Chemical Constituents and Biological Activities of Galinsoga parviﬂ ora Cav. (Asteraceae) from Egypt

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Introduction

The genus Galinsoga belonging to the fami- ly Asteraceae is represented with about 13 spe- cies in North and South America (Boulos, 2002).

Galinsoga parvifl ora Cav. has recently been intro- duced and naturalized as a weed in the Nile delta of Egypt (Boulos, 2002). Some of the relevant reports from different localities revealed variation of its chemical constituents. In China, the isolation of diterpenes that belong to the kaurenoic acid group and sterols such as β-sitosterol, stigmasterol, and α-spinasterol has been reported (Pan et al., 2007). Pakistanian plants revealed the presence of sterols such as β-sitosterol and β-sitosterol-3- O-β-D-glucopyranoside, phenolic com pounds like 4-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and gallic acid (Tariq et al., 2008), fl avonoids such as galinsoside A, galinsoside B, and 7,3′,4′-trihy- droxyfl avanone (Ferheen et al., 2009), and the aromatic esters galinosoates A – C (Afza et al., 2012).

Recently, 88 essential oil constituents were reported in the plant growing in Columbia with (Z)-3- hexen-1-ol, β-caryophyllene, and 6-demethoxy- ageratochrome as major compounds (Pino et al., 2010). The aim of this study was to investigate the

chemical composition of the Egyptian G. parvi- ﬂ ora as well as its hepatoprotective, hypoglycemic, antioxidant, cytotoxic, and antimicrobial activities.

Material and Methods Plant materials

Galinsoga parvifl ora Cav. was collected in the fl owering stage in December 2008 from plants growing naturally in the fi elds in the vicinity of Benha, Qalubiya province, Egypt. The identifi cation was verifi ed by Dr. H. Abdel Baset, Profes- sor of Botany, Faculty of Science, Zagazig Uni- versity, Zagazig, Egypt. A voucher specimen is deposited in the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Zagazig Uni- versity, Zagazig, Egypt. The plants were shade- dried and ground by an electric mill to a moder- ately fi ne powder.

Materials for chemical study

Melting points were determined on a Bibby Sturt scientiﬁ c melting point apparatus (Digital, Stone, Staffordshire, UK) and are uncorrected.

Infrared spectral analyses were carried out using

of Galinsoga parviﬂ ora Cav. (Asteraceae) from Egypt

Islam Mostafa, Ehsan Abd El-Aziz, Samia Hafez, and Assem El-Shazly*

Pharmacognosy Department, Faculty of Pharmacy, Zagazig University,

44519 Zagazig, Egypt. Fax: 002 055 230 3266. E-mail: assemels2002@yahoo.co.uk

* Author for correspondence and reprint requests

Z. Naturforsch. 68 c, 285 – 292 (2013); received July 8, 2012/June 1, 2013

The phytochemical investigation of an aqueous ethanolic extract of Galinsoga parviﬂ ora Cav. (Asteraceae) resulted in the isolation and identiﬁ cation of eleven compounds namely:

triacontanol, phytol, β-sitosterol, stigmasterol, 7-hydroxy-β-sitosterol, 7-hydroxystigmasterol, β-sitosterol-3-O-β-D-glucoside, 3,4-dimethoxycinnamic acid, protocatechuic acid, fumaric acid, and uracil. Furthermore, 48 volatile constituents were identiﬁ ed in the hydrodistilled oil of the aerial parts. The ethanolic extract at a content of 400 mg/kg body weight (BW) exerted 87% reduction in the alanine aminotransferase enzyme level in cirrhotic rats compared with the standard silymarin (150 mg/kg BW) and also exerted a reduction in the blood glucose level equivalent to that of glibenclamide (5 mg/kg BW) in diabetic rats. The ethanolic extract, light petroleum and ethyl acetate fractions exhibited substantial antimicrobial activity against Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Aspergillus niger, and Candida albicans. The ethyl acetate fraction showed strong antioxidant activity at a concentration of 150 mg/mL as compared with 0.1 M ascorbic acid. The cytotoxic effect against the MCF-7 cell line was found to be weak.

