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Chemical constituents and biological activities

of Cichorium intybus L.

Article in Natural Product Sciences · April 2004

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Natural Product Sciences
10(2): 69-73 (2004)

Chemical Constituents and Biological Activities of Cichorium intybus L.

Abdalla M. EI-Lakany1•*, Maha A. Aboul-Ela1, Mohamed M. Abdul-Ghani2, and Hattem Mekky1
1
Pharmacognosy Department, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
2
Department of Organic Chemistry, Faculty of Science, Beirut Arab University, Beirut, Lebanon

Abstract - Continuation of a phytochemical study of Cichorium intybus L. (Astraceae) growing in Egypt, resulted in
the isolation and identification of a new sesquiterpene lactone 3, 4-dihydrolactucin, in addition to the eight known
compounds; kaempferol, isoscutellarin, cichoriin, umbelliferone, lupeol, lupeol acetate, P-sitosterol, and P-sitosterol-
3-0-glucoside. Chemical structures of the isolated compounds were assigned based on different physical, chemical,
and spectroscopic techniques including IR, UV, MS, lD- and 2D-NMR spectra. Furthermore, the antimicrobial,
and spasmogenic activities of some fractions and isolates were also assessed.
Key words - Cichorium intybus L.; Lactuaceae; Asteraceae; chemical constituents; new sesquiterpene lactone;
antimicrobial, spasmogenic activities.

Introduction Experimental

Lactuaceae (Asteraceae) is a well-known tribe belonging Cichorium intybus - L. used in this study was collected
to subfamily liguliflorae. Plants of the tribe were reported to in April, 2001 from the flowering plants growing wildly in
possess diverse pharmacological actions, such as antidiabetic, Abyss province, South of Alexandria. The identity of the
anticarcinogenic, and hepatoprotective activities (Steven and plant was confirmed at the Faculty of Science, University
Tyler, 1992; Bertog et al., 1992). Flavonoids, coumarins, of Alexandria, Alexandria, Egypt.
and sesquiterpene lactones are among the most common General - Melting points were determined on a Griffin
secondary metabolites of this tribe. Cichorium intybus L. melting point apparatus and were uncorrected. UV spectra
(Lactuaceae) is a perennial herb with historical use as were recorded on a Perkin Elmer double beam spectro-
laxative, diuretic, and in the treatment of gallstones, hepatic photometer Model 550S, attached to a Hitachi recorder
disorders, and indigestion (Tyler et al., 1988; Crellin and Model 561. IR spectra were determined on a Jasco infrared
Philpatt, 1990). Previous work on this plant has lead to the spectrophotometer, Model (FT/IR-300E), in KBr pellets.
isolation of isoquercetrin (Amer, 1974), 10-hydroxyguaia- NMR analyses were recorded on a Bruker Avance 300 MHz
4,11(13)-diene-6,12-olide, its 11,13-dihydro derivative (El- instrument. MS spectra were recorded on a GC coupled with
Masry et al., 1984 ), desacetylmatricarin, aloe-emodin, a Shimadzu 8080A Mass Spectrometer. Solvents used were
esculetin, 3,4-dihydroxymethylbenzoate, caffeic acid, and of analytical grade. Silica gel for CC and silica gel GF254 for
quercetin (Aboul-Ela, et al., 2002). TLC (Merck) were used. Shift reagents for UV spectral
In this communication, we report on the isolation and analysis are: 5% CH30Na, 5% AIC13, cone. HCI, anhydrous
identification of the new sesquiterpene lactone, 3,4- NaOAc and CH30H. Pharmacological effects were recorded
dihydrolactucin, and the known compounds, kaempferol, on a chymograph.
isoscutellarin, cichoriin, umbelliferone, lupeol, lupeol acetate, Extraction and isolation - A. Air-dried and powdered
~-sitosterol, and ~-sitosterol-3-0-glucoside. Antibacterial roots of C. intybus (2.5 kg) were macerated in 90% EtOH.
and antifungal screening for some pure isolates was conducted. The concentrated hydroalcoholic extract was successively
Furthermore, the spasmogenic activity of some fractions and extracted with petroleum ether, CHC13, EtOAc and n-BuOH.
isolates were performed using rabbit intestine preparation. The EtOAc fraction (6 g) was subjected to CC using CHCh
and MeOH mixtures . Crystallization of fractions obtained
using 8-10% MeOH in CHC13 afforded 10 mg of compound
*Author for correspondence 1. PTLC (Solvent, EtOAc: CHCb: MeOH, 5:2:1) of fractions
Fax: 009611818402, E-mail: [email protected] eluted using 14-16% MeOH in CHCb afforded 100 mg of

