ORIGINAL PAPER

Phytotoxic activity of Cachrys pungens Jan, a mediterraneanspecies: separation, identification and quantification of potentialallelochemicals

Fabrizio Araniti • Mariangela Marrelli •

Antonio Lupini • Francesco Mercati •

Giancarlo Antonio Statti • Maria Rosa Abenavoli

Received: 11 August 2013 / Revised: 27 December 2013 / Accepted: 31 December 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014

Abstract In continuous research for bioactive com-

pounds obtained from plants to use for weed control in

sustainable agriculture, the aerial parts of Cachrys pungens

Jan (Umbelliferae) were extracted with methanol and then

fractionated using hexane, chloroform (CHCl3) and ethyl

acetate (AcOEt). The potential phytotoxicity of total

methanolic extract and each fraction was assayed in vitro

on seed germination and root elongation of lettuce (Lact-

uca sativa L.) and the most active fractions were assayed

on three of the most common weeds (Lolium perenne,

Amaranthus retroflexus, Echinochloa crus-galli). Non lin-

ear regression that allowed to obtain the ED50 index for

both physiological processes was applied. The fraction

bioassays indicated the following hierarchy of phytotox-

icity for both processes: CHCl3 C AcOEt [ hexane.

Moreover, in the present work was chemically character-

ized for the first time (through HPTLC) the polar fraction

of this species pointing out the high presence of flavonoids

and phenolic acids. In particular six of them have been

chemically characterized and quantified (naringin, querce-

tin, catechin, caffeic acid, ferulic acid and gallic acid).

These results make C. pungens Jan a potential source of

natural compounds employable for an eco-friendly

agriculture.

Keywords Amaranthus retroflexus � Apiaceae � Bio-

herbicide � Lactuca sativa L. � Natural products � Root

growth � Seed germination � Echinochloa crus-galli �Lolium perenne � Weed management

Introduction

In the past decades, the search and discovery of new

natural products from plants, fungi or microbes to use for

weed and pest management in sustainable agriculture

have attracted a lot of attention from allelopathy

researchers (Macias et al. 2007). In particular, allelo-

chemicals, secondary metabolites implied in plant–plant

communications, could be considered as potential source

of new natural compounds and/or as potential template to

develop new herbicides with different modes of action

and less harmful than synthetic chemicals, currently used

in agriculture practices. Several studies have shown that

production of allelochemicals is largely increased when

plants are exposed to biotic or abiotic stress (Chaves and

Escudero 1999). The mediterranean area, characterized by

a long and dry season and important pool of biodiversity,

is a promising ecosystem on which to focus the research

on plant with high phytotoxic potential (Araniti et al.

2012b; Selmar and Kleinwachter 2013). Nowadays few

studies on phytotoxicity of plants belonging to the med-

iterranean flora have been done, increasing the interest on

this subject.

The genus Cachrys (Apiaceae) is widely distributed in

the mediterranean basin (Tutin et al. 1968; Zohary 1972). It

is endemic to southern Europe and northern Africa where it

is represented by three or four different species. In Italy,

C. libanotis, C. sicula, C. ferulacea and C. pungens are the

most representative species especially concentrated in the

Communicated by O. Ferrarese-Filho.

F. Araniti � A. Lupini � F. Mercati � M. R. Abenavoli (&)

Dipartimento di AGRARIA, Facolta di Agraria-Salita Melissari,

Universita degli Studi ‘‘Mediterranea’’ di Reggio Calabria,

89124 Reggio Calabria, RC, Italy

e-mail: mrabenavoli@unirc.it

M. Marrelli � G. A. Statti

Dipartimento di Farmacia e Scienze della Salute e della

Nutrizione, Universita della Calabria, 87030 Rende, CS, Italy

123

Acta Physiol Plant

DOI 10.1007/s11738-013-1482-8

southern regions, where they colonize arid places and

edges.

Phytochemical composition of the essential oils and the

non polar constituents of different Cachrys species have

been reported (Ignateva et al. 1972; Grande et al. 1986;

Abad et al. 2001; Menichini et al. 2012). Many coumarins

and in particular furocoumarins among which bergapten

and imperatorin have been identified in these species as the

major secondary metabolites. Their role as antimicrobial,

antitumoral and anti-aggregant and potent antioxidants

have been reported (Hoult and Paya 1996). However, no

information concerning the phytotoxic potential of the

extracts obtained from plants belonging to Cachrys genus

are available yet.

In this respect, the total extract of C. pungens was

assayed on L. sativa, a sensitive species to phytotoxic

compounds. Then, through bioguided fractionation, the

most active fractions were identified and successively

assayed on three of the most common weeds (Lolium

perenne, Amaranthus retroflexus, Echinochloa crus-galli).

The total content in phenolic compounds and flavonoids

was also evaluated and the polar fraction was investigated,

for the first time, through HPTLC to identify and quantify

bioactive molecules.

