Submit manuscript...
eISSN: 2379-6367

Pharmacy & Pharmacology International Journal

Research Article Volume 9 Issue 3

Chemical characterization and antibacterialantifungal activity of Rutaceae family essential oils from different plants on probiotic microorganisms

Alper Cimik

Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, Turkey

Correspondence: Alper Cimik, Department of Pharmacognosy, Faculty of Pharmacy, Anadolu University, 26470, Eskisehir, Turkey, Tel +90(537)2217460

Received: May 30, 2021 | Published: June 22, 2021

Citation: Cimik A. Chemical characterization and antibacterial-antifungal activity of Rutaceae Family Essential oils from different plants on probiotic microorganisms. Pharm Pharmacol Int J. 2021;9(3):120-125. DOI: 10.15406/ppij.2021.09.00336

Download PDF

Abstract

Lemon, lime, orange, grapefruit, bergamot, mandarin and bitter orange species which have major characteristic specialities of Rutaceae family, have antimicrobial activities on pathogene microorganisms. Probiotic microorganisms have valuable effects on human body and inhibition of probiotics causes many diseases. In this present study, it was aimed to determine indicate probiotic resistance against natural antimicrobial agents (as essential oils) compare to pathogenes in previous studies. Analysis of essential oils (Eos) from were analyzed by GC-FID and GC/MS, analysis of Eos antimicrobial and antifungal activity from were analyzed by Microdilution test. Limonene (%95.29) and Linalool (%34.94) were found as major compounds of EOs respectively. All essential oils have antimicrobial activities on probiotic microorganisms.

Keywords: essential oil, lamiaceae, antimicrobial, antifungal, characterization

Introduction

The Rutaceae are a large, widely distributed family of trees and other woody plants comprising about 150 genera and some 180 species.1 The genus Citrus has been variously described as consisting of from 1 to 162 species.2,3 The most widely accepted taxonomic systems today are those of Swingle (1946) and Tanaka (1977) who recognized 16 and 162 species, respectively. Relationships among taxa are complicated by several factors such as a high frequency of bud mutation, a long history of cultivation, and wide cross-compatibility. In species that are grown primarily for fruit, sports may be vegetatively propagated and maintained by budding, which can lead to small, mutation-based differences among varieties within cultivated species.4 For example, little genetic variation was detected within the important cultivated species C. sinensis and C. paradisi when examined by microsatellite-based markers.5-7 In medicine, Citrus fruits are used in the treatment of various diseases. Research shows that the intake of Citrus fruits can reduce the incidence of gastric cancer. In addition, some isolated compounds from these fruits have effects on the central nervous system. For example, limonene, which is present in high concentrations in Citrus aurantium, showed a strong anxiolytic effect when tested in both animals and humans (Table 1).8

Citrus species

Pharmacological action8

Citrus aurantium L.

Gastrointestinal stimulant and general tonic. Treatment of central nervous system disorders like insomnia, anxiety, and hysteria.

Relieve stomach cramps and constipation, combat stomach acidity.

Hypoglycemic effect.

Anti‐inflammatory.

Anxiolytic effect.

Citrus sinensis L.                         

Sedative action.

Anthelmintic properties.

Treatment of liver cirrhosis.

Antidiabetic properties.

Anxiolytic effect.

Citrus bergamia L.

Antibacterial. Antifungal. Anti‐inflammatory. Analgesic. Antiproliferative and anticancer properties. Neuropsychopharmacological. Neuroprotective.

Anxiolytic activity.

Hypoglycemic and hypolipidemic activities.

Citrus limon L.

Analgesic. Anti‐anemic. Anti‐sclerotic. Antipyretic. Antiseptic.

Emollient and moisturizer properties.

Anti‐diarrheal. Diuretic. Intestinal mucosa protector. Local hemostatic.

Vascular stimulant and protector.

Antioxidant. Antiallergic. Antiviral. Anti‐inflammatory. Antiproliferative, antimutagenic, and anticancer activities.

