Wight ex Duthie Gamble Karunamoorthy Kiruthiga 1, Jothiramshekar Saranya



Yüklə 73.45 Kb.
Pdf просмотр
tarix19.08.2017
ölçüsü73.45 Kb.

Chemical Composition, Antimicrobial, Antioxidant and Anticancer Activity

of Leaves of Syzygium benthamianum 

(Wight ex Duthie) Gamble

Karunamoorthy Kiruthiga 

1

, Jothiramshekar Saranya 

1

,

Palanisami Eganathan 

1

*, Puthiyapurayil Sujanapal 

2

, Ajay Parida 

1

Plant Tissue Culture and Bioprospecting Laboratory, M.S. Swaminathan Research



Foundation, 3

rd

 Cross Street, Taramani, Chennai- 600113, India



Community Agrobiodiversity Centre, M.S. Swaminathan Research

Foundation, Wayanad- 673121, Kerala, India

Abstract: 

The study was carried out on antimicrobial, antioxidant and anticancer activity of Syzygium



benthamianum leaf extract. Chemical compositions of the leaf were analysed using GC/MS technique. A total

of 24 compounds were identified among which 4-(4-ethylcyclohexyl)-1-pentyl-Cyclohexene (24.07 %) and

Linoleic acid (

15.16 %


) are the major constituents.

 Antimicrobial activity of the leaf was observed on six

bacterial and three fungal species, whose MIC values ranged from 100 to 500 

μ

g/ml. At higher concentration,



the extract exhibits higher scavenging activity (94.7 %) that was comparable with standard BHT. It was also

observed that the leaf sample were able to effectively inhibit the growth of Hep 2 cells.



Keywords:  Syzygium benthamianum, ethyl acetate extract, antimicrobial, antioxidant,

anticancer activity.



Introduction

Syzygium is a genus of flowering plants 

1

belonging to family Myrtaceae comprising of



about 1200 species, and spread across tropical

Africa, subtropical and tropical Asia, Australia,

New Caledonia, New Zealand, Pacific island;

among these 80 species have been reported from

China 

2

 and more than 75 species from India 



3

.

Syzygium benthamianum is one of the species

that has been categorized as vulnerable tree

species under the IUCN 

4

 red list of threatened



species. Syzygium species have been reported to

exhibit antidiabetic 

5,6

, antifungal 



7,8

, antiinflam-

matory 

9

, antibacterial 



10

, antioxidant 

11

, antihyper-



lipidemic 

12

 and growth inhibitory effects against



oral pathogens 

13

Syzygium species are also found



to posses antihyperglycemic activity 

14

, cytotoxic



15

, antiangiogenic 

15

, Antinociceptive activity 



16

.

Till date there is no literature available on the



medicinal properties of Syzygium benthamianum.

Therefore, the present study was undertaken to

analyze some of the medicinal properties of

Syzygium benthamianum leaf extract constituents

and their biological activities.



Materials and methods

Plant material and extraction

The plant material was collected from



Journal of Biologically Active Products from Nature

ISSN Print: 2231-1866 Online: 2231-1874

www.jbappn.com

*Corresponding author (Palanisami Eganathan)

E-mail: < 

eganathan@gmail.com

 >

      © 


2011, Har Krishan Bhalla & Sons

Received 05 

June 

2011; accepted in revised form 28 



June

 2011


JBAPN 1 (4) 2011 pp 273 - 278

273


Kurichiarmala, Wayanad District in the state of

Kerala, India, and identified as Syzygium



benthamianum by Community Agrobiodiversity

Centre (CAbC), M.S. Swaminathan Research

Foundation, Wayanad, Kerala and voucher

specimens deposited in the herbarium. The shade

dried leaves were powdered and subjected to

solvent extraction. Compound extraction was

carried out using soxhlet apparatus 

17

 with ethyl



acetate as solvent at 77°C for 4 hrs. Solvent was

collected and evaporated using rotary evaporator

at 77°C. The dried extracts were dissolved in

dimethyl sulphoxide (DMSO) and evaluated for

their efficacy against micro organisms.