Key words: Galinsoga parviﬂ ora, Terpenes, Biological Activity

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a Jasko FT/IR-6100 type A spectrophotometer (Tokyo, Japan). Mass spectra were obtained on a mass spectrometer model SSQ 7000 produced by Finnigan (San Jose, CA, USA) and operated at 70 eV, and on a JEOL JMS 500 spectrometer (To- kyo, Japan) operated at 70 eV. ¹H and ¹³C NMR spectra were obtained using a JEOL instrument at 500 and 125 MHz, respectively. Chemical shifts are given in ppm with tetramethylsilane (TMS) as internal standard using CDCl3 and CD3OD as solvents.

Column chromatography (CC) was carried out using silica gel (60 to 230 mesh; Fluka, Buchs, Switzerland). Thin-layer chromatography (TLC) was performed on silica gel-coated aluminum plates (silica gel 60 GF254; Fluka). Developed chromatograms were visualized by spraying with anisaldehyde/sulfuric acid reagent followed by heating at 100 °C for 10 min or under UV light.

The solvents used for extraction, separation, and/

or detection were of analytical grade.

Materials for biological activity study

CCl4, parafﬁ n oil, 1% acetic acid, sulforhoda- mine B (SRB) protein dye, 40% trichloroacetic acid (TCA) stock solution, 10 mM Tris-EDTA buffer (pH 10.5), gum acacia, silymarin, glibenclamide, streptozocin, RPMI medium, fetal bovine serum (FBS), L-glutamine, penicillin G sodium, streptomycin sulfate, trypsin, EDTA, dimethyl sulfoxide (DMSO), 2,2-diphenyl-1-picryl hydra- zyl (DPPH), ascorbic acid, methanol, nutrient agar media, Sabouraud’s agar media, cefotaxin, and nystatin were from Sigma/Aldrich (St. Louis, MO, USA). Glucomen-glyco^® blood glucose meter was from Menarini Biotech (Rome, Italy). The human breast adenocarcinoma cell line (MCF-7) was obtained from the Serum and Vaccine Re- search Institute, Giza, Egypt.

Animals

All animal care and experimental procedures were conducted in accordance with the guidelines of the animal ethics committee of the Fa- culty of Pharmacy, Zagazig University, Zagazig, Egypt (approval number P1-5, 24 May 2010) and were handled following the International Animal Ethics Committee Guidelines, ensuring minimum animal suffering. The Faculty of Medicine, Cairo University, Cairo, Egypt provided the experimental animals. All animals were held under standard

laboratory conditions in the animal house of the Faculty of Medicine, Cairo University at 27 °C with a 12-h/12-h light/dark cycle. They were fed laboratory diet and water ad libitum. Adult male albino rats, weighing 150 – 180 g, were used in hepatoprotective and hypoglycemic studies.

Statistical analysis

Signiﬁ cance of the hepatoprotective and hypoglycemic effects of the plant extract were calculated using Student’s t-test at P < 0.05.

Extraction and isolation of compounds

The air-dried whole powdered plants of G. parviﬂ ora (4 kg) were exhaustively extracted with 80% aqueous ethanol (3 x 10 L). The ethanolic extract was ﬁ ltered and then concentrated under vacuum to yield 400 g of a viscous green- ish residue. The residue was suspended in water and partitioned between light petroleum (b.p.

60 – 80 °C, 5 x 0.5 L), chloroform (5 x 0.5 L), and ethyl acetate (5 x 0.5 L), successively. The organic solvents were evaporated under vacuum to yield 105, 2.9, and 6.5 g of ﬁ nal residues, respectively.

Isolation of seven compounds from the light petroleum fraction

The light petroleum fraction (50 g) was redis- solved in dichloromethane (75 mL), and the re- sulting solution was mixed with silica gel (35 g).

The dry mixed initial zone was chromatographed on a silica gel column (100 cm x 5 cm, 450 g) at room temperature. The column was eluted with a gradient of a mixture of light petroleum/dichloromethane/methanol as mobile phase to afford seven compounds.

Isolation of four compounds from the ethyl acetate fraction

A dry mixed initial zone was obtained by dissolving 6 g of the ethyl acetate fraction in methanol (10 mL) mixed with 3 g silica gel. The dry zone was chromatographed on a silica gel column (75 cm x 3.5 cm, 240 g). The column was eluted with a gradient of a mixture of benzene/ethyl acetate as mobile phase to give four compounds.

