ASIAN J. EXP. BIOL. SCI.

VOL 2(3) 2011: 842-486

© Society of Applied Sciences

ORIGINAL ARTICLE

A Novel Anti-Oxidant Lemon grass Oil Mouth Wash-a clinical trial

1 1 1 2 3
MeenaAnand K , Ruchika Goyal , SubrayaBhat G , Shobha Kamath , Anand K M ,Madhur
2 2 2 2
Aggarwal , Meghna A. Bhandarkar Mahima BS and Sonal Sukreeth
1
Dept. of Periodontics, Manipal College of Dental Sciences, Manipal
2
Dept. of Biochemistry, Kasturba Medical College, Manipal
3
Department of Microbiology, Melaka Manipal Medical College, Manipal.
ABSTRACT
Natural antioxidants are presumed to be safe since they occur in plant foods, and are seen as more desirable than their
synthetic counter parts. Plants also possess enzymatic systems that protect them against H2O2 and other harmful reactive
oxygen species; these include superoxide dismutase (SOD) and catalase. Antioxidants are those substances which when
present in minimum quantities prevents the oxidation of a substrate. Recently, there has been a considerable interest in finding
natural antioxidants from plants
The aim of our study was to compare the efficacy of lemongrass oil mouthwash for anti oxidant property by estimation of
superoxide dismutase levels before and after administration of lemongrass oil mouthwash. A total of 40 subjects were included
in this study. Subjects were divided into 4 groups i.e. 3 test groups and one control group. Initially, saliva and gingival
crevicular fluid (GCF) were collected and sent for analyzing the antioxidant level levels.Lemongrass oil mouthwash was
administered at three different concentrations and estimation of antioxidants was done after 15 days by collecting saliva and
gingival crevicular fluid. The subjects in the case group showed increased antioxidant levels when compared to the control
group. There was no statistical difference in the antioxidant levels within the case groups. The lemongrass oil mouthwash was
found to have anti oxidant activity at all the three concentrations levels..
KEY WORDS: Lemongrass oil mouthwash, saliva, gingival crevicular fluid, oxidative stress, superoxidedismutase

INTRODUCTION
A growing number of consumers are embracing the philosophy that natural products are better for their health and the
environment. As such, they are seeking products they perceive to be safer, healthier and without toxic chemical or
synthetic ingredients.Research has also proven its efficacy, breathing new life into it. Laboratory analysis has shown
that herbs contain vital vitamins, minerals, and natural chemicals that may be essential to curing a diseased body.
Natural antioxidants are presumed to be safe since they occur in plant foods, and are seen as more desirable than their
synthetic counter parts. These natural antioxidants occur in all higher plants, and in all parts of plants. Typical
compounds that exhibit antioxidant activity include vitamins, carotenoid and phenolic compounds [1].Plants also
possess enzymatic systems that protect them against H2O2 and other harmful reactive oxygen species; these include
superoxide dismutase (SOD) and catalase[2].
Human body is subject to numerous biologic stresses. This may be due to the environmental or pathological. The
mode of stress which has been a speculative subject of interest in the recent times is “oxidative stress phenomenon”
this is believed in part to be responsible for the inflammatory conditions. This may affect the periodontium which
manifests as gingivitis and periodontitis.
The botanical genus name cymbopogan for lemongrass is derived from Greek Cymbo – boat and pogon – beard. The
essential oil of lemon grass consists mainly of citral. Citral is a mixture of two stereoisomericmonterpene aldehydes;

482 ASIAN J. EXP. BIOL. SCI .VOL 2(3) 2011

A Novel Anti-Oxidant Lemon grass Oil Mouth Wash-a clinical trial ................................................................................................MeenaAnand K et al.

in lemon grass oil, the trans isomer geranial and isomer neral (25 to 38%). Further terpenoids in lemon grass oil are
nerol, limonene, linalool and β-caryphyllene. The content of myrcene is low, but still enough to make the oil
susceptible to oxidative polymerization. Based on important features of the herb an attempt to incorporate antioxidant
micronutrient in the form of mouthwash a small trial was done by using lemongrass oil at various concentrations.