69
70 Natural Product Sciences

compound 2. Hz, H-8). 13C NMR (8, CD 30D): 144.3 (C-2), 136.8 (C-3),
B. Air-dtied and powdered aerial parts of C. intybus (21 170.9 (C-4), 164.4 (C-5), 99.5 (C-6), 168.0 (C-7), 92.3 (C-8),
Kg) were macerated in 90% alcohol. The concentrated 149.5 (C-9), 107.8 (C-10), 125.3 (C-1'), 130.9 (C-2'), 116.1
hydroalcoholic extract was successively extracted with (C-3'), 158.6 (C-4'), 116.1 (C-5'), 130.9 (C-6').
petroleum ether, CHC13 , EtOAc, and n-BuOH. The CHCl 3 Compound (4): Dark yellow crystals, m.p. 300-301 °C.
fraction (250 g) was subjected to CC, using petroleum ether. UV An,ax (MeOH): 282, 332 nm. Degradation occurs with
The polarity was increased by gradual addition of CHCh, all shift reagents. EIMS m/z (rel. abund. % ): 286 ( 100) [M+,
then by CH30H. PTLC (Solvent, CHCl 3 : MeOH, 9: 1) of C 15 H 100 6], 258 (47), 257 (9), 168 (80), 140 (52), 118 (38),
fractions obtained using 11-19% MeOH in CHC13 afforded 112 (5.4) . 1H NMR (8, CD 30D): 7.06 (2H, d, J = 8.5 Hz,
23 mg of compound 3 and 10 mg of compound 4. In addition, H-2', H-6'), 6.77 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.08 (IH, s,
lupeol, lupeol acetate, ~-sitosterol and ~-sitosterol-3-0- H-3), 5.59 (IH, s, H-6).
glucoside were also isolated and identified. The EtOAc Compound (5): Transparent prisms, m.p. 2 15°C. It showed
fraction (15 g) was subjected to CC using CHCb, EtOAc an intense blue fluorescence under UV lamp and gave a
and MeOH mixtures. Crystallization of fractions obtained positive Molisch's test. UV A,nax (MeOH): 234, 289, 347 nm,
using 60-90% EtOAc in CHC1 3 from MeOH afforded 0.8 (MeOH + NaOMe): 249, 306, 390 nm, (MeOH +AlCh):
g of compound 5. 234, 289, 347 nm, (MeOH + AlCh + HCl): 234, 289, 347 nm,
Compound (1): White amorphous powder. UV Amax (MeOH + NaOAc): 252, 282, 352 nm. EIMS. mlz (rel.
(MeOH): 246 nm. IR Vrnax (KBr): 1782, 1678, 1635, 1620 abund. %): 340 (2) [M+, C 15 H 160 9], 320 (2), 293 (72), 179
and 1215 cm- '. EIMS m/z (rel. abund. % ): 278 (2) [M+, (90), 178 (97), 167 (90), 149 (100), 127 (68), 97 (56). 1H
C 15H 18 0 5], 262 (2.9), 247 (3.1), 233 (9.15), 219 (3.6), 203 NMR (8, CDC13 + CD 30D): 7.72 (lH, d, J = 9.5 Hz, H-4),
(3.4), 177 (9.5), 167 (8), 149 (32), 133 (19), 111 (17), 57 7.11 (IH, s, H-8), 6.94 (lH, s, H-5), 6.19 (IH, d, J = 9.4 Hz,
(100). 1H NMR (8, CD30D): 2.46 (3H, s, H-14), 2.49 (IH, m, H-3), 4.88 (lH, d, J = 7.3Hz, H-1'), 3.31-3.85 (m, 6H, H-2'-
H-9~), 2.54 (2H, d, J = 7.4 Hz, CHr3), 2.52 (IH, m, H-4), H-6'). 13 C NMR (8, CDC13 + CD 300): 163.0 (C-2), 114.1
2.74 (H, t, J = 11.0 Hz, H-9a), 3.02 (lH, t.t, J = 10.1, 3.0 Hz, (C-3), 144.9 (C-4), 141.3 (C-4a), 113.3 (C-5), 148.9 (C-6),
H-7), 3.11 (lH, t, J =2.9 Hz, H-5), 3.63 (]H, dd, J = 10.9, 149.8 (C-7), 104.7 (C-8), 149.99 (C-8a), 102.4 (C-1'), 76.0
