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Dill seed oil as a possible contraceptive agent: antiangiogenic effects on endothelial cells

Abstract

Dill (Anethum graveolens L.) essential oil is wide spread in the food, beverage and pharmaceutical sectors. Dill is a member of the Apiaceae (Umbelliferae) family. It has the following biological activities: antioxidant, antifungal, antibacterial, antimicrobial, antihyperlipidemic, antihypercholesterolemic, antispasmodic, antiproliferative and anti-inflammatory. Aqueous extract of dill seed has reported effects on sex hormones and infertility potential. Moreover, boiled dill seed has an impact on reducing labor duration in giving birth. Implantation and placentation are necessary for a healthy pregnancy in the early stages. Angiogenesis is responsible for these essential processes. This study aimed to investigate dill seed oil’s cytotoxic and antiangiogenic effects on rat adipose tissue endothelial cells (RATECs). Dill seed oil showed dose-dependent cytotoxicity on RATECs. It disrupted endothelial tube formation and depolymerized F-actin stress fibers. According to this study, depolymerization of F-actin stress fiber by dill seed oil could inhibit angiogenesis by suppressing endothelial cell proliferation, tube formation and motility. In other words, dill seed oil can be a new anti-angiogenic agent and a novel contraceptive.

Keywords:
Anethum graveolens; Angiogenesis; Dill seed oil; Endothelial cells; Contraceptive