AIMAND OBJECTIVE:
The purpose of the present study was to investigate the role of intrinsic superoxide dismutase antioxidants in
periodontal environment in gingivitis, after nonsurgical treatment, nonsurgical treatment with using lemongrass oil
mouthwash with various concentrations (0.1%, 0.25% and 0.5%) respectively.

MATERIALSAND METHODS:
The present study was done in Department of Periodontics, Manipal in collaboration with department of biochemistry
Manipal. A total of 40 subjects were recruited for the study after taking the informed consent. Subjects were divided
into 2 groups cases and controls. 30 subjects were included in case group, which was again divided into 3 groups
based on the concentration of the lemongrass oil used in the mouthwash.Atotal of 10 subjects were included in control
group. Lemongrass oil mouthwash was prepared using the standard protocol at various concentrations (0.1%, 0.25%
and 0.5%) in Department of Pharmaceutics, MCOPS, Manipal.
All the patients were examined clinically, subjects with moderate to severe gingivitis were included in the study.
Exclusion criteria were regular users of mouthwash, patients who had undergone antimicrobial therapy and scaling in
past three months. The age of the patients ranged between 20 -35 years. There were 25 male and 15 female included in
the study. Initially saliva and Gingival crevicular fluid was collected using the standard protocol and sent for
superoxide dismutase antioxidant estimation after which the scaling was done. The case group patient received the
lemongrass oil mouthwash of any one concentration. The patients were advised to use 15ml mouthwash twice daily
th
for 15 days. On 15 day the patients were recalled, saliva and gingival crevicular fluid is collected and sent for the
estimation of anti oxidant.
Sampling of the saliva was done by collecting the whole saliva in glass beakers and transferred into salivette /
eppendorf tubes and centrifuged at 3000 rpm at 4° C for 5 min, the supernatant was stored at -80°C until analysis.
Gingival crevicular fluidsampling was performed between 8:00 and 10am. The area was isolated with cotton rolls and
gently air dried. Care was taken to eliminate salivary contamination. The samples were collected by standardized
periopaper strips using intra crevicular method given by Loe and Holm Pedersen [3]. Total 12 strips were placed
successfully for 1 minute each at the entrance of the sulcus or pocket and the fluid seeping out was collected. Any
paper contaminated with blood was discarded and collection was repeated. To ensure sufficient assay sensitivity 12
strips were used, six for each antioxidant thus standardizing the procedure and keeping them at the pocket or sulci for
equal duration (i.e. 1 minute each). The GCF strips were pooled with 1ml Tris-HCL buffer (PH 6.5) and eluted for 30
minutes and stored till analysis for SOD assay. 600 microliter phosphate buffer saline eluted for 30 minutes and stored
till analysis for protein thiol assay. Thiol was analyzed in samples by DTNB (dithionitrobenzoic acid) which reacts
with accessible thiol groups and reduces them to stable compounds. DTNB is reduced to MNB
(mercaptonitrobenzoate) Superoxide dismutase activity was analyzed by the reduction of NBT (nitrobluetetrazolium)
by Xanthin / xanthin oxidase system. The fromedformazan was detected spectrophotometrically at 560nm and
compared to standard. It was measured in units/ ml. Statistical analysis was done collecting the data which was fed
into a computer and analyzed using the statistical package SPSS/PC+.

RESULTS:
Superoxide dismutase levels in saliva were analyzed and the results showed that group one two three and group four
had the median score 28.98 /ml, 23.42 /ml, 26.69 /ml, 13.01 /ml respectively. (Table1). The superoxide dismutase
levels group 1 when it was compared with group2 there was no statistical difference between the group (p= 0.24).
When group 1 was compared with group 3 there was statistical significant difference between the groups (p=0.023).
When group 1 was compared with group 4 there was statistical significant difference between the groups (p=0.000).
When group 2 was compared with group 3 there was no statistical significance difference between the groups
(p=0.35). When group 2 was compared group 4 there was statistical significant difference between the groups
(p=0.000). When group 3 was compared with group 4 there was statistical difference between the groups (p=0.000).
(Table 1)

ASIAN J. EXP. BIOL. SCI .VOL 2(3) 2011 483

A Novel Anti-Oxidant Lemon grass Oil Mouth Wash-a clinical trial ................................................................................................MeenaAnand K et al.

