Turkish Journal of Biology

Volume 33 Number 2 Article 10

1-1-2009

Antioxidant and Antibacterial Activity of Diospyros ebenum Roxb.

Leaf Extracts
YOGESH BARAVALIA

MITAL KANERIA

YOGESHKUMAR VAGHASIYA

JIGNA PAREKH

SUMITRA CHANDA

Follow this and additional works at: https://journals.tubitak.gov.tr/biology

Part of the Biology Commons

Recommended Citation
BARAVALIA, YOGESH; KANERIA, MITAL; VAGHASIYA, YOGESHKUMAR; PAREKH, JIGNA; and CHANDA,
SUMITRA (2009) "Antioxidant and Antibacterial Activity of Diospyros ebenum Roxb. Leaf Extracts," Turkish
Journal of Biology: Vol. 33: No. 2, Article 10. https://doi.org/10.3906/biy-0805-34
Available at: https://journals.tubitak.gov.tr/biology/vol33/iss2/10

This Article is brought to you for free and open access by TÜBİTAK Academic Journals. It has been accepted for
inclusion in Turkish Journal of Biology by an authorized editor of TÜBİTAK Academic Journals. For more
information, please contact academic.publications@tubitak.gov.tr.
 Turk J Biol
33 (2009) 159-164
© TÜBİTAK
doi:10.3906/biy-0805-34

Antioxidant and Antibacterial Activity of Diospyros ebenum Roxb.

Leaf Extracts

Yogesh BARAVALIA, Mital KANERIA, Yogeshkumar VAGHASIYA, Jigna PAREKH, Sumitra CHANDA
Phytochemical, Pharmacological and Microbiological Laboratory, Department of Biosciences, Saurashtra University,
Rajkot-360 005, Gujarat, INDIA

Received: 28.05.2008

Abstract: The therapeutic properties of plants, including antioxidant and antibacterial activity, have been investigated. In the present
study the petroleum ether, ethyl acetate, methanol, and aqueous extracts of Diospyros ebenum Roxb. were evaluated for DPPH (2,2-
diphenyl-1-picryl-hydrazyl) free radical scavenging (antioxidant) activity and antibacterial activity. Antibacterial activity was
determined against Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC29737, Pseudomonas aeruginosa ATCC27853,
Salmonella typhimurium ATCC23564, and Enterobacter aerogenes ATCC13048 using the agar well diffusion method. Total phenolic
and flavonoid content of these extracts was determined as the gallic acid and quercetin equivalent, respectively. Results show that
the methanol extract was active against all 5 bacterial strains, whereas the aqueous extract was active against none. The methanol
extract had the highest total phenolic content and DPPH free radical scavenging activity (IC50 = 20 μg/ml). The antioxidant and
antibacterial potential of the methanol extract of D. ebenum observed in the present study indicates that most active constituents of
this plant may be phenolics.

Key Words: Diospyros ebenum, antioxidant activity, antibacterial activity, total phenol content, flavonoid content

Diospyros ebenum Roxb. Yapraklarındaki Çözücü Özütlerin Antioksidant ve

Antibakteriyel Aktiviteleri

Özet: Bitkilerin antioksidant ve anti-enfeksiyon gibi tedavi edici özellikleri, güçlü farmokolojik faaliyetlerinden dolayı, dünyadaki son
bilimsel gelişmelerin ışığı altında araştırılmaktadır. Bu çalışmada, Diospyros ebenum Roxb. ‘un petrol eteri, etil asetat ve su
özütlerinin, DPPH (2,2-difenil-1-pikril-hidrazil) serbest radikal temizleme ve antibakteriyel aktiviteleri değerlendirilmiştir.
Antibakteriyel aktivite, Bacillus subtilis ATCC6633, Staphylococcus aureus ATCC29737, Pseudomonas aeruginosa ATCC27853,
Salmonella typhimurium ATCC23564 ve Enterobacter aerogenes ATCC13048 ‘e karşı agar kuyu difizyon metodu kullanılarak
belirlenmiştir. Bu özütlerin toplam fenolik ve flavonoid içeriği sırasıyla gallik asit ve quercetin eşdeğeri olarak belirlenmiştir. Sonuç,
su özütünün çalışılan bütün mikrobial suşlara karşı inaktif iken, metanol özütünün kullanılan beş bakteri suşuna karşı aktif olduğunu
göstermiştir. Toplam fenolik içeriği ve DPPH serbest radikal temizleme aktivitesi metanol özütünde (IC50 = 20 μg/ml) daha fazla
bulunmuştur. Bu çalışmada belirtilen D. ebenum’un metanol özütünün antioksidant ve antibakteriyel potansiyeli bu bitkinin aktif
içeriğinin çoğunun fenolik bileşenler olabileceğini göstermektedir.

