Analysis of Component and Antibacterial Activity of Ethanol Extract and Etyl Acetate Fraction of Pagoda (Clerodendrum paniculatum L.) Leaves against Pseudomonas aeruginosa and MRSA

 

Dewi Pertiwi1, Panal Sitorus1, Ihsanul Hafiz2, Denny Satria1

1Department of Pharmaceutical Biology, Faculty of Pharmacy,

Universitas Sumatera Utara, Medan, 20155, Indonesia.

2Department of Pharmacology, Faculty of Pharmacy and Health,

Institut Kesehatan Helvetia, Medan, 20124, Indonesia.

*Corresponding Author E-mail: dewipertiwi@usu.ac.id

 

ABSTRACT:

The current study aimed to determine chemical constituent and antibacterial activity of ethanol extract and ethyl acetate fraction of Clerodendrum paniculatum. Component of ethanol extract and ethyl acetate fraction analyzed by LC-MS and antibacterial activity to inhibit Pseudomonas aeruginosa and Meticilin Resistant Staphylococcus aureus (MRSA) use disk diffusion method. Dried powder and ethanol extract of C. paniculatum contains of flavonoid, glycoside, tannin and steroid/triterpenoid in phytochemical screening. Analysis of component by LC-MS show that ethanol extract of C. paniculatum contains of Erucamid, Cafeic acid, 7-Hidroxycoumarin dan linamarin. Ethyl acetate fraction of C. paniculatum contains of Erucamid, Apigenin, Cafeic acid, Kynurenic acid, Apigenin 7-O-glucuronide, 6-O-Methylscutellarin, Apigetrin, 4-Methoxycinnamic acid, 4-Coumaric acid, Scutellarin dan 7-Hidroxycoumarin. Inhibition value of ethanol extract of C. paniculatum against P. aeruginosa is 12.13±0.057; 10.83±0.058; 9.63±0.057; 8.3±0.058 in concentration of 300, 200, 100 and 50ppm. Inhibition value of ethyl acetate fraction of C. paniculatum against P. aeruginosa is 14.50±0.100; 13.73±0.058; 12.87± 0.057; 11.83±0.058 in concentration of 300, 200, 100 and 50 ppm. Inhibition value of ethanol extract of C. paniculatum against MRSA is 8.67±0.057; 7.80±0.100; 7.06±0.057; 6.43±0.057 in concentration of 300, 200, 100 and 50 ppm. Inhibition value of ethyl acetate fraction of C. paniculatum against MRSA is 9.77±0.057; 8.03 ±0.057; 7.33±0.057; 6.70±0.100 in concentration of 300, 200, 100 and 50ppm.

 

KEYWORDS: Clerodendrum paniculatum, analysis of component, LC-MS, Pseudomonas aeruginosa, MRSA.

 

 


INTRODUCTION:

Plant substances are widely distributed, including alkaloids, organosulfur compounds, phenolic acids, flavonoids, carotenoids, coumarins, terpenes, tannins, and some primary metabolites (amino acids, peptides, organic acids) exhibit antibacterial and antifungal activity1-5. Recently, there has been increasing interest in the use of natural antibacterial and antifungal agents, due to the necessity of finding safer treatment against bacterial and fungal infections6.

 

 

The Clerodendrum paniculatum (pagoda flower), which has tall, pyramid shape inflorescences, is one of the most spectacular Clerodendrum species. It is widely cultivated and often establishes as a garden7. Several studies found that C. paniculatum have antibacterial and antifungal activity8,9,10. because its chemical constituent such as flavonoid, steroid, terpenoid and glycocide11,12. The present investigation aimed to determination chemical constituent and evaluated antibacterial activity of ethanol extract and ethyl acetate fraction of C. paniculatum against gram positive bacteria (Pseudomonas aeruginosa) and gram negative bacteria (Meticilin Resistant Staphylococcus aureus/MRSA). P. aeruginosa is a pathogen bacteria can cause severe, acute and chronic infection in burns, wounds, urinary and pulmonary tracts13. MRSA is resistance bacteria whose presence is a problem that threatens human life14 and the major cause for so many skin infections15. Although many antimicrobial drugs are available, its use is limited by a number of factors such as low potency, drug toxicity and drug resistense strains. Thus, the biologically active, safer and more effective antimicrobial agents are needed to explore16.