Gas chromatography-Mass spectrometry

analysis

The GC (Agilent 6890) conditions were as

follows: DB-5 column (30 m X 0.25 mm X

0.25


μm), injection in split mode (50:1),

temperatures were maintained at 250°C in the

injector and detector, helium was used as carrier

gas at a flow rate of 1 ml/min; oven temperature

was initially maintained at 100°C for 5 min and

then raised to 220°C at a rate of 10°C/min and

held at 220°C for 18 min. The MS (Agilent 5973

inert MSD) electron multiplier 2188.2 V, mass

spectra data were acquired in the scan mode in

m/z range 58-550. The compounds of the leaf

extract were identified by comprising their

retention indices (RI), with those on the stored

in NIST (National Institute of Standards and

Technology) library and by comparing their mass

spectra with the data already available in

literature 

18

.

Microbial isolates



Six Gram negative (Pseudomonas aeroginosa,

Proteus vulgaris, Proteus mirabilis, Vibrio

cholera, Klebsiella pneumoniae and Escherishia

coli) and two Gram positive (Staphylococcus

aureus and Bacillus subtilis) bacterial strains and

three fungal (Aspergillus niger, Alternaria



alternata and Pencillium chrysogenum) species

were used in this study. The bacterial stock

cultures were maintained on nutrient agar

medium and fungal culture on potato dextrose

agar medium, and were stored at 4°C.

Determination of antimicrobial activity

The extracts were tested for their antimicrobial

activity by Disc diffusion method. Bacterial

species were sub-cultured on nutrient agar

medium and fungal species on potato dextrose

agar medium, which were then incubated at

37°C for 24 h and 27°C for 48 h respectively.

The test solutions of the dried extracts at the

concentrations of 1000 

μg, 500 μg, 250 μg, 100

μg/ml were impregnated on sterile discs.

Streptomycin and Nystatin were used as positive

controls. The disc impregnated with ethyl

acetate was used as negative control. The discs

were placed on the surface of the nutrient agar

for bacteria and incubated at 37°C for 24 h. The

discs were placed on the surface of potato

dextrose agar for fungi and incubated at 27°C

for 48 h. Inhibition zones were calculated as the

difference between disc diameter (6mm) and the

diameters of inhibition 

19

. The antibacterial



activities were evaluated by the determination

of minimum inhibitory concentration and

minimum lethal concentration by micro broth

dilution assay 

20

.

Determination of antioxidant activity with



2,2 diphenyl-1-picrylhydrazyl (DPPH)

radical scavenging method

The free radical scavenging activity of the

sample was evaluated using DPPH 

21

. The sample



was prepared to the concentration of 1mg/ml

using methanol. Pure methanol was taken as blank

and 0.016 % butylated hydroxyl toluene (BHT)

was taken as the standard. 2.7 ml of methanol,

100 

μl of sample and 200 μl of DPPH reagent



(1mg/ml) were added and these mixtures were

kept in dark incubation at RT for 30 mins.

Samples were visualized in UV-VIS spectro-

photometer at wavelength of 517nm.

Percentage of DPPH radical scavenging activity

of the sample was calculated as:

= [(A

DPPH


 - A

S

)/A



DPPH

] x100.


Where, A

S

 is the absorbance of the solution



when the sample extract is added at a particular

level and A

DPPH

 is the absorbance of the DPPH



solution 

22

.



Palanisami Eganathan 

et al. / JBAPN 1 (4) 2011 pp 273 - 278

274


Anticancer activity: MTT Assay

The anticancer activity of the sample was

measured using 3-(4,5-dimethyl thiazol-2yl)-2,5-

diphenyl tetrazolium bromide (MTT) assay 

23

.

The monolayer culture of Hep2 cells at a



concentration of 10 cells/ml/well were seeded in

24 well titre plates. Cells were permitted to adhere

for 24 h, and then treated with different dilution

(1:1 to 1:64) of the extract for 24 h; 200 

μl of

MTT (5 mg/ml in PBS) were added to each well,



and the cells were incubated for a further 6-7 hrs

in 5 % CO

2

 incubator. After removal of the



medium, 1ml of DMSO was added to each well.

The absorbance was recorded at the wavelength

of 570 nm. The effect of the extracts on cell

growth inhibition was assessed as percent cell

viability, where vehicle-treated cells were taken

as 100 % viable.