TABLE: 1COMPARISION OF SUPEROXIDE DISMUTASE LEVELS IN SALIVA BETWEEN GROUPS

Comparison of SOD levels in Saliva between groups

NO. Median Std Std error IQR 95% confidence
deviation interval
Lower Upper
bound bound
Group 1
10 28.98 10.89 3.44 21.34 16.99 32.59

Group 2
10 23.422 8.84 2.79 10.56 17.97 30.62

Group 3 10 26.69 31.22 9.87 20.70 1.19 45.87

Group 4 10 13.01 7.99 2.52 12.92 5.72 17.16

Comparison 1 v/s 2 1v/s 3 1 v/s 4 2 v/s 3 2 v/s 4 3 v/s 4

Mann – WhitneyU 34.0 20.50 2.00 37.00 6.00 4.00

Wilcoxon W 89.0 75.50 57.00 92.00 61.00 59.00

Asymp sig (2 -
0.22 0.026 0.000 0.32 0.001 0.000
tailed)
Exact
sig[2*(1 -tailed 0.24 0.023 0.000 0.35 0.000 0.000
sig.)]

Superoxide dismutase in gingival crevicular fluid were analyzed and the results showed that group one two three and
group four had the median score and 30.21 /ml, 23.80 /ml, 16.59 /ml and 3.71 /ml respectively. (Table 2) The
superoxide dismutase levels of gingival crevicular fluid when group 1 was compared with group 2 there was no
statistical difference between the groups (p=0.35). When group 1 was compared with group 3 there was no statistical
significant difference between the groups (p=0.015). When group 1 was compared with group 4 there was statistical
significant difference between the groups (p=0.000). When group 2 was compared with group 3 there was no
statistical difference between the groups (p= 0.43). When group 2 was compared with group 4 there was statistical
difference between the groups (p=0.002). When group 3 was compared with group 4 there was statistical difference
between the groups (p=0.023). (Table 2)

DISCUSSION:
Antioxidants orchestrate many biologic responses to inflammation and immunity, they function as signaling
mechanisms for redox regulation, even minimal levels of oxidative stress is highly sensed and the protective
antioxidant mechanism is set into action which is essential for the maintenance of the structural integrity of proteins
thus explaining why their levels must have increased in the present study.
In the present study the subjects selected were having gingivitis. Superoxide dismutase levels increased when
compared with the initial values in all the 4 groups. The increase may be due to the initial periodontal therapy in group
4 and in group 1,2and 3 the increase in superoxide dismutase was due to initial therapy and use of lemongrass oil
mouthwash. This indicates that as the giniigvitis reduces, antioxidant levels increase and oxidative stress reduces
which is in correlation to the reported literature by Bartold PM et al [4], Battino, M et. al [5], Halliwell,B.[6], Van
dyke, T.E et al. [7], Waddington, R.J[8]. In group 4 there was increase in the

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A Novel Anti-Oxidant Lemon grass Oil Mouth Wash-a clinical trial ................................................................................................MeenaAnand K et al.

TABLE: 2COMPARISION OF SUPEROXIDE LEVELS IN GINGIVAL CREVICULAR FLUID BETWEEN

GROUPS

Comparison of SOD levels in Gingival crevicular fluid between groups

NO. Median Std Std error IQR 95% confidence interval
deviation Lower Upper
bound bound
Group 1 10 30.21 6.55 2.07 7.71 23.58 32.95

Group 2 10 23.80 11.88 3.75 22.00 14.96 31.96

Group 3 10 16.59 16.46 5.20 11.02 3.03 26.58

Group 4 10 3.71 12.31 3.89 15.15 -4.25 13.36
Comparision 1 v/s 2 1v/s 3 1 v/s 4 2 v/s 3 2 v/s 4 3 v/s 4
Mann – Whitney 37.0 18.0 5.0 39.0 11.0 20.0
U
Wilcoxon W 92.0 73.0 60.0 94.0 66.0 75.0