 

MATERIALS AND METHODS:

Plant and chemicals material:

Fresh leaves of C. paniculatum were collected from Brastagi, Medan, Indonesia. C. paniculatum was identified in Lembaga Ilmu Pengetahuan Indonesia (No 752/IPH.101/If.07/V/2015). The chemicals used were distilled water, ethanol, ethylacetate, n-hexane and DMSO.

 

Preparation of fractions:

The air-dried and powdered leaves of C. paniculatum (500g) were repeatedly fractionated by maceration with n-hexane (3×3 day, 7.5L). The powder was dried in the air and fractionated with ethylacetate (3×3 day, 7.5L) at room temperature with occasional stirring. Then, the powder was dried in the air and fractionated with ethanol (3×3day, 7.5 L). The filtrate was collected, evaporated under reduced pressure to give a viscous fraction, and then freeze-dried.

 

Phytochemical Screening:

Phytochemical screening was conducted using a standard method for detection of alkaloids, flavonoids, tannins, glycosides, saponins and steroids/triterpenoids17,18.

 

Phytochemical constituent analysis with LC-MS/MS:

LC-MS is selectively detect and confirm molecular identity combining the separating power of LC along with MS19. This analytical method has good recovery, precision and sensitivity as well as analytical procedures that can be completed in a shorter time20. Analysis of phytochemicals from ethylacetate fraction (EAF) was carried out with TSQ Exactive (Thermo) (LSIH, Brawijaya University) with mobile phase A (0.1% formic acid in water) and phase B (0.1% formic acid in acetonitrile) following the gradient method. A flow rate of 40μL/min was used for the Hypersil GOLD aQ column, 50 1mm 1.9μm, and the time for analysis was 70 min. The results were analyzed using the Compound Discoverer software with mzCloud21,22.

 

Antibacterial Activity:

The antimicrobial activity of the isolated compound was performed by the paper disk method against Pseudomonas aeruginosa and Methicilin Resistent Staphylococcus aureus (MRSA). C. paniculatum ethanol extract and etil acetate fraction was prepared in the concentration of 300, 150, 100 and 50ppm using distilled water that has been sterilized.  0.3µL of C. paniculatum  solution was pipetted into sterile paper disk on the surface of the medium containing bacteria test strain. Paper disks were also inoculated with distilled water (10 µL) as a negative control. Paper disks that had already impregnated with clindamycin (30µg) were used as a positive control.  The plates were places in an incubator at 37oC for approximately 24 hours23,24. Inhbition zones were measured and recorded. Screening of antibacterial activity was repeated twice23.

 

Statistical analysis:

Data were expressed as mean±SD. All statistics were analyzed using the SPSS 22 program.

 

RESULT AND DISCUSSION:

Table 1. Phytochemical Screening of dried powder and ethanol extract of C. paniculatum

From the result in No

Phytochemical Constituent

Dried Powder

Ethanol extract

1

Alkaloids

-

-

2

Flavonoids

+

+

3

Glycocides

+

+

4

Saponins

-

-

5

Tannins

+

+

6

Steroids/triterpenoids

+

+

+ = The secondary metabolite was available in sample

-  = The secondary metabolite was not available in sample

 

From the result in table 1, we know that dried powder and ethanol extract of C. paniculatum consist of flavonoids, glycocides, tannins and steroid/triterpenoids.

 

Phytochemical constituent analysis of ethanol extract and ethyl acetate fraction of C. paniculatum was carried out with LCMS/MS to obtain information about its compounds. The results were presented in Table 2 and Table 3.

 

Table 2: Phytochemical constituent analysis of C. paniculatum ethanol extract with LC-MS/MS

No

Name

Formula

Moleculer Weight

Retention Time (min)

1.

Erucamide

C22 H43 N O

337.3347

26.382

2.

Caffeic acid

C9 H8 O4

180.0426

1.026

3.

7-Hydroxycoumarine

C9 H6 O3

162.032

7.58

4.

Linamarin

C10 H17 N O6

247.106

1.162

 

Constituent of ethanol extract of C. paniculatum are Erucamid, Cafeic acid, 7-Hidroxycoumarin dan linamarin.

 

Constituent of ethyl acetate fraction of C. paniculatum are Erucamid, Apigenin, Cafeic acid, Kynurenic acid, Apigenin 7-O-glucuronide, 6-O-Methylscutellarin, Apigetrin, 4-Methoxycinnamic acid, 4-Coumaric acid, Scutellarin dan 7-Hidroxycoumarin.

