Pc

DHa

b

c

d

a

ARRA

KCFHNPS

1

fvhmasttspmctPol2

(

0h

Industrial Crops and Products 55 (2014) 43–48

Contents lists available at ScienceDirect

Industrial Crops and Products

jo ur nal home p age: www.elsev ier .com/ locate / indcrop

hytotoxicity and cytotoxicity of disesquiterpene and sesquiterpeneoumarins from Ferula pseudalliacea

ara Dastana, Peyman Salehia,∗, Faezeh Ghanatib,∗∗, Ahmad Reza Gohari c,ossein Maroofid, Naba Alnajarb

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C. Evin, Tehran, IranDepartment of Plant Biology, Faculty of Biological Science, Tarbiat Modares University (TMU), POB: 14115-154, Tehran, IranMedicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, IranResearch Center of Agriculture and Natural Resources, Forked Road of Jame-Jam, Sanandaj, Iran

r t i c l e i n f o

rticle history:eceived 26 October 2013eceived in revised form 28 January 2014ccepted 30 January 2014

a b s t r a c t

Relative toxicity of a new disesquiterpene- and five sesquiterpene coumarins from the roots of Ferulapseudalliacea was investigated on tobacco cells, as a plant model cell line. The effects of these compoundson the germination of certain weeds and crop plants (from solanaceae) were evaluated as well. Thecytotoxic effects of these compounds were also evaluated on human cancer cell line, HeLa. The highestinhibitory effect on the growth of tobacco cells was observed by sanandajin and farnesiferol B. Sanandajin

eywords:ytotoxicityerula pseudalliaceaela cellsicotiana tabacum

also remarkably inhibited seed germination of all tested weeds and plants. Sanandajin, farnesiferol B,and kamolonol acetate displayed the highest potency against HeLa cells with IC50 of 2.2, 6.7, and 4.9 �M,respectively. The results of the present investigation indicated that disesquiterpene and sesquiterpenecoumarins isolated from F. pseudalliacea root extract can be considered as potent herbicides and cancer

hytotoxicityesquiterpene coumarins

chemopreventive agents.

. Introduction

Noxious weeds and non-native invaders are of ever growingactors which limit crop production and are usually controlled byast application of herbicides. Increased application of herbicidesowever, is considered a risk to human health and the environ-ent as well. The capability of accurately predicting the herbicidal

ctivity and site of action of a new chemical class without exten-ive laboratory studies would be worth tens of millions of dollarso the herbicide industry (Duke, 1990). Selective herbicides con-rol specific weeds, protect crops and allow the intensification andpread of key crops such as corn, rice, soybeans and wheat. Theyrovide a highly efficient, cost-effective, flexible and convenientethod of in-crop weed control. Several plant materials and their

onstituents have been reported to have sufficient phytotoxicityo act as natural herbicides or allelochemicals (Duke et al., 2000;andey, 1996; Pandey et al., 2005). These compounds may directly

r indirectly influence on other plants through leaching from roots,eaves or volatile emissions (Taiz and Zeiger, 2010; Weir et al.,004). Their phytotoxicity is attributed to their ability to change

∗ Corresponding author. Tel.: +98 2129904049; fax: +98 2122431783.∗∗ Corresponding author. Tel.: +98 21 82884717; fax: +98 21 82884717.

E-mail addresses: p-salehi@sbu.ac.ir (P. Salehi), ghangia@modares.ac.irF. Ghanati).

926-6690/$ – see front matter © 2014 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.indcrop.2014.01.051

© 2014 Elsevier B.V. All rights reserved.

the normal metabolic processes in other plants, including respira-tion, cell division, growth and development, and enzyme activity(Zeng et al., 2008). Oxidative phosphorylation uncouplers, mitosisinhibitors, fatty acid, amino acid, and lipid biosynthesis inhibitorsare of major categories of herbicides whose their mode of actionhave been widely tested (Duke, 1990).

Application of natural herbicides has been recently increasedas safer and more environmental friendly for weed control. So farsome natural products have been used as herbicides (Duke et al.,2000; Macías, 1995). For example, Bialaphos was a commercialnatural herbicide produced by fermentation of Streptomyces hygro-scopicus and Streptomyces viridochromeogenes (Murakami et al.,1986). Trans, trans-germacranolide sesquiterpene lactones cos-tunolide, parthenolide, and their 1,10-epoxy and 11,13-dihydroderivatives showed inhibitory effects on the growth and germi-nation of certain mono and dicotyledonous species in a similarmanner to that of the commercial herbicide Logran (Macías et al.,2000). Nonetheless, herbicidal potential of a large number of plantconstituents has not been studied adequately.