Percentage of viable cell concentration was

calculated thus

24

:

Viability (%) = (Optical Density of sample x



optical density of control) x 100

Results and discussion

Crude extract of leaf showed a total of 24

compounds among which 4-(4-ethyl-

cyclohexyl)-1-pentyl-Cyclohexene (24.07 %)

followed by 

Linoleic acid (

15.16 %

), 


2,6,10,-

14,18-Penta-methyl-2,6,10,14,18-eicosapen-

taene (10.27 %), 9,17-Octadecadienal,(z)- (9.96

%),


 

Z,E-3,13-Octadecadien-1-ol (7.14 %)

 and

7-Pentadecyne



 (7.36 %) are the major consti-

tuents (Table 1).



Table 1. Compound composition of Syzygium benthamianum leaves extract

Compound

RT 

a

RI 

b

%

Copaene


7.363

1376


0.19

α-trans-bergamotene

7.820

1436


0.13

(-)-isocaryophyllene

8.007

1404


0.47

cis-

β-Farnesene

8.324

1443


0.21

α-Caryophyllene

8.537

1454


0.17

1-Decene


8.789

991


0.72

α

-selinene



9.058

1494


0.16

cis-

α-Bisabolene

9.232

1504


0.27

Dodecyl acrylate

12.283

-

0.44



cis-Pinane

14.817


983

4.45


1-Tridecyne

15.273


-

0.80


5-Nonadecen-1-ol

15.621


-

1.21


(R)-(-)-14-Methyl-8-hexadecyn-1-ol

19.228


-

0.65


(-)-

β-citronellene

19.345

-

0.28



1,2-15,16-Diepoxyhexadecane

19.441


-

0.22


Linoleic acid

24.743


2173

15.16


Decahydro-8a-ethyl-1,1,4a,6-tetramethylnapthalene

26.455


-

5.96


Cyclohexene,4-(4-ethylcyclohexyl)-1-pentyl-

28.711


-

24.07


7-Pentadecyne

29.615


-

7.36


2,6,10,14,18-Pentamethyl-2,6,10,14,18-eicosapentaene

29.710


-

10.27


Z,E-3,13-Octadecadien-1-ol

30.671


-

7.14


9,17-Octadecadienal,(z)-

30.927


-

9.96


Eicosane

33.383


2000

4.22


3,6-Dimethyl-5-oxo-1,2,3,5-tetrahydroimidazol[1,2-a]pyrimidine 34.122

-

5.00



a

Compounds are listed and elution from DB5 column

b

Values calculated from published literature (DB5 column)



Palanisami Eganathan 

et al. / JBAPN 1 (4) 2011 pp 273 - 278

275


The MIC values of the plant extract against the

tested bacterial isolates ranged from 100 to 500

μ

g/ml


. Plant extract at the concentration of 100

μ

g/ml inhibits the growth of Proteus vulgaris and



Proteus mirabilis whereas,  the growth of

Staphylococcus aureus was inhibited at the

concentration of 500 

μ

g/ml. All other microbial



species used in this study showed minimum

inhibitory concentration at 250 

μ

g/ml 


(Table 2)

.

The ethyl acetate extract of Syzygium



benthamianum  leaves were found to act as

potent free radical scavengers in comparison

with BHT, a commercial antioxidant. At higher

concentration (400 

μ

g/ml) the extract has



significant inhibition of DPPH radical

scavenging activity (Table 3). Syzygium



benthamianum  shows comparable scavenging

activity with Syzygium cumini fruit 

25

 and


Syzygium aromaticum buds 

11

.



MTT assay to evaluate the effect of the extract

on cell viability of Hep2 cells was used. The ethyl

acetate extract treatment to these cell lines

resulted in a remarkable dose-dependent inhi-

bition of cell growth. The extract showed maxi-

mum cell inhibition at higher concentration

(Table 4). The extract of Syzygium benthamianum

showed higher activity on cancer cell lines and

this result correlated with the activity exhibited

by Syzygium cumini on AML cells 

25

.

Thus from the present study it was proven that



the leaves of Syzygium benthamianum possess

effective biological properties against pathogens

and cancer cells. In addition also it exhibits high

scavenging activity against free radical

comparable with that of standard available drugs.