 

Tabel 3: Phytochemical constituent analysis of ethyl acetate fraction of C. paniculatum with LC-MS/MS

No

Name

Formula

Molecular Weight

Retention Time (min)

1.

Erucamide

C22H43NO

337.3349

21.738

2.

Apigenin

C15H10O5

270.0534

11.56

3.

Caffeic acid

C9H8O4

180.0425

8.019

4.

Kynurenic acid

C10H7NO3

189.0431

4.455

5.

Apigenin 7-O-glucuronide

C21H18O11

446.0857

8.618

6.

6-O-Methylscutellarin

C22H20O12

476.0966

9.676

7.

Apigetrin

C21H20O10

432.1065

8.532

8.

4-Methoxycinnamic acid

C10H10O3

178.0634

21.198

9.

4-Coumaric acid

C9H8O3

164.0478

4.958

10.

Scutellarin

C21H18O12

462.0804

7.825

11.

7-Hydroxycoumarine

C9H6O3

162.0319

0.876

 

Antimicrobial activity of ethanol extract and ethyl acetate fraction of C. paniculatum to Pseudomonas aeruginosa

 

Table 4. Inhibition value of ethanol extract and ethyl acetate fraction of C. paniculatum to P. aeruginosa

No

Sample

Treatment

Inhibition (Mean ± Std)

1.

Ethanol extract of

C. paniculatum

Positive control

28.7 ± 0.000

Negative control

0

300 ppm

12.13 ± 0.057

200 ppm

10.83 ± 0.058

100 ppm

9.63 ± 0.057

50 ppm

8.33 ± 0.058

2.

Ethyl acetate fraction of

C. paniculatum

Positive control

28.7 ± 0.000

Negative control

0

300 ppm

14.50 ± 0.100

200 ppm

13.73 ± 0.058

100 ppm

12.87 ± 0.057

50 ppm

11.83 ± 0.058

 

Table 5. Inhibiton value of ethanol extract and ethyl acetate fraction of C. paniculatum to MRSA

No

Sample

Treatment

Inhibition (Mean ± Std)

1.

Ethanol extract of

C. paniculatum

Positive control

15.20 ± 0.100

Negative control

0

300 ppm

8.67 ± 0.057

200 ppm

7.80 ± 0.100

100 ppm

7.06 ± 0.057

50 ppm

6.43 ± 0.057

2.

Ethyl acetate fraction of

C. paniculatum

Positive control

15.20 ± 0.100

Negative control

0

300 ppm

9.77 ± 0.057

200 ppm

8.03 ± 0.057

100 ppm

7.33 ± 0.057

50 ppm

6.70 ± 0.100

 

The statistical test with ANOVA using SPSS program was carried out to see the significant average comparison value between diameter of inhibition at various concentrations on bacteria. The result showed that a significant difference (p<0,05) between the extract and fraction in concentration of 300,200,100,50 ppm compared to negative and positive control.

 

 

Ethanol extract                                          Ethyl acetate fraction

Picture 1. Antibacterial activity of ethanol extract and ethyl acetate fraction of C. paniculatum against P.aeruginosa

 

 

Ethanol extract                                  Ethyl acetate fraction

Picture 2. Antibacterial activity of ethanol extract and ethyl acetate fraction of C. paniculatum against MRSA

 

Based on the result of diameter inhibition in table 4 and table 5, it showed that the inhibition of ethanol extract and ethyl acetate fraction of C. paniculatum against gram negative bacteria (P. aeruginosa) is greater compared to gram positive bacteria (MRSA). Then, the ethanol extract and ethyl acetate fraction of C. paniculatum are more sensitive against gram negative bacteria. The difference in activity is due to differences in the structure and components of the walls on bacteria cells. The peptidoglycan layer on the gram negative bacteria’s cell wall are thinner, whereas in gram positive bacteria the peptodiglycan layer is thicker 25.

 

The antimicrobial activity caused by ethanol extract and ethyl acetate fraction can occur due to the content of secondary metabolites such as flavonoids, phenolics and terpenoids. Flavonoids can inhibit bacterial growth through inhibition of DNA gyrase, thus inhibits cytoplasmic membrane function 26.

 

Through hydrogen bonds, flavonoid compounds have the ability to form complexes with bacterial cell proteins. The structure of the cell wall and bacterial cytoplasmic membrane contains of proteins. Because the protein structure of bacterial cells is damaged due to hydrogen bonds with flavonoids and becomes unstable, so that the bacterial cell protein loses its biological activity, the function of bacterial cell permeability is disrupted and results in bacterial cell death27-29.

