Microbiological

Communication

Biosci. Biotech. Res. Comm. 10(1): 205-212 (2017)

Antimicrobial activity of aqueous and ethanolic

extracts of

Heracleum persicum

Myrtus

and

Lemon verbena

against

Streptococcus mutans

Aniseh Abdoli, Jaleh Mohajeri Borazjani and Parham Roohi

Department of Chemical Engineering, Boushehr Branch, Islamic Azad University, Boushehr, Iran

ABSTRACT

Regarding prevalence of oral bacteria and their complications and effects on increase of bacterial resistance toward

chemical medications, identi cation and application of herbals for  ghting bacteria has been of crucial importance from

the past. The aim of this study is to investigate the antimicrobial activity of aqueous and alcohol extract of Heracleum

persicum, Myrtus and Lemon verbena against Streptococcus mutans. For this purpose, disk and well methods were

applied and MIC and MBC of each extract were determined by broth micro-dilution method. In both methods, the best

treatment of Streptococcus mutans was ethanol extract of Myrtus at 600 mg/mL concentration which did not have sig-

ni cant difference with the hallow diameter induced by Amoxicillin antibiotic (21/33). In this study the minimum inhi-

bition concentration and minimum bactericidal concentration of Myrtus ethanolic extract were 3.12 mg/mL and 6.25

mg/mL, respectively. After that, Lemon verbena aqueous extract with 6.25 mg/mL and 12.5 mg/mL was in the second

rank. Among the studied extracts, Myrtus ethanolic extract and aqueous extract of Lemon verbena were the strongest

agents, respectively and it is recommended, after further investigation of medical formulation, to be used as a substitu-

tion for antibiotics as they have lower side effects. The can also be used as mouthwash and antibacterial chewing gums.

KEY WORDS: DISK DIFFUSION, MINIMUM BACTERICIDAL CONCENTRATION, MINIMUM INHIBITION CONCENTRATION, WELL

205

ARTICLE INFORMATION:

*Corresponding Author: anis.abdoli@yahoo.com

Received 11

Jan, 2017

Accepted after revision 21

March, 2017

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC and Crossref Indexed Journal

NAAS Journal Score 2017: 4.31 Cosmos IF : 4.006

reserved.

Online Contents Available at: http//www.bbrc.in/

INTRODUCTION

History of oral diseases goes back to the human his-

tory. About 500 bacterial species exist in mouth some

of which can cause oral infectious disease. Reduction

in pathogenic oral microbes can be very important in

improvement of oral ulcers and infections (Mozafari et

al 1995). Dental cavity is an infectious disease; it means

that bacteria play role in its creation. If hygiene meas-

ures were not properly taken, protein materials of the

saliva would precipitate on the teeth and in case of con-

tacting with food, the possibility of the bacterial growth

206 ANTIMICROBIAL ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Abdoli, Borazjani and Roohi

and sedimentation will increase. By increase of bacte-

rial population and more sedimentation of food parti-

cles and saliva content, dental plaque will be created.

Bacteria of dental plaque, by degrading sugar materials,

produce acids, the more the number of these bacteria,

the higher the amount of acid will be. This acid may

cause degradation of dent enamel and release its mineral

content and therefore weakens.

In this content, Streptococcus mutans is the most

common oral bacterium which is the main cause of

dental cavity. High levels of S. mutans put the person

in high risk group; these groups varies in each country

and race according to amount of S. mutans which is a

facultative anaerobic, gram-positive coccus commonly

found in human oral cavity. In oral health domain, to

reduce oral microbial  ora and prevent from dental cav-

ity and gingival disease, application of antimicrobial

agents, especially herbal ones (due to lower side effects)

is of crucial important are increasing as application of

chemical drugs results in bacterial resistance and other

side effects which may be even more dangerous than the

disease itself, (Mimica 2010, Vogel 2011, Amin et al 2012

and Karikalan and Mohankumar (2016).

In recent years, application of herbal drugs has become

more common in oral hygiene as they have antimicrobial,

antifungal and anticancer effects and lower side effects

(Ryan 2004). Extract of many herbs have effective com-

pounds against fungi, bacteria, viruses, and insects and

also show antioxidant behavior (Kordali 2005; Burt 2004).