Anahtar Sözcükler: Diospyros ebenum, antioksidant aktivite, antibakteriyel aktivite, toplam fenol içeriği, flavonoid içeriği

Introduction compounds from natural sources has always been of great

Nature has been a source of medicinal agents for interest to scientists looking for new sources of useful
thousands of years and an impressive number of modern drugs for treating infectious diseases.
drugs have been isolated from natural sources; much of Oxidation processes are very important in all
this isolation was based on the uses of these agents in organisms. The uncontrolled production of oxygen free
traditional medicine (1). The study of biologically active radicals and the unbalanced mechanism of antioxidant

159
Antioxidant and Antibacterial Activity of Diospyros ebenum Roxb. Leaf Extracts

protection results in cancer, diabetes, and coronary heart wool, and then kept on a rotary shaker at 190-220 rpm
diseases (2-4). The antioxidant properties of plant for 24 h. Then the extract was filtered with 8 layers of
extracts have been attributed to their polyphenolic muslin cloth, and the residue was dried and used for
content (5,6); therefore, plants containing high levels of extraction in another solvent. While the filtrate was
polyphenols are of great importance as natural centrifuged at 5000 g for 10 min, the supernatant was
antioxidants. Many byproducts and wastes generated by collected, and the solvent was evaporated. The dried
the agro-industry contain polyphenols, which can be extract of each solvent was stored at 4 °C in airtight
potentially used as food antioxidants and preventive bottles. Extraction was performed in triplicate and the
agents against some diseases (7). mean values are presented.
Infectious diseases caused by bacteria, fungi, viruses, Total Phenol determination
and parasites remain a major threat to public health,
Total phenolic content of the extracts was determined
despite tremendous progress in human medicine. Their
according to the Folin-Ciocalteu reagent method (11),
impact is particularly great in developing countries
with some modification. Plant extract (1 ml) was mixed
because of the relative unavailability of medicines and the
with Folin-Ciocalteu reagent (0.1 ml, 1 N) and allowed to
emergence of widespread drug resistance (8).
stand for 15 min. Then 5 ml of saturated Na2CO3 was
Diospyros ebenum Roxb. belongs to the family added. The mixtures were allowed to stand for 30 min at
Ebenaceae. This plant is distributed throughout India in room temperature and total phenolic content was
deciduous forests. Terminal buds are absent and determined spectrophotometrically at 760 nm. A
branchlet tips sometimes form a spine. The leaves are calibration curve was made by preparing gallic acid (10 to
1
alternate, occasionally slightly translucent, dotted or with 100 μg ml ) solution in distilled water. Total phenol
gland pits. The fruits of this plant are edible; the bark is values are expressed in terms of the gallic acid equivalent
–1
astringent (9) and its decoction is used to treat diarrhea (mg g of extracted compound).
and dyspepsia. The leaves are diuretic laxative,
Flavonoid Determination
carminative, and styptic. The dried flowers are used to
treat urinary and skin infections. The aluminum chloride colorimetric method (12), with
some modification, was used to determine flavonoid
The aim of the present study was to assess the in vitro content. Plant extract (1 ml) in methanol was mixed with
antioxidant and antibacterial activity of different solvent 1 ml of methanol, 0.5 ml of aluminum chloride (1.2%),
extracts of Diospyros ebenum leaves. and 0.5 ml of potassium acetate (120 mM). The mixture
was allowed to stand for 30 min at room temperature
Materials and Methods and the absorbance of the reaction mixture was measured
at 415 nm. The calibration curve was made by preparing
Collection of Plant Material 1
a quercetin (5 to 60 μg ml ) solution in methanol.
The leaves of Diospyros ebenum Roxb. were collected Flavonoid content is expressed in terms of the quercetin
–1
from Rajkot, Gujarat, India in September 2007 and equivalent (mg g of extracted compound).
identified by Dr. N.K. Thakrar, Department of Biosciences, DPPH Free Radical Scavenging Activity
Saurashtra University, Rajkot, Gujarat, India (voucher
specimen no. PSN430). The leaves were thoroughly The free radical scavenging activity of the D. ebenum
washed with tap water, air dried, homogenized to a fine extracts was measured using the modified method of
powder, and stored in airtight bottles. McCune and Johns (13). Various concentrations of the
stock solution (1 ml) were mixed with freshly prepared
Extraction DPPH (0.3 mM, 1 ml) in methanol to produce a final
The dried D. ebenum leaf powder was extracted DPPH concentration of 0.1 mM. The mixture was
successively in petroleum ether, ethyl acetate, methanol, vigorously shaken and left to stand for 10 min in the
and distilled water via the cold percolation method (10). dark, and its absorbance was measured at 517 nm. The
Ten grams of dried D. ebenum leaf powder was placed in concentration of each sample required for 50%
100 ml of solvent in a conical flask, plugged with cotton scavenging of DPPH free radicals (IC50) were determined