Preparation and analysis of the essential oil Fresh aerial parts of G. parviﬂ ora were sub- jected to hydrodistillation for 4 h using a Clev-

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enger-type apparatus. The yield was 0.1% (v/w) of a yellow coloured oil. The oil was analysed by gas chromatography/mass spectrometry (GC/MS) using a Clarus 600 gas chromatograph (Shelton, CO, USA) equipped with a fused silica column Rtx^®-5MS (equivalent to DB5; 30 m x 0.23 mm ID, 0.25 μm ﬁ lm thickness). The capillary column was directly coupled to a quadruple mass spectrometer Clarus 600 T. EI-mass spectra were re- corded at 70 eV.

The conditions were as follows: injector tem- pera ture, 150 °C; oven temperature program:

initial temperature, 45 °C, 2 min isothermal, 45 – 300 °C at 4 °C/min, 20 min isothermal; split ratio, 1:20; carrier gas, He (1.2 mL/min). Reten- tion indices (RI) were calculated with respect to a set of co-injected standard n-alkanes.

Biological evaluation Hepatoprotective effect

Twenty four male rats weighing 150 – 180 g were used in this study. Elevated liver enzyme levels were induced by subcutaneous (s.c.) injection of 0.2 mL/100 g body weight (BW) of 40 mL/L CCl4 dissolved in paraffi n oil. The injection was given twice a week for 6 weeks (Zhao et al., 2005). The rats were divided into 4 groups (n = 6). Group I received liquid paraffi n oil only (normal control group), while the other groups (II–IV) received CCl4. Thereafter, group I received the vehicle (10% gum acacia), while group II (negative control) were cirrhotic rats that received the vehicle. Group III (positive control) were cirrhotic rats treated with the standard silymarin suspended in vehicle in a dose of 150 mg/kg BW, and group IV (intoxicated) were cirrhotic rats treated with the total ethanolic extract of G. parvifl ora in a dose of 400 mg/kg BW. The treatment period continued for 7 d. Blood samples were collected from the orbital sinus of fasted (for at least 8 h) rats. Se- rum was separated by centrifugation at 1370 x g for 15 min to determine the biological param- eters. Liver enzymes, alanine aminotransferase (ALT) and proteins (albumin), were measured in the collected plasma.

Hypoglycemic effect

Twenty four adult male rats (150 – 180 g) were used in this study. Diabetes was induced in rats by intraperitoneal (i.p.) injection of streptozo-

cin (STZ) in a single dose of 75 mg/kg BW according to the method described by Sokeng et al.

(2005). Rats became diabetic after 5 d of inject- ing STZ, their blood glucose levels ranging from 250 to 288 mg/100 mL. Rats were divided into four groups (n = 6). The ﬁ rst group was the non- diabetic group and received gum acacia mucilage (10%), it served as a control; the second was the diabetic group and received only the vehicle (10% gum acacia); the third and fourth diabetic groups received orally by gavage glibenclamide (5 mg/kg BW) and the total ethanolic extract of G. parviﬂ ora (400 mg/kg BW), respectively. Blood glucose levels were determined using a glucomen- glyco^® blood glucose meter, 24 sensor strips.

Cytotoxic activity

Cytotoxicity of the ethanolic extract was measured against MCF-7 cells using the SRB assay reported by Skehan et al. (1990). Cells were seeded in a 96-well plate in fresh medium and left for 24 h at 37 °C under 5% CO2 atmosphere, to at- tach to the wall of the plate. Thereafter cells were incubated with appropriate concentrations of the test extract (0.1, 1, 10, 100, 1000 μg/mL). The vol- umes were completed to 200 μL/well using fresh medium, and incubation was continued for 72 h.

Control cells were treated with vehicle alone. The absorbance of each well was measured at 564 nm using an ELISA reader. Cell survival was measured as the percentage absorbance compared to the control (non-treated cells). The experiment was triplicated.

Antioxidant activity

The scavenging activity of the DPPH radicals was investigated according to the method of Peiwu et al. (1999). Stock solutions of the crude ethanol extract and fractions (light petroleum, ethyl acetate) were prepared by dissolving 150 mg each in 1 mL of methanol. Methanolic DPPH solution (2.95 mL, 4.5 mg/100 mL) was added to 50 μL of test material in a disposable cuvette. The absorbance was measured at 517 nm at regular intervals of 15 s for 5 min. Ascorbic acid (at 0.1 M) was used as a standard as described by Govindarajan et al. (2003).