Therefore, considering therapeutic methods of Iranian tra-

ditional medicine,  nding new pharmaceutical resources

for oral disease treatment seems essential. Among these

herbals, Myrtus, Heracleum persicum and Lemon verbena

can be mentioned. Myrtus is an ever-green plant whose

leaves have pharmaceutical effects. Myrtus has been

locally used for treatment of herpes and an antiseptic

agent and also to treat the nasal mucus in ammation.

Lemon Verbena is a plant in pale green whose leaves have

been used for therapeutic purposes its  ower and leaves

has been also used as tea and  avoring; its leaf essence

is also employed for making perfumes. Heracleum persi-

cum is an aromatic plant which has been long used for

 avoring the foods. Heracleum persicum has numerous

properties including strong antimicrobial and disinfecting

agent. In this study, the effects of Heracleum persicum,

Lemon verbena and Myrtus on S. mutans has been inves-

tigated and their ability in reduction or removal of harm-

ful oral bacteria is examined.

MATERIAL AND METHODS

First, sampling was done; Heracleum persicum, Myr-

tus and Lemon verbena leaves were collected from Fars

province and evaluated by an expert to identify them.

Before, grinding, leaves were dried in shadow exposed

to free air. Extraction was performed by wetting and

then application of rotary machine. Ethanol 96% was

used for preparing he ethanolic extract and distilled

water was employed for preparation of aqueous extract.

Standard strain of S. mutans (PTCC 1683) was provided

from Persian Type Culture Collection in lyophilized form.

Then by application of a physiologic serum, a micro-

bial suspension equals to 0.5 McFarland (1.5 



CFU/

ml) was prepared. Antibacterial test was performed by

two methods of disk and well. Minimum inhibition

concentration (MIC) was determined in 96-well micro-

plates by standard broth micro-dilution method (dilu-

tion n liquid medium). Minimum bactericidal concentra-

tion (MBC) was determined based on MIC values of each

extract. Statistical analyses of the data were performed

by MINITAB 16 software. To examine signi cance of the

effects and compare alcoholic and aqueous extracts in

different concentrations, the mentioned software (one-

way variance test based on Toochi test with P<0.05) was

employed. All the measurements were repeated for three

times and the results were reported in the form of aver-

age standard deviation.

RESULTS AND DISCUSSION

Results of well method are listed in table 1 as average

diameter of S. mutants inhibition zone hallow ± stand-

ard deviation according to table 1 and P-values. All the

data were normal. As table 2 shows, C7 related to anti-

biotic (amoxicillin) is in group A and C1, related to etha-

nolic extract of Myrtus against S. mutants is in group A

and B; C4 including aqueous extract of Heracleum per-

sicum is in group C. therefore, it can be said that amoxi-

cillin and aqueous extract of Myrtus do not have sig-

ni cant difference. However, they both have signi cant

difference with aqueous extract of Heracleum persicum.

C6, C2, C5 and C3 included aqueous extract of Lemon

verbena, aqueous extract of Myrtus, ethanolic extract of

Lemon verbena and ethanolic extract of Heracleum per-

sicum against S. mutans, respectively; and are located in

group B and C. they don’t have statistically signi cant

difference with each other and also ethanolic extract

of Myrtus and aqueous extract of Heracleum persicum;

however, they have signi cant difference with amoxicil-

lin antibiotic.

Results of well method are listed in table 3 as average

diameter of s. mutants inhibition zone hallow standard

deviation according to table 3, the data of zero imply

that the extract had no impact on S. mutans. Based on

the data of this table and P-values, all the data were nor-

mal. As it can be seen in table4,

C7 including antibiotic

(amoxicillin) against S. mutans is in group A and C1,

related to ethanolic extract of Myrtus against S. mutants

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIMICROBIAL ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS 207

Abdoli, Borazjani and Roohi

Table 1. average diameter of

S. mutants

inhibition zone hallow (mm) standard deviation

in three different concentrations of aqueous and ethanolic extracts of Heracleum

persicum, Myrtus and Lemon verbena by well method.