Materials and methods

Plant materials

Aerial parts of Cachrys pungens Jan were collected on

August 2008 in Southern Italy (Calabria) at the end of the

flowering period. Leaves and stem were cut, air dried and

then stored at room temperature till use. The identification

of the plant species was carried out by Dr. Carmen Gangale

from the Botanical Garden, located at the University of

Calabria, and a voucher specimen was deposited at the

Herbarium of the Natural History Museum of Calabria

(CLU).

Crude extracts preparation and fractionation

The crude extract preparation and its fractionation was

carried out as described by Araniti et al. (2013) with some

modifications. Air-dried plant materials (682 g) of Cachrys

pungens Jan were exhaustively extracted through macera-

tion in methanol for 72 h and then filtered (the maceration

was repeated three times). The extraction procedure was

carried out at room temperature (25 ± 1 �C) and in dark

conditions to avoid molecular photo-degradation. The

methanolic extract was evaporated to dryness through a

rotary evaporator (35 �C) and then completely dried under

nitrogen gas. After yield calculation (Table 1), the

methanolic extract was fractionated through liquid–liquid

chromatography technique, using three organic solvents

characterized by increasing polarity: n-hexane, chloroform

(CHCl3) and ethyl acetate (AcOEt). The fractions obtained

were dried and their extraction yields were also calculated

(Table 1).

Bioassays with methanolic extract and its fractions

Seed germination bioassay on L. sativa

The methanolic extract and its fractions were assayed at the

following concentrations: 0, 0.625, 1.25, 2.5, 5, 7.5,

10 mg mL-1. Two milliliters of each test sample, previ-

ously dissolved in methanol, were added to a double sheet

of filter paper in Petri dish (6 cm Ø) and dried to allow the

solvent evaporation. Then the extracts were appropriately

diluted with 2 mL of sterile deionized water. Deionized

water represented the control (0 mg mL-1). Successively,

for each concentration, 10 seeds of Lactuca sativa previ-

ously sterilized [15 % (v/v) NaClO solution for 15 min],

were distributed to each Petri dish on a double layer of

filter paper. The experiments were carried out in a growth

chamber at 25 ± 1 �C, 70 % relative humidity, with 16/8 h

light/dark. The number of germinated seeds was counted

(rupture of seed coat and emergence of radicle at least

1 mm long) each day for 4 days after which no further seed

germination occurred. Total germination index [GT (%)]

was calculated using the equation proposed by Chiapusio

et al. (1997). To evaluate the viability of seeds treated with

the methanolic extracts, at the end of the treatment, the

ungerminated seeds were carefully washed and placed in a

Petri dish containing filter paper wetted with deionized

water.

Root elongation bioassay on L. sativa

For the root elongation bioassay, five seedlings of L. sativa

chosen for uniformity in root length were treated in the

same conditions previously described in ‘‘Seed germination

bioassay on L. sativa’’. After 48 h of treatment, an image of

seedlings roots for each treatment was captured by scanner

Table 1 Yield of methanolic extract and its fractions of C. pungens

dry material

Species Dry

weight (g)

Solvents Total

weight (g)

Yield %

C. pungens 682 Methanolic TOT 84.36 12.4

n-hexane 7.5 1.1

CHCl3 23.9 3.5

AcOEt 3.4 0.5

Acta Physiol Plant

123

(Epson Expression 800, Regent Instruments, Quebec,

Canada) and the Total root length [TRL (%)] was measured

using the WinRhizo Pro System v. 2002a Software

(Instruments Regent Inc., Quebec, Canada).

Bioassays on weeds

The most active fractions, CHCl3 and AcOEt, individu-

ated during the bioassays carried on L. sativa, were

evaluated for their inhibitory activity on seed germina-

tion and root growth of three common weeds, L. per-

enne, A. retroflexus and E. crus-galli. The procedure and

concentrations assayed were similar to that described

above (‘‘Bioassays with methanolic extract and its frac-

tions’’) (A. retroflexus was incubated at 30 ± 1 �C)

except for number of seeds that was 15. Because of seed

germination scalability, the number of germinated seeds

was counted each day for 7 days. Then ungerminated

seeds were tested for their viability as previously

described in ‘‘Bioassays with methanolic extract and its

fractions’’.

Determination of total phenolic content

Total phenolic content of both methanolic extract and the

AcOEt fraction was determined using the Folin–Ciocalteu

method with chlorogenic acid as standard (Conforti et al.

2009). Fifty milligrams of each sample were poured in a

tube and extracted with 25 mL of the following solvent

solution: 40 mL acetone: 40 mL methanol: 20 mL water:

0.1 mL acetic acid. The samples, heated at 60 �C for 1 h,

were homogenized for 30 s and then cooled at room tem-

perature. Successively, 200 lL of solution were collected

and 1 mL of Folin–Ciocalteu’s reagent, and 1 mL of

sodium carbonate solution (7.5 %) was added. After vor-

texing, the samples were allowed to stand for 2 h in dark

conditions. Absorption of each sample was measured at

726 nm (Perkin-Elmer Lambda 35 UV/VIS spectropho-

tometer) and total phenolic content was expressed as

chlorogenic acid equivalents in mg g-1 of dry plant

material.