Many phytotoxins produced by plants are secondary metabo-lites i.e., terpenes, tannins, steroids, quinines, flavonoids andcoumarins (Li et al., 2010). Cytostatic activity of coumarins in plant

cells in vitro was discovered (Gawron and Glowniak, 1987). Phy-totoxic effects of methoxycoumarins from Myrraya paniculata wasreported by Jiwajinda et al. (2000). Inhibition of ATPases and acidphosphatases activities accompanied by ultrastructural damages

4 ops an

wh(psA

aPcaFfbL

g(gcseeofecasaIaev

dawphddpttc

2

2

SiaRM

2

ci(a

4 D. Dastan et al. / Industrial Cr

ere induced in meristematic cells of Allium cepa root tips by 4-ydroxycoumarin, 7-hydroxycoumarin, psoralen, and xanthotoxinPodbielkowska et al., 1996). Inhibitory effects of some synthetichosphorus-containing coumarin derivatives on the growth of thehoots of pea, wheat, and cucumbers have been also reported byleksieva et al. (1995).

Secondary metabolites of the plants have also been regardeds potential chemotherapeutic agents for combating cancers.odophyllotoxin, Taxol, and Topotecan are major instances of suchompounds (Cragg and Newman, 2009). There are some studiesbout the cytotoxic activity of sesquiterpene coumarins from genuserula, such as umbelliprenin from F. szowitsiana, farnesiferol Crom F. assa-foetida, umbelliprenin, farnesiferol A, gummosin andadrakemone from F. persica var. persica (Barthomeuf et al., 2008;ee et al., 2010; Shahverdi et al., 2006).

The genus Ferula (Apiaceae) comprises about 180 species whichrow mainly in central Asia, the Middle East, and central EuropePimenov and Leonov, 2004). This genus is well documented as aood source of biologically active compounds such as monosac-harides, sulfur-containing derivatives, coumarins, sesquiterpenes,esquiterpene coumarins, sesquiterpene lactones and daucanesters (Abd El-Razek et al., 2001; Iranshahi et al., 2007; Kajimotot al., 1998; Kapoor, 1990). Sesequiterpene coumarins are built upf a common coumarin group and a sesquiterpene moiety, there-ore more extensive and promising biological properties can bexpected from this class of natural compounds. The sesquiterpeneoumarins isolated from genus Ferula have wide range of biologicalctivities such as anticoagulant, antibacterial, antiviral (anti HIV),pasmolytic, anti-inflammatory, P-glycoprotein (P-gp) inhibitorynd cytotoxic properties (Abd El-Razek et al., 2003; Nazari andranshahi, 2011). Therefore, further investigations on biologicalctivities of coumarin derivatives of F. pseudalliacea seem to be nec-ssary for completion of the knowledge about the potentials of thisaluable class of natural products.

The aim of the present study was to evaluate the effects ofisesquiterpene and sesquiterpene coumarins from F. pseudalli-cea on the growth of tobacco cells and germination of certaineeds and crop plants seeds. The cytotoxic effects of these com-ounds were also evaluated on human cancer cell line, HeLa. Weave previously reported the extraction and identification of a newisesquiterpene- and five sesquiterpene coumarins from F. pseu-alliacea roots (Dastan et al., 2012). It opens new approaches onossible use of these compounds as herbicide in management ofhe weed and to our knowledge; this is the first report on the phy-otoxicity and cytotoxicity of disesquiterpene and sesquiterpeneoumarins.

. Materials and methods

.1. Plant material

Different parts of F. pseudalliacea plants were collected fromanandaj, Kurdistan province, Iran, in September 2012. They weredentified and a voucher specimen (MPH-1197) was depositedt the Herbarium of the Institute of Forests and Rangelandsesearches, Sanadaj, Iran. Plant specimen was identified by Hosseinaroufi from the same institute.

.2. Extraction and isolation of natural compounds

The roots of F. pseudalliacea were separated and their

onstituents were extracted, purified, and identified follow-ng the previously described method with a few modificationsDastan et al., 2012). In brief, root samples (1 kg) were crushednd extracted with 3 L of n-hexane during 24 h, and the

d Products 55 (2014) 43–48

extraction procedure was repeated three times. Evaporation ofsolvent under reduced pressure afforded 30 g of a light browngum. Aliquots of this gum (25 g) was fractionated on a sil-ica gel column (5 cm × 70 cm) eluted with n-hexane/CHCl3/EtOAcmixtures of increasing polarity (100/0/0, 8/2/0, 5/5/0, 0/100/0,0/9/1, 0/3/7, respectively) to give fractions 1–11. A pre-liminary thin layer chromatography test for coumarins wasconducted using anisaldehyde reagent as well as observationunder UV 254 and 366 nm. Based on the results of this exper-iment, fraction 4 (5 g) was selected and purified on a silicagel column (3 cm × 50 cm) [n-hexane/EtOAc (19/1) to EtOAc],to afford nine fractions (4.1–4.9). Silica gel chromatography(2 cm × 120 cm) [n-hexane/CHCl3/EtOAc (12/7/1–2/9/9)] of frac-tion 4.5 afforded five fractions (4.5.1–4.5.5). Gel permeationchromatography (GPC) of fraction 4.5.1 gave sanandajin (1)(10 mg). Column chromatography on silica gel (1.5 cm × 120 cm)[n-hexane/CHCl3/EtOAc (8/11/1–4/8/8)] of fraction 4.5.3 gave fourfractions (4.5.3.1–4.5.3.4). Methyl galbanate (2) (18 mg) was iso-lated from fraction 4.5.3.1 on a reversed-phase (C18) column(1 cm × 170 cm) [C3H6O/H2O (8/2)].