extract

Concentration (mg/ml)

p-value 200 p-value 400 p-value 600

Ethanolic Myrtus 0.07 14±1.73 >0.15 14.6±71.52 >0.15 18.67±1.52

Aqueous Myrtus >0.15 13±2 >0.15 13.33±1.52 0.07 14.33±0.57

ethanolic

Heracleum

persicum

>0.15 9 >0.15 11.33±1.52 >0.15 13.66±2.08

Aqueous

Heracleum

persicum

0.07 6.33±0.57 0.07 7.33±0.57 0.07 9.66±0.57

Ethanolic Lemon

Verbena

0.07 10.67±1.15 >0.15 11.67±1.52 0.07 14.33±1.15

Aqueous Lemon

Verbena

0.07 9.33±1.15 >0.15 14.67±2.08 0.07 17.33±0.57

Table 2. comparison of hallow diameter

averages with each other and hallow

induced by amoxicillin antibiotic by well

method through Toochi method.

groups average number

A 21.333 3 C7

A B 15.780 3 C1

B C 13.777 3 C6

B C 13.553 3 C2

B C 12.223 3 C5

B C 11.330 3 C3

C 7.773 3 C4

Table 3. Average diameter of S. mutants inhibition zone hallow (mm) ± standard

deviation in three different concentrations of aqueous and ethanolic extracts of

Heracleum persicum, Myrtus and Lemon verbena by disk diffusion method.

Extract

Concentration (mg/ml)

p-value 200 p-value 400 p-value 600

Ethanolic Myrtus >0.15 11.66±2.08 >0.15 13.33±2.51 >0.15 14±2.64

Aqueous Myrtus >0.15 7±1 >0.15 8±1 >0.15 9±1

ethanolic

Heracleum

persicum

0.000 0.00 0.00 0.00 >0.15 8±2

Aqueous

Heracleum

persicum

0.00 0.00 0.00 0.00 0.00 0.00

Ethanolic Lemon

Verbena

0.07 6.33±0.57 0.07 7.66±1.15 0.07 11±1.73

Aqueous Lemon

Verbena

0.00 0.00 0.07 8.33±0.57 >0.15 11.33±1.52

is in group A and B; C3 and C4 including ethanolic and

aqueous extracts of Heracleum persicum, respectively,

are in group C. Therefore, it can be said that amoxicillin

and ethanolic extract of Myrtus do not have signi cant

difference. However, they both have signi cant differ-

ence with aqueous and ethanolic extracts of

Heracleum

persicum. C5, C2 and C6 included ethanolic extract of

Lemon verbena, aqueous extract of Myrtus and aque-

ous extract of Lemon verbena against S. mutans, respec-

tively; and are located in group B and C. they don’t have

statistically signi cant difference with each other and

also ethanolic extract of Myrtus and aqueous extract of

Heracleum persicum; however, they have signi cant dif-

ference with amoxicillin antibiotic.

Regarding table 1 and 3, except aqueous extract of

Heracleum persicum which had hallow diameter of 0 in

three different concentration, the rest of extract showed

antimicrobial behavior and the most effective treatment

for gram positive bacterium of S. mutans in both meth-

ods (well and disk diffusion) was by ethanolic extract of

Myrtus whose average hallow diameter in well method

Abdoli, Borazjani and Roohi

208 ANTIMICROBIAL ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

and at concentration of 600 mg/ml was equal to 18.67

mm. this diameter was not signi cantly different with

the hallow induced by amoxicillin (a common antibiotic)

which was 21.33 ± 0.57 mm.