Determination of flavonoids

Total flavonoid content was estimated in both methanolic

and AcOEt extracts using a colorimetric method, based on

the formation of a flavonoid–aluminum complex (Quettier-

Deleu et al. 2000). Absorbance was measured at 430 nm

(Perkin-Elmer Lambda 35 UV/VIS spectrophotometer) and

the values were calculated from a calibration curve

obtained with quercetin and expressed as quercetin equiv-

alents in mg g-1 of dry plant material.

Chemical characterization of phenolic compounds

through HPTLC

The chemical characterization and quantification of phe-

nolic compounds was carried out through high-perfor-

mance thin-layer chromatograph (HPTLC). The utilized

HPTLC apparatus (CAMAG, Muttenz, Switzerland) con-

sisted of a Linomat five sample applicator using 100 lL

syringes and a Camag TLC Visualizer linked to winCATS

software. Filtered solutions were applied on normal phase

glass plates 20 9 10 cm (VWR International s.r.l., Mi-

lano, Italy) with glass-backed layer silica gel 60 (2 lm

thickness) previously washed in methanol and dried for

3 min at 100 �C. The solution application was done

through a nitrogen flow with the following operating

conditions: syringe delivery speed, 150 nl s-1; injection

volume, 1 lL; band width, 8 mm; distance from bottom,

15 mm; solvent front position, 9 mm. For the detection of

quercitrin, naringin, catechin, caffeic acid and rutin,

HPTLC plates were developed with a mobile phase

characterized by the following ratio and composition:

AcOEt (100); dichloromethane (25); acetic acid (10);

formic acid (10); water (11) (v/v/v/v/v). For the identifi-

cation of quercetin, kaempferol, naringenin, hesperetin

and ferulic acid the mobile phase was AcOEt (100);

dichloromethane (31.25); acetic acid (1.25); formic acid

(1.25); water (1.25) (v/v/v/v/v).

A mobile phase composed of AcOEt (100); dichloro-

methane (31.25); acetic acid (0.25); formic acid (0.25); and

water (0.25) (v/v/v/v/v) was utilized for the detection of

cinnamic and gallic acids. Developed layers were deriva-

tized with Natural Product Reagent (NPR) (1 g diphenyl-

borinic acid aminoethylester in 200 mL of AcOEt) and

anisaldehyde (1.5 mL p-anisaldehyde, 2.5 mL H2SO4,

1 mL AcOH in 37 mL EtOH). Before and after derivati-

zation the TLC plates were inspected, through a Camag

TLC visualizer, under a UV light (254 and/or 366 nm) or

under white light upper and lower (WRT).

The AcOEt fraction of C. pungens was dissolved in

methanol to have a final concentration of 40 mg mL-1.

This fraction was used for TLC fingerprinting and co-

chromatography with marker compounds and applied in

triplicate on the TLC plates. For qualitative determinations,

standards at 3 mg mL-1 concentration in methanol (or

ethanol for naringin, caffeic acid, kaempferol, naringenin

and hesperetin) have been used.

For quantification of identified compounds, working

stock solutions were prepared by dilution with methanol

(for quercitrin, catechin, ferulic acid and gallic acid) or

ethanol (naringin and caffeic acid) at 0.5, 1, 2, 3, 4, 6, 8,

10 mg mL-1 final concentrations. Standard solutions of

each compound were spotted on HPTLC plate to give

absolute amounts of 0.5, 1, 2, 3, 4, 6, 8, 10 lg/band. The

Acta Physiol Plant

123

calibration curves were prepared using absolute amount

(lg/band) as independent variable (X) and the peak area of

standards as dependent variable (Y), and they confirmed

linear relationship between the working concentration and

the peak areas. Quantification of compounds was per-

formed using regression equations. The correlation coeffi-

cients (R2) were found to be [0.98. Regression analyses

test of the compound was performed using GraphPad Prism

Software (San Diego, CA, USA).

Experimental design and statistical analysis

A completely random design with five replications was

adopted to evaluate the effects of methanolic extracts and

the three solvent fractions on germination and root elon-

gation processes of L. sativa, L. perenne, E. crus-galli and

A. retroflexus. Germination and root elongation responses

to different doses of methanolic extract and solvent frac-

tions were evaluated by a nonlinear regression model, as

previously described by Araniti et al. (2013), using a log-

logistic function to calculate the ED50 parameter which

defines the effective dose required to reduce 50 % of the

total response. The comparison of the phytotoxicity of

methanolic extract and solvent fractions was performed by

one-way ANOVA using the ED50 as a variable and the

extraction solvent as main factor. The ED50 data were first

checked for deviations from normality (Kolmogorov–

Smirnov test) and tested for homogeneity (Leven Median

test). Tukey’s test comparison was utilized to compare the

mean values of ED50 among crude extract and its fractions

(P \ 0.05).

