Introduction 2. Materials and Methods
1. Introduction 2. Materials and Methods
Gomphocarpus sinaicus (Boiss.) is atoxic plant,
2.1 Plant Material
due to its high content of cardiac glycosides. G. Aerial parts of Gomphocarpus sinaicus (Boiss.) sinaicus (Boiss.) plant which grows wild in the sandy
were collected form Sinai in may, 2005 at the flowering mountains regions in South Sinai Governorate, Egypt
and fruiting stage. The plants were identified according under the common name “Hargel”. It is rich in a
to the taxonomic characters of Tackhlom [3]. The number of cardenolides, in particular cardenolide
samples were air-dried at room temperature and then glucosides with unusual doubly linked sugars finely powdered.
El-Askary et al. [1, 2]. Therefore, the present work was designed to isolate and identify the major active 2.2 Instruments
cardenolides from the methanol extract of G. sinaicus IR spectra were performed with Perkin Elmer 1430 and to evaluate their fungicidal activity against some
Ratio Recording Infrared Spectrometer in KBr disks. of the soil borne and post harvest fungi. Also,
1 H and 13 C NMR spectra were recorded at 500MHz and investigation of the effects of these compounds on
125 MHz respectively, on a JEOL JNM ECD 500 certain fungi enzymes in an attempt to throw some
Spectrophotometer in CDCl 3 .
light on their mode of action.
2.3 Test Organisms
Corresponding author: Moustafa A. Abbassy, professor, research fields: chemistry of botanical pesticides, toxicology of
Four fungal pathogens were used in this study. Two pesticides. E-mail: [email protected].
Isolation and Identification of Cardenolide Compounds of Gomphocarpus sinaicus and Their Fungicidal Activity Against Soil Borne and Post Harvest Fungi
soil born fungi, Rhizoctonia solani (Kuhn.) and (15 × 15 mL), MeOH-CHCl 3 70% (15 × 15 mL), Fusarium oxysporium (Schiech.), and two post harvest
MeOH-CHCl 3 90% (15 × 15 mL) controlled by TLC fungi (Rhizopus stolonifer (Ehren) and Penicillium
was performed on silica gel plates (PolyGram Sil digtatum (Pres.) sacc, were used. The fungi were
G/UV 254, Germany) using solvent system maintained during the course of the experiments on
CHCl 3 -MeOH 10%, CHCl 3 -MeOH 15%, Czapek-Dox Agar (CDA) at 25 ºC.
CHCl 3 -MeOH 20%. Detection was done by kedde’s reagent. Evaporation of the solvents of eluted fractions
2.4 Isolation and Identification of Cardenolide was carried under reduced pressure to yield liquid card. Compounds from G. sinaicus Extracts
genin (6 mL).
Batches of air dried aerial parts of G. sinaicus each
2.5 Antifungal Assay
of (1 kg) were percolated with MeOH and the extracts were concentrated, decanted from an insoluble wax,
The antifungal activity of the isolated cardenolide
compounds, as well as the standard fungicides, was was triturated with petroleum ether and the residue was
diluted with H 2 O and extracted with CHCl 3 . The wax
tested using the radial growth inhibition technique [4, added to the CHCl 3 extract, which was then filtered
5]. Appropriate volumes of the stock solutions of the under reduced pressure through powdered charcoal.
tested compounds in Triton X-100 were added to
molten nutrient agar medium (Czapek-Dox Agar; CDA) combined filtrates were concentrated under reduced
The charcoal was washed with CHCl 3 and the
to obtain a range of concentrations (10, 50, 100, 500, pressure to give a dark brown gum. The gum in CHCl 3 and 1,000 μg/mL before pouring into the Petri dishes
was fractionated by chromatography column (CC) of (9.0 cm in diameter) at 40-45 °C. Each concentration
silica gel and eluted with MeOH-CHCl 3 10% to give
was tested in triplicate. Parallel controls were
10 eluates (each of 100 mL), controlled by TLC, and maintained with Triton X-100 mixed with CDA grouped as follows: the solvent of the first six eluates
medium. The discs of mycelial felt (0.5 cm diameter) was evaporated under reduced pressure to yield
of the plant pathogenic fungi, taken from 8-day-old fraction I and the second four eluates yield fraction II.