Numerous studies have reported antibacterial activity

of herbal extracts. Many researchers have assigned anti-

bacterial properties of Myrtus to its poly phenolic com-

pounds (Cakir et al 2004).In a study by Montoro et al in

2005, the antimicrobial effects of Myrtus extract were

attributed to poly phenolic compounds. This compound

is often antibacterial and two important compounds

named Mirtocomolone A and B would be released from

that which have antimicrobial effects especially on

gram positive bacteria such as S. mutans (Montoro et

al 2005). Rotstein investigated the antibacterial effects

of the compounds in Myrtus extract on gram positive

and gram negative bacteria and shows that Myrtus has

not a signi cant impact on gram negative bacteria and

the antibacterial properties of this extract, especially on

gram positive bacteria, could be attributed to Mirtoko-

molone A (Rotstein et al 1974). Gram negative bacteria,

due to having saccharide lipo-poly external membrane

and also some channels involving in material trans-

portation, are inherently more resistant against toxins,

hydrophilic dyes and antibiotics (Ebrahimi et al 2009).

More sensitivity of gram positive bacterial to the extracts

can be due to their different cell wall and its compound

(Taskin et al 2007).

Saeidi et al (2012) investigated antibacterial proper-

ties of Myrtus extract and essence against antibiotic-

resistant strains of Staphylococcus aureus. They con-

cluded that high concentrations of Myrtus extract have

antibacterial properties which are in complete agreement

with the results of this study which examined the effect

of high concentration of Myrtus extract on S. mutans.

Therefore, these compounds can be applied for pharma-

ceutical therapies.The results of Carlo et al in 2010 about

Myrtus indicated that the aqueous and ethanolic extract

of this plant have highest percent of extractive com-

pound. Also this extract has highest phenolic compound

as well. As S. mutans showed highest sensitivity to the

ethanolic extract, it can be concluded that its ethanolic

extract has highest extractive compounds and phenolic

compounds which can be an explanation for antimicro-

bial activity of Myrtus plant.

As table 1 shows, in well method and foe lower con-

centrations, the impact of aqueous and ethanolic extract

of Myrtus on S. mutans did not differ, therefore, it can

be said that in lower concentrations, Myrtus extract

dissolved in water can have better impact. According

to table 1 and 3, in both methods (disk and well) and

among all the investigated treatments, gram positive

bacterium of S. mutans exhibited the highest sensitiv-

ity to ethanolic extract of Myrtus; after that, aqueous

extract of Lemon verbena had the highest impact; which

did not have signi cant difference with the former

extract this could be due to the type and compounds

of aqueous extracts; which can explain the reason for

different effective compounds of aqueous and ethanolic

extract of Lemon verbena.

Due to possessing different metabolites in its essence

(especially terpenoids and citral) Lemon verbena, has

attracted considerable attention in terms of antimicro-

bial effects. Different studies have mentioned its effect

in removal of oral microbial  ora especially gram posi-

tive bacteria and controlling diarrhea. Terpenoids can

be easily dissolved in different solvents such as water,

methanol and ethanol (Zaferanieh 2003). Therefore, it

can be claimed that in this research, water was more

successful in dissolving terpenoids of Lemon verbena

in high concentration therefore it was more effective

on gram positive bacterium of S. mutans. Based on the

data in table 1 and 3, in both methods of well and disk

diffusion and in low concentrations of Lemon verbena

extract, ethanolic extracts induced larger inhibition hal-

lows in comparison with the aqueous extracts against S.

mutans this can be explained by bactericidal activity of

alcohol in lower concentrations.

Table 4. comparison of hallow diameter averages

with each other and hallow induced by amoxicillin

antibiotic by disk diffusion method through Toochi

method.

groups average number

A 21.333 3 C7

12.997 3

B C 8.330 3 C5

B C 8.000 3 C2

B C 6.553 3 C6

B C 2.667 3 C3

C 0.000 3 C4

Table 5. MIC and MBC values (in mg/ml) for ethanolic

and aqueous extracts of Myrtus, Heracleum persicum

and Lemon verbena against Streptococcus mutans.

Extract MIC (mg/ml) MBC (mg/ml)

Ethanolic Myrtus 3.12 6.25

Ethanolic Heracleum

persicum

100 200

Ethanolic Lemon verbena 12.5 25

Aqueous Myrtus 12.5 25

Aqueous Heracleum

persicum

100 200

Aqueous Lemon verbena 6.25 12.5

Abdoli, Borazjani and Roohi

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIMICROBIAL ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS 209

In this research, regarding the values in tables 1 and

3, in both methods, S. mutans showed highest resistiv-

ity toward Heracleum persicum extract especially its

aqueous extract which had signi cant difference with

ethanolic extract of Myrtus and amoxicillin antibiotic.