Acknowledgement

Authors are thanking to the Department of

Biotechnology, GOI, New Delhi for financial

support.


Table 2. Minimum Inhibition Concentration (MIC) of Syzygium benthamianum

leaf extract against bacterial and fungal isolates

Bacterial and Fungal isolates

MIC 

μμμμμg/ml



Escherichia coli

250


Pseudomonas aeruginosa

250


Klebsiella pneumonia

250


Proteus vulgaris

100


Staphylococcus aureus

500


Bacillus subtilis

250


Vibrio cholera

250


Proteus mirabilis

100


Alternaria alternata

250


Penicillium  chrysogenum

250


Aspergillus niger

250


Table 3. Antioxidant activity of Syzygium benthamianum leaf extract

S. No.

Sample

Concentration (

μμμμμg/ml)



% Antioxidant activity

1

Blank



0

0

2



Standard

400


95.7

± 1.2


3

Sample


50

78.08 ± 1.65

100

85.7


± 2.5

200


89.3

± 3.1


400

94.7


± 2.12

Palanisami Eganathan 



et al. / JBAPN 1 (4) 2011 pp 273 - 278

276


References

1. Eliot Rodger, W., Jones, D.L., Blake, T. (2010). Encyclopaedia of Australian Plants Suitable

for Cultivation. Vol. 9 – Sp-Z. Port Melbourne: Lothian Press. pp. 160-61.

2. Jie, C., Craven, L. A.  (2007). Syzygium. Flora of China, Vol. 13: 335.

3. Anand, A., Srinivasa Rao, C., Balakrishna, P. (1999). In vitro propagation of Syzygium

travancoricum Gamble-an endangered tree species, Plant Cell, Tissue and Organ Culture, 56(1):

59-63.


4. IUCN. (1998). Syzygium benthamianum. In: IUCN 2011. IUCN Red List of Threatened Species.

Version 2011.1. www.iucnredlist.org.

5. Nonaka, G., Aiko, Y., Aritake, K., Nishioka, I. (1992). Tannins and related compounds. CXIX:

Samarangenins A and B, novel proanthocyanidins with doubly bonded structures, from Syzygium



samarangens and Syzygium aqueum, Chemical and Pharmaceutical Bulletin, 40: 2671-2673.

6. Kumar, A., Ilavarasan, R., Jayachandran, T., Deecaraman, M., Aravindan, P., Padmanabhan,



N., Krishan, M.R.V. (2008). Anti-diabetic activity of Syzygium cumini and its isolated compound

against streptozotocin-induced diabetic rats, Journal of Medicinal Plants Research, 2: 246-249.

7. Park, M.J., Gwak, K.S., Yang, I., Choi, W.S., Jo, H.J., Chang, J.W. (2007). Antifungal acti-

vities of the essential oils of Syzygium aromaticum (L.) Mer. Et Perry and Leptospermum



petersonii Bailey and their constituents against various dermatophytes, Journal of Microbiology,

45: 460-465.

8. Ayoola, G.A., Lawore, F.M., Adelowotan, T., Aibinu, I.E., Adenipekun, E., Coker H.A.B.,

Odugbemi, T.O. (2008). Chemical analysis and antimicrobial activity of the essential oil of

Syzygium aromaticum (Clove), African Journal of Microbiology Research, 2: 162-166.

9. Chaudhuri, A.K.N., Pal, S., Gomes, A., Bhattacharya, S. (1990). Anti-inflammatory and related

actions of Syzygium cuminii seed extract, Phytotherapy Research, 4: 5-10.

10. Shyamala G.S., Vasantha, K. (2010). Phytochemical screening and antibacterial activity of



Syzygium cumini (L.) (Myrtaceae) leaves extracts, International Journal of PharmTech Research,

2: 1569-1573.

11. Nassar, M.I., Gaara, A.H., Ghorab, A.H.E., Farrag, A.R.H., Shen, H., Huq, E., Mabry, T.J.

(2007). Chemical constituents of clove (Syzygium aromaticum, Fam. Myrtaceae) and their

antioxidant activity, Revista Latinoamericana de Quimica, 35: 47-57.