160
 Y. BARAVALIA, M. KANERIA, Y. VAGHASIYA, J. PAREKH, S. CHANDA

graphically by plotting the percentage of DPPH decrease Results and Discussion

as a function of the sample concentration: The use of medicinal plants plays a vital role in
meeting basic health requirements in developing
% DPPH radical scavenging = [(B – A)/B] × 100 countries. These plants may be a new source of
antibacterial, antifungal, and antiviral agents with
significant activity against infective microorganisms
where B is the absorbance of the blank (DPPH plus (15,16).
methanol) and A is the absorbance of the sample (DPPH, In the present study the extractive yield of D. ebenum
methanol plus sample). leaf varied among the different solvents used. The
Test Microorganisms methanol extract had the highest extractive yield
(12.37%) and the ethyl acetate extract had the lowest
The bacterial strains used were identified strains
(2.70%). Parekh et al. (17) and Vaghasiya and Chanda
obtained from the National Chemical Laboratory (NCL),
(10) reported similar results.
Pune, India. In all, 2 gram-positive (Bacillus subtilis
ATCC6633 and Staphylococcus aureus ATCC29737) and A number of studies have focused on the biological
3 gram-negative (Pseudomonas aeruginosa ATCC27853, activity of phenolic compounds, which are potential
Salmonella typhimurium ATCC23564, and Enterobacter antioxidants and free radical scavengers (18,19). In the
aerogenes ATCC13048) bacterial strains were studied. present study total phenolic content was highest in the
methanol extract and lowest in the petroleum ether
Antibacterial Assay
extract, whereas flavonoid content was highest in the
A loop full of each strain was inoculated in 25 ml of petroleum ether extract and lowest in the aqueous extract
nutrient broth in a conical flask and then incubated at (Figure 1).
room temperature on a rotary shaker for 24 h in order
Various assays are used to test antioxidant activity,
to activate the test bacteria. The final cellular
but the most widely used methods are those that involve
concentration was 1 × 10 cfu/ml. Muller Hinton agar (Hi
8
generation of free radical species that are then neutralized
Media) was used to determine antibacterial susceptibility. by antioxidant compounds (20,21). The DPPH radical is
Bacterial assays were performed using the agar well commonly used as a substrate to evaluate antioxidant
diffusion method (14). Media and test bacterial cultures activity; it is a stable free radical that can accept an
were poured into petri dishes (Hi-Media). Each test strain electron or hydrogen radical to become a stable molecule.
(200 μl) was inoculated into the media when the
temperature was 40-42 °C. Care was taken to ensure
        !" ## $

proper homogenization. After media were solidified a well 

was made in the plates with the help of a cup-borer (8.5  
mm). The well was filled with 100 μl of extract (500 μg  

well 1) and the plates were incubated overnight at 37 °C.
Bacterial growth was determined according to the
diameter of the zone of inhibition. The experiments were 
performed 3 times and mean values are presented. For
each bacterial strain, controls were maintained, in which
pure solvent (DMSO) was used instead of the extract. The 
results of all the solvent extracts were compared with the
  

standard antibiotics amikacin (30 μg) and piperacillin

(100 μg) (Hi Media). 

Statistical Analysis  

Figure 1. Total phenol and flavonoid content of the solvent extracts of

All the experiments were performed in triplicate and
Diospyros ebenum leaf. Pe: Petroleum ether extract; Ea: ethyl
results are presented as mean ± SEM (standard error acetate extract; Me: methanol extract; Aq: aqueous extract.
mean). Values are expressed as mean ± SEM, n = 3.