In disk diffusion test, in addition to aqueous extract of

Heracleum persicum, its ethanolic extract had also sig-

ni cant difference with ethanolic extract of Myrtus and

amoxicillin antibiotic.In disk diffusion method, aqueous

extract of Heracleum persicum showed no inhibition

zone against the growth of S. mutans for different con-

centration and only its ethanolic extracts showed anti-

microbial properties in high concentrations. Nazemi et al

in (2005) had examined antimicrobial activity of Hera-

cleum persicum aqueous extracts against 5 pathogenic

bacteria including Bacillus subtilis, Bacillus polymixa,

Staphylococcus, Nocardia aureus and Enterococcus fae-

calis by disk diffusion test. Heracleum persicum extract

and showed no inhibition hallow for the mentioned bac-

teria which is in complete agreement with the results of

present study on S. mutans.

Regarding tables 1 and 3, the largest average hal-

low diameter that Heracleum persicum extract induced

against S. mutans is related to its ethanolic extract

obtained by well method which is equal to 13.66 in

concentration of 600 mg/ml. In contrary to disk diffu-

sion test, in well method, ethanolic extract of Heracleum

persicum had no signi cant difference with ethanolic

extract of Myrtus and amoxicillin antibiotic; however its

antimicrobial activity was lower than the other extracts,

but it still exhibited antimicrobial properties. Khorshidi

et al (2014), proved existence of tannin and saponin

compounds in Iranian Heracleum persicum by phyto-

chemical tools. They also showed inhibitory effects of

these compounds on Escherichia coli, Salmonella antry-

dys, Staphylococcus aureus and Bacillus cereus. They

claimed that this herbal can be applied as an antimicro-

bial plant.

Useful pharmaceutical effects of plants are often due to

their secondary products. In herbals, these compounds are

mainly secondary metabolites such as alkaloids, steroids,

tannins and phenolic compounds produced by the plant

and restored in some of their speci c parts. These com-

pounds have antimicrobial properties in laboratory con-

dition. Such complicated compounds can be found in spe-

ci c groups, families and species (Balandrin et al 1985).

As  gures 1 and 2 show, in both methods (well and

disk diffusion), by increasing concentration of Myrtus,

Heracleum persicum and Lemon verbena, antibacterial

activity enhanced as increasing the concentration will

increase the permeability of antibacterial extract agent

to bacterium cell wall. Only aqueous extract of Hera-

cleum persicum, with hallow diameter of zero, had no

increase at any concentration which indicated the impact

of solvent on extracting the effective compounds. There-

fore, it can be said that water, in comparison with etha-

nol, has no ability to derive the effective compounds of

Heracleum persicum. Generally, in this study, antibacte-

rial activity decreased by reducing the concentration of

applied extracts.

According to  gures 3 and 4, S. mutans exhibited

more sensitivity toward ethanolic extracts (except for

Lemon verbena extracts whose aqueous extract had

more antimicrobial properties at high concentrations)

and showed larger hallows. Presence of alcohol resulted

in derivation of more polar compounds; therefore, more

sensitivity of gram positive bacteria can be attributed to

polarity of extracts as polar compounds can easily pen-

etrate through gram positive cell membranes and end to

their death (i.e. Streptococcus mutans).

Streptococcus mutans showed sensitivity to all the

extracts in well method which varied from 6.33 mm for

FIGURE 1. comparison of Streptococcus mutans growth inhibition hallow

diameter for different concentrations of Heracleum persicum, Myrtus and

Lemon verbena extracts by well method.

Abdoli, Borazjani and Roohi

210 ANTIMICROBIAL ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

FIGURE 4. comparison of Streptococcus mutans growth inhibition

hallow diameter for Heracleum persicum, Myrtus and Lemon verbena

ethanolic and aqueous extracts by disk diffusion method.

FIGURE 2. comparison of Streptococcus mutans growth inhibition hallow

diameter for different concentrations of Heracleum persicum, Myrtus and

Lemon verbena extracts by disk diffusion method.