cultures on CDA plates, were transferred aseptically to Fraction I (3 × 3 g) was again subjected to CC on
the centre of Petri dishes. The treatments were silica gel (30 g, 15 mL fractions) (15 × 15 mL) and
incubated at 25 °C in the dark. Colony growth diameter
was measured after the fungal growth in the control fractions were controlled by TLC. Detection of
eluted with CH 3 C(O)CH 3 -CHCl 3 (1:4). The eluated
treatments had completely covered the Petri dishes. cardenolides by TLC was performed by using kedde’s
Percentage of mycelial growth inhibition was reagent (1:1 mixture of 5% 3,5-dinitrobenzoic acid in
calculated from the formula: Mycelial growth MeOH and 2 N KOH) and the solvent systems
inhibition = [(DC −DT)/DC] × 100 [6], where DC and CHCl 3 -MeOH 10%, CHCl 3 -MeOH 15%, DT are average diameters of the fungal colonies for CHCl 3 -MeOH 20%. Evaporation of the solvents of
control and treatments, respectively. The concentration eluted fractions was carried under reduced pressure to
of the compound inhibiting fungal mycelial growth by yield card. glycoside. Recrystallization of this 50% (EC 50 ) was calculated using probit analysis. compound from acetone yielded pure prisms crystals
2.6 Biochemical Measurements
6.2 g.
2.6.1 Pectin Methyl Esterase (PME) Activity CC on silica gel (30 g, 15 mL Fractions.) (15 × 15 mL),
Fraction II was purified with MeOH-CHCl 3 20% by
Pectin methyl esterase enzyme was secreted from
MeOH-CHCl 3 30% (15 × 15 mL), MeOH-CHCl 3 50%
the tested fungi that were grown on Czapeck Dox
Isolation and Identification of Cardenolide Compounds of Gomphocarpus sinaicus and
Their Fungicidal Activity Against Soil Borne and Post Harvest Fungi
medium supplemented with 1% pectin (apple pectin: the 10, 50, 100, 500 and 1,000 ppm of each tested apipectin 150 SAG), as recommended by El-Gorrani et
compound and tested fungicides (flutolanil or copper al. [7].
oxychloride) was prepared in conical flasks. The The activity of PME was measured according to the
inoculums discs of the tested fungi were located on the method described by Smith [8], Talboys and Busch [9]
surface of the medium in the flasks. The flasks then with some modifications.
incubated until—the hyphal growth in untreated flasks The reaction mixture was prepared from the
(control) was completed, then the contents of flasks following components: pectin (0.5 g), sodium chloride
were filtrated under vacuum. The filtrate was used to (0.58 g) bromothymol blue solution (0.05%) 2.5 mL,
measure the enzyme activity. The filtrates were and distilled water up to 1,000 mL chloroform (4 mL)
centrifuged for 15 minutes at 4,000 rpm. Aliquots of was added to reaction mixture to avoid bacterial
supernatants were assayed for polyphenol oxidase contamination. The reaction mixture was immediately
activity using spectrophotometer at 575 nm. The adjusted at pH 7.0 with 0.05 N sodium hydroxide
reaction mixture consisted of 2.0 mL of borate buffer solutions.
(pH 9.0), 1.0 mL of 1% P-aminobenzoic (acid Flasks (100 mL) containing a total definite volume
alcoholic solution), 2.0 mL of 1% catechol, 1.0 mL of (10 mL) consisted of 7.0 mL reaction mixture (pH 7) +
filtrate. The same reaction mixture was prepared
2.0 mL of crude enzyme + 1.0 mL of a 10, 50, 100, 500 without enzyme source and considered as a blank. or 1,000 ppm concentration of each tested compound
Enzyme activity was measured as optical density and tested fungicide (flutolanil or copper oxychloride).
(absorbance) after one hour incubation in a shaking Each mixture was incubated at 30 °C for 24 h, and then
water bath at 45 °C.