The difference in the absorbance between the test sample and blank was expressed as percent inhibition taken as the activity: inhibition (%) = [(AB – AA)/AB] · 100, where AB is the absorbance

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of the blank sample and AA is the absorbance of the tested extracts.

Antimicrobial activity

The cup-plate method (Woods and Washington, 1995) was used to detect the preliminary antimicrobial activity of the total ethanolic extract and of different fractions (light petroleum and ethyl acetate) of G. parviﬂ ora. The samples were dissolved in dimethyl formamide (DMF) at a concentration of 100 mg/mL. Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Staphylococ- cus epidermidis ATCC 12228, Micrococcus spp.

ATCC 10240, and Bacillus subtilis ATCC 6633), Gram-negative bacteria (Pseudomonas aerugino- sa ATCC 9027 and ATCC 27853, Klebsiella pneu- moniae ATCC 27736, Salmonella typhimurium ATCC 14028, and Escherichia coli ATCC 10536), and fungi (Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231) were the used standard strains obtained from the Department of Microbiology, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt.

The nutrient agar or Sabouraud’s agar (40 g/L dextrose, 10 g/L peptone, 20 g/L agar) were seeded at about 10⁶ microbial cells. Each cup was ﬁ lled with about 100 μL from each extract. Cefotaxin and nystatin (5 mg/mL) were used as standard antibacterial and antifungal, respectively. The plates were incubated overnight at 37 °C for bacteria and 30 °C for fungi. Zones of inhibition were measured (in mm).

Results and Discussion Identiﬁ cation of compounds

Silica gel column chromatography of the light petroleum fraction of G. parviﬂ ora resulted in the isolation of seven compounds including: triacontanol (Galala, 2009), phytol (Kim et al., 2005), β-sitosterol, stigmasterol (Goad and Akihisa, 1997), 7-hydroxy-β-sitosterol, 7-hydroxystigmasterol (McCarthy et al., 2005; Qiang et al., 2011), and β-sitosterol-3-O-β-D-glucoside (Bayoumi et al., 2010). The yields were 20, 1000, 550, 100, 5, 5, and 35 mg, respectively. Furthermore, column chromatography of the ethyl acetate fraction afforded four compounds including 3,4-dimethoxycinnamic acid (Torres et al., 2003; Nawwar et al., 2009), protocatechuic acid (Yu et al., 2006), fumaric acid (He et al., 2011; Zhang et al., 2010), and uracil (Srivastava and Gupta, 2010). The

yields were 5, 30, 15, and 6 mg, respectively. Com- pounds were identiﬁ ed from their spectral data (IR, MS, ¹H and/or ¹³C NMR), physical properties, and comparison with the data reported in the literature.

Except β-sitosterol, stigmasterol, β-sitosterol- 3-O-β-D-glucoside, and protocatechuic acid, all other compounds are reported for the ﬁ rst time from this plant and the genus Galinsoga.

This result differs from those reported else- where in the literature. In contrast to previous reports (Pan et al., 2007; Tariq et al., 2008; Ferheen et al., 2009; Afza et al., 2012), we obtained no evi- dence for α-spinasterol, diterpenes with a kau- rene skeleton, 4-hydroxybenzoic acid, gallic acid, galinsoside A, galinsoside B, 7,3′,4′-trihydroxyﬂ a- vanone, and galinosoates.

Essential oil constituents

Altogether 48 identifi ed compounds covered 83.13% of all peaks observable in the chroma- togram of the hydrodistilled oil of G. parvifl ora (Table I) with (Z)-γ-bisabolene being the most abundant component (45.66%), followed by (E)-caryophyllene (4.99%), (Z)-bisabolol-11-ol (4.95%), and phytol (4.39%). Identifi cation of the compounds was based on their mass spectra, retention indices, and comparison with published data (Adams, 2007; El-Shazly and Hussein, 2004;

El-Shazly et al., 2002, 2004). On the other hand, we could not ﬁ nd any traces of (Z)-3-hexen-1-ol, β-caryophyllene, and 6-demethoxy-ageratochrome which have been reported as major compounds in the essential oil of leaves from G. parviﬂ ora growing in Colombia (Pino et al., 2010).

This divergence in the chemical proﬁ le of G. parviﬂ ora may be attributed to either geo- graphical and ecological factors, or be an indica- tion of the existence of different chemotypes of this plant.