Table 1 Pharmacological action table of widely-used Citrus sp. fruits in previous studies

Antibiotics are drugs that have the ability to prevent or destroy the growth of various microorganisms. The antibiotic era began when Alexander Fleming (1881-1955) discovered penicillin in 1928. Louis Pasteur, in his work on the fermentation of lactic acid (1857), mentioned the existence of certain substances capable of showing antimicrobial effects. In that fact, probiotic microorganisms so much important for indicate pathology of infections of pathogene microorganisms. Generally probiotics are more resistant than pathogene microorganisms and they inhibit them in competetive inhibition tests (Table 2).9

Essential oil

Inhibited Pathogene Microorganisms

Citrus limon L                                                  

Bacillus cereus, Mycobacterium smegmatis, Listeria monocytogenes, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae, Pseudococ cusper, Aspergillus niger, A. flavus, Penicillium verrucosum, P. chrysogenum, Kluyveromyces fragilis, Rhodotorula rubra, Candida albicans, Hanseniaspora guillermoni10,11

Citrus aurantifolia L.

Bacillus subtilis ATCC 6633, Enterococcus durans ED010, Enterococcus hirae ATCC 10541, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis ATCC 49134, Enterobacter cloacae EC02, Proteus mirabilis PM02, Pseudomonas aeruginosa ATCC 9721, Escherichia coli ATCC 10536, Serratia marcescens ATCC 19980 and Salmonella tiphi ATCC 13311, Candida albicans ATCC 10231, Candida parapsilosis ATCC 221912

Citrus sinensis L.

Staphylococcus aureus, Listeria monocytogenes, Vibrio parahaemolyticus, Salmonella typhimurium, Escherichia coli,  Pseudomonas aeruginosa, Aspergillus flavus, A. fumigatus, A. niger, A. terreus, Alternaria alternata, Cladosporium herbarum, Curvularia lunata, Fusarium oxysporum, Helminthosporium oryzae, Penicillium chrysogenum, P. verrucosum, Trichoderma viride13,14

Citrus paradisi L.

Bacillus cereus, Enterococus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudococcus sp., Shigella flexneri, Staphylococcus aureus, Cladosporium cucumerinum, Penicillium digitatum, P. italicum, P. chrysogenum11,15

Citrus bergamia L.

Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella enterica, S. typhimurium, Pseudomonas putida, Arcobacter butzleri, Enterococcus faecium, E. faecalis, Listeria monocytogenes, Hanseniaspora guilliermondii, Debaryomyces hansenii, Kluyveromyces fragilis, Rhodotorula rubra, Candida albicans, Aspergillus niger, A. flavus, Penicillium italicum, Fusarium solani, F. sporotrichioides, F. oxysporum, Curvularia lunata, Verticillium dahliae, Phomopsis sp., Phoma sp., Myrothechium verrucaria16

Citrus reticulata L.

Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Staphylococcus aureus, Penicillium italicum, P. digitatum, P. chrysogenum, Aspergillus niger, A. flav. Alternaria alternata, Rhizoctonia solani, Curvularia lunata, Fusarium oxysporum, Helminthosporium oryzae17

Citrus aurantium amara L.

Bacillus subtilis, B. cereus, Staphylococcus aureus, S. epidermis, Enterococcus faecalis, Micrococcus luteus, Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Aspergillus niger, A. flavus, A. nidulans, A. fumigatus, Fusarium graminearum, F. oxysporum, F. culmorum, Alternaria alternata18,19

Table 2 Antimicrobial activity of investigated Rutaceae essential oils on pathogene microorganisms in previous studies

Materials and methods

Plant material

Pharmacopeae essential oils were used as standarts of Rutaceae plants. EOs were selected from Anadolu University, Faculty of Pharmacy, Pharmacognosy Research Laboratory essential oil collection. Microorganisms were bought from Christian Hansen©.

GC-MS analysis

The GC-MS analysis was carried out with an Agilent 5975 GC-MS system. Innowax FSC column (60m, 0.25mm film thickness) was used with helium as carrier gas (0.8ml/min). GC oven temperature was kept at 60°C for 10 min and programmed to 220°C at a rate of 4°C/min, and kept constant at 220°C for 10 min. Then, programmed to 240°C at a rate of 1°C/min. Split ratio was adjusted at 40:1. The injector temperature was set at 250°C. Mass spectra were recorded at 70eV. Mass range was from m/z 35 to 450.

GC analysis

The GC analysis was carried out using an agilent GC system. FID detector temperature was 300°C to obtain the same elution order with GC-MS, simultaneous auto-injection was done on a duplicate of the same column applying the same operational conditions. Relative percentage amounts of the separated compounds were calculated from FID chromatograms.

Identification of components

Characterization of the essential oil components was carried out by comparison of their retention times with those of authentic samples or by comparison of their Linear Retention Indices (LRI) to a series of n-alkanes. Computer matching against commercial Wiley GC/MS library (MacLafferty and Stauffer, 1989), MassFinder 3 Library (Koenig et al., 2004) and in house “Baser Library of Essential Oil Constituents” built up by genuine compounds and components of known oils, as well as MS literature data (Joulain and Koenig, 1998: ESO, 2000) was used for the identification.