Asymp sig (2 - 0.32 0.016 0.001 0.40 0.003 0.023

tailed)
Exact sig[2*(1 - 0.35 0.015 0.000 0.43 0.002 0.023
tailed sig.)]

antioxidant level, but it was lesser than that of the other groups. This may imply that the lemongrass oil
mouthwash may have an additive effect on the treatment outcome, when it was used along with scaling.
The antioxidant activity of the lemongrass oil was also studied by Rabbani, S.I et al. [9] studied the antioxidant
activity of citral by in vitro, which showed its anti oxidant activityby superoxide scavenging method. The result
showed that citral significantly inhibited the formation of micronuclei induced by nickel. Superoxide scavenging
activity of the citral suggests that the antioxidant action could be responsible for anti-clastogenic effect of citral
against nickel chloride.
The isoform CuZnSOD which is extracellular form of superoxide dismutase enzyme is reported to be surface bound to
collagen fibrils of gingival and periodontal fibroblasts and protects against the powerful oxidizing agent superoxide.
During oxidative stress like states of inflammation the superoxide dismutase gets temporarily inactivated or depleted.
On reducing the bacterial load, neutrophil induced release and fibroblast induced release of superoxide is reduced thus
reactivating and replenishing the suppressed superoxide dismutase enzyme [10].
Saliva is a glandular secretion unlike GCF which is an interstitial fluid but since saliva has protein in its composition
anti-oxidants will be expressed as a part of its protein system. In the present study the superoxide dismutase
antioxidants were present in saliva.Superoxide dismutase levels were reduced before treatment and were increased
after the non-surgical treatment and also by using lemongrass oil mouthwash. The study is in accordance with the
study done by Diab –Ladki et al. [11] who showed reduced antioxidant level in gingivitis and periodontitis group
when compared with the healthy group.
Lemongrass oil has antibacterial[12], anti inflammatory[13], and also superoxide scavenging property[9]. Reduction
in the bacterial load, decrease in inflammation and reduction of the oxidative stress will bring about the overall health
of the tissues. Based on above property, lemongrass oil mouthwash can be used as an adjunct along with the non
surgical therapy.

CONCLUSION:
Lemongrass oil mouthwash can be used as an adjunct along with the non surgical therapy. Further investigations are
required to emphasize the relevance of these intrinsic antioxidants and beneficial properties of lemongrass oil mouth
wash need to be done.

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REFERENCES:
[1]. Chopra, R.N.(1985) Indigenous drugs of India. Dhur& Sons. Pvt. Ltd. Calcutta, India. .
[2]. Perry, L.M.(1980) Medicinal Plants of East and South EastAsia. The MIT Press, London, pp. 164-5;
[3]. Loe H.and Holm. Pederson, P.( 1965)Absence and presence of fluid from normal and inflamed gingiva. Period ontology; 3: 171 – 7.
[4]. Bartold, P.M and Page, R.C.(198 6) The effect of chronic inflammation on gingival connective tissue proteoglycans and hyaluronic acid.
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[10]. Jacoby, B.H.and Davis, L.W.(1991) The electron microscopic immunolocalization of a copper zinc superoxide dismutase in association
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[11]. Diab –Ladki, R., Pellat, B. and Chahine, R.(2003) Decrease in the total anti oxidant activity of saliva in patients with periodontal diseases.
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[12]. Prabuseenivasan, S., Jayakumar, M and Ignacimuthu, S.(2006) In vitro antibacterial activity of some plant essential oils. BMC
Complementary and Alternative Medicine , 6: 39
[13]. Carbajal, D., Casaco, A., Arruzazabala, L., Gonzalez, R.and Tolon, Z. (1989) Pharmacological study of Cymbopogon citrates leaves. J.
Ethnopharmacology; 25: 103-107.

Correspondence to Author : Dr.K.MeenaAnand , Dept. of Periodontics ,Manipal College of Dental Sciences ,Manipal –
576104 Karnataka, INDIAE-Mail: drmeenaanand@yahoo.com, drmeenanand@gmail.com

486 ASIAN J. EXP. BIOL. SCI .VOL 2(3) 2011