Biological evaluation Hepatoprotective effect

As shown in Table II, the total ethanolic extract of G. parviﬂ ora (400 mg/kg BW) and the standard silymarin (150 mg/kg BW) signiﬁ cantly reduced the level of ALT activity compared to the CCl4-treated group. The effect of the total ethanolic extract (400 mg/kg BW) was equivalent to 87% and 83% of ALT and total albumin,

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respectively, provoked by the standard silymarin (150 mg/kg BW).

Hypoglycemic effect

As shown in Table III, the potency of the total ethanolic extract of G. parviﬂ ora at a content of 400 mg/kg BW was nearly equivalent to that of glibenclamide (5 mg/kg BW) in reducing the blood glucose levels of diabetic rats.

Cytotoxic activity

The total ethanolic extract of G. parviﬂ ora at concentrations of 0.1, 1, 10, and 100 μg/mL ex- erted only a weak cytotoxic activity against the MCF-7 cell line. The percentage cell viabilities were 99.23, 98.21, 92.58, and 91.53, respectively. However, the extract in high concentration (1000 μg/mL) killed all cells. So, the extract was considered to be cytocidal rather than cytotoxic (Houghton et al., 2007).

Antioxidant activity

In comparison with the standard 0.1 M ascorbic acid, the ethyl acetate fraction of G. parviﬂ ora exhibited strong antioxidant activity at a concentration of 150 mg/mL, the ethanolic extract showed moderate antioxidant activity at a concentration of 150 mg/mL, while the light petro leum fraction showed weak antioxidant activity at the same concentration. Our results are in agreement with those reported for G. parviﬂ ora from Chile ( Ranilla et al., 2010) and Poland (Bazylko et al., 2012).

Antimicrobial activity

All extracts had a weak antibacterial effect against all tested Gram-positive bacteria, except B. subtilis (Table IV). For the tested Gram-negative bacteria, all extracts exhibited a weak antibacterial effect against K. pneumoniae and S. typhimurium, and a signiﬁ cant effect against E. coli and P. aer- uginosa, relative to the standard cefotaxin. The ex- tracts were strongly antifungal against A. niger and C. albicans, relative to the standard nystatin.

The presence of 3,4-dimethoxycinnamic acid, protocatechuic acid, and fumaric acid, as well as the high content of phytol and sterols as constituents of G. parviﬂ ora may contribute to the ap- preciable hepatoprotective, hypoglycemic, antioxidant, and antimicrobial effects (Fred-Jaiyesimi et al., 2009; He et al., 2011; Nagavamsikrishna et al., 2012; Ranilla et al., 2010; Radhiga and Pugalendi, 2011; Harini and Pugalendi, 2010).

Table I. Essential oil constituents of G. parviﬂ ora aerial parts.

No. Compound^a RI^b Content (%)

1 6-Methyl-5-hepten-2-one 991 0.46

2 Mesitylene 993 0.30

3 n-Decane^c 998 0.36

4 3(E)-Hexenyl acetate^c

5 3(Z)-Hexenyl acetate 1009 0.22

6 Methyl heptanoate 1026 0.13

7 Benzene acetaldehyde 1043 0.10

8 p-Tolualdehyde 1083 0.16

9 trans-Sabinene hydrate 1098 0.23

10 n-Nonanal 1101 0.11

11 1,3,8-p-Menthatriene 1114 0.10

12 Terpinen-4-ol 1178 0.05

13 Naphthalene 1179 0.06

14 Dihydro-carveol 1193 0.15

15 exo-Fenchyl acetate 1232 0.12

16 Ethyl-2-octynoate 1285 0.09

17 α-Longipinene 1359 0.16

18 n-Undecanol 1371 0.16

19 β-Maaliene 1380 0.37

20 α-Isocomene 1387 0.31

21 Phenyl ethyl isobutanoate 1394 0.20

22 β-Isocomene 1405 0.25

23 (E)-Caryophyllene 1421 4.99

24 α-neo-Clovene 1453 0.32

25 α-Humulene 1457 0.51

26 Sesquisabinene 1462 0.23

27 (Z)-γ-Bisabolene 1513 45.66

28 (E)-γ-Bisabolene 1527 2.37

29 α-Cadinene 1537 0.24

30 cis-Sesquisabinene hydrate 1545 0.63

31 Germacrene B 1553 0.25

32 (Z)-Dihydro-apofarnesol 1567 2.42 33 Caryophyllene oxide 1591 2.65 34 (Z)-Bisabolol-11-ol 1614 4.95 35 10-epi-γ-Eudesmol 1617 1.41