Antimicrobial and antifungal activities with microdilution methods

This technique helps to determine MIC (minimal inhibitory concentration) and MLK (minimal lethal concentration) values ​​of antimicrobial drugs. For this purpose, 2 or 10-fold dilutions of antimicrobial drug in Mueller-Hinton Broth are made and dilutions of dense concentrations of drugs are obtained. Ex. drug 256, 128, 64, 32, 16, 8, starting at 256 32g in 1 ml. 4, 2, 1, 0.5, 0.25, 0.12 /g/mL are gradually diluted in three layers. The isolated test is seeded in 100µL of the 24-48 hours liquid culture of the microorganism and incubated at 37°C for 24-48 hours. The reproduction in the tubes is evaluated by the eye. Thus, the final dilution without reproduction is accepted as MIC value. However, in order to be precise, it is appropriate to perform the test in three parallel. The average of the most recent results is the MIC or MLK obtained. Essential oil fractions of Citrus sp. were dissoluted in %10(v,v) DMSO(Merck©, CAS: 67-68-5) and emulsufied in distilled water. Resazurin sodium(Sigma-Aldrich©,CAS No:62758-13-8) is used as indicator for determination of MIC values.  Chloramphenicol (Sigma-Aldrich©, CAS: 57-75-7) was used as positive control as indicated in the Clinical Laboratory Standards Institute guide.20,21 In this study we calculated MIC values as other MIC studies in the literature.

Results and discussion

As shown in Table 2, in total 36 constituents were identified. The main components were limonene and linalool in Esseintial Oils as 68,7%, 95,299%, 73,101% and 34,94% respectively.  β-pinene, myrcene, γ-terpinene and linalyl acetate were the second major component in EOs 10,644%, 1,417%, 16,048% and 13,561% resp. The third major component were γ-terpinene, α-pinene, p-cymene and α-pinene in EOs 1,921%, 0,503%, 2,819% and 12,032% resp. The contents of these EOs show us limonene is most widely chemical compound in this study (Table 3 & 4).

Compound Name (EOs)

C. limonum       

C.  sinensis       

C. reticulata        

C. aurantium         

α-pinene

1.63

0.503

1.923

12.032

linalyl acetate

-

-

-

13.561

β-pinene

10.644

-

1.524

-

sabinene

1.734

0.383

0.275

1.198

myrcene

1.424

1.417

1.691

1.748

caryophylene oxide

-

-

-

-

β-caryophylene

 

