Effects of Varying Concentrations of Oregano (Origanum vulgare) Extract in the Cellular

Respiration Using Mouse (Mus musculus) Liver Homogenate1

Submitted by:

Manglicmot, Kareen Joy B.

BIO 120 S-3L
Group 3

Date Submitted:

18 November 2015

1
A scientific paper submitted to Professor Cheryl A. Natividad in partial fulfillment of the
requirements in BIO 120: Cell Biology laboratory, 1st Semester, AY 2015-2016.
 ABSTRACT

In this study, we investigated the effect of varying

concentrations of oregano (Origanum vulgare) extract to
cellular respiration. Mouse liver homogenate, succinate
solution and varying concentrations of oregano extract were
mixed in four different setups. The rate of respiration was
determined spectrophotometrically. The absorbance of DPIP
was measure at 605 nm for 10 minutes at 2 minute intervals.
Results showed that a high concentration of oregano extract
had increasing absorbance which means that there was no
dye reduction thus cellular respiration did not occur. A low
concentration of the oregano extract on the other hand had a
slight decrease in absorbance which means that there was a
slight DPIP reduction thus cellular respiration occurred at a
very slight extent. Oregano extract contain carvacrol and
thymol which are known for their antimicrobial properties.
These antimicrobial property of carvacrol and thymol may
also be associated with oregano extracts inhibitory effect on
cellular respiration. Although further studies should be
conducted in order to identify the mechanism behind the
observed effect of oregano extract in cellular respiration.

INTRODUCTION

Cellular respiration is the process wherein free energy in sugars and other molecules

derived from food is released. It is the chemical transformation of energy rich compounds in the

diet such as glucose and starch into ATP, the energy currency of the cell (Lodish et al., 2003).

Cellular respiration has three main metabolic stages namely glycolysis, tricarboxylic acid cycle or

Krebs cycle and oxidative phosphorylation (electron transport and chemiosmosis). In a eukaryotic

cell, glycolysis occurs in the cytosol, the tricarboxylic acid cycle or Krebs cycle takes place in the

mitochondrial matrix while the site of the oxidative phosphorylation (electron transport and

chemiosmosis) is the inner mitochondrial membrane (Campbell and Reece, 2008). Thus, the

mitochondrion is also termed as the “powerhouse” of the cell because it serves as the cell’s energy

transducing system (Reamillo et al., 2009).

 In order to carry on the processes in cell respiration, a group of proteins are needed to

catalyze reactions. These proteins are specifically called enzymes. Enzymes have extraordinary

catalytic power which is often far greater than that of synthetic or inorganic catalysts. They are

specific for their substrates and accelerate chemical reactions tremendously. Enzymes catalyze the

hundreds of stepwise reactions that degrade nutrient molecules, conserve and transform chemical

energy, and make biological macromolecules from simple precursors. Through the action of

regulatory enzymes, metabolic pathways are highly coordinated to yield a harmonious interplay

among the many activities necessary to sustain life (Nelson and Cox, 2008). However, the catalytic

activities of these enzymes may be inhibited in a different ways. Inhibition refers to the decrease

on the extent or rate of enzymatic activity. These inhibitors may bind to either the free enzyme or

enzyme-substrate complex. Inhibition may be classified as competitive, noncompetitive and

uncompetitive (Reamillo et al., 2009).

Researchers have now given interest in herbal plants for their pharmaceutical properties.

In this study, we focused on oregano as a possible inhibitor of cellular respiration. Oregano

(Origanum vulgare) is a species under the mint family. It is an herb that is used in culinary and

folk medicine. Oregano contains polyphenols, including numerous flavones (Dragland et al.,

2003). Ingesting large amount of essential oil of O. vulgare results to stimulation of blood flow in

the pelvic area and uterus and abortion (Enrst, E., 2002). In Austrian folk medicine, oregano was

used internally (as tea) or externally (as ointment) for treatment of disorders of the gastrointestinal

tract, respiratory tract, and nervous system (Vogl et al., 2013).

In this study, we aim to determine the effect of oregano extract in cellular respiration,

specifically its effect on the activity of succinate dehydrogenase by measuring

spectrophotometerically measuring the rate of reduction of the dye 2,6-dichlorophenolindophenol

(DPIP). It is hypothesized that varying concentration of oregano extract have varying effects on

the rate of respiration in the liver mitochondria of mice. The study was done to give insights on

the effect of extract of herbal leaves like Oregano in cellular respiration.

The study was conducted at D-332, Institute of Biological Sciences, University of the

Philippines Los Baños on November 11, 2015 at 10 AM to 1 PM.

