CLINICAL AND BIOCHEMICAL EFFECTS OF COCOA CONSUMPTION … PDF 2017/65-74.pdf · Kumar K N, Srinath...

Kumar K N, Srinath C, Mohan P C, Narayana B R, Sunder S S, Swathi V. Clinical and Biochemical

effects of Cocoa consumption in moderate Chronic Periodontitis . J Periodontal Med Clin Pract 2016;03: 65-74.

1 2 3 4 5 6Dr. Kiran Kumar. N, Dr. C. Srikanth, Dr. P. Chandra Mohan, Dr. B. Rama Narayana, Dr. S. Shyam Sunder, Dr. V.Swathi.

Clinical And Biochemical Effects Of Cocoa Consumption In Moderate Chronic Periodontitis

Original Research

Affiliation

1. Professor, Department of periodontics, Mamata Dental College, Khammam, India

2. Professor & Head of Department, Department of periodontics, Mamata Dental College,

Khammam, India

3. Professor, Department of periodontics, Mamata Dental College, Khammam, India

4. Senior lecturer, Department of periodontics, Mamata Dental College, Khammam, India

5. Senior lecturer, Department of periodontics, Mamata Dental College, Khammam, India.

6. Post Graduate Trainee, Department of periodontics, Mamata Dental College, Khammam, India

Corresponding Author:

Dr.Kiran Kumar. N

Department of Periodontics, Mamata Dental College, Khammam.

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ABSTRACT

Cocoa is produced from the seeds of the tropical tree

Theobroma cacao. Cocoa in the dark chocolate is

proven to have properties like antioxidant, anti-

inflammatory, anti-carcinogenic, anti-cariogenic,

anti-bacterial and anti-viral agent. Cocoa enriched

diet reduces the oxidative stress induced

periodontitis.

AIM: To evaluate the effect of cocoa consumption

in the treatment of patients with chronic

periodontitis.

MATERIALS AND METHODS: Study group

consists of 40 patients who are randomly divided in

to two groups namely, treatment group and control

group. Treatment group received 30gms/day dark

chocolate with cocoa and control group received

30gms/day white chocolate without cocoa for

4weeks after initial scaling and root planning.

Clinical parameters evaluated are gingival index,

modified papillary bleeding index, probing pocket

depth, clinical attachment loss. Saliva samples were

collected from patients at baseline and 4 weeks after

eating chocolate for total antioxidant status,

glutathione and lipid peroxidation of saliva.

RESULTS: Intra group comparison showed there

is significant decrease in clinical parameters in both

the groups after 4 weeks, with more significant

decrease in the test group. Test group showed there

is additional significant reduction in lipid

peroxidation when compared to the control group.

Total anti-oxidant status and glutathione levels

increased significantly in the dark chocolate group

than control group.

CONCLUSION: Consuming cocoa increases total

anti-oxidant status of saliva and decreases lipid

peroxidation, gingival bleeding and inflammation.

INTRODUCTION:

Periodontitis is a chronic inflammatory disease of

tooth supporting structures, initiated by oral

microbiota and their products. Polymorphonuclear

Vol-III, Issue - II, May-Aug 2016

leukocytes (PMN) act as first line of defense and

they release large amount of reactive oxygen

species (ROS). ROS are highly reactive molecules

originated from molecular oxygen, containing one 1or more unpaired electrons . These ROS are

neutralised by body's natural antioxidants, if not

leads to damage namely oxidative stress.

Polyunsaturated fatty acids (PUFA) in the

membrane lipids are the main targets of ROS

which thereby causes lipid peroxidation.

Peroxidation of PUFA results in the formation of

Malondialdehyde which is used as a biomarker of 2lipid peroxidation .

An antioxidant is any substance that when present

at concentrations below those of their oxidizable

substrate significantly delays or prevents

oxidation of that substrate. The different possible

mechanisms by which antioxidants may offer

protection against free radical damage include

:Inhibit the formation of free radicals, Scavenge

radicals to inhibit chain initiation and break chain

propagation, Repair of the damage caused by free 3radicals with the help of ''de novo'' enzymes .

Dark chocolate has been proven to be the diet with

maximum amount of antioxidants due to the

presence of 70-99% of cocoa. Cocoa is a

Polyphenolic flavonoids. Flavonoids possess

antioxidant, anti-allergic, anti-inflammatory, 4cytoprotective and antibacterial activity . Thus

dark chocolate has been discovered to have a

number of health benefits like alleviation of

cardiovascular disease, regulation of blood sugar,

antioxidant protection, alleviation of cold and

cough, reduced cancer risk, slowing aging, 5increased immune function etc .

Hence the aim of the study is to evaluate the effects

of cocoa rich dark chocolate on lipid peroxidation

and antioxidant status in saliva of chronic

periodontitis patients.