161
Antioxidant and Antibacterial Activity of Diospyros ebenum Roxb. Leaf Extracts

Reductions in the DPPH radical induced by antioxidants 

was determined by the decrease in its absorbance at 517
nm. The concentration of a sample at which the inhibition 
percentage reaches 50% is its IC50 value. The IC50 value is

+<> ? 
negatively related to antioxidant activity, as it expresses *
the amount of antioxidant needed to decrease its radical
concentration by 50%. The lower the IC50 value, the '
higher the antioxidant activity of a tested sample.
In the present study the methanol extract had the &
highest antioxidant activity (IC50 = 20 μg/ml). This activity
was very near to that shown by the standard (ascorbic 
acid, IC50 = 11.4 μg/ml), while the petroleum ether  
 
extract had the lowest antioxidant activity (IC50 = 145
μg/ml) (Figure 2). Liu and Ng (22), and Siriwardhana et Figure 2. DPPH free radical scavenging activity (IC50 values) of the
solvent extracts of Diospyros ebenum leaf. Pe: Petroleum
al. (23) reported a high correlation between DPPH radical
ether extract; Ea: ethyl acetate extract; Me: methanol extract;
scavenging activity and total polyphenolic content. In the Aq: aqueous extract; Asc: ascorbic acid.
present study the methanol extract of D. ebenum had the Values are expressed as mean ± SEM, n = 3.
highest phenolic content, as well as the highest DPPH free
radical scavenging activity.
The past 3 decades have seen a dramatic increase in spread of antibiotic-resistant pathogens (25). The
resistance to antimicrobial agents, leading to the repeated aqueous extract tested in the present study did not have
use of antibiotics and insufficient disease control (24). any activity against the bacterial strains studied. The
Due to the increasing prevalence of antibiotic-resistant methanolic extract demonstrated activity against all 5
pathogens in hospitals and homes, a deliberate search is bacterial strains and a large zone of inhibition against P.
in progress for alternative treatments to combat further aeruginosa (Figure 3). The ethyl acetate extract showed


 ^



X Z +\ !" " $






@ Q Q
@  
Figure 3. Antibacterial activity of the solvent extracts of Diospyros ebenum leaf. Bs: Bacillus subtilis; Sa: Staphylococcus aureus; Pa: Pseudomonas
aeruginosa; St: Salmonella typhimurium; Ea: Enterobacter aerogenes. Pe: Petroleum ether extract; Ea: ethyl acetate extract; Me: methanol
extract; Aq: aqueous extract; Ak: amikacin; Pc: piperacillin. Values are expressed as mean ± SEM, n = 3.

162
 Y. BARAVALIA, M. KANERIA, Y. VAGHASIYA, J. PAREKH, S. CHANDA

activity against 3 bacterial strains—B. subtilis, S. aureus, The results of the present study show that the
and P. aeruginosa—while the petroleum ether extract methanol extract of D. ebenum leaf contained a high total
showed activity only against S. aureus. The standard phenolics level, and is a good source of antioxidant as well
antibiotic piperacillin showed activity only against the as antibacterial agents; therefore, it can be considered
gram-positive bacteria. All the extracts, except the potentially useful for medicinal application.
aqueous extract, were more potent against S. aureus than
was the standard amikacin. The methanol extract was
more active against P. aeruginosa and S. typhimurium Corresponding Author:
than was amikacin. Chanda SUMITRA

Many studies have shown that natural antioxidants in Phytochemical,

plants are closely related to their biofunctionality, such as Pharmacological and Microbiological Laboratory,
the reduction of chronic diseases and inhibition of Department of Biosciences,
pathogenic bacterial growth, which are often associated
Saurashtra University, Rajkot-360 005, Gujarat - INDIA
with the termination of free radical propagation in
biological systems (26). E-mail: svchanda@gmail.com