36 5-Cedranone 1632 0.75

37 Valerianol 1650 0.33

38 Cedr-8(15)-en-10-ol 1658 0.75

39 epi-β-Bisabolol 1672 0.98

40 8-Hydroxy-isobornyl

isobutanoate 1678 0.58

41 α-Bisabolol 1685 0.53

42 n-Hexadecanol 1869 0.54

43 Cyclohexadecanolide 1922 0.80

44 Phytol 2137 4.39

45 n-Tricosane 2318 0.31

46 n-Pentacosane 2502 0.60

47 n-Heptacosane 2720 0.95

48 n-Nonacosane 2911 0.90

Total 83.13

a In elution order from an Rtx^®-5MS (equivalent to DB5) column.

b Retention index.

c Compounds co-eluted; this value refers to the sum of both compounds.

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In addition to our phytochemical and biological studies of G. parviﬂ ora, close monitoring of hepatic and renal safety variables as well as rigorous long-term clinical follow-up studies are required before extracts from this plant can be used as a natural therapy.

Acknowledgement

The authors thank Mr. Nader Shawky (Depart- ment of Microbiology, Faculty of Pharmacy, Zaga- zig University, Zagazig, Egypt) for his assistance in the determination of the antimicrobial activities.

Table III. Effect of the total ethanolic extract of G. parviﬂ ora and glibenclamide on blood glucose levels of STZ- induced diabetic rats.

Group Blood glucose level

(mean ± SEM) [mg/100 mL]

Effect

(%) Change

(%) Relative potency

Control group 92.17 ± 3.5 – – –

Diabetic group 270.9 ± 6.7 100.00 0.00 –

Diabetic + glibenclamide (5 mg/kg BW) group 213.2 ± 4.6 78.7 –21.3 1 Diabetic + plant extract (400 mg/kg BW) group 215.7 ± 12.8 79.62 –20.38 0.96 Test of signiﬁ cance using Student’s t-test. P < 0.05.

Table II. Effect of the total ethanolic extract of G. parviﬂ ora on the levels of serum albumin and alanine aminotransferase (ALT).

Group Serum albumin level [mg/100 mL] Serum ALT level [U/L]

Relative

potency Change (%) Effect

(%)

Mean ± SEM Relative

potency Change (%) Effect

(%)

Mean ± SEM

Normal control rats – – – 4.6 ± 0.3 – – – 49 ± 3.9

Negative control (cirrhotic) rats (CCl4)

– 0 100 3.6 ± 0.2 – 0 100 84.3 ± 4.3

Positive control group [CCl4 +

silymarin (150 mg/kg BW)] 1 –16.67 83.33 4.2 ± 0.24 1 –25.27 74.73 63 ± 1.5 Intoxicated treated group

[CCl4 + plant extract (400 mg/kg BW)]

0.83 –13.89 86.11 4.1 ± 0.1 0.87 –21.95 78.05 65.8 ± 2.6

Test of signiﬁ cance using Student’s t-test. P < 0.05.

Table IV. Antimicrobial activity of different extracts of G. parviﬂ ora. Inhibition zone diameter in mm.

Tested material Fungi Gram-negative bacteria Gram-positive bacteria

C. albicans ATCC 10231 A. niger ATCC 16404 E. coli ATCC 10536 S. typhimurium ATCC 14028 K. pneumoniae ATCC 27736 P. aeruginosa ATCC 27853 P. aeruginosa ATCC 9027 B. subtilis ATCC 6633 Micrococcus spp. ATCC 10240 S. epidermidis ATCC 12228 S. aureus ATCC 6538

Total ethanolic extract 18 25 20 20 21 25 25 20 11 17 20

Light petroleum fraction 17 20 20 22 20 25 22 20 13 14 20

Ethyl acetate fraction 17 25 18 19 20 20 26 15 15 17 22

Cefotaxin – – 25 40 37 33 30 22 43 58 34

Nystatin 20 25 – – – – – – – – –

The total plant extract and solvent fractions were applied at a concentration of 100 mg/mg DMF, while the used standards (cefotaxin and nystatin) were applied at a concentration of 0.5 mg/mL.

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