Results and discussion

Bioassays with C. pungens methanolic extract

and its solvent fractions

The methanolic extract from the aerial part of C. pungens

showed strong inhibitory effects on lettuce seed germi-

nation (Fig. 1). According to many authors (Chung and

Miller 1995; Araniti et al. 2012b), the degree of inhibition

increased along with increasing extract concentration

reaching at 7.5 mg mL-1 after 48 h of exposure, the

highest inhibitory level (Table 2; Fig. 1). The fractions,

obtained through the bio-guided fractionation of the

methanolic extract, showed a different activity. Chloro-

formic fraction appeared to be the most active since it

significantly inhibited (-37 %) lettuce seed germination

at the lowest concentration (0.6 mg mL-1), reaching, at

1.2 mg mL-1 concentration, 97 % of the inhibition and

causing the complete block at higher doses (Table 2;

Fig. 1). Although less active, hexane and AcOEt fractions

inhibited lettuce seed germination. In particular, the

AcOEt fraction reduced (-37 %), at 5 mg mL-1 con-

centration (Table 2; Fig. 1). This inhibition was increasing

at higher doses reaching the complete inhibition when

seeds were treated with 10 mg mL-1. Also hexane frac-

tion caused a statistically significant inhibition at

5 mg mL-1 concentration (*15 %), reaching 93 % of

germination block after treatment with 10 mg mL-1

(Table 2; Fig. 1).

After 48 h of treatment, the ungerminated seeds were

carefully washed allowing them to recover in deionized

water to verify the reversible/irreversible effects of meth-

anolic extract and its fractions. Interestingly, seeds treated

with the methanolic extract and the hexane and AcOEt

fractions were unable to germinate; conversely seeds

exposed to the chloroformic fraction promptly germinated

in less than 24 h (data not shown). This ability to recover

after chloroformic fraction treatment could be due to the

high presence of coumarins, already characterized and

quantified in the chloroformic fraction of C. pungens

(psoralene, xanthotoxin, bergapten, 3-methylsuberosine,

isopimpinellin and isooxypeucedanin) (Menichini et al.

2012). These compounds are known to retard seed germi-

nation and to extend the dormancy acting on seed coat and

aleurone layer, which precedes radicle protrusion (Aliotta

et al. 1993, 1994). Furthermore, it has been demonstrated

that, after coumarin treatment, seed rehydration, during

phase I, allowed that the germination program could be

successfully executed (Abenavoli et al. 2006). Conversely,

the incapacity of lettuce seeds to germinate after metha-

nolic, hexane and AcOEt fraction treatment highlighted

that they contained different phytotoxins able to irrevers-

ibly block seed germination. Similar effects were already

observed by Araniti et al. (2013) on lettuce seeds treated

with Calamintha nepeta extract and its fractions. Indeed,

they observed that the most active molecules were mainly

partitioned in the hexane and AcOEt fractions particularly

rich in terpenoids and phenolic acids, respectively.

As for the seed germination, the root elongation process

pointed out a strong dose-dependent inhibitory effect in

lettuce. In fact, after only 48 h of exposure, the methanolic

extract, at the highest concentration (10 mg mL-1),

strongly inhibited the root elongation of lettuce seedlings

with a reduction about of 79 % compared with control

(Table 2; Fig. 1). Among the fractions, the most active

were the chloroformic and the AcOEt ones. They signifi-

cantly inhibited root growth of lettuce seedlings already at

the lowest concentration (0.6 mg mL-1) (Table 2; Fig. 1)

and this effect increased along with increasing doses

reaching, at the highest concentrations (10 mg mL-1), 82

and 70 % of inhibition with chloroformic and the AcOEt

fractions, respectively. The hexane was the less active

fraction causing 30 % of inhibition at the highest

Acta Physiol Plant

123

concentration and significantly stimulated lettuce root

growth (?25 % compared to the control) at lowest con-

centration (0.6 mg mL-1) (Table 2; Fig. 1). This stimula-

tory effect, caused by natural and synthetic chemicals, at

very low concentrations, is known as ‘‘hormesis’’. Rizvi

and Rizvi (1992) observed this phenomenon with some

natural organic compounds which were able to promote, at

low concentrations, lettuce root growth. Moreover, as

reported by several authors (Baghestani et al. 1999), this

positive effect could be related to the ability of some

species to exploit this material as a source of nutrition or,

as sustained by Rice (1984), such stimulation might not

depend on allelopathic compounds but due to an organic

matter enrichment of growth substrate. Raw data regarding

seed germination and root elongation process were fitted

through a non-linear regression model using a log-logistic

response equation largely employed in weed management

for the research of several herbicides and allelochemicals

Fig. 1 Dose–response curves of

total germination index (GT %)

and total root length (TRL, cm)