INTRODUCTION

Apium plants belong to the Apiaceae family. They are among the herbs used in traditional medicine (Salehi et al., 2019Salehi B, Venditti A, Frezza C, Yücetepe A, Altuntaş Ü, Uluata S, et al. Apium Plants: Beyond simple food and phytopharmacological applications. Applied Sciences. 2019; 9(17):3547.). There are about 20 types of flowering plants in this genus. (Sowbhagya, Srinivas, Krishnamurthy, 2010Sowbhagya HB, Srinivas P, Krishnamurthy N. Effect of enzymes on extraction of volatiles from celery seeds. Food Chem . 2010;120(1):230-234.; Salehi et al., 2019Salehi B, Venditti A, Frezza C, Yücetepe A, Altuntaş Ü, Uluata S, et al. Apium Plants: Beyond simple food and phytopharmacological applications. Applied Sciences. 2019; 9(17):3547.). They are medium to tall, biennial and perennial plants. They grow to one meter in swampy areas in subtropical and temperate regions. The leaves are pinnate or bipinnate with small white flowers. These plants are grown worldwide for their green leaves, bulbous roots, seeds (fruits), and petioles (Malhotra, 2012Malhotra SK. Celery. In Handbook of Herbs and Spices, (2nd edn). UK; Woodhead publishing limited: Sawston; 2012.; Salehi et al., 2019Salehi B, Venditti A, Frezza C, Yücetepe A, Altuntaş Ü, Uluata S, et al. Apium Plants: Beyond simple food and phytopharmacological applications. Applied Sciences. 2019; 9(17):3547.). It contains the phytochemical components bergapten, flavonoids, glycosides, furanocoumarins, furocoumarin, limonene, psoralen, xanthotoxin, and selinene (Salehi et al., 2019Salehi B, Venditti A, Frezza C, Yücetepe A, Altuntaş Ü, Uluata S, et al. Apium Plants: Beyond simple food and phytopharmacological applications. Applied Sciences. 2019; 9(17):3547.). Anethum graveolens is commercially cultivated for its leaves (celery, smallage), roots (celeriac), seeds, and essential oil because of its characteristic smell and health benefits (Mencherini et al., 2007Mencherini T, Cau A, Bianco G, Della Loggia R, Aquino RP, Autore G. An extract of Apium graveolens var. dulce leaves: Structure of the major constituent, apiin, and its anti-inflammatory properties. J Pharm Pharmcol. 2007;59(6):891-897.; Salehi et al., 2019Salehi B, Venditti A, Frezza C, Yücetepe A, Altuntaş Ü, Uluata S, et al. Apium Plants: Beyond simple food and phytopharmacological applications. Applied Sciences. 2019; 9(17):3547.). Anethum graveolens has various applications in food production as a flavoring ingredient or spice owing to its unique aroma and essential oil. Essential oils have been common constituents of plant foods and spice mixtures for centuries due to their therapeutic effects (Sowbhagya, Sampathu, Krishnamurthy, 2007Sowbhagya HB, Sampathu SR, Krishnamurthy N. Evaluation of size reduction on the yield and quality of celery seed oil. J Food Eng. 2007;80(4):1255-1260.; Sowbhagya, Srinivas, Krishnamurthy, 2010Sowbhagya HB, Srinivas P, Krishnamurthy N. Effect of enzymes on extraction of volatiles from celery seeds. Food Chem . 2010;120(1):230-234.; Salehi et al., 2019Salehi B, Venditti A, Frezza C, Yücetepe A, Altuntaş Ü, Uluata S, et al. Apium Plants: Beyond simple food and phytopharmacological applications. Applied Sciences. 2019; 9(17):3547.). One of the members of the plant family Apiaceae, dill, is an annual or biennial herb (Sintim et al., 2015Sintim HY, Burkhardt A, Gawdea A, Cantrell CL, Astatkiec T, Obour AE, et al. Hydrodistillation time affects dill seed essential oil yield, composition, and bioactivity. Ind Crops Prod. 2015;63:190-196.) Dill grows up to 90-120 cm tall (Shyu et al., 2009Shyu YS, Lin JT, Chang YT, Chiang C, Yang DJ. Evaluation of antioxidant ability of ethanolic extract from dill (Anethum graveolens L.) flower. Food Chem. 2009;115(2):515-521.). It has slender branched stem, finely divided leaves, small umbels (2-9 cm diameter) of yellow flowers, and long spindle-shaped roots. The dill seed is aromatic, carminative, stomachic, mildly diuretic, and stimulant. Most of the time, it is used while flavoring in meats, stews, pastries, and vinegar as a whole or ground (Sintim et al., 2015Sintim HY, Burkhardt A, Gawdea A, Cantrell CL, Astatkiec T, Obour AE, et al. Hydrodistillation time affects dill seed essential oil yield, composition, and bioactivity. Ind Crops Prod. 2015;63:190-196.; Weisany, Raei, Ghassemi-Golezani, 2016Weisany W, Raei Y, Ghassemi-Golezani K. Funneliformis mosseae alters seed essential oil content and composition of dill in intercropping with common bean. Ind Crops Prod . 2016;79:29-38.). Carvone and phellandrene are the most significant EO compounds in this plant, and in the fully grown seeds, d-carvone and d-limonene are the most critical compounds (Weisany, Raei, Ghassemi-Golezani, 2016Weisany W, Raei Y, Ghassemi-Golezani K. Funneliformis mosseae alters seed essential oil content and composition of dill in intercropping with common bean. Ind Crops Prod . 2016;79:29-38.). The quantity of carvone and α-phellandrene largely determines the properties of dill oil, with the characteristics of an herb oil predominating if the carvone content is less than 35% (Callan et al., 2007Callan NW, Johnson DL, Westcott MP, Welty LE. Herb and oil composition of dill (Anethum graveolens L.): Effects of crop maturity and plant density. Ind Crops Products. 2007;25(3):282-287.). The role of medicinal plants in human health services worldwide is undeniable. Moreover, some essential oil (EO) components of the medicinal plants are used in the industry (Weisany, Raei, Pertot, 2015Weisany W, Raei Y, Pertot I. Changes in the essential oil yield and composition of dill (Anethum graveolens L.) as response to arbuscular mycorrhiza colonization and cropping system. Ind Crops Prod . 2015;77:295-306.).