Fraction 4.8 was more purified on a silica gel column(2 cm × 100 cm) [CHCl3/EtOAc (9/1–4/6)], to afford ethyl galbanate(3) (15 mg) and four other fractions (4.8.1–4.8.4). Fekrynol acetate(4) (10 mg) was obtained from fraction 4.8.3 on a semi-preparativeRP-HPLC using MeOH in H2O (75–100% MeOH) as mobile phase.Silica gel column chromatography (3 cm × 70 cm) [n-hexane/EtOAc(7/3–4/6)] of fraction 8 (2.0 g) afforded five fractions (8.1–8.5).Farnesiferol B (5) (12 mg) was obtained from fraction 8.1 on asilica gel column (2 cm × 150 cm) [n-hexane/EtOAc (19/1–1/1)] fol-lowed by semi-preparative RP-HPLC using MeOH in H2O (80–100%MeOH) as mobile phase. Kamonolol acetate (6) (20 mg) was puri-fied from fraction 8.4 on a silica gel column (1.5 cm × 120 cm)[n-hexane/CHCl3 (8/2–1/9)].

2.3. Phytotoxicity bioassay

Phytotoxicity of the purified compounds extracted from theroots of F. pseudalliacea was assessed on suspension-culturedtobacco (Nicotiana tabacum L. cv. Burley 21) cells as a plant modelcell line. Suspension cultures established from calli of tobaccocells (N. tabacum L. cv. Burley 21) that had been maintainedin our laboratory for 252 subcultures. Both calli and subse-quent suspensions were grown in a modified MS (Murashigeand Skoog, 1962) medium without glycine and containing 3%sucrose. The medium was contained: NH4NO3, 20.61 mM; KH2PO4,1.25 mM; CaCl2, 2.99 mM; MgSO4, 1.50 mM; MnSO4, 0.1 mM; Fe-EDTA, 0.1 mM; H3BO3, 0.1 mM; CoCl2, 0.11 mM; CuSO4, 0.1 mM;Na2MoO4, 1.03 mM; ZnSO4, 29.91 mM, KI, 5 mM, at pH 5.8. Allchemicals were purchased from Sigma (Tokyo, Japan). Suspension-cultured cells were grown at 25 ◦C in darkness on an orbital shakerat 120 rpm and were sub-cultured every 7 days, when they werestill in their logarithmic growth phase (Ghanati et al., 2001). Fre-quent subcultures provided us homogenous and undifferentiatedbatches of tobacco cells. Seven day old tobacco cells were treatedwith 0.83 ppm of each compound for 1 h. After these periods, thecells were harvested on Bukner funnel using nylon mesh (42 �m)under reduced pressure, washed 3 times with fresh media andreturned to the new control MS media. The same procedure offiltration and washing was conducted on control cells. The cellswere allowed to grow in control media for further 1 week in orderto determine the inhibitory effect of tested compounds on their

growth (Yamamoto et al., 1994). The cells were then harvestedand weighed. Phytotoxicity of the tested compounds was deter-mined regarding to the change of fresh weight of tobacco cells inthe control media.

ops an

2

ddoposPIaPCtoa

2

cdwtttm(aFmsw0asAo

F

t

2

ouo1aCt32dTcwbg

I

D. Dastan et al. / Industrial Cr

.4. Seed germination bioassay

Effect of purified compounds extracted from the roots of F. pseu-alliacea was conducted under laboratory conditions using Petriish test. Petri dishes (9-cm diameter) were lined with two layersf filter papers. The filters were moistened with or without com-ounds (0.083 ppm). Forty seeds of common purslane (Portulacaleracea), red-root amaranth (Amaranthus retroflexus), pepper (Cap-icum annum), and eggplant (Solanum melongena) were placed peretri dish. The seeds were obtained from IRIPP (Iranian Researchnstitute of Plant Protection). Three Petri dishes were maintaineds replicates for each treatment in a completely randomized design.etri dishes were placed in a growth chamber (cool White 840limacell 707, Munich, Germany) at 27 ◦C, 12 h/12 h dark/light pho-operiod light intensity of 100 �mol m−2 s−1, and relative humidityf ∼80%. The percentage of seeds germination was measured 7 daysfter treatment.