MATERIAL & METHODS:

PATIENT SELECTION:

This cross sectional observational study was

conducted on a total of 40 subjects reporting to the

department Periodontology, Mamata Dental

College, Khammam. The participants enrolled in

the study were belonged to the age group of 35-50

years. Regular dental check-ups were done on

consecutive series of patients and divided in to two

groups namely control (white chocolate) group

and test (dark chocolate) group. All the subjects

were explained about the study and written

informed consent were obtained.

INCLUSION CRITERIA:

1. Patients of age between 35-50 years.

2. Patients diagnosed with moderate form of

chronic periodontitis.

3. Patients without any history of previous

periodontal therapy.

Periodontal diseases classification is based on the

American Academy of Periodontology 1999 6classification system . The main diagnostic

criteria is clinical attachment loss (CAL) 3 – 4mm.

EXCLUSION CRITERIA:

1. Patients who took any medication in the

last 3 months.

2. Patients taking any nutritional and vitamin

supplements.

3. Patients with any other systemic diseases.

4. Pregnant and Lactating women.

5. Smokers and Alcoholic patients.

6. Patients allergic to cocoa products.

INTERVENTION:

All patients received phase I periodontal treatment

i.e., scaling and root planing along with oral

hygiene instructions. One week later, the patients

with less than 30% plaque index participated in the

study. Participants were assigned to two groups.

Group I participants received 30 grams of white



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chocolate without cocoa (MARCO company,

India) and group II receives 30 grams dark

chocolate rich in cocoa (MARCO company,

India) three times a day for 4 weeks. Chocolates

were packed in encoded packets.

Clinical parameters evaluated are gingival index

(GI), modified papillary bleeding index (MPB),

probing pocket depth (PD), clinical attachment

loss (CAL). Measurements are done with a UNC

15 probe. All the parameters were evaluated at

baseline and after 4 weeks.

SALIVA SAMPLE COLLECTION:

Prior to the collection procedure, the participants

began by rinsing their mouths thoroughly several

times with water and then resting quietly for a

while. Un-stimulated whole saliva (3 ml) was

collected by means of the spitting method. The

collection started with the instruction to rid the

mouth of saliva by swallowing. Subsequently,

saliva was allowed to accumulate in the floor of

the mouth, without stimulation of saliva secretion

by means of oro-facial movements. The

participant then spit the accumulated saliva into

an ice-chilled sterilized containers. All the

collected saliva samples are stored under -80̊C.

BIOCHEMICAL INVESTIGATIONS:

Malondialdehyde levels of saliva were estimated

using TBARS assay (Tiuborbituric acid reactive

substances), total anti-oxidant status of saliva

using FRAP assay (ferric reducing antioxidant

power) and glutathione (GSH) levels by DTNB

(5,5/-dithiobis-2 nitrobenzoic acid) through

spectrophotometric assay.

TBARS are measured at 532 nm after boiling with 7the thiobarbituric acid and FRAP after reaction

with 2,4,6-tripyridyl-s-triazinein hydrochloric 8acid, acetate buffer and ferric chloride at 593 nm .

GSH based on reduction of 5,5/-dithiobis-2 9nitrobenzoic acid at 412 nm .

Figure: LABINDIA – UV 3200 DOUBLE BEAM SPECTROPHOTOMETER.



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STATISTICAL ANALYSIS:

Intra group comparison of clinical parameters and

biochemical parameters were analysed using

paired t test and Inter group using independent

sample t test.

RESULTS:

The mean values of the clinical parameters of

both the groups i.e., white chocolate and dark

chocolate groups at baseline and after 4 weeks are

shown in tables 1a and 1b respectively.

Significant difference was observed from base

line to 4 weeks in GI, MPB, FRAP, GSH and

TBARS values. There is decrease in GI, MPB,

TBARS and increase in FRAP values in both the

groups. No significant difference was observed in

terms of PD and CAL. Inter group comparison at

baseline and after weeks are shown in tables 2a

and 2b respectively. At baseline when both the

groups were compared significant difference was

observed only in FRAP values. Inter group

comparison after 4 weeks showed there is

significant difference in MPBI, FRAP, GSH and

TBARS.

Group Baseline 28 days p-value

Mean SD Mean SD

White GI 1.65 .16 1.29 .10 <0.001; Sig

MPB 1.55 .10 1.20 .08 <0.001; Sig

PD 2.97 .30 2.90 .23 0.11; NS

CAL 3.16 .19 3.14 0.14 0.526; NS

FRAP 238.86 16.95 263.81 12.62 <0.001; Sig

TBARS .32 .02 .29 .02 <0.001; Sig

GSH 0.59 0.06 0.61 0.14 <0.002; Sig

Table 1a: Intra-group.