Dill has the following properties: antioxidant (Sintim et al., 2015Sintim HY, Burkhardt A, Gawdea A, Cantrell CL, Astatkiec T, Obour AE, et al. Hydrodistillation time affects dill seed essential oil yield, composition, and bioactivity. Ind Crops Prod. 2015;63:190-196.; Satyanarayana et al., 2004Satyanarayana S, Sushruta K, Sarma GS, Srinivas N, Subba Raju GV. Antioxidant activity of the aqueous extracts of spicy food additives-evaluation and comparison with ascorbic acid in in-vitro systems. J Herb Pharmacother. 2004;4(2):1-10.; Kazemi, 2015Kazemi M. Phenolic profile, antioxidant capacity and anti-inflammatory activity of Anethum graveolens L. essential oil. Nat Prod Res. 2015;29(6):551-553.; Oshaghi et al., 2016Oshaghi EA, Khodadadi I, Tavilani H, Goodarzi MT. Aqueous extract of Anethum Graveolens L. has potential antioxidant and antiglycation effects. Iran J Med Sci. 2016;41(4):328-333.), antifungal (Fatope et al., 2006Fatope MO, Marwah G, Onifade AK, Ochei JE, Al Mahrogi YKS. C-13 NMR analysis and antifungal and insecticidal activities of Oman dill herb oil. Pharm Biol. 2006;44(1):44-49; Kaur, Arora, 2009Kaur GJ, Arora DS. Antibacterial and phytochemical screening of Anethum graveolens, Foeniculum vulgare and Trachyspermum ammi. BMC Complementary Altern Med. 2009;9:30.; Ma et al., 2015Ma B, Ban X, Huang B, He J, Tian J, Zeng H, et al. Interference and mechanism of dill seed essential oil and contribution of carvone and limonene in preventing sclerotinia rot of rapeseed. PLoS One. 2015;10(7):e0131733.), antimicrobial (Delaquis et al., 2002Delaquis PJ, Stanich K, Girard B, Mazza G. Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int J Food Microbiol. 2002;74(1-2):101-109.; Wahba, Ahmed, Ebraheim, 2010Wahba NM, Ahmed AS, Ebraheim ZZ. Antimicrobial effects of pepper, parsley,anddilland their roles in the microbiological quality enhancement of traditional Egyptian Kareish cheese. Foodborne Pathog Dis. 2010;7(4):411-418.), antihyperlipidemic and antihypercholesterolemic (Yazdanparast, Alavi, 2001Yazdanparast R, Alavi M. Antihyperlipidaemic and antihypercholesterolaemic effects of Anethum graveolens leaves after the removal of furocoumarins. Cytobios. 2001;105(410):185-191.; Monsefi et al., 2014Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem. 2014;96(9):1402-1413.; Danesi et al., 2016Danesi F, Govoni M, D’Antuono LF, Bordoni A. The molecular mechanism of the cholesterol-lowering effect of dill and kale: The influence of the food matrix components. Electrophoresis. 2016;37(13):1805-1813.; Zhenjing et al., 2018Zhenjing L, Yibin X, Mengxian L, Qingbin G, Yaxin S, Changlu W, Cheng L. The Antioxidation of Different Fractions of Dill (Anethum graveolens) and Their Influences on Cytokines in Macrophages RAW264.7. J. Oleo Sci. 2018; 67, (12) 1535-1541.), antibacterial (Ma et al., 2015Ma B, Ban X, Huang B, He J, Tian J, Zeng H, et al. Interference and mechanism of dill seed essential oil and contribution of carvone and limonene in preventing sclerotinia rot of rapeseed. PLoS One. 2015;10(7):e0131733.; Delaquis et al., 2002Delaquis PJ, Stanich K, Girard B, Mazza G. Antimicrobial activity of individual and mixed fractions of dill, cilantro, coriander and eucalyptus essential oils. Int J Food Microbiol. 2002;74(1-2):101-109.), antiinflammatory (Kazemi, 2015Kazemi M. Phenolic profile, antioxidant capacity and anti-inflammatory activity of Anethum graveolens L. essential oil. Nat Prod Res. 2015;29(6):551-553.) and antiproliferative (Monsefi et al., 2014Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem. 2014;96(9):1402-1413.; Nakano et al., 1998Nakano Y, Matsunaga H, Saita T, Mori M, Katano M, Okabe H. Antiproliferative constituents in Umbelliferae plants II. Screening for polyacetylenes in some Umbelliferae plants, and isolation of panaxynol and falcarindiol from the root of Heracleum moellendorffii. Biol Pharm Bull. 1998;21(3):257-261.; Tsyganov et al., 2016Tsyganov DV, Krayushkin MM, Konyushkin LD, Strelenko YA, Semenova MN, Semenov VV. Facile synthesis of natural Alkoxynaphthalene analogues from plant Alkoxybenzenes. J Nat Prod. 2016; 79(4):923-928.). Dill is useful in treating several gastrointestinal diseases such as flatulence, gas, indigestion, stomachache, and colic as a folk remedy (Norman, 1990Norman J. The Complete Book of Spices, Dorling Kindersley, New York; 1990.; Duke, 2001Duke JA. Handbook of medicinal herbs. (2nd edn). London: CRC Press; 2001.; Zhenjing et al., 2018Zhenjing L, Yibin X, Mengxian L, Qingbin G, Yaxin S, Changlu W, Cheng L. The Antioxidation of Different Fractions of Dill (Anethum graveolens) and Their Influences on Cytokines in Macrophages RAW264.7. J. Oleo Sci. 2018; 67, (12) 1535-1541.). There is an antispasmodic effect of dill seed oil on the gastrointestinal tract (Fleming, 2000Fleming T. PDR for herbal medicines, New Jersey: Medical Economics Company. 2000.). Moreover, in nursing mothers, dill fruit enhances milk production (Norman, 1990Norman J. The Complete Book of Spices, Dorling Kindersley, New York; 1990.; Zhenjing et al., 2018Zhenjing L, Yibin X, Mengxian L, Qingbin G, Yaxin S, Changlu W, Cheng L. The Antioxidation of Different Fractions of Dill (Anethum graveolens) and Their Influences on Cytokines in Macrophages RAW264.7. J. Oleo Sci. 2018; 67, (12) 1535-1541.). The aromatic water of dill fruit (concentrated dill water) has a soothing effect on the digestive system. It is benefical in relieving hiccups and colic in babies, (Ishikawa, Kudo, Kitajima, 2002Ishikawa T, Kudo M, Kitajima J. Water-soluble constituents of dill. Chem Pharm Bull. 2002;50(4):501-507.), bad breath, cough, cold, flu, and menstrual cramp pains (Weisany, Raei, Ghassemi-Golezani, 2016Weisany W, Raei Y, Ghassemi-Golezani K. Funneliformis mosseae alters seed essential oil content and composition of dill in intercropping with common bean. Ind Crops Prod . 2016;79:29-38.). It has been reported that dill seed extracts in mice have antisecretory and mucosal protective effects (Hosseinzadeh et al., 2002Hosseinzadeh H, Reza-Gholam K, Ameri M. Effects of Anethum graveolens L. Seed extracts on experimental gastric irritation models in mice. BMC Pharmacol. 2002;2:21.). Its antibacterial activity against a panel of rapidly growing mycobacteria with minimum inhibitory concentration (MIC) was in the range of 2-128 µg/ml (Stavri, Gibbons; 2005Stavri M, Gibbons S. The antimycobacterial constituents of dill (Anethum graveolens). Phytother Res . 2005;19(11):938-941.). Dill is beneficial while lowering blood cholesterol levels in hypercholesterolemic animals and humans (Danesi et al., 2016Danesi F, Govoni M, D’Antuono LF, Bordoni A. The molecular mechanism of the cholesterol-lowering effect of dill and kale: The influence of the food matrix components. Electrophoresis. 2016;37(13):1805-1813.). Dill seed has an appetite-enhancing effect (Kazemi, 2015Kazemi M. Phenolic profile, antioxidant capacity and anti-inflammatory activity of Anethum graveolens L. essential oil. Nat Prod Res. 2015;29(6):551-553.).