.5. Radical scavenging capacity and reducing activity

Reducing power of the cells before and after treatment withompounds was determined by an assay that has been previouslyescribed (Bemani et al., 2013). In brief, 1 mL of tobacco cell extractsas mixed with 1.25 mL of 1% potassium ferricyanide. The mix-

ure was incubated at 50 ◦C for 20 min and then 1.25 mL of 10%richloroacetic acid was added to the mixture followed by cen-rifugation at 3000 rpm for 10 min. The supernatant (1.25 mL) was

ixed with distilled water (1.25 mL) and ferric chloride solution0.1%, 0.25 mL). The absorbance was measured at 700 nm. Ascorbiccid at concentration of 10 mg/mL was used as positive control.ree radical scavenging capacity of the cell extracts was deter-ined using DPPH (2,2′-diphenylpicrylhydrazyl) method, with

ome modifications (Bemani et al., 2013). Initially, 0.002% DPPHas dissolved in methanol and 1.5 mL of this solution was added to

.5 mL of the cell extract. The solution mixture was kept in dark atmbient temperature for 30 min, and then its absorbance was mea-ured at 517 nm against a blank of 1.5 mL of 0.002% DPPH solution.scorbic acid (10 mg/mL) was used as positive control. The capacityf cell extract to scavenge free radicals was calculated as follows:

ree radical scavenging capacity (%) = A blank − A sampleA blank

× 100

where A blank is the absorbance of the control and A sample ishe absorbance of the sample.

.6. Evaluation of cytotoxicity

Cytotoxicity of the natural compounds extracted from the rootsf F. pseudalliacea was assessed by determination of their IC50ssing HeLa-60 cell line. The ovarian cancer cell line (HeLa-60) wasbtained from the Pasteur Institute of Iran and was cultured in RPMI640 medium, supplemented with 10% FBS, 100 U/mL penicillin,nd 100 �g/mL streptomycin. The cells were incubated at 37 ◦C, 5%O2, and were subcultured every 4 days. The cytotoxic effects ofhe compounds were investigated by colorimetric bioassay using-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) in a4-well plate (triple holes) (Bemani et al., 2013). Compounds wereissolved in dimethyl sulfoxide (DMSO) to make a stock solution.he DMSO concentration was kept below 0.05% throughout the cellulture and treatment periods. After incubation for 4 h, cells growthas measured by measuring the absorbance of samples at 492 nm

y ELISA Reader (Anthuos 2020, Australia). The percentage of cell

rowth inhibition was calculated as follows:

nhibition % of cancer cells growth = A − B

A× 100

d Products 55 (2014) 43–48 45

where A is optical density of control cancer cells and B is opticaldensity of compound-treated HeLa cells. Since the DMSO concen-tration was kept below 0.05% and did not exert any detectable effecton cell growth or cell death, no inhibition was detected in controlgroup (Meng et al., 2013).

2.7. Statistical analyses

All of the experiments were carried out with independent rep-etitions three times with three samples each. Statistical programSPSS (version 16, Chicago, IL, USA) was used and the significanceof differences between treatments was evaluated using LSD test atlevel of p ≤ 0.05.

3. Results

3.1. Isolated and purified coumarins

Chemical structures of the isolated coumarins from F. pseudalli-acea roots i.e. sanandajin (1), methyl galbanate (2), ethyl galbanate(3), fekrynol acetate (4), farnesiferol B (5) and kamonolol acetate(6) are depicted in Fig. 1. It is noteworthy that sanandajin (1) is thefirst and only disesquiterpene coumarin that has been reported yet.

3.2. Phytotoxicity and seed germination

Inhibitory effects of 1 h treatment with 0.83 ppm of each com-pound on further growth of tobacco cells are shown in Fig. 2. Thehighest inhibitory effect was observed by sanandajin (1), followedby farnesiferol B (5), kamonolol acetate (6), fekrynol acetate (4), andethyl galbanate (3), respectively. The rate of reduction of tobaccocells growth by these compounds were 93%, 82%, 49%, 45%, and39%, respectively (Fig. 2). Pretreatment with methyl galbanate (2)did not inhibit further growth of tobacco cells in normal medium(Fig. 2).

Table 1 shows the percentage of seed germination of two weedand two crop plants of solanaceae (same family of tobacco) aftertreatment with Ferula extracted compounds. Sanandajin remark-ably inhibited seed germination of pepper, eggplant, and commonpurslane with 57%, 83%, and 54%, respectively. This compound alsocaused a ∼30% reduction in red-root amaranth seed germination.While kamonolol acetate, fekrynol acetate, ethyl galbanate, andmehyl galbanate did not significantly inhibit germination of red-root amaranth, inhibitory effect of farnesiferol B was outstanding.All compounds inhibited the germination of eggplant seeds signif-icantly (Table 1).