METHODOLOGY

I. Extraction of Juice from Oregano Leaves

10 grams of fresh oregano leaves which had been previously washed with distilled

water were weighed and cut into small pieces. The shredded leaves were placed in a pre-

cooled mortar and ground thoroughly using the pestle. The extract was then strained through

a double-layer of cheesecloth into an ice cold beaker.

II. Homogenization of Liver Tissue from Mouse

A mouse was sacrificed by pulling its tail firmly and quickly while securing the mouse

at its neck region. The abdominal and thoracic cavities were opened with an incision at the

midline with care to avoid damage on the liver. The liver was then removed rapidly. The liver

was weighed and immersed in cold 0.05 M phosphate buffer, with pH 7.3. (50 mL of 0.05 m

phosphate buffer was used for every 1 g of liver).The liver with phosphate buffer was then

blended at high speed in a cold blender for 30 seconds then cooled the jar in an ice bucket for

another 30 seconds. This was done three times. The resulting homogenate was then placed in

a cold beaker and maintained in an ice bath.

III. Purification of the Liver Mitochondria

The liver homogenate was decanted into four 25 mL centrifuge tubes with care not to

include the debris that settled. The homogenate was spun using the centrifuge at the Genetics

Laboratory at 2500 rpm for 10 minutes at 0-4°C to allow sedimentation of debris and nuclei.

The supernatant was decanted again into pre-chilled 15 mL centrifuge tubes while the pellet

was discarded. The supernatant was spun again using the centrifuge at the MBB Laboratory

at 10,000 x g for 15 minutes at 0-4°C. The supernatant was discarded after. 5 mL of 0.05 M

phosphate buffer was added to each mitochondrial pellet. It was resuspended and collected

into a small beaker.

IV. Measurement of Respiration by Dye Reduction

1 mL of succinate solution was placed in each of the 4 test tubes and appropriate

reagents and amount of each as indicated in Table 1. For each tube, a specific blank was used.

Concentrated DPIP and liver homogenate were added prior to reading the absorbance of each

mixtures. The absorbance was read at 605 nm at 2 minute interval for 10 minutes. The

mixtures were shaken and the cuvettes were wiped before each reading. Absorbance readings

were recorded in proper table in Appendix A. The absorbance was plotted against time in

Figure 1.
Table 1. Test tubes contents (in mL) for the measurement of respiration by dye reduction
Tube/ Liver Succinate Water Oregano DPIP
Blank no. homogenate Solution Extract solution
1 1 1 1 1 1
Blank 1 1 2 1 0
2 1 1 1.5 0.5 1
Blank 1 1 2 1 0
3 1 1 1.75 0.25 1
Blank 1 1 2 1 0
4 1 1 2 0 1
Blank 1 1 3 0 0

RESULTS AND DISCUSSION

Results showed that varying concentrations of oregano extract have varying effects on the

rate of cellular respiration which was measured spectrophotometrically by dye reduction of

concentrated DPIP. Test tube 1 contained concentrated oregano extract (1mL) while test tube 2

contained 0.5 mL oregano extract, test tube 3 contained 0.25 mL oregano extract while test tube 4

did not contain oregano extract which also served as the negative control. Figure 1 shows the

absorbance of DPIP in four setups containing varying concentrations of oregano extract, water,

succinate solution and concentrated DPIP at 605 nm for 10 minutes at 2 minute interval. Based on

the trendlines constructed, test tube 1 which contained 1mL of oregano extract had the highest

increase in absorbance followed by test tube which contained 0.50mL oregano extract. Test tube

3 with 0.25 mL oregano extract had a slight decrease in absorbance while test tube 4 which did not

contain oregano extract had a dramatic decrease in absorbance.

A decrease in absorbance indicates that respiration took place which means DPIP was

reduced which is evident in its change in colour from blue to colourless because there was a change

in its molecular structure. On the other hand, an increase in absorbance indicates that respiration
 did not take place because DPIP did not change its colour from blue to colourless because a transfer

of electrons did not occur.

0.18
0.16
0.14
0.12
0.1
Absorbance

0.08
0.06
0.04
0.02
0
-0.02 0 2 4 6 8 10 12

-0.04

Legend: 1 2 3 4 Linear (1) Linear (2) Linear (3) Linear (4)

Time (min)
Figure 1. Absorbance of DPIP in four setups containing varying concentrations of oregano
extract, water, succinate solution and concentrated DPIP at 605 nm for 10 minutes at 2 minute
interval.

Results obtained can be related to the chemical compounds present in oregano carvacrol

are thymol (Mockute et al., 2001). Carvacrol is present in the essential oil of oregano and is

a monoterpenoid phenol that gives oregano its characteristic pungent, warm odor (De Vincenzi et

al., 2004). Carvacrol inhibits the growth of several bacterial strains such as E. coli (Du et al., 2008)

and Bacillus cereus. In Pseudomonas aeruginosa, carvacrol causes damages to the cell

membrane of these bacteria and, unlike other terpenes, inhibits their proliferation (Di Pasqua et

al., 2007). The cause of the antimicrobial properties is believed to be disruption of the bacteria

membrane (Cristani et al., 2007).