of L. sativa exposed to different

fractions of C. pungens obtained

from the bio-guided

fractionation of the C. pungens

methanolic extract. All the

dose–responses curves pointed

out a significance level of

P \ 0.001

Acta Physiol Plant

123

(Nielsen et al. 2004; Petersen et al. 2001). This model

allowed to obtain the ED50, a specific parameter which

defines the effective dose causing 50 % of total response in

the population. All the data obtained for both seed germi-

nation and root growth were characterized by a high sta-

tistical significance (P \ 0.001) (Table 2). The ED50

values in both seed germination and root growth confirmed

the strong inhibitory effects of C. pungens methanolic

extract and its fractions. In particular, the ED50 values of

seed germination evidenced that the chloroformic fraction

was the most phytotoxic (0.64 mg mL-1) followed by the

methanolic extract (1.64 mg mL-1), particularly active,

the AcOEt (6.7 mg mL-1) and the n-Hexane

(9.7 mg mL-1) fractions (Table 2). Concerning the root

growth, the methanolic extract was the most active

(0.4 mg mL-1) followed by the chloroformic

(1.3 mg mL-1) and the AcOEt (6.8 mg mL-1) fractions

(Table 2). The less active was the n-Hexane fraction for

which the ED50 was not detectable (ND). Comparing the

ED50 of the methanolic extract on both processes (data not

shown) was possible to confirm that, as reported by several

authors (Araniti et al. 2012a; Chon et al. 2002), the root

growth was a more sensitive process than seed germination

to allelochemicals. Furthermore, except for the chlorofor-

mic fraction the ED50 values of lettuce seeds and seedlings

treated with the other fractions were higher than values

observed with methanolic extract. This could be due to the

heterogeneous chemical composition of methanolic extract

that, although at lower concentrations than in the fractions,

contained many different classes of chemical compounds

that interacting synergistically determined higher inhibi-

tory effect. Similar responses were also observed by

Araniti et al. (2012b) with Artemisia arborescens extract

where the methanolic extract was more active than its

fractions. However, nearly all fractions were able to inhibit

with different strength both germination and root elonga-

tion processes.

Bioassays on weeds

The experiments carried out on weeds pointed out species-

specific, extract- and dose-dependent inhibitory effects

(Tables 3, 4; Figs. 2, 3). Among all the weed species

assayed, E. crus-galli appeared to be the less sensitive to

both fractions (CHCl3 and AcOEt). In fact, the highest

concentration of the chloroformic and AcOEt fractions

caused 64 and 43 % of inhibition, respectively (Table 3;

Fig. 2). The germination of L. perenne was also strongly

inhibited by both fractions. However, while the chloro-

formic fraction caused, at the highest concentration, a 70 %

of inhibition of the germination, the AcOEt fraction

determined a 98 % inhibition already at 5 mg mL-1 con-

centration, reaching a complete block of germination at

higher concentrations (Table 3; Fig. 2). Amaranthus ret-

roflexus seemed to be the most sensitive species to both

fractions showing a complete inhibition of the germination

Table 2 ED50 (mg mL-1) values estimated by the log-logistic equations for the seed germination and root growth responses of L. sativa to C.

pungens methanolic extract and to different solvent fractions obtained from the bio-guided fractionation of the C. pungens methanolic extract

Concentration (mg mL-1) L. sativa

GT (%) TRL (cm)

MeOH n-Hexane CHCl3 AcOEt MeOH n-Hexane CHCl3 AcOEt

0 100a 100a 100a 100a 1.15a 1.65a 1.87a 1.51a

0.6 82.5b 96.7a 63.3b 96.7a 0.46b 2.06b 1.3b 1.26b

1.2 75c 93.3ab 3.3c 96.7a 0.34c 1.77a 1.01b 1.34b

2.5 10d 96.7a 0d 100.0a 0.31d 1.63a 0.51c 1.06c

5 2.5e 86.7b 0d 63.3b 0.29e 1.61c 0.39d 0.89d

7.5 0f 90.0b 0d 56.7b 0.27ef 1.54d 0.36e 0.79e

10 0f 6.7c 0d 0c 0.26f 1.13e 0.34e 0.45f

ED50 (mg mL-1)

GT (%) TRL (cm)

MeOH 1.64 (±0.07)a 0.4 (±0.03)a

n-Hexane 9.7 (±0.6)d ND

CHCl3 0.64 (±0.2)b 1.3 (±0.14)b

AcOEt 6.7 (±0.4)c 6.8 (±0.8)c

Different letters along the column indicate statistically significant differences at P \ 0.05 (Tukey test). No Detectable (N.D.). Values within the

brackets indicate standard error (SD); (N = 5)

Acta Physiol Plant

123

at of 7.5 (CHCl3) and 5 (AcOEt) mg mL-1 concentrations

(Table 3; Fig. 2).