-

-

camphora

-

-

-

-

thymol

-

-

-

-

α-thujene

-

0.78

-

limonene

68.7

95.299

73.101

9.692

limonene-4-ol

0.064

-

-

1,8-cineole

-

-

-

-

carvacrol

-

-

-

-

(Z)-β-ocimene

-

-

-

-

(E)-β-ocimene

-

-

-

5.553

p-cymene

1.921

-

2.819

-

terpinolene

-

-

0.748

-

methyl acetate

-

-

-

-

bicyclogermanilen

-

-

-

-

carvacrol

-

-

-

-

linalool

-

0.239

-

34.94

Δ-3-karnen

-

0.265

-

-

Δ-terpineol

-

-

-

-

γ-muurolan

-

-

-

-

α-terpinene

-

-

0.293

-

bornyl acetate

-

-

-

-

geranyl acetate

0.669

-

-

3.185

terpinen-4-ol

-

-

-

-

β-caryophyllene

0.363

-

-

-

geranyl isobutirate

-

-

-

-

geraniol

1.414

-

-

2.784

geranial

-

0.08

-

-

β-phellandrene

-

0.168

0.212

-

p-cymene-8-ol

-

-

-

-

neryl acetate

-

-

-

1.657

nerol

-

-

-

1.035

neral

0.777

-

-

-

(E)-nerolidole

-

-

-

2.494

menthone

-

-

-

-

dimethyl antranilate

-

-

0.585

-

germacrene D

-

-

-

-

isomenthone

-

-

-

-

neomenthole

-

-

-

-

isopulegon

-

-

-

-

menthole

-

-

-

-

cis-p-mentha-1-ol

-

0.157

-

-

trans-p-mentha-2,8-diene

-

0.153

-

-

pulegon

-

-

-

-

menthofurane

-

-

-

-

isopulegol

-

-

-

-

camphene

-

-

-

-

α-kapaen

-

-

-

-

γ-terpinene

9.178

-

16.048

-

trans-carveol

-

0.07

-

-

cis-carveol

-

0.178

-

-

tricyclene

-

-

-

-

α-tuyen

-

-

-

-

cis-1,2-limonene-epolisite

-

0.311

-

-

trans-1,2-limonene-epolisite

-

0.177

-

-

terpineolene

-

-

-

-

trans- sabinene- hydrite

-

-

-

-

camphor

-

-

-

-

γ-terpineol

-

-

-

-

α-humulene

-

-

-

-

α-terpineol

-

0.037

-

3.657

α-terpinyl acetate

-

-

-

-

decanal

 

0.05

-

-

menthyl acetate

-

-

-

-

borneole

-

-

-

-

farnesol

-

-

-

3.477

octanal

-

0.09

-

-

valensen

-

0.165

-

-

p-cymen-8-ol

-

-

-

-

bicyclogermanilene

-

-

-

-

Imalol

-

-

-

-

Imalil acetate

-

-

-

-

sabinyl acetate

-

-

-

-

Total %

98.454

99.806

99.999

97.013

Table 3 Chemical components of Rutaceae essential oil

Microorganism

La-5

La-14

L.reu.

L.rh.

L.fer

B.coa.

B.N.

B.cl.

S.sal.

S.ther.

S.b.

S.c.

BB-12

Essential oil (mg/L)

C. limonum

>128   

0.25>   

>128   

>128   

>128   

>128   

>128   

0.25>   

>128   

0.25>   

>128   

>128   

96    

C. aurantifolia

>128

>128

128

64

64

64

>128

96

0.25>

>128

0.25>

>128

64

C. sinensis

>128

>128

>128

128

96

>128

>128

0.25>

0.25>

>128

0.25>

>128

>128

C. paradisi

>128

>128

128

32

32

>128

>128

2

0.25>

>128

0.25>

96

0.5

C. bergamia

>128

>128

>128

>128

>128

>128

8

12

>128

8

>128

32

>128

C. aurantium

>128

>128

>128

>128

>128

>128

>128

0.25>

>128

0.25>

>128

>128

>128

Ketoconazole

4

4

8

0,37

12

16

12

0.5

12

0.25>

-

-

0.25

Chloramphenicol

-

-

-

-

-

-

-

-

-

-

1

1

-

Table 4 MIC table of Rutaceae family Eos
La-5,  Lactobacillus acidophilus La-5;  La-14, Lactobacillus acidophilus La-14; L.fer., Lactobacillus fermentum CECT- 5716; L.reu., Lactobacillus reuteri DSM 17938; L.rh., Lactobacillus rhamnosus GG; B.coa., Bacillus coagulans SNZ 1969; B.cl., Bacillus subtilis var. clausii ATCC9799; B.N., Bacillus subtilis var. natto BN; S.sal., Streptococcus salivarius K12; S.ther., Streptococcus thermophilus TH-4; S.b., Saccharomyces cerevisae var. boulardii ATCC–MYA976; S.c., Saccharomyces cerevisae ATCC–MYA9763; BB-12, Bifidobacterium bifidum BB-12

For these results, Citrus aurantifolia, Citrus paradisi and Citrus bergamia essential oils are most effective EOs against probiotic microorganisms. If they are used on gastrointestinal microflora directly, they can inhibit many microorganisms and cause many gastroinstestinal problems. All of the EOs in this study effect Saccharomyces cerevisae var. boulardii ATCC–MYA976. This microorganism isn’t resistant against EOs without Citrus limonum, Citrus bergamia and Citrus aurantium . C. aurantium didn’t show any antimicrobial activity against probiotic microorganisms without Streptococcus thermophilus and Bacillus clausii.  When compared all data’s about this study probiotic microorganisms generally resistant against Rutaceae EOs. As indicated in the pathogens microorganism’s table section, many microorganisms inhibited with Rutaceae family EOs but probiotic microorganisms are generally resistant on related EOs. This is important to protecting human body against bacterial and fungal infections with symbiotic microorganisms and their fundamental seconder metabolites. This study shows us probiotic microorganisms abilities to protect human body when natural antimicrobial compounds are taken.

In the other hand, probiotic microorganisms can use with antimicrobial agents in the same drug formulations to solve resistant pathogens super infectious agents in the future.

Acknowledgments

None.

Conflicts of interest

Author declares that there is no conflict of interest.