 

CONCLUSION:

In conclusion, the constituent of ethanol extract of C. paniculatum are Erucamid, Cafeic acid, 7-Hidroxycoumarin dan linamarin. Constituent of ethyl acetate fraction of C. paniculatum are Erucamid, Apigenin, Caffeic acid, Kynurenic acid, Apigenin 7-O-glucuronide, 6-O-Methylscutellarin, Apigetrin, 4-Methoxycinnamic acid, 4-Coumaric acid, Scutellarin dan 7-Hidroxycoumarin. The antibacterial activity of ethyl acetate fraction of C. paniculatum against P. aeruginosa and MRSA greater than its ethanol extract.

 

ACKNOWLEDGEMENTS:

The authors are thankful to Rector of Universitas Sumatera Utara, through “TALENTA” 2020 for the research funding provided.

 

REFERENCES:

1.      Chandra H, Bishnoi P, Yadav A, Patni B, Mishra AP, Nautiyal AR, Antimicrobial resistance and the alternative resources with special emphasis on plant-based antimicrobials–A review. Plants, 2017; 6(16). DOI: 10.3390/plants6020016

2.      Özçelik B, Kartal M, Orhan I, Cytotoxicity, Antiviral and antimicrobial activities of alkaloids, flavonoids, and phenolic acid, Pharm. Biol, 2011; 49:396–402. DOI: 10.3109/13880209.2010.519390

3.      Barbieri R, Coppo E, Marchese A, Daglia M, Sobarzo-Sánchez E, Nabavi SF, Nabavi, SM, Phytochemicals for human disease: An update on plant-derived compounds antibacterial activity, Microbiol. Res, 2017; 196:44–68. DOI: 10.1016/j.micres.2016.12.003

4.      Fialova S, Rendekova K, Mucaji P, Slobodnikova L, Plant natural agents: Polyphenols, alkaloids and essential oils as perspective solution of microbial resistance, Curr. Org. Chem, 2017; 21:1875–1884. DOI: 10.2174/1385272821666170127161321

5.      Khameneh B, Iranshahy M, Soheili V, Bazzaz BSF, Review on plant antimicrobials: A mechanistic viewpoint, Antimicrob. Resist. Infect. Control, 2019; 8, 118. DOI: 10.1186/s13756-019-0559-6

6.      Srinivasa U, Divakar K, Kamath K and Shabaraya, AR, Antimicrobial activity of Clerodendrum phlomidis leaf extracts and its isolated fractions. International Journal of Research in Pharmacy and Biosciences, 2018; 5(3):10-11.

7.      Hafiz I, Jansen S, Rosidah, Antioxidant and Anti-inflammatory Activity of Pagoda Leaves (Clerodendrum paniculatum L.) Ethanolic Extract in White Male Rats (Rattus novergicus). International Journal of PharmTech Research, 2016; 9.

8.      Joseph J, Bindhu AR, Aleykutty NA, Antimicrobial Activity of Clerodendrum paniculatum Linn, IJRAP, 2011; 2(3):1003-1004.

9.      Leena PN, Aleykutty NA, Comparative Study on Antibacterial Activities of Clerodendron paniculatum Linn and Clerodendron paniculatum Linn Root Extract. IJAPBC, 2012; 1(3): 325-327.

10.   Praveen M, Kumar DK, Padmaja V, Matthew A, Kumar AP, Preliminary Phytochemical, Antimicrobial and Toxicity Studies on Clerodendrum paniculatum Linn Leaves, Hygea.J.D.Med, 2012; 4(1): 41-50.

11.   Shrivastava N, Patel T, Clerodendrum and Healthcare: An Overview. Medicinal and Aromatic Plant Science and Biotechnology, 2007; 1(1): 142-150.

12.   Priyanka K, Kuppast I. J, Gururaj S. V, Chethan I. A. Screening of Aerial Parts of the Plant Clerodendrum paniculatum Linn for Anti-Convulsant Activity. Res. J. Pharmacology and Pharmacodynamics.2019; 11(1):01-04. doi: 10.5958/2321-5836.2019.00001.6

13.   Wissam Zam, Rim Harfouch, Salwa Bittar, Meray Sayegh. Antibacterial activity of various Syrian honey types against Pseudomonas aueruginosa. Res. J. Pharmacognosy and Phytochem. 2017; 9(2): 73-76. doi: 10.5958/0975-4385.2017.00013.9

14.   Manjunath Sangappa, Padma Thiagarajan. Isolation and Screening of Soil Penicillium sp VIT-2012 Metabolites against Methicillin Resistant Staphylococcus aureus. Research J. Pharm. and Tech. 6(12): Dec. 2013; Page 1340-1349.