100 A
B A
(2) total sodium hydroxide solution consumed by the blank
titrated to pH 7.0 with 0.01 N sodium hydroxide. The
gave the actual consumption of sodium hydroxide where I % is inhibition percent; A is absorbance of the solution due to pectin methyl esterase activity on the
control sample at 575 nm; B is absorbance of the substrate. Similar mixtures for each treatment except
treatment sample at 575 nm.
the crude enzyme (which was substituted by
2.6.3 Cellulase Activity
inactivated enzyme kept in a boiling water bath for 10 Cellulase enzyme was secreted from the tested fungi minutes) was prepared concurrently and considered as
that were grown on Czapeck Dox medium amended
a blank treatment. The flasks containing the blank were with 3% w/v concentration of carboxy methyl cellulose titrated with 0.01 N sodium hydroxide solutions to the
as the sole carbon source and autoclaved (1.5 lb/in2) pH 7.0. Each treatment was replicated three times. The
for 15 min only as recommended by Kavitha et al. [11]. inhibition percentage of the activity was calculated by
Then cultures were inoculated with the fungus, the following equation:
incubated at 28 °C for 12 days and then filtrated
I % A B
through Whatman No. 1. The filtrate was considered as
a source of crude enzyme which was immediately used where I% is inhibition percent; A is mL of NaOH (0.01
for assaying the enzyme activity.
N) in the control treatment; B is mL of NaOH (0.01 N) The activity of cellulase was monitored by in the treatment.
estimation of reducing sugar using the dinitrosalicylic
2.6.2 Polyphenol Oxidase (PPO) Activity
method [12].
Determination of polyphenol oxidase activity was The reaction mixture was prepared from the carried out according to the method that described by
following components (containing 10 g of Broesch [10]. The czapeck-Dox medium containing
3,5-dinitrosalicylic acid, 10 g NaOH, 20 mL of phenol,
Isolation and Identification of Cardenolide Compounds of Gomphocarpus sinaicus and Their Fungicidal Activity Against Soil Borne and Post Harvest Fungi
0.5 g of sodium sulfate with a final volume of 1,000 positive results for both chromogenic spray reagents. mL).
The identifies of the isolated compounds were In a test tube, 2 mL of a solution (containing 1 g of
established by comparing their infrared, IR spectra, CMC, 100 mL of buffer, 0.2 M HAC-NaAC) with pH
mass spectrum, MS, and nuclear magnetic
4.8 was added to 2 mL of CMC-NaAC buffer. The resonance— 1 H-NMR and 13 C-NMR with the literature mixture was kept in a water bath at 50 ºC for 5 min,
data by El-Askary et al. [13], Abdel-Azim et al. [14], then 1 mL of crude enzyme was added and the mixture
Abdel-Azim [15] as well as the corresponding data for was warmed in a water bath at 50 ºC for 30 min. Then 1
various molecules given by Weast [16], Robien et al. mL of 10, 50, 100, 500 or 1,000 ppm of each tested
compound and tested fungicides (flutolanil or copper Results in Table 1 show that card. glycoside had the oxychloride) was added and incubated at 28 ºC for 24 h.
molecular formula C 29 H 45 O 10 deduced from its mass Finally 3 mL of the previously prepared reaction
spectrum and the presence of 29 carbons observed in mixture solution was added. The absorbance of
the 13 C NMR spectrum. The MS spectrum revealed a
2 O }, spectrophotometer after 15 min incubation in a shaking
CMCase was measured at 575 nm with a + molecular ion peak at M/Z 553 {M} , 517 {M-2H
3 H 7 OH} + , 381 {M-H 2 O-CH 3 } , 272 water bath at 50 ºC. Blanks were prepared similarly (3
414 {M-C
2 O-CO} , 213 {M-OCH 3 } + and a base peak at replicates per treatment) but without the addition of the + M/Z 159 {M-CH
{M-2H
2 =CH 2 } .
enzyme sample during incubation. Table 2 shows the IR spectrum revealed several
I % A B 100
absorption bands at 4205-4335, 3061-3740, 1219-1536, (3)
907-1190, 1653-1682 and 1682-1939 cm -1 which where I% is inhibition percent; A is absorbance of the
indicated the presence of CH, OH(stretching), control sample at 575 nm; B is absorbance of the
OH(bending), CO, C=C and C=O. treatment sample at 575 nm.
Table 3 shows the 1 H NMR spectrum, the presence
of a signal at δ 2.49 indicated the presence of 3’-hydroxyl group. A signal at δ 0.818 indicated the