12. Modi Dikshit, C., Rachh, P.R., Nayak, B.S., Shah, B.N., Modi, K.P., Patel, N.M., Patel, J.K.

(2010). Antihyperlipidemic acitivity of Syzygium cumini Linn. Seed extract on high cholesterol

fed diet rats, International Journal of Pharmaceutical Sciences, 1: 330-332.

13. Cai, L., Wu, C. D. (1996). Compounds from Syzygium aromaticum possessing growth inhibitory

activity against oral pathogens, Journal of Natural Products, 59: 987-990.

14. Rekha, N., Balaji, R., Deecaraman, M. (2010). Antihyperglycemic and antihyperlipidemic

effects of extracts of the pulp of Syzygium cumini and bark of Cinnamon zeylanicum in strepto-

zotocin-induced diabetic rats, Journal of Applied Bioscience, 28: 1718-1730.

Table 4. Anticancer activity of Syzygium benthamianum leaf extract

S.No.

Concentration (

μμμμμg/ml)



Anticancer activity %

1

50



50.75

2

100



57.64

3

250



68.15

4

500



75.44

5

1000



84.76

Palanisami Eganathan 



et al. / JBAPN 1 (4) 2011 pp 273 - 278

277


15. Aisha, A.F.A., Nassar, Z.D., Siddiqui, M.J., Abu Salah, K.M., Alrokayan, S.A., Ismail, Z.,

Abdul Majid, A.M.S. (2011). Evaluation of antiangiogenic, cytotoxic and antioxidant effects

of Syzygium aromaticum L. extracts, Asian Journal of Biological Sciences, 4(3): 282-290.

16. Avila Pena, D., Pena, N., Quintero, L., Suarez Roca, H. (2007). Antinociceptive activity of

Syzygium jambos leaves extract on rats, Journal of Ethnopharmacology. 112(2): 380-385.

17. Alade, P.I., Irobi, O.N. (1993). Antimicrobial activities of crude leaf extract of Acalypha



wilkensiana, Journal of Ethnopharmacology, 39: 171-174.

18. Adams, R.P. (1995). Identification of essential oil components by Gas Chromatography/Mass

Spectrometry. 4

th

 Ed, Allured Publishing, Carol Stream, Illinois.



19. Hewitt, W., Vincent, S. (1989). Theory and application of Microbiological assay. Academic

Press, San Diego.

20. NCCLS. (2009). Methods for dilution antimicrobial susceptibility tests for bacteria that grow

aerobically. Approved Standard M7-A8, Eighth Edition, Wayne, Pennsylvania.

21. Negi, P.S., Chauhan, A.S., Sadia, G.A., Rohinishree, Y.S., Ramteke, R.S. (2005). Antioxidant

and antibacterial activities of various seabuckthorn (Hippophae rhamnoides L.) seed extracts,

Food Chemistry, 92, 119-124.

22. Barros, L., Baptista, P., Ferreira, I.C.F.R. (2007). Effect of Lactarius piperatus fruiting body

maturity stage on antioxidant activity measured by several biochemical assays, Food and Chemical

Toxicology, 45: 1731-1737.

23. Mosmann, T. (1983). Rapid colorimetric assay for cellular growth and survival: application to

proliferation and cytotoxicity assays, Journal of Immunological Methods, 65: 55-63.

24. Rahman, S., Salehin, F., Iqbal, A. (2011). In vitro antioxidant and anticancer activity of young

Zingiber officianale against human breast carcinoma cell lines, BMC Complementary and

Alternative Medicine, 11: 76.

25. Afify, A.M.R., Fayed, S.A., Shalaby, E.A., El Shemy, H.A. (2011). Syzygium cumini (pomposia)

active principles exhibit potent anticancer and antioxidant activities, African Journal of Pharmacy

and Pharmacology, 5(7): 948-956.

Palanisami Eganathan 



et al. / JBAPN 1 (4) 2011 pp 273 - 278

278



Поделитесь с Вашими друзьями:


Verilənlər bazası müəlliflik hüququ ilə müdafiə olunur ©www.azkurs.org 2019
rəhbərliyinə müraciət

    Ana səhifə