According to Monsefi et al. (2014Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem. 2014;96(9):1402-1413.) dill seed stimulated progesterone release and affected the menstrual cycle. Flavonoids such as kaempferol, myristicin, and vicenin are present in dill seeds. Among them kaempferol and vicenin have phytoestrogen features (Monsefi et al., 2014Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem. 2014;96(9):1402-1413.; Jana, Shekhawat, 2010Jana S, Shekhawat GS. Phytochemical analysis and antibacterial screening of in vivo and in vitro extracts of indian medicinal herb: anethum graveolens. Res J Med Plant. 2010;4(4):206-212.; Gómez-Coronado et al., 2004Gómez-Coronado DJ, Ibañez E, Rupérez FJ, Barbas C. Tocopherol measurement in edible products of vegetable origin. J Chromatogr A. 2004;1054(1-2):227-233.). Phytoestrogens are nonsteroidal components similar to natural estrogens, an example of which is 17β-estradiol. They attach to alpha and beta estrogen receptors, and result in biological effects such as cell growth, differentiation, and maintaining general homeostasis of reproductive and other systems (Hall, Couse, Korach, 2001Hall JM, Couse JF, Korach KS. The multifaceted mechanisms of estradiol and estrogen receptor signaling. J Biol Chem. 2001;276(40):36869-36872.; Ososki and Kennely, 2003Ososki AL, Kennelly EJ. Phytoestrogens: a review of the present state of research. Phytother Res . 2003;17(8):845-869.)