5.6 Hz, H-15a), 3.75 (IH, dd, J = 10.l, 4 Hz, H-6), 3.77 (lH, (C-2'), 77.8 (C-3'), 74.0 (C-4'), 78.0 (C-5'), 61.7 (C-6').
dd, J = 10.9, 4.0 Hz, H-15b), 3.79 (lH, dt, J = 4.2, 6.6 Hz, Antimicrobial activity - Antibacte1ial and antifungal
H-8), 6.16 (lH, dd, J = 3.1 , 1.2 Hz, H- 13a), 6.23 (lH, dd, assays were caffied out using the agar diffusion technique
J = 3.1, 1.2 Hz, H-13b). 13C NMR (8, CD30D): 153.3 (C-1), (Jian and Kar, 1971) against one Gram-positive bacterium;
208.5 (C-2), 43.7 (C-3), 38.4 (C-4), 48.0 (C-5), 83.6 (C-6), Staphylococcus aureus, two Gram-negative bacteria; Escherichia_
59.2 (C-7), 68.9 (C-8), 51.0 (C-9), 135.6 (C-10), 140.2 (C- coli and Pseudonwnas aeroginosea, and the Fungus; Candida
l I), 176.4 (C-12), 123.4 (C-13), 23.6 (C-14), 66.1 (C-15). albicans. These organisms were local isolates provided from
Compound (2): Colorless needles, m.p. 228°C. It showed Department of Microbiology, Faculty of Pharmacy, University
intense blue fluorescence under UV lamp and gave a negative of Alexandria.
Molisch's test. EIMS m/z (rel. abund. %): 162 (8) [M+, Procedure. 1 ml of 24 hours broth culture of each of
C9H60 3] , 149 (25), 138 (13), 121 (7), 110 (100), 94 (90), the tested organisms was separately inoculated into 100
81 (41), 66 (75), 55 (89). 1H NMR (8, CDCh + CD 30D): ml of sterile molten nutrient agar maintained at 45°C. The
7.59 (lH, d, J = 15.8 Hz, H-4), 7.07 (IH, br.s, H-8), 6.97 (IH, inoculated medium is well mixed and poured into sterile
br.d, J = 8.1 Hz, H-6), 6.80 (lH, d, J = 8.1 Hz, H-5), 6.30 10 cm diameter Petri dishes, receiving 15 ml. After setting,
(lH, d, J = 15.8 Hz, H-3). 13C NMR (8, CDC13 + CD30D): ten cups, each 8 mrn in diameter, were cut in the agar medium
167.3 (C-2), 114.5 (C-3), 144.8 (C-4), 147.7 (C-4a), 114.7 (Oxoid). Five milligrams of each fraction or isolate, accurately
(C-5), 121.2 (C-6), 144.2 (C-7), 113.4 (C-8), 145.3 (C-8a). weighed, were dissolved in 1 ml DMF. The solutions were
Compound (3): Yellow crystals, m.p. 304-306°C. UV Amax inserted in the cups and incubated at 37°C for 24 hours. The
(MeOH): 370, 266 nm, (MeOH + NaOMe): 408, 277 nm, sensitivities were observed by measuring the Inhibited
(MeOH + AlCl3): 400, 278 nm, (MeOH + AlCh + HCl): Zones (IZ) in mrn of some isolates with those of the control
400, 278 nm, (MeOH + NaOAc): 397, 277 nm. EIMS m/z (Table I). Minimum Inhibitory Concentration (MIC) and
(rel. abund. %): 286 (3) [M+, C 15HI006 ] , 256 (7), 128 (100), Minimum Bactericidal Concentration (MBC) of some
118 (33), 113 (67), 97 (95). 1H NMR (8, CD 30D): 7.09 (2H, isolates were also conducted (Table 2).
d, J = 11.4 Hz, H-2', H-6'), 6.74 (2H, d, J = 11.4 Hz, H-3', Spasmogenic activities: A preliminary pharmacological
H-4'), 5.95 (lH, d, J = 3.0 Hz, H-6), 5.14 (lH, d, J = 3.0 study was carried out on some isolates and fractions of C.
Vol. 10, No. 2, 2004 71