3.3. Free radical scavenging and reducing capacity of tobacco cells

Fig. 3 shows the capacity of tobacco cells to scavenge DPPHbefore and after 1 h treatment with natural compounds isolatedfrom F. pseudalliacea roots. The capacity of those cells which werepretreated with kamonolol acetate (6), farnesiferol B (5), fekrynolacetate (4), ethyl galbanate (3), and mehyl galbanate were identicalto that of negative controls, while the capacity of radical scaveng-ing of tobacco cells which were pretreated with sanandajin (1) wassignificantly lower than that of control group (Fig. 3).

Similarly, in comparison with control cells, the ferric ion reduc-ing activity of tobacco cells was significantly changed just after 1 htreatment with sanadajin and was not changed in treatment withother compounds (Fig. 4).

3.4. Cytotoxicity

Cytotoxic effects of the six compounds extracted from F. pseu-dalliacea roots on HeLa cancer cells are depicted in Fig. 5. As shown,

46 D. Dastan et al. / Industrial Crops and Products 55 (2014) 43–48

F sananB

t(a

4

SwagkE1eecb

ig. 1. Chemical structures of the isolated coumarins from F. pseudalliacea roots, (1), and (6) kamonolol acetate.

he least IC50 (the highest cytotoxicity) was observed by sanandajin1) followed by kamonolol acetate (6), farnesiferol B (5), fekrynolcetate (4), ethyl galbanate (3), and mehyl galbanate, respectively.

. Discussion

The chemistry of genus Ferula has been widely studied.esquiterpene derivatives, especially sesquiterpene coumarinshich are stored in the roots of the plant are well documented

s biologically active compounds. Fekrynol acetate (4), ethylalbanate (3), methyl galbanate (2), and farnesiferol B (5) are well-nown sesquiterpene coumarins of certain Ferula species (Abdl-Razek et al., 2003; Iranshahi et al., 2007; Veselovskaya et al.,981), which have been detected in F. pseudalliacea as well (Dastan

t al., 2012). Phytotoxic and inhibitory effects of simple coumarins.g., methoxycoumarins, psoralen and xanthotoxin produced fromertain plant roots on the growth of the shoots of other crops haveeen reported (Podbielkowska et al., 1996).

dajin, (2) methyl galbanate, (3) ethyl galbanate, (4) fekrynol acetate, (5) farnesiferol

Pretreatment of suspension-cultured tobacco cells with1.38 �M of disesquiterpene coumarin sanandajin (1), just for 1 h,remarkably inhibited further growth of the cells in normal media.The detrimental effect of sanandajin (1) was also evidenced bydiminished radical scavenging capacity and antioxidant activityof tobacco cells after pretreatment with this compound. Reactiveoxygen species (ROS) are formed during normal metabolism ofthe plant cells by partial reduction of molecular oxygen. They areoverproduced when the cells encounter to different stresses, e.g.,phytotoxic compounds of F. pseudalliacea roots. Scavenging anddetoxification of ROS, is performed by the activity of enzymatic andnon-enzymatic antioxidants and protect their cells from oxidativedamage. In addition to crucial roles in defense system, antioxidantscoordinate growth and development. Most importantly, there is a

relationship between cellular redox state, and regulation of geneexpression associated with biotic and abiotic stress responsesand cell survival. In other words, the lower radical scavengingcapacity of treated tobacco cells, the lower potential to growth and

D. Dastan et al. / Industrial Crops and Products 55 (2014) 43–48 47

0

0.5

1

1.5

2

2.5

FW o

f tob

acco

cel

ls (g

)

a a

bb

b

cd

Δ

Fig. 2. Phytotoxic and inhibitory effects of disesquiterpene and sesquiterpenecoumarins from F. pseudalliacea on the growth of the tobacco cells. Data are pre-sa

wsecdh

nc(eogr5esfo

dcwsl

mtHcese

b

70

74

78

82

86

90

Rad

ical

scav

engi

ng c

apac

ity

(DPP

H%

) b b b b b c

a

Fig. 3. The capacity of tobacco cells to detoxify DPPH free radicals before and aftertreatment with coumarins of F. pseudalliacea roots. Data are presented as mean ± SD,n = 3. Different letters show significant differences at p ≤ 0.05 according to LSD.

0.0

0.4

0.8

1.2

Ferr

ic io

ns r

educ

ing

capa

city

(

Abs

700

nm

)

a a

aa b bc

Fig. 4. The ferric ion reducing activity of tobacco cells. Data are presented asmean ± SD, n = 3. Different letters show significant differences at p ≤ 0.05 accordingto LSD.

0

4

8

12

16

20

24

IC50

(M

)

a

b b c c

d

by accumulation of free amino acids and ammonium in the cells.