Successful implantation between a mother and an embryo is a complicated operation. Uterine, immunologic, thrombophilic, and embryonic factors affect this complex process. Successful implantation permits the provision of oxygen and nutrients and leads to adequate placental development. Implantation and placentation are necessary for the early stages of a healthy pregnancy (Jung et al., 2016Jung YW, Kim JO, Rah H, Kim JH, Kim YR, Lee Y, et al. Genetic variants of vascular endothelial growth factor are associated with recurrent implantation failure in Korean women. Reprod BioMed Online. 2016;32(2):190-196.). The process in which new capillaries grow from pre-existing capillaries and postcapillary venules is called angiogenesis. This is a tightly controlled process. Apart from wound healing, the development of the corpus luteum, and embryonic development, this process seldom occurs under normal conditions. However, persistent unregulated angiogenesis causes many problems (Wang et al., 2004Wang S, Zheng Z, Weng Y, Yu Y, Zhang D, Fan W, et al. Angiogenesis and anti-angiogenesis activity of Chinese medicinal herbal extract. Life Sci. 2004;74(20):2467-2478.).

The present study investigated the cytotoxic and antiangiogenic effects of dill seed oil in RATECs. The purpose of this study is to evaluate the potential use of dill seed oil as an angiogenic agent and as a contraceptive.

MATERIAL AND METHODS

Plant material

Ripe seeds of dill (Anethum graveolens L.) were purchased. Kemal Hüsnü Can Başer established the authenticity of the dill seeds and a sample was deposited at the Department of Pharmacognosy Depository with number 2004:34. Crushed seeds were hydrodistilled using a Clevenger type apparatus with 3.6% oil yield on a dry weight basis.

Analysis of the essential oil

The oil was analyzed by capillary GC (Gas Chromatography) and GC/MS using an Agilent GC-MSD (Gas Chromatography - Mass Spectrometry) system.

GC and GC/MS analysis

GC-MS conditions:

The oils were analyzed by capillary GC/MS using an Agilent GC-MSD system (Agilent Technologies Inc., Santa Clara, CA). The column was an HP-Innowax FSC column (Hewlett-Packard-HP, U.S.A.) (60 m × 0.25 mm i.d., with 0.25 μm film thickness). Helium was the carrier gas (0.8 mL/min). The GC oven temperature was kept at 60°C for 10 min and raised 240°C at a rate of 1°C/min. The split flow was at 40 mL min−1 with a 40:1 split ratio. The injector temperature was 250 °C. Mass spectra were taken at 70 eV with a mass range of m/z 35-450.

GC conditions:

The GC instrument was an Agilent 6890N GC system fitted with a FID detector at 300°C. To obtain the same elution order with GC-MS, there was a simultaneous autoinjection on a duplicate of the same column with the same operational conditions.

Identification of compounds

Essential oil components were identified by comparison of their mass spectra with those in the Baser Library of Essential Oil Constituents, Wiley GC/MS Library, Adams Library, and MassFinder Library and confirmed by comparison of their retention indices. A homologous series of n-alkanes were the reference points in the calculation of relative retention indices (RRIs). The relative percentages of the separated compounds were calculated from FID chromatograms.

The analysis results are expressed as the mean percentage as listed in Table 1.