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Group Baseline 28 days p-value

Mean SD Mean SD

Dark GI 1.73 .13 1.26 .08 <0.001; Sig

MPB 1.60 .12 1.13 .10 <0.001; Sig

PD 3.06 .14 2.98 .24 0.107; NS

CAL 3.23 .19 3.2 0.2 0.156; NS

FRAP 256.76 9.63 500.43 17.26 <0.001; Sig

TBARS .32 .03 .26 .05 <0.001; Sig

GSH 0.54 0.05 0.78 .08 <0.001; Sig

Table 1b: Intra-group



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Group p-value

White Dark

Mean SD Mean SD

Baseline GI 1.65 .16 1.73 .13 0.097; NS

MPB 1.55 .10 1.60 .12 0.121; NS

PD 2.97 .30 3.06 .14 0.207; NS

CAL 3.16 .19 3.23 .19 0.213; NS

FRAP 238.86 16.95 256.76 9.63 <0.001; Sig

TBARS .32 .02 .32 .03 0.59; NS

GSH 0.59 0.06 0.54 0.05 0.203;NS

Table 2a: Inter-group

Group p-value

White Dark

Mean SD Mean SD

28 days GI 1.29 .10 1.26 .08 0.336; NS

MPB 1.20 .08 1.13 .10 0.02; Sig

PD 2.90 .23 2.98 0.24 0.302; NS

CAL 3.14 .14 3.2 0.2 0.293; NS

FRAP 263.81 12.62 500.43 17.26 <0.001; Sig

TBARS .29 .02 .26 .05 0.044; Sig

GSH 0.61 0.14 0.78 .08 0.031; Sig

Table 2b: Inter-group



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DISCUSSION:

Only few studies evaluated the effect of cocoa

enriched diet on periodontium. This study has been

done as an attempt to correlate cocoa with

periodontal parameters. Daily consumption of

cocoa enriched diet can significantly reduce the

lipid peroxidation and also increase the total anti-

oxidant capacity. The results showed there is

decrease in gingival inflammation and bleeding in

both groups which may be due to scaling and root

planing along with oral hygiene instructions. But

this decrease was more significant in test group

those who have consumed dark chocolate. This is

because gut bacteria breaks down and ferments the

components in dark chocolate, converting them

into anti-inflammatory compounds like flavanols, 10catechins etc. with properties like scavenging

activity, inhibit lipid peroxidation, reduction in 11oxidative stress.

There is significant increase in the total

antioxidant and glutathione levels and significant

decrease in the lipid peroxidation in both the

groups. Cocoa in dark chocolate is rich source of

anti-oxidants that protect DNA by preserving the

cell membranes by scavenging the free radicals.

Procyanidins and their monomeric precursors,

epicatechin and catechin, contribute to the 12antioxidant activity of cocoa.

These results are in accordance with a similar 13study by Roodgaryan et al on moderate form of

chronic periodontitis patients. MPBI and GI were

significantly decreased in treatment group

compared to the control in the weeks of 4th, 6th thand 8 . Treatment group showed the increase in

FRAP, and decrease in TBARS, which were

statically significant when compared with control

group.14Takaaki Tomofuji et al investigated the levels of

8-hydroxydeoxyguanosine and reduced/oxidized

glutathione ratio to evaluate gingival oxidative

damage and antioxidant status, respectively. Rats

fed with cocoa enriched diet did not show any

change in 8-hydroxydeoxyguanosine and

reduced/oxidized glutathione ratio, whereas rats

with regular diet showed increased 8-

hydroxydeoxyguanosine level and decreased

reduced/oxidized glutathione ratio. Thus

supporting cocoa-enriched diet hampers

periodontitis-induced oxidative stress.15Wang et al studied the effect of Chocolate

consumption on plasma epicatechin and oxidative

Damage. Results showed that there is a direct

relationship between consumption of dark

chocolate and plasma concentration of

procyanidin and also the rise in plasma epicatechin

levels contributed to the ability of plasma to

dampen lipid peroxidation by preventing the free

radicals. The effect of Chocolate Consumption on 16Plasma Oxidation Status by Rein et al proved

that Consumption of chocolate can result in

significant increase in plasma epicatechin

concentrations and decreases in plasma oxidation

products.

Daily consumption of cocoa enriched diet resulted

in significantly higher levels of epicatechin and

increased resistance of human low density 17 18lipoproteins to oxidation . Dietrich Rein et al

stated that 2hours after consumption of chocolate

there was a significant increase in plasma total

antioxidant capacity by 31% and a decrease of in

plasma 2-thiobarbituric acid reactive substances

by 40%.

CONCLUSION:

Dark chocolate with cocoa enriched-polyphenol

have various properties, which endow them with

various positive effects. These properties inhibits

lipid peroxidation and increases total anti-oxidant

capacity of saliva in chronic periodontitis patients.



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Competing interest / Conflict of interest The author(s) have no competing interests for financial support, publication of this research, patents and royalties through this collaborative research. All authors were equally involved in discussed research work. There is no financial conflict with the subject matter discussed in the manuscript.Source of support: NIL

Copyright © 2014 JPMCP. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



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