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and coworkers studied the in vitro penetration enhancement effect of eugenol on

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2.1.11. Other pharmacological actions

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Activity studied Experimental Effect (Dose/Concentration) Ref.

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Jadhav et al. (2004) reported the development and evaluation of controlled-release

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Poly(I:C)-, and pam3CSK-activated macrophages. Pa-EE strongly down-regulated LPS-

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eudesmadien-12,6-olide) (compound 1 ), as a bioactive marker. The patent also covered

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osteoprotegerin (OPG) in osteoblasts, preventing alveolar bone from destruction by

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Plant/Herb Dosage Form References

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egg lysozyme (antimicrobial enzyme) was covalently attached to two types of

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