TABLE I
Main components of the dill seed oil. [3]

Cell culture

Growth of Rat Adipose Tissue Endothelial Cells (RATECs (Koparal et al., 2004Koparal AT, Yamaguchi H, Omae K, Torii S, Kitagawa Y. Differential effect of green tea catechins on three endothelial cell clones isolated from rat adipose tissue and on human umbilical vein endothelial cells. Cytotechnology. 2004;46(1):25-36.) occurred in Dulbecco’s Modified Eagle’s Media DMEM (Sigma, St.Louis, MO, USA) which contains 10% heat-inactivated fetal calf serum (Sigma), 9.2% NaHCO3 and 1% penicillin/ streptomycin (Sigma). RATECs were cultured in a humified atmosphere, containing 5% CO2 at 37°C.

In vitro cytotoxicity assay

The colorimetric MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay (Mossmann, 1983Mossmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65(1-2):55-63.) determined the cytotoxic effects of dill seed oil. First, stock solutions of dill seed oil were prepared in dimethyl sulfoxide (DMSO), stored at 4oC, and diluted in a fresh, complete medium. Cells (3x103/ well) were seeded in 96-well microtiter culture plates in a final volume of 100 µl. After attachment for 24 h, the culture medium was removed, and the cells were treated with different concentrations of dill seed oil. There were eight replicate wells per concentration. Untreated controls (medium) and solvent controls (DMSO at a final concentration of 0.1% v/v) were conducted in parallel. After removal of the supernatant fluid, 200 µl/well DMSO was added and the mixture was shaken for 5 min. The absorbance was measured at 570 nm using a using a microplate reader (Bio-Tek, ELX808IU, USA). There were three or more replications in all experiments. Statistical analysis of the MTT assay was performed with SPSS (Statistical Package for Social Sciences) software. One way ANOVA (Analysis of Variance), and Tukey test followed this evaluation. A value of p<0.05 was the threshold for statistical significance.

In vitro angiogenesis assay

Koparal et al. (2004Koparal AT, Yamaguchi H, Omae K, Torii S, Kitagawa Y. Differential effect of green tea catechins on three endothelial cell clones isolated from rat adipose tissue and on human umbilical vein endothelial cells. Cytotechnology. 2004;46(1):25-36.) indicated that RATEC colonies showed the potential to form capillary-like structures when cultured on Matrigel. The matrigel tube formation process was as previously described (Quchi et al., 2004Quchi N, Kobayashi H, Kihara S, Kumada M, Sato K, Inoue T, et al. Adiponectin stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signaling in endothelial cells. J Biol Chem . 2004;279(2):1304-1309.). RATECs were serum starved by culturing in endothelial cell basal medium-2 (EBM-2; Cambrex Bio Sciences, CC3156) with 1% FBS for 4 h. Serum starved cells were plated at a density of 1,5 104 cells/well on Matrigel. It coated the wells of 96-well plates after equilibration with EBM-2 medium (containing dill seed oil when indicated). Endothelial cells formed a network structure at 12 h, when they were cultured on Matrigel. Photographs were taken as representatives by using an Olympus CKX41 microscope.

Cytoskeletal immunofluorescence

Fluorescent phalloidin stained the F-actin cytoskeleton by the method described by Rubin et al., (1991Rubin LL, Hall DE, Porter S, Barbu K, Cannon C, Homer HC, et al. A cell culture model of the blood-brain barrier. J Cell Biol. 1991;115(6):1725-1735.) with slight modifications. Untreated control, solvent control, and dill seed oil-treated RATEC monolayers grown on glass coverslips (Marienfeld, Germany) were fixed with 3.7% (w/v) paraformaldehyde-phosphate buffer saline (PBS) for 15 min at 37 oC. The cells were then permeabilized with 0.5% Triton X-100 (v/v) in PBS for 5 min at 37 oC. Preparations were washed three times with PBS. 5 µg/ml tetramethylrhodamine B isothiocyanate (TRITC)-labelled phalloidin (Sigma) stained the cells for 1 h at 37 oC. RATECs were rinsed with PBS. Fluorescent images were viewed using an Olympus fluorescence microscope.