The comparison of ED50 for seed germination con-

firmed that E. crus-galli was the less sensitive species

exhibiting the ED50 values calculable only in presence of

the chloroformic fraction (6.27 mg mL-1). This con-

firmed the less sensitiveness of this weed to allelochem-

icals, as previously reported (Kil and Yun 1992; Araniti

et al. 2012b). On the contrary, A. retroflexus was the most

sensitive to both fractions showing lower ED50 values

(Table 3), whereas L. perenne was extremely sensitive to

the AcOEt fraction (2 mg mL-1) and less to the chloro-

formic fraction (7 mg mL-1) (Table 3). As observed in

lettuce, all ungerminated weeds treated with AcOEt

fraction, then washed and placed on filter paper moist-

ened with distilled water, were unable to germinate,

Table 3 ED50 (mg mL-1) values estimated by the log-logistic

equations for the response of seed germination of L. perenne,

Echinochloa crus-galli and Amaranthus retroflexus to chloroform

(CHCl3) and ethyl acetate (AcOEt), the most active fractions,

obtained from the bio-guided fractionation of the C. pungens

methanolic extract

Concentration (mg mL-1) GT (%)

L. perenne E. crusgalli A. retroflexus

CHCl3 AcOEt CHCl3 AcOEt CHCl3 AcOEt

0 95.6a 93.3a 94.6a 93.3a 97.8a 93.3a

0.6 86.7b 91.1ab 97.8b 97.8a 100a 95.6a

1.2 84.4bc 86.7b 93.3a 93.3a 80b 46.7b

2.5 80c 20c 91.1c 93.3a 64.4c 24.4c

5 60d 2.2d 53.3d 86.7ab 6.7d 0d

7.5 37.8e 0e 37.8e 73.3b 0e 0d

10 28.9f 0e 35.6e 66.7c 0e 0d

ED50 (mg mL-1)

CHCl3 7.04 (±0.77)c 6.27 (±0.52)c 2.92 (±0.15)b

AcOEt 1.99 (±0.12)a ND 3.21 (±0.23)b

Different letters along the GT (%) column and among the ED50 values indicate statistical significative differences at P \ 0.05 (Tukey’s test). No

Detectable (N.D.). Values into the brackets indicate standard error (SE); (N = 5)

Table 4 ED50 (mg mL-1) values estimated by the log-logistic

equations for the root growth response of L. perenne, Echinochloa

crus-galli and Amaranthus retroflexus to chloroform (CHCl3) and

ethyl acetate (AcOEt), the most active fractions, obtained from the

bio-guided fractionation of the C. pungens methanolic extract

Concentration (mg mL-1) TRL (cm)

L. perenne E. crusgalli A. retroflexus

CHCl3 AcOEt CHCl3 AcOEt CHCl3 AcOEt

0 2a 2.1a 2.1a 2.4a 2.4a 3.4a

0.6 1.8b 2ab 2b 2.3a 2.3a 2b

1.2 1.5c 1.9b 1.7c 2.4a 1.6b 1.7c

2.5 0.7d 1.2c 1.1d 1.6b 1c 1.3d

5 0.3e 0.7d 0.7e 1.3c 0.2d 0.4e

7.5 0.3ef 0.2e 0.6ef 0.8d 0.1e 0.2f

10 0.2f 0.2f 0.5f 0.6e 0.1e 0.1g

ED50 (mg mL-1)

CHCl3 2.06 (±0.17)b 3.1 (±0.4)c 1.94 (±0.15)b

AcOEt 3.16 (±0.32)c 4.97 (±0.39)d 1.14 (±0.13)a

Different letters along the GT (%) column and among the ED50 values indicate statistically significant differences at P \ 0.05 (Tukey’s test). No

Detectable (N.D.). Values into the brackets indicate standard error (SE); (N = 5)

Acta Physiol Plant

123

whereas those treated with the chloroformic fraction,

except for A. retroflexus, were able to recover their ger-

minative capacity (data not shown) underlying the high-

est toxic activity of the AcOEt fraction. The extremely

high phytotoxicity, also observed by different authors

(Imatomi et al. 2013; Araniti et al. 2013; Djurdjevic

2004; An et al. 2000), could be attributed to its marked

capacity to extract some phenolic acids and flavonoids

largely known for their inhibitory activity (Reigosa et al.

2007; Weston and Mathesius 2013; Hassan and Mathe-

sius 2012).

Root growth data indicated that all the weed species

were extremely sensitive to both fractions (Fig. 3). In

particular, as observed for germination, A. retroflexus was

the most sensitive species showing the lowest ED50 values,

although here, the AcOEt fraction was more active than the

chloroformic (1.94 vs 1.14 mg mL-1, Table 4). On the

contrary, the chloroformic was the most active fraction on

L. perenne and E. crus-galli showing 2.06 and 3.1

(mg mL-1) ED50 values, respectively (Table 4).

Evaluation of total phenolics and total flavonoids

content

In the present study, total phenolics were quantified on the

AcOEt fraction only because the quantification in the

methanolic extract of C. pungens has been already pointed

out in previous work (Menichini et al. 2012). During the

fractionation, AcOEt appeared to be the solvent on which

phenolic compounds were better solubilized (An et al. 2000;

Djurdjevic et al. 2004; Zhu and Mallik 1994). Indeed, phe-

nolic compounds were mostly partitioned in this fraction

showing a value of 581.9 mg g-1 of extract (corresponding

to 2.9 mg g-1 of DW) considering that methanolic extract

exhibited 145.6 mg g-1 of extract [corresponding to

18 mg g-1 of dry plant material (DW)] (Menichini et al.