Table 1. Antibacterial and antifungal activity of some isolates of the molecular formula to be C, 5H, 80 5 . MS showed the
C. intybus L.
molecular ion peak at m/z 278, while 13C-NMR indicated
Inhibition Zones (IZ) in mm the presence of 15 carbons. DEPT experiment indicated
Bacteria Fungi these carbons to be: 1CH3 , 4CH 2 , SCH, and 5 quaternary
Isolate G-Positive G-Negative carbons. IR spectrum showed the presence of absorption
P_ C. bands characteristic for a, ~-unsaturated carbonyl (1678
S. aureus E. coli aeroguws albicans cm- 1) and a carbonyl for y-lactone ring (1782 cm- 1). The
ea
guaianolide nucleus was inferred from the 1H-NMR spectrum
Desacetyl matricarin* 14 18 12 17
Caffeic acid* 15 19 12 17 where the chemical shifts and coupling constants of the
Quercetin* 19 18 16 17 observed signals supported this suggestion (El-Masry et al.,
Aloe-emodin* 17 18 17 20 1984). 1H-NMR spectrum showed two doublets of doublets
Esculetin 14 18 16 17
Isoquercetrin * 15 18 14 20 (J = 3.1 , l.2Hz) at 8 6.16 and 8 6.23, both corresponding
Cichoriin 14 20 14 20 to a single vinylic carbon appearing at 8 140.2; as indicated
Control (DMF) 14 18 12 17 by DEPT-135° and HMQC experiments; characteristic for
Weak activity : Control<Observed value by 2 units. the exocyclic a -methylene y-lactone moiety. Furthermore,
Moderate activity: Control+2<0bserved value>Control+5.
Strong activity: Observed value>Control+5.
the spectrum revealed the presence of a vinylic methyl
*Previously isolated (Aboul-Ela et al. , 2002). signal at 8 2.46 due to Cl:h-14 and a hydroxymethyl group
CH20H-15, appearing as two doublets of doublets at 8
3.63 (] = 10.9, 5.6 Hz) and 8 3.77 (J = 10.9, 4.0 Hz).
intybus L. using rabbit intestine (Berry, 1970). Furthermore, H-6 appeared as a doublet of doublet at 8
Materials and reagents: Rabbit intestine, Tyroid solution, 3.75 (J = 10.1, 4.0 Hz). The large coup]jng constant(] = 10.1
50 ml organ bath, chymograph, DMSO, BuOH, quercetin, Hz) between H-6 and H-7 supported the presence of 6,7-
esculetin and cichoriin. The response of the rabbit intestine trans-lactone ring (Williams and Fleming, 1980; Kisiel et
to different isolates and fractions from the aerial parts is al. , 1997). 13 C-NMR spectrnm indicated the occurrence of
shown in Table 3. two carbonyl carbons at 8 208.5 and 8 176.4 assigned to
C-2 and C-12 of the lactone moiety, respectively. Three
Results and discussion oxygenated carbon signals were resonating at 8 83.6, 68.9
and 66.1, were due to C-6, C-8 and C-15, respectively.
13
C-NMR and MS analyses of compound 1 established Comparison of the observed data of compound 1 with

Table 2. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of some isolates from C. intybus L.
Isolate MIC (µg/ml) MBC (µg/ml)
Number. S. aureus E. coli P aeroginosea C. albicans S. aureus E.coli P aeroginosea C. albicans
Quercetin* 62.5 62.5 12.5 62.5
Aloe-emodin* 31.5 500 >500 31.5
Isoquercetrin* 125 500 125 >500
Cichoriin 62.5 62.5 62.5 >500
No activity:> 125 Moderate activity: 62.5.
Weak activity: 125 Strong Activity: 31.5.
*Previously isolated (Aboul-Ela et al., 2002).