RESULTS AND DISCUSSION

Chemical analysis of dill seed oil

The GC-GC/MS analysis results are available in Table I. Limonene and carvone were the main components of dill seed oil, with 38% and 56% ratios, respectively.

Cytotoxicity

The effect of dill seed oil on the proliferation of RATECs was examined. The mitochondrial dehydrogenase (MTT) assay, evaluated the effect of dill seed oil on cell proliferation. The percentage of growth inhibition by dill seed oil at various concentrations on RATECs was determined as the percentage of viable-treated cells compared to viable cells of untreated controls. Dill seed oil treatment showed a remarkable dose-dependent decrease in cell viability.

The cells were incubated with increasing doses of dill seed oil (12,5µM, 25µM, 50µM and 100 µM). At the end of four days of treatment, the cell quantity was counted using a tetrazolium salt-based assay. According to the results, the inhibitory effect on cell proliferation was dose and time -dependent (Figure 1).

FIGURE 1
Dill seed oil inhibits cell proliferation. Cell proliferation was measured using the MTT colorimetric assay after one, two, three and four days of exposure. The results are expressed as the mean ± SD. * Indicates a statistically significant difference from the control group by the Tukey test (p< 0.05).

In vitro antiangiogenic activity

Endothelial cells must rearrange themselves into a tube in the later stages of angiogenesis to create a new small blood vessel. The endothelial cell tube formation assay was used here as an in vitro model of this process. In the control and solvent control groups, RATECs on a Matrigel substratum displayed high motility and differentiated into well-defined network-like structures within 12 h. Dose-dependent inhibition of tube formation was observed when dill seed oil (12,5-100 µM) was added at the time of Matrigel planting. Photographs of cellular morphology were taken in four arbitrary areas per well to quantify the inhibitory effect of dill seed oil (Figure 2).

FIGURE 2
Effect of dill seed oil on rat adipose tissue endothelial cell (RATEC) tube formation. (a) Control cells and (b) solvent control cells. RATECs were treated with 12,5 µM dill (c), 25 µM dill (d), 50 µM dill (e), 100 µM dill (f). The images are representative of independent triplicate assays. Scale bar:125 µm.

Disruption of the endothelial cytoskeleton

The organization of the cytoskeletal structure of F-actin filaments is necessary to maintain and modulate cellular morphology, structural integrity, and permeability of the endothelium (Carbajal, Schaeffer, 1999Carbajal JM, Schaeffer RC. RhoA inactivation enhances endothelial barrier function. Am J Physiol. 1999;277(5 Pt 1):955-964.). Therefore, the impacts of dill seed oil on the cytoskeletal architecture were investigated with specific staining of F-actin filaments after 24 h of treatment. F-actin filaments in the center position were the stress fibers, and the periphery of the cells was the dense peripheral bands before applying dill seed oil.

RATECs were grown on coverlips. The endothelial cytoskeleton of the RATECs was in a normal distribution. Dill seed oil treatment caused dramatic disruption of the organization of F-actin filaments within the cells. There were reductions in the density of F actin filaments both in the center and peripheral regions of the cells along with the increased concentration of dill seed oil. According to the F-actin staining, apart from unaligned F-actin bundles along the periphery of the cell, dill seed oil treatment led to the disorganization of straight, parallel stress fibers (Figure 3).

FIGURE 3
Fluorescence microscopic images of F-actin. F-actin filaments were stained with (TRITC)-labeled phalloidin. (a) Control cells and (b) solvent control cells demonstrate stress fibers and normal actin distribution in bundles along the periphery of the cell. Increasing doses of dill seed oil c: 12,5 µM, d: 25 µM, e: 50 µM and f: 100 µM disrupts F-actin filaments. The images are representative of independent triplicate assays. Scale bar: 125 µm.

DISCUSSION

A search for endothelial cell growth inhibitors was the first experimental strategy to examine new antiangiogenic compounds. Activation of endothelial cells, which rest in the parent vessel, occurs by an angiogenic signal. Then, these cells are stimulated to synthesize and release degradative enzymes that allow endothelial cells to migrate, proliferate and finally differentiate to make way for capillary tubules. There is a possibility for any of these steps to be a potential target for pharmacological intervention (Quesada, Muñoz-Chápuli, Medina, 2006Quesada AR, Muñoz-Chápuli R, Medina MA. Anti-angiogenic drugs: from bench to clinical trials. Med Res Rev. 2006;26(4):483-530.)