2012). Conversely, the total flavonoids quantification was

evaluated on both methanolic extract and AcOEt fraction.

Also the flavonoid content followed the same trend, showing

a higher concentration in the AcOEt fraction by

305.04 mg g-1 of extract (1.52 mg g-1 of DW) compared

Fig. 2 Dose–response curves of

total germination index (GT, %)

of Amaranthus retroflexus,

Echinochloa crus-galli and

L. Perenne exposed to different

fractions of C. pungens obtained

from the bio-guided

fractionation of the C. pungens

methanolic extract. All the

dose–responses curves pointed

out a significance level of

P \ 0.001

Acta Physiol Plant

123

with 28.3 mg g-1 of extract (3.5 mg g-1 of DW) determined

in the methanolic extract. Moreover, because of the polar

affinity of some of these compounds with CHCl3 (Hashim

and Devi 2003), the quantification of both total phenolics and

flavonoids was carried out also on the chloroformic fraction,

but their presence was not quantifiable (data not shown). The

high concentration of polyphenolic compounds in the AcOEt

fraction could explain its high phytotoxic activity on almost

all species (Tables 2, 3). As previously described by Weir

et al. (2004) phenolic compounds were involved in growth

inhibition of various plant species interfering with their

biochemical and physiological processes such as the dis-

ruption of the activity of metabolic enzymes that are involved

in glycolysis and the oxidative pentose phosphate pathways.

Moreover, Kaur et al. (2005) observed cellular changes in

roots and a reduction in growth on plants of Sinapis alba

treated with phenolic compounds. Similar observations were

recorded by Anjum and Bajwa (2010) on Chenopodium

album and Rumex dentatus treated with the crude aqueous

extracts of Helianthus annuus particularly rich in phenolics.

HPTLC

The polyphenol compositions of the AcOEt fraction

obtained from C. pungens Jan methanolic extract were

assessed using HPTLC. The HPTLC is a sophisticated

technique widely used for the analyses of medicinal drugs,

dietary supplements and nutraceuticals that allows an

excellent separation, qualitative and quantitative analysis

of a wide range of compounds (Rashmin et al. 2011; Ahire

et al. 2013; Kikowska et al. 2012). Furthermore, it provides

a better separation of compounds also from complex

mixtures that characterizes natural products (Nicoletti

2011) because of the optimized material and improved

sample application. For these reasons, it has been utilized

in the present study. The presence of 12 phenols, i.e.

quercitrin, naringin, catechin, caffeic acid, rutin, quercetin,

kaempferol, naringenin, hesperetin, ferulic acid, cinnamic

acid and gallic acid, was verified in the AcOEt fraction of

C. pungens Jan. Six of these compounds were identified:

catechin, a flavonoid; naringin and quercitrin, two

Fig. 3 Dose–response curves of

total root length (TRL, cm) of

Amaranthus retroflexus,

Echinochloa crus-galli and

L. Perenne exposed to different

fractions of C. pungens obtained

from the bio-guided

fractionation of the C. pungens

methanolic extract. All the

dose–responses curves pointed

out a significance level of

P \ 0.001

Acta Physiol Plant

123

flavonoid glycosides; caffeic and ferulic acids, two cin-

namic acids and gallic acid, a phenolic acid. In Fig. 4 are

reported the chromatographic profiles of investigated

extract and the first four components, quercitrin, naringin,

catechin and caffeic acid, identified by comparison with

corresponding standards. The elution of the HPTLC plate

with the mobile phase AcOEt (100); dichloromethane

(31.25); acetic acid (1.25); formic acid (1.25); and water

Fig. 4 HPTLC chromatograms of ethyl acetate fraction of C.

pungens Jan and utilized standards. Mobile phase: AcOEt/CH2Cl2/

CH3COOH/HCOOH/H2O (100: 25: 10: 10: 11; v/v/v/v/v). a ethyl

acetate fraction of C. pungens, b naringin (Rf = 0.25), c quercitrin

(Rf = 0.38), d catechin (Rf = 0.87), e caffeic acid (Rf = 0.95)

Fig. 5 Chromatographic profiles of ethyl acetate fraction of C.

pungens Jan and ferulic acid. Mobile phase: AcOEt/CH2Cl2/

CH3COOH/HCOOH/H2O (100: 31.25: 1.25: 1.25: 1.25; v/v/v/v/v).

a ethyl acetate fraction of C. pungens, b ferulic acid (Rf = 0.87)

Fig. 6 HPTLC chromatograms of ethyl acetate fraction of C.

pungens Jan and gallic acid. Mobile phase: AcOEt/CH2Cl2/

CH3COOH/HCOOH/H2O (100: 31.25: 0.25: 0.25: 0.25; v/v/v/v/v).

a ethyl acetate fraction of C. pungens, b gallic acid (Rf = 0.43)

Acta Physiol Plant

123

(1.25) (v/v/v/v/v) allowed to verify the presence of a fifth

compound, ferulic acid (Rf = 0.87, Fig. 5). Further ana-

lysis allowed the identification of gallic acid, another

phenolic compound whose corresponding chromatographic

profiles are reported in Fig. 6 (Rf = 0.43).