References

  1. Stace HM, Armstrong JA, James SH. Cytoevolutionary patterns in Rutaceae. Plant Systematics and Evolution. 1993;187(1):1–
  2. Malik MN. A new concept in Citrus classification. Pak J Sci Res. 1973;25:268–271.
  3. Tanaka T. Fundamental discussion of Citrus classification. Stud Citrol. 1977;14:1–6.
  4. Frost HB, Soost RK. Seed production: development of gametes and embryos. In: Reuther W, Webber HJ, Batchelor LD (eds) The citrus industry, vol 2. University of California: Berkeley; 1968. 290–324 p.
  5. Kijas JMH, Fowler JCS, Thomas MR. An evaluation of sequence tagged microsatellite site markers for genetic analysis within Citrus and related species. Genome. 1995;38(2):349–
  6. Luro F, Laigret F, Bove JM, et al. DNA amplified fingerprinting, a useful tool for determination of genetic origin and diversity in Citrus. Hortscience. 1995;30(5):1063–1067.
  7. Fang DQ, Roose ML. Identification of closely related Citrus cultivars with inter-simple sequence repeat markers. Theor Appl Genet. 1997;95:408–417.
  8. Pimenta FCF, Cunha-Tavares NA, Chaves-Neto G, et al. Pharmacological Actions of Citrus Species. Citrus Pathology; Gill H, Garg H, Eds; 2017. 197–211 p.
  9. Latour B. Pasteur on lactic acid yeast: a partial semiotic analysis. Configurations. 1993;1(1):129–
  10. Oshaghi MA, Ghalandari R, Vatandoost H, et al. Repellent effect of extracts and essential oils of Citrus limon (Rutaceae) and Melissa officinalis (Labiatae) against main malaria vector, Anopheles stephensi (Diptera: Culicidae). Iran J Public Health. 2003;32(4):47–52.
  11. Viuda-Martos M, Ruiz-Navajas Y, Fernández-López J, et al. Antifungal activity of lemon (Citrus limon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.) and orange (Citrus sinensis L.) essential oils. Food Control. 2008;19(12):1130–1138.
  12. Costa R, Bisignano C, Filocamo A, et al. Antimicrobial activity and chemical composition of Citrus aurantifolia (Christm.) Swingle essential oil from Italian organic crops. Journal of Essential Oil Research. 2014;26(6):400–408.
  13. Franco-Vega A, Reyes-Jurado F, Cardoso-Ugarte GA, et al. Sweet Orange (Citrus Sinensis) Oils. Elsevier Inc.; New York, NY, USA; 2015.
  14. Sharma N, Tripathi A. Effects of Citrus sinensis (L.) Osbeck epicarp essential oil on growth and morphogenesis of Aspergillus niger (L.) Van Tieghem. Microbiol Res. 2008;163(3):337–344.
  15. Yan F, Polk DB. Probiotics: progress toward novel therapies for intestinal diseases. Curr Opin Gastroenterol. 2010;26(2):95–101.
  16. Avila-Sosa R, Navarro-Cruz AR, Sosa-Morales ME, et al. Bergamot (Citrus bergamia) oils. In: Essential oils in food preservation, flavor and safety. Academic Press; 2016. 247–252 p.
  17. Chutia M, Deka Bhuyan P, Pathak MG, et al. Antifungal activity and chemical composition of Citrus reticulata Blanco essential oil against phytopathogens from North East India. LWT Food Sci Technol. 2009;42(3):777–780.
  18. Hsouna AB, Hamdi N, Halima NB, et al. Characterization of essential oil from Citrus aurantium L. flowers: antimicrobial and antioxidant activities. Journal of Oleo Science. 2013;62(10):763–772.
  19. Ammar AH, Bouajila J, Lebrihi A, et al. Chemical composition and in vitro antimicrobial and antioxidant activities of Citrus aurantium L. flowers essential oil (Neroli oil). Pak J Biol Sci. 2012;15(21):1034–1040.
  20. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved Standard, NCCLS document M27-A2. 2nd ed. Wayne: National Committee for Clinical Laboratory standards; 2002.
  21. National Committee for Clinical Laboratory Standards. Methods for dilution microbial susceptibility tests for bacteria that grow aerobically. Approved Standard, NCCLS document M7-A7. 7th ed. Wayne: National Committee for Clinical Laboratory standards; 2006.
Creative Commons Attribution License

©2021 Cimik. This is an open access article distributed under the terms of the, which permits unrestricted use, distribution, and build upon your work non-commercially.