 On the other hand, thymol is a natural monoterpene phenol and is isomeric with carvacrol,

which gives oregano its pleasant aromatic odor and strong antiseptic properties. Thymol has

shown antibacterial activity against bacterial strains including Aeromoans

hydrophila and Staphylococcus aureus (Dorman and Deans, 2000). This antibacterial activity is

caused by inhibiting growth and lactate production, and by decreasing cellular glucose uptake

(Evans and Martin, 2000).

These antimicrobial property of carvacrol and thymol may also be associated with oregano

extracts inhibitory effect on cellular respiration. Although further studies should be conducted in

order to identify the mechanism behind the observed effect of oregano extract in cellular

respiration.

SUMMARY AND CONCLUSION

Cellular respiration is the conversion of the energy found in sugar and other food stuff into

ATP. Several enzymes are involved in this stepwise process. There are also inhibitors that can

decrease or even stop the activity of these enzymes thus also decreasing the amount of ATP

produced. In this study, we investigated the effect of varying concentrations of oregano extract to

cellular respiration. It was hypothesized that varying concentration of oregano also have varying

effect on respiration.

Mouse liver homogenate, succinate solution and varying concentrations of oregano extract

were mixed in four different setups. The rate of respiration was determined by dye reduction using

concentrated DPIP. Results showed that a high concentration of oregano extract had increasing

absorbance which means that there was no dye reduction thus cellular respiration did not occur. A
low concentration of the oregano extract on the other hand had a slight decrease in absorbance

which means that there was a slight DPIP reduction thus cellular respiration occurred at a very

slight extent.

Oregano extract contain carvacrol and thymol which are known for their antimicrobial

properties. These antimicrobial property of carvacrol and thymol may also be associated with

oregano extracts inhibitory effect on cellular respiration. Although further studies should be

conducted in order to identify the mechanism behind the observed effect of oregano extract in

cellular respiration.

LITERATURE CITED

Campbell, NA & JB Reece. 2009. Biology. 8th ed. USA: Pearson Education, Inc.,

Cristani M, D'Arrigo M, Mandalari G; et al. (2007). "Interaction of four monoterpenes contained

in essential oils with model membranes: implications for their antibacterial activity". J.
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De Vincenzi M, Stammati A, De Vincenzi A, Silano M (2004). "Constituents of aromatic plants:

carvacrol". Fitoterapia 75 (7–8): 801–4

Di Pasqua R, Betts G, Hoskins N, Edwards M, Ercolini D, Mauriello G (2007). "Membrane

toxicity of antimicrobial compounds from essential oils". J. Agric. Food Chem. 55 (12):
4863–70

Dorman, H.J.D.; Deans, S.G. (2000). "Antimicrobial agents from plants: antibacterial activity of
plant volatile oils". J. Appl. Microbiol 88

Dragland, Steinar; Senoo, Haruki; Wake, Kenjiro; Holte, Kari; Blomhoff, Rune (2003). "Several
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Du WX, Olsen CE, Avena-Bustillos RJ, McHugh TH, Levin CE, Friedman M (2008). "Storage
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Evans, J.; Martin, J. D. (2000). "Effects of thymol on ruminal microorganisms". Curr.

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Appendix A. Absorbance of DPIP in four setups containing varying concentrations of oregano
extract, water, succinate solution and concentrated DPIP at 605 nm for 10 minutes
at 2 minute interval.

ABSORBANCE
Time (min) Tubes
1 2 3 4
0 0.043 0.005 0.155 0.127
2 0.040 0.011 0.147 0.084
4 0.069 0.018 0.140 0.017
6 0.083 0.032 0.148 0.003
8 0.091 0.030 0.150 0.002
10 0.114 0.042 0.137 0.002
 Tube/ Liver Succinate Water Oregano DPIP
Blank no. homogenate Solution Extract solution
1 1 1 1 1 1
Blank 1 1 2 1 0
2 1 1 1.5 0.5 1
Blank 1 1 2 1 0
3 1 1 1.75 0.25 1
Blank 1 1 2 1 0
4 1 1 2 0 1
Blank 1 1 3 0 0

ABSORBANCE
Time (min) Tubes
1 2 3 4
0 0.043 0.005 0.155 0.127
2 0.040 0.011 0.147 0.084
4 0.069 0.018 0.140 0.017
6 0.083 0.032 0.148 0.003
8 0.091 0.030 0.150 0.002
10 0.114 0.042 0.137 0.002