TEr

ented as mean ± SD, n = 3. Different letters show significant differences at p ≤ 0.05ccording to LSD.

ithstand against applied coumarines. Disesquiterpen coumrineanandajin also remarkably inhibited seed germination of pepper,ggplant, and common purslane. Therefore, it seems that thisompound causes cell death in all phases of plant growth andevelopment and more likely can be introduced as a none-selectiveerbicide.

The second compound in F. pseudalliacea root with phytotoxicityear to that of sanandajin (1) was farnesiferol B (5). Although thisompound is also a sesquiterpene coumarin, as kamonolol acetate6), fekrynol acetate (4), and ethyl galbanate (3), its phytotoxicffect was much higher than those of the others, probably becausef its structural differences with others especially a polar hydroxylroup. Farnesiferol B remarkably inhibited seed germination of red-oot amaranth, eggplant, common purslane and pepper with 76%,0%, 39% and 9%, respectively. Regard to remarkable suppressorffects on germination of red-root amaranth, which is very aggres-ive and quickly becoming a significant weed problem in crop fields,arnesiferol B can be suggested as a potential herbicide for controlf invasive weeds.

The level of phytotoxicity of isolated compounds from F. pseu-alliacea roots prompted us to assess their cytotoxicity on humanancer cells. Interestingly, the order of cytotoxicity of compoundsas more or less similar to that of their phytotoxicity. So that

anadajin and methyl galbanate (2) showed the highest and theowest cytotoxicity with IC50 of 2.2 and 18.5 �M, respectively.

Phytotoxicity of sanandajin (1) on tobacco cells and seed ger-ination on pepper, eggplant, and common purslane was better

han the other compounds, as well as its highest potency againsteLa cells. Sanandajin (1) as the first disesquiterpene coumarinontain a common coumarin group and a two sesquiterpene moi-ty showed significant results and highlights the importance oftudying the biological and pharmaceutical properties of dis-

squiterpene coumarins as lead compound.

Exposure to micromolar levels of coumarins caused severe inhi-ition of growth of suspension-cultured carrot cells accompanied

able 1ffect of disesquiterpene and sesquiterpene coumarins from F. pseudalliacea on seed germetroflexus.

Germination (% ± SE)

Ctrl Sanandajin Methyl galbanate Eth

Capsicum annum 66.6 ± 6 28.3 ± 6 63.3 ± 5 58Solanum melongena 81.6 ± 3 13.3 ± 8 35.1 ± 3 16Portulaca oleracea 51.6 ± 7 23.3 ± 4 40.1 ± 6 54Amaranthus retroflexus 30.3 ± 5 20.6 ± 5 26.6 ± 4 26

Fig. 5. The cytotoxic effects of disesquiterpene and sesquiterpene coumarins fromF. pseudalliacea on HeLa human cancer cell line. Data are presented as mean ± SD,n = 3. Different letters show significant differences at p ≤ 0.05 according to LSD.

These effects were interpreted in terms of the stimulation of proteincatabolism and/or interference with protein biosynthesis inducedby coumarins (Abenavoli1 et al., 2003). Inhibition of growth and

ination of Capsicum annum, Solanum melongena, Portulaca oleracea, and Amaranthus

yl galbanate Fekrynol acetate Farnesiferol B Kamonolol acetate

.3 ± 6 58.3 ± 5 60.2 ± 6 64.6 ± 4

.6 ± 4 15.2 ± 3 41.6 ± 3 30 ± 6

.1 ± 3 50.5 ± 3 31.6 ± 3 45.1 ± 4

.6 ± 2 26.6 ± 5 6.6 ± 2 23.3 ± 5

4 ops an

cao

ibhtttdanacv2oo

cochikwofoaafh

5

sdp

R

A

A

A

A

B

B

B

B

C

8 D. Dastan et al. / Industrial Cr

ell cycle progression and interaction with double helix of DNA aremongst other proposed mechanisms for cytotoxic effects of furan-coumarins (Lacy and O’Kennedy, 2004; Taiz and Zeiger, 2010).

It has been shown that some furanocoumarins stronglynhibited CYP3A4 which are related to drug excretion from theody or its detoxification in the cells (Iwanaga et al., 2010). Itas been hypothesized that the most prominent biological fea-ures of sesquiterpene coumarins of the genus Ferula are relatedo the inhibition of ABC transporters by these compounds. Inhibi-ion of human P-gp and similar efflux pumps of bacteria resulting inecrease of resistance to antibiotics have been documented (Nazarind Iranshahi, 2011). Bocca et al. explained the possible mecha-ism for cytotoxic activity of ferulenol, a sesquiterpene coumarin,nd proved that ferulenol follows the same mechanism as similaroumarin compounds tested before. So that it causes cytotoxicityia impairing the microtubular system of tumor cells (Bocca et al.,002; Schiff et al., 1979). Lee et al. attributed the antitumor activityf farnesiferol C to the inhibition of cell proliferation and inhibitionf vascular endothelial growth factor (Lee et al., 2010).