15.   Sharma. Shalini, Upadhayay U. M, Mistry Sunil. Antimicrobial Activity of Extracts against MRSA. Research J. Pharm. and Tech 2016; 9(12):2283-2286. doi: 10.5958/0974-360X.2016.00460.1

16.   Shanker K., Krishna Mohan G., Bhagavan Raju M., Divya L., Sanjay B. Efficacy of leaves extract of Acacia nilotica against Pseudomonas aeruginosa with reference to Disc diffusion method. Res. J. Pharmacognosy & Phytochem. 2014; 6(2): 96-98.

17.   Harborne JB, Phytochemical Method, Chapman and Hall Ltd, London, 1984.

18.   Madhuri M. Deshmukh, Chhaya S. Ambad, Nutan Kendre, Navnath G. Kashid. Biochemical Screening, Antibacterial and GC-MS Analysis of Ethanolic Extract of Hemidesmus indicus (L) R.Br. root. Res. J. Pharmacognosy and Phytochem. 2019; 11(2):73-80. doi: 10.5958/0975-4385.2019.00014.1

19.   T M Kalyankar, A C Jadhav, S M Mhetre. Recent Advances in Coupling Technology in Analysis of Natural Product. Research J. Pharm. and Tech. 5(9): September 2012; Page 1145-1153.

20.   Tine Y, Yang Y, Renucci F, Costa J, Wele A, Paolini J. LC-MS/MS analysis of flavonoid compounds from Zhanthoxylum zanthoxyloides extracts and their antioxidant antivities, Nat. Prod. Comm, 2017; 12(12):1865-1868. DOI:10.1177/1934578X1701201213

21.   Gomathy Subramanian, Subramania Nainar Meyyanathan, Byran Gowramma, Dhanabal S. Palanisamy. Liquid chromatography-mass spectrometric method for simultaneous estimation of apigenin and luteolin from Achillea millefolium Linn. Asian J. Research Chem. 2016; 9(12): 629-632. Doi: 10.5958/0974-4150.2016.00086.9

22.   K. Mahendra, Y.L. N. Murthy, C.V. Narasimha Rao, K. Bala Murali Krishna. Quantitative Determination of Solanesol in Tobacco by Liquid Chromatography–Mass Spectrometry (LC-MS). Asian J. Research Chem. 4(7): July, 2011; Page 1125-1130.

23.   Octaviani M, Fadhli H, Yuneistya E. Antimicrobial Activity of Ethanol Extract of Shallot (Allium cepa L.) Peels Using the Disc Diffusion Method. Pharmaceutical Sciences and Research (PSR), 2019; 6(1):62 – 68.

24.   Sam Jebaraj A, Gopinath P. Antibacterial Activity of Honey Against Clinical Isolates of Pseudomonas aeruginosa. Research J. Pharm. and Tech 2016; 9(8):1174-1176. doi: 10.5958/0974-360X.2016.00224.9

25.   Allison D, Gilbert P, Pharmaceutical Microbiology (7th ed), Blackwell Science Massachusets, USA, 2004.

26.   Chusnie TTP, Lamb AJ, Antimicrobial activity of flavonoid. International Journal of Antimicrobial Agents, 2005; 26(5):343-356. DOI: 10.1016/j.ijantimicag.2005.09.002

27.   Górniak I, Bartoszewski R, Króliczewski J, Comprehensive review of antimicrobial activities of plant flavonoids, Phytochem Rev, 2019; 18:241–72. DOI:10.1007/s11101-018-9591-z

28.   Kumar S, Pandey AK. Chemistry and biological activities of flavonoids: an overview. Sci World J, 2013; 2013:1–16. https://doi.org/10.1155/2013/162750

29.   Nijveldt RJ, Van Nood ELS, Van Hoorn DE, Boelens PG, Van Norren K, Van Leeuwen PAM. Flavonoids: a review of probable mechanisms of action and potential applications, Am J Clin Nutr, 2001; 74:418–25. DOI: 10.1093/ajcn/74.4.418

 

 

 

 

Received on 18.05.2021           Modified on 02.11.2021

Accepted on 05.01.2022         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(7):3047-3050.

DOI: 10.52711/0974-360X.2022.00509