References
1. Cragg GM, Newman DJ. Medicinals for the millennia. Ann NY Acad 11. McDonald S, Prenzler PD, Antolovich M et al. Phenolic content and
Sci 953: 3-25, 2001. antioxidant activity of olive extracts. Food Chem 73: 73-84,
2001.
2. Erlund I, Alfthan G, Maenpaa J et al. Tea and coronary heart
disease: The flavonoid quercetin is more bioavailable from rutin in 12. Chang C, Yang M, Wen H et al. Estimation of total flavonoid
women than in men. Arch Intern Med 161: 1919-1920, 2001. content in propolis by two complementary colorimetric methods.
J Food Drug Anal 10: 178-182, 2002.
3. Yang YC, Lu FH, Wu JS et al. The protective effect of habitual tea
consumption on hypertension. Arch Intern Med 164: 1534-1540, 13. McCune LM, Johns T. Antioxidant activity in medicinal plants
2004. associated with the symptoms of diabetes mellitus used by the
indigenous peoples of the North American boreal forest. J
4. Yam MF, Basir R, Asmawi MZ et al. Antioxidant and
Ethnopharmacol 82: 197-205, 2002.
hepatoprotective activities of Elephantopus tomentosus ethanol
extract. Pharm Biol 46: 199-206, 2008. 14. Perez C, Paul M, Bazerque P. An antibiotic assay by the agar well
diffusion method. Acta Biol Med Exp 15: 113-115, 1990.
5. Lu Y, Foo YL. Antioxidant activities of polyphenols from sage
(Salvia officinalis). Food Chem 75: 197-202, 2001. 15. Munoz-Mingarro D, Acero N, Llinares F et al. Biological activity of
extracts from Catalpa bignonioides Walt. (Bignoniaceae). J
6. Murthy KNC, Singh RP, Jayaprakasha GK. Antioxidant activities of
Ethnopharmacol 87: 163-167, 2003.
grape (Vitis vinifera) pomace extracts. J Agric Food Chem 50:
5909-5914, 2002. 16. Coelho de Souza G, Haas APS, von Poser GL et al.
Ethnopharmacological studies of antimicrobial remedies in the
7. Torres JL, Varela B, Garcia MT et al. Valorization of grape (Vitis
south of Brazil. J Ethnopharmacol 90: 135-143, 2004.
vinifera) byproducts. Antioxidant and biological properties of
polyphenolic fractions differing in procyanidin composition and 17. Parekh J, Karathia N, Chanda S. Evaluation of antibacterial activity
flavonol content. J Agric Food Chem 50: 7548-7555, 2002. and phytochemical analysis of Bauhinia variegata L. bark. Afr J
Biomed Res 9: 53-56, 2006.
8. Okeke IN, Laxminarayan R, Bhutta ZA et al. Antimicrobial
resistance in developing countries. Part 1: recent trends and 18. Kahkonen MP, Hopia AI, Vuorela HJ et al. Antioxidant activity of
current status. Lancet Infect Disease 5: 481-493, 2005. plant extracts containing phenolic compounds. J Agric Food Chem
47: 3954-3962, 1999.
9. Duraipandiyan V, Ayyanar M, Ignacimuthu S. Antimicrobial activity
of some ethnomedicinal plants used by Paliyar tribe from Tamil 19. Sugihara N, Arakawa T, Ohnishi M et al. Anti and pro-oxidative
Nadu, India. BMC Comp Alter Med 6: 35-41, 2006. effects of flavonoids on metal induced lipid hydroperoxide-
dependent lipid peroxidation in cultured hepatocytes loaded with
10. Vaghasiya Y, Chanda SV. Screening of methanol and acetone
-linolenic acid. Free Radical Bio Med 27: 1313-1323, 1999.
extracts of fourteen Indian medicinal plants for antimicrobial
activity. Turk J Biol 31: 243-248, 2007. 20. Arnao MB, Cano A, Acosta M. The hydrophilic and lipophilic
contribution to total antioxidant activity. Food Chem 73: 239-
244, 2001.

163
Antioxidant and Antibacterial Activity of Diospyros ebenum Roxb. Leaf Extracts

21. Masoko P, Eloff JN. Screening of twenty four South African 24. Vaghasiya Y, Nair R, Chanda S. Investigation of some Piper species
Combretum and six Terminalia species (Combretaceae) for for anti-bacterial and anti-inflammatory property. Int J Pharmacol
antioxidant activities. Afr J Trad CAM 4: 231-239, 2007. 3: 400-405, 2007.
22. Liu F, Ng TB. Antioxidative and free radical scavenging activities of 25. Olukoya DK, Idika N, Odugbemi A. Antibacterial activities of some
selected medicinal herbs. Life Sci 66: 725-735, 2000. plants from Nigeria. J Ethonopharmacol 39: 69-72, 1993.
23. Siriwardhana N, Lee KW, Jeon YJ et al. Antioxidant activity of 26. Covacci V, Torsello A, Palozza P et al. DNA oxidative damage
Hizikia fusiformis on reactive oxygen species scavenging and lipid during differentiation of HL-60 human promyelocytic leukemia
peroxidation inhibition. Food Sci Technol Int 9: 339-346, 2003. cells. Chem Res Toxicol 14: 1492-1497, 2001.

164