None of these mechanisms have been tested for sesquiterpeneoumarins so far. While all above-mentioned mechanisms may bef relevance for inhibitory effects of disesqui- and sesquiterpeneoumarins of F. pseudalliacea on both plant and human cancer cells,owever further investigations are needed to clarify them. It was

nteresting that sanandajin (and to lower extent, farnesiferol B andamolonol acetate), not only inhibited seed germination of testedeeds and crops, but also suppressed the growth and metabolism

f cultured tobacco cells and the viability of HeLa cells. There-ore, it seems that these compounds inhibit fundamental processf growth and metabolism of the cells e.g., microtubule couplingnd interrupting mitosis. In this respect, these compounds functions dinitroaniline, phosphoramidate, chlorpropham, and prophamorm K category of herbicides according HRAC classification oferbicides) (Vaughn and Lehnen, 1991; Boger et al., 2000).

. Conclusion

In conclusion, the results of the present study suggest thatesqui- and disesquiterpene coumarins may be appropriate can-idates as lead compound, for natural herbicides and potentharmaceuticals for cancer therapy.

eferences

bd El-Razek, M.H., Ohta, S., Ahmed, A.A., Hirata, T., 2001. Monoterpene coumarinsfrom Ferula ferulago. Phytochemistry 57, 1201–1203.

bd El-Razek, M.H., Ohta, S., Hirataa, T., 2003. Terpenoid coumarins of the genusFerula. Hetrocycles 60, 689–716.

benavoli1, M.R., Sorgonà, A., Sidari, M., Badiani, M., Fuggi, A., 2003. Coumarininhibits the growth of carrot (Daucus carota L. cv Saint Valery) cells in suspensionculture. J. Plant Physiol. 160, 227–237.

leksieva, V., Karanov, E., Nikolova, R., Bojilova, A., 1995. Plant growth regulat-ing activity of some phosphorus derivatives of coumarin. J. Plant Physiol. 21,45–51.

arthomeuf, C., Lima, S., Iranshahi, M., Chollet, P., 2008. Umbelliprenin from Ferulaszowitsiana inhibits the growth of human M4Beu metastatic pigmented malig-nant melanoma cells through cell-cycle arrest in G1 and induction of caspasedependent apoptosis. Phytomedicine 15, 103–111.

emani, E., Ghanati, F., Rezaei, A., Jamshidi, M., 2013. Effect of phenylalanine onTaxol production and antioxidant activity of extracts of suspension-culturedhazel (Corylus avellana L.) cells. J. Nat. Med. 67, 446–451.

occa, C., Gabriel, L., Bozzo, F., Miglietta, A., 2002. Microtubule interacting activity

and cytotoxicity of prenylated coumarin ferulenol. Planta Med. 68, 1135–1138.

oger, P., Matthes, B., Schmalfuß, 2000. Towards the primary target of chloroac-etamides – new findings pave the way. Pest Manage. Sci. 56, 497–508.

ragg, G.M., Newman, D.J., 2009. Nature: a vital source of leads for anticancer drugdevelopment. Phytochem. Rev. 8, 313–331.

d Products 55 (2014) 43–48

Dastan, D., Salehi, P., Gohari, A.R., Zimmermann, S., Kaiser, M., Hamburger,M., Khavasi, H.R., Ebrahimi, S.N., 2012. Disesquiterpene and sesquiterpenecoumarins from Ferula pseudalliacea, and determination of their absolute con-figurations. Phytochemistry 78, 170–178.

Duke, S.O., 1990. Overview of herbicide mechanisms of action. Environ. Health Per-spect. 87, 263–271.

Duke, S.O., Dayan, F.E., Romagni, J.G., Rimando, A.M., 2000. Natural products assources of herbicides: current status and future trends. Weed Res. 40, 99–111.

Gawron, A., Glowniak, K., 1987. Cytostatic activity of coumarins in vitro. Planta Med.529, 53–526.

Ghanati, F., Morita, A., Yokota, H., 2001. Selection and partial characterization of aboron tolerant tobacco cell line. Soil Sci. Plant Nutr. 47, 405–410.

Iranshahi, M., Arfa, P., Ramezani, M., Jaafari, M.R., Sadeghian, H., Bassarello, C., pia-cente, S., pizza, C., 2007. Sesquiterpene coumarins from Ferula szowitsiana andin vitro antileishmanial activity of 7-prenyloxycoumarins against promastig-otes. Phytochemistry 68, 554–561.

Iwanaga, K., Hayashi, M., Hamahata, Y., Miyazaki, M., Shibano, M., Taniguchi, M.,Baba, K., Kakemi, M., 2010. Furanocoumarin derivatives in kampo extractmedicines inhibit cytochrome P450 3A4 and P-glycoprotein. Drug Metab. Dis-pos. 38, 1286–1294.