FIGURE 3. comparison of Streptococcus mutans growth inhibition

hallow diameter for Heracleum persicum, Myrtus and Lemon verbena

ethanolic and aqueous extracts by well method.

Abdoli, Borazjani and Roohi

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS ANTIMICROBIAL ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS 211

aqueous extract of Heracleum persicum to 18.67 mm for

ethanolic extract of Myrtus. This trend didn’t hold in

disk diffusion test. According  gures 5 and 6, extracts

induced smaller inhibition hallows in comparison to well

method; moreover, some extracts showed no antimicro-

bial activity against Streptococcus mutans in disk dif-

fusion test and induced no hallow of growth inhibition.

Quantitative comparison of these two methods indicated

that well method, in comparison with disk diffusion

scheme, showed higher inhibition effect on microorgan-

ism growth. This difference can be attributed to the fact

that in well method, more amount of herbal extract is

directly added to the wells while in disk diffusion test,

the disks would be impregnated with the extracted for

15 minutes and put on agar surface indirectly. Therefore

it is possible that the disks did not absorbed adequate

amount of the extracts and do not show high propaga-

tion properties.

According to table 5, MIC of this study varied in 3.12-

100 mg/ml range and variation range of MBC was 6.25-

200 mg/ml. lower amounts would be neglected. It must

be also noted that the reported MIC is related to MIC of

the extracts. Hence, if the effective compounds of the

extracts were separated, MIC values would de nitely

be lower than the reported values; as extract contains

numerous compounds and only a few of them have anti-

bacterial behavior.

For these experiments, double dilution series of the

extracts were prepared in 96-well micro-plates in range

of 0.09 mg/ml to 200 mg/ml. Among the studied treat-

FIGURE 6. comparison of Streptococcus mutans growth inhibition

hallow diameter obtained by both methods for different concentra-

tions of Heracleumpersicum, Myrtus and Lemon verbena aqueous

extracts.

FIGURE 5. comparison of Streptococcus mutans growth inhibition

hallow diameter obtained by both methods for different concentra-

tions of Heracleumpersicum, Myrtus and Lemon verbena ethanolic

extracts.

Abdoli, Borazjani and Roohi

212 ANTIMICROBIAL ACTIVITY OF AQUEOUS AND ETHANOLIC EXTRACTS BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

ments, Myrtus ethanolic extract Lemon verbena aqueous

extract exhibited lowest MIC (3.12 and 6.25 mg/ml) and

lowest MBC (6.25 and 12.5 mg/ml) indicating higher sen-

sitivity of Streptococcus mutans to this extract. Aqueous

and ethanolic extracts of Heracleum persicum had high-

est MIC (100 mg/ml) and MBC (200 mg/ml), suggesting

more resistance of Streptococcus mutans toward these

extracts. These results coincide with the results obtained

from disk diffusion and well methods. MIC and MBC

of aqueous Myrtus extract and ethanolic Lemon verbena

extract were the same and were equal to 12.5 mg/ml and

25/mg/ml, respectively.

CONCLUSION

Recently, side effects of antibiotics and microorganisms’

resistance toward them have resulted in more attention to

herbal extracts with speci c biologic properties. Herbal-

based antimicrobial compounds have numerous thera-

peutic uses. They are not only effective in treatment of

infectious diseases, but they can simultaneously reduce

some of the side effects of conventional antimicrobial

agents. Therefore, regarding the signi cant impacts of

herbals reported in traditional medicine, this study rec-

ommend that all these stages to be performed on other

pathogenic oral bacteria and those exists in natural oral

 ora. In this way the optimized concentration of the

mentioned extracts will be obtained which in addition to

protecting form disease, do not disturb oral microbial bal-

ance. By collaborative cooperation of different centers,

more comprehensive studies on this  led can be expected;

which would have considerable impacts on treatment of

antibiotic-resistant oral infections and reduction of oral

health problems. Such studies can also pave way for mass

production of herbal-based mouthwash and antibacterial

chewing gums with minimum side effects.

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