Table 3. Response of the rabbit intestine

Isolates and Fractions Dose I ml Response
DMSO 0.5 ml and 1 ml No response
Quercetin * 10 mg I 1 ml 40% enhancement followed by 60% relaxation
Esculetin 10 mg I 0.5 ml 33% enhancement
Cichoriin 10 mg I 0.5 ml 83% enhancement
CHCh Fraction 10 mg I 0.5 ml 40% relaxation followed by 110% enhancement
5 mg I 0.25 ml 33% relaxation followed by 40% enhancement
Bu OH l ml Relaxation 100 %
BuOH Fraction 10 mg I 1 ml BuOH Relaxation 100%
*Previously isolated (Aboul-Ela et al., 2002).
72 Natural Product Sciences

those reported for lactucin (Khalil et al., 1991), indicated lactucin, suggested that compound 1 is a dihydroderivative
their basic structural similaiity. The appearance of the molecular of lactucin. However, the absence of the characteristic H-3
ion peak at m/z 278, i.e. 2 mass units higher than that of signal, which was recorded at 8 6.15 in 1H-NMR spectrum
of lactucin, and the appearance of a doublet (J = 7.4 Hz),
integrated for 2H, at 8 2.52, suggested that compound 1
could be a 3,4-dihydroderivative. HMBC experiment (Fig.
1) allowed the confirmation of the final structure to be the
new sesquiterpene lactone; 3,4-dihydrolactucin.
Compounds 2, 3, 4, and 5 were identified as umbelliferone
(Yamoguchi, 1970), kaempferol (Markham et al., 1978),
isoscutellarin (Jay and Gonnet, 1973), and cichoriin (Abdel-
Salarn et al., 1986, respectively) by comparing their spectral
data with those previously published.
Results of the antimicrobial . screening indicated that,
quercetin, aloe-emodin showed moderate activity against
0 S. aureus, while caffeic acid and isoquercetrin showed weak
3, 4-Dihydrolactucin (1) activity. Cichoriin and caffeic acid exhibited weak activity
against E. Coli. Isoquercetrin and cichoriin showed weak
activity against P. aeroginosea, while quercetin, aloe-emodin,
R~ esculetin showed moderate activity. This points to the
stonger activity of aglycones over their glycosides. Aloe-
R10M0Ao emodin, isoquercetrin, and cichoriin showed moderate
Umbelliferone (2): R=H, R 1=H antifungal activity (Table 1). MIC and MBC determinations
Cichoriin (5): R=OH, R 1=Glc. showed that, aloe-emodin is the only compound having
strong fungicidal activity and the strongest bacteriostatic
OH activities against P. aeroginosea. Quercetin showed moderate
bacteriostatic activity against S. aureus and E. Coli. Cichoriin
HO showed moderate bacteriostatstic and bactericidal action
against P. aeroginosea (Table 2).
Only quercetin, showed relaxation effect on the rabbit
intestine, while the other fractions and isolates showed
OHO
spasmogenic activity. This founding concedes with the laxative
Kaempferol (3): R=H, R 1=0H
Isoscutellarin (4):R=OH, R 1=H effect of Chicory found in the literature (Fetrow and Avila,
1999; Crellin and philpatt, 1990; Tyler et al., 1988).
Isolated compounds
Acknowledgment
Authors thank Dr. NabilaM. Ghazy, Prof. of Pharmacognosy,
Pharmacognosy Department, Faculty of Pharmacy, University
of Alexandria, Egypt for her unlimited help. They also
thanks Prof. Dr. Mohamed Anwar, Head of Pharmaceutical
Microbiology Department, and Dr. Ahmed El-Mallah,
Assistance Professor of Pharmacology, Faculty of Pharmacy,
University of Alexandria, Egypt, for carrying out the anti-
microbial screening and measuring the spasmogenic activities,
respectively.

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