Jiwajinda, S., Santisopasry, V., Ohigashi, H., 2000. Coumarin related compoundsas plant growth inhibitors from two rutaceous plants in Thailand. Biosci. Bio-technol. Biochem. 64, 420–423.

Kajimoto, T., Yahiro, K., Nohara, T., 1998. Sesquiterpenoid and disulphide derivativesfrom Ferula assafoetida. Phytochemistry 28, 1761–1763.

Kapoor, L.D., 1990. Handbook of Ayurvedic Medicinal Plants. CRC Press, Boca Raton,FL.

Lacy, A., O’Kennedy, R., 2004. Studies on coumarins and coumarin related com-pounds to determine their therapeutic role in the treatment of cancer. Curr.Pharm. Des. 10, 3797–3811.

Lee, J.H., Choi, S., Lee, Y., 2010. Herbal compound farnesiferol C exerts antiangiogenicand antitumor activity and targets multiple aspects of VEGFR1 (Flt1) or VEGFR2(Flk1) signaling cascades. Mol. Cancer Ther. 9, 389–399.

Li, Z.H., Wang, Q., Ruan, X., Pan, C.D., Jiang, D.A., 2010. Phenolics and plant allelopathy.Molecules 15, 8933–8952.

Macías, F.A., 1995. Allelopathy in the Search for Natural Herbicide Models ACS Sym-posium Series, vol. 582. American Chemical Society, Washington, DC.

Macías, F.A., Galindo, J.C., Castellano, D., Velasco, R.F., 2000. Sesquiterpene lactoneswith potential use as natural herbicide models (I): trans, trans-germacranolides.J. Agric. Food Chem. 48, 5288–5296.

Meng, H., Li, G., Huang, J., Zhang, K., Wang, H., Wang, J., 2013. Sesquiterpene coumarinand sesquiterpene chromone derivatives from Ferula ferulaeoides (Steud.) Korov.Fitoterapia 86, 70–77.

Murakami, T., Anzai, H., Imai, S., Satoh, A., Kozo, N., Thompson, C.J., 1986. Thebialaphos biosynthetic genes of Streptomyces hygroscopicus: molecular cloningand characterization of the gene cluster. Mol. Gen. Genet. 205, 42–53.

Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bio-assayswith tobacco tissue cultures. Physiol. Plant. 15, 473–497.

Nazari, Z.E., Iranshahi, M., 2011. Biologically active sesquiterpene coumarins fromFerula Species. Phytother. Res. 25, 315–323.

Pandey, D.K., 1996. Phytotoxicity of sesquiterpene lactone parthenin on aquaticweeds. J. Chem. Ecol. 22, 151–160.

Pandey, D.K., Mishra, N., Singh, P., 2005. Relative phytotoxicity of hydroquinoneon rice (Oryza sativa L.) and associated aquatic weed green musk chara (Charazeylanica Willd.). Pesticide Biochem. Physiol. 83, 82–96.

Pimenov, M.G., Leonov, M.V., 2004. The Asian umbelliferae biodiversity database(ASIUM) with particular reference to South-West Asian taxa. Turk. J. Bot. 28,139–145.

Podbielkowska, M., Walenza, M., Dobrzynska, K., Zobel, A.M., 1996. Effect of twofuranocoumarins and three other coumarins on ultrastructure ATPases and acidphosphatases in meristematic cells of Allium cepa root tips. J. Pharmacognosy.34, 96–104.

Schiff, P.B., Fant, J., Horwitz, S.B., 1979. Promotion of micotubular assembly by taxol.Nature 22, 665–667.

Shahverdi, A.R., Saadat, F., Khorramizadeh, M.R., Iranshahi, M., Khoshayand, M.R.,2006. Two matrix metalloproteinases inhibitors from Ferula persica var. persica.Phytomedicine 13, 712–717.

Taiz, L., Zeiger, E., 2010. Plant Physiology, 5th ed. Sinauer Associates, Massachusetts.Vaughn, K.C., Lehnen, L.P., 1991. Mitotic disrupter herbicide. Weed Sci. 39,

450–457.Veselovskaya, N., Sklyar, Y., Savina, A., 1981. Fekrynol and its acetate from Ferula

krylovii. Khim. Prir. Soedin. 17, 589.Weir, T.L., Park, W.-S., Vivanco, J.M., 2004. Biochemical and physiological mecha-

nisms mediated by allelochemicals. Curr. Opin. Plant Biol. 7, 472–479.Yamamoto, Y., Rikiishi, S., Chang, Y., Ono, K., Kasai, M., Matsumoto, H., 1994.

Quantitative estimation of aluminum toxicity in cultured tobacco cells: corre-lation between aluminum uptake and growth inhibition. Plant Cell Physiol. 35,575–583.

Zeng, R.S., Mallik, A.U., Luo, S.M., 2008. Allelopathy in Sustainable Agriculture andForestry. Springer, New York.