The quantification of identified phenolic compounds

was performed using regression equations. Among these

molecules, the most abundant was catechin, with an

amount of 150.78 ± 10.96 mg g-1 of extract (corre-

sponding to 0.75 ± 0.05 mg g-1 of dry weight of plant

material, Table 5). HPTLC analyses revealed that the

AcOEt fraction of C. pungens contained also flavonoid

glycoside quercitrin at 82.74 ± 5.11 mg g-1 of extract

(Table 5). Gallic acid concentration was 43.20 (±1.84)

mg g-1 of extract (Table 5). Lower amounts were

observed for naringin (34.81 ± 1.77 mg g-1 of extract,

0.17 ± 0.01 mg g-1 of plant material) and ferulic and

caffeic acids (34.86 ± 1.59 and 29.94 ± 0.13 mg g-1 of

extract, corresponding to 0.18 ± 0.01 and

0.12 ± 0.01 mg g-1 of plant material, respectively)

(Table 5). All the flavonoids and phenolic acids identified

in C. Pungens extract were well known for their biological

activity. For example, Bais et al. (2003) reported the high

phytotoxicity of catechin on germination and growth of

different weeds and crops (Centaurea diffusa, Kochia

scoparia, Linaria dalmatica, Lycopersicon esculentum,

Triticum aestivum and Arabidopsis thaliana). The flavo-

noid quercitrin, isolated from the leaves of the noxious

perennial weed Pluchea lanceolata, has been shown to

inhibit the growth of the legume asparagus bean (Vigna

unguiculata) in a concentrations range 10-4–10-3 M (In-

derjit and Dakshini 1995), whereas the flavones naringin

promoted the root elongation of Raphanus sativus and

Lepidium sativum when assayed at the concentration of

10-5 M (De Martino et al. 2012). Moreover, Patterson

(1981) reported that 10–30 lmol L-1 caffeic and ferulic

acids significantly inhibited the growth of soybean

(Glycine max) strongly reducing the photosynthetic pro-

ducts and chlorophyll content. Gallic acid, at 1 mM, sig-

nificantly inhibited the germination of Phaseolus mungo

L. (Sasikumar et al. 2002) and as observed by Bubna et al.

(2011), the exogenous application of caffeic acid increased

the formation of lignin monomers that solidified the cell

wall and inhibited root growth of Glycine max. Other

studies reported that ferulic acid significantly reduced, after

6 days of treatment, root length and fresh weight of Zea

mays seedlings and the activities of hydrolase, maltase,

phospholipase and protease (Devi 1992).

Conclusion

The present work highlighted the high phytotoxic potential

of the genus Cachrys. Moreover, it confirmed the bio-

guided fractionation bioassay as a useful and rapid tool for

the identification of chemicals characterized by a strong

biological activity. The results evidenced, for the first time,

that Cachrys genus, largely studied in pharmacological

field as source of terpenes and coumarins present in the non

polar fraction, showed a strong biological activity on its

polar fraction. It appeared particularly rich in phenolic

compounds, well known for their phytotoxic activity on

plant species, and probably, responsible of germination and

root elongation inhibition observed on lettuce seedlings

and weeds. As already reported, root elongation was more

sensitive than germination in all species to all active

fractions.

Additional work is required (i) to deeply characterize the

polar fraction and to identify other compounds, already

revealed from chromatograms; (ii) to individuate which

compounds, alone or in combination, are responsible of the

biological effects observed; (iii) to investigate the physio-

logical, biochemical and molecular targets affected by the

most interesting active chemicals identified as well as their

mode of action.

Author contribution The work presented here was car-

ried out in collaboration between all authors. Araniti Fab-

rizio designed experimental setup, carried out the

laboratory experiments, analyzed the data and wrote the

paper. Mariangela Marrelli carried out the phytochemical

analysis and contributed to paper writing. Lupini Antonio

and Mercati Francesco co-worked in laboratory. Statti

Giancarlo Antonio co-designed experiments and analyzed

the data. Abenavoli Maria Rosa co-designed experiments,

interpreted and discussed the results and made a critical

revision of the manuscript.

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Compound Amount

(mg g-1 of

extract)

Amount (mg g-1

of DW of plant

material)

Naringin 34.81 (±1.77) 0.17 (±0.01)

Quercitrin 82.74 (±5.11) 0.41 (±0.03)

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Caffeic acid 24.94 (±0.13) 0.12 (±0.01)

Ferulic acid 34.86 (±1.59) 0.18 (±0.01)

Gallic acid 43.20 (±1.84) 0.22 (±0.01)

Data are expressed as mean ± SD (N = 3). DW dry weight

Acta Physiol Plant

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