Many plants are natural sources of antiangiogenic compounds (Liu et al., 2006Liu Z, Schwimer J, Liu D, Lewis J, Greenway FL, York DA, et al. Gallic acid is partially responsible for the antiangiogenic activities of Rubus leaf extract. Phytother Res. 2006;20(9):806-813.). Dill seed oil is a natural plant extract traditionally used as a food additive. Dill seeds flavor cakes and pastries, soups, salads, potatoes, meats, sauerkraut, and pickles (Preedy, 2015Preedy VR. Essential oils in food preservation, flavor and safety. London: Academic Press; 2015.).

According to Monsefi et al. (2014Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem. 2014;96(9):1402-1413.), dill seed oil extract led to the survival of corpus luteum, and enlarged its granulosa lutein cells smooth endoplasmic reticulum (SER), which reacts to the high secretion of progesterone. Dill seed induced prolongation of the diestrus phase of the estrous cycle, and it inhibited ovulation in the following cycle (Monsefi et al., 2006 (a)Monsefi M, Ghasemi M, Bahaoddini A. The effects of anethum graveolens L. on female reproductive system of rats. DARU. 2006(a);14:131-135.; Monsefi et al., 2006 (b)Monsefi M, Ghasemi M, Bahaoddini A. The Effect of Anethum graveolens L. on Female reproductive system. Phytother Res . 2006(b);20(10):865-868.). These results and steroidal contraceptive pill performance are similar, so it is likely that the aqueous extract of dill seed directly acts on the ovary and leads to an increase in progesterone levels by affecting the progesterone-producing cells of the corpus luteum (Monsefi et al. 2014Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem. 2014;96(9):1402-1413.).

The contractive effects of Dill seeds, which have been in the laboratory, on myometers have been observed in an earlier study (Lis-Balchin, Hart, 1997Lis-Balchin M, Hart SA. Preliminary study of the effect of essential oils on skeletal and smooth muscle in vitro. J Ethnopharmacol. 1997;58(3):183-187.; Hekmatzadeh et al., 2014Hekmatzadeh SF, Bazarganipour F, Malekzadehc J, Goodarzi F, Aramesh S. A randomized clinical trial of the efficacy of applying a simple protocol of boiled Anethum Graveolens seeds on pain intensity and duration of labor stages. Complementary Ther Med. 2014;22(6):970-976.). Additionally, oxytocin, which plays an important role in uterine contraction, is released with dill seeds (Hekmatzadeh et al., 2014Hekmatzadeh SF, Bazarganipour F, Malekzadehc J, Goodarzi F, Aramesh S. A randomized clinical trial of the efficacy of applying a simple protocol of boiled Anethum Graveolens seeds on pain intensity and duration of labor stages. Complementary Ther Med. 2014;22(6):970-976.). The report of Zagami et al. (2012Zagami SE, Golmakani N, Kabirian M, Shakeri MT. Effect of dill (Anethum graveolens Linn.) seed on uterus contractions pattern in active phase of labor. Indian J Tradit Knowl. 2012;11(4):602-606.) has shown that dill seed infusion (1 tablespoon whole dill seed seeped in a half or whole cup boiling water for 3-4 min) leads to an increase in the number of contractions and a reduction in the duration of the first stage of labor. The benefits of this plant during the postpartum period were reported by Mahdavian et al. (2001Mahdavian M, Golmakani N, Mansoori A, Hoseinzadeh H, Afzalaghaee M. An investigation of effectiveness of orall Dill extracts on postpartum hemorrhage. J Women Midwifery Infertil Iran. 2001;78(4):19-26.).

According to the report of Hekmatzadeh et al. (2014Hekmatzadeh SF, Bazarganipour F, Malekzadehc J, Goodarzi F, Aramesh S. A randomized clinical trial of the efficacy of applying a simple protocol of boiled Anethum Graveolens seeds on pain intensity and duration of labor stages. Complementary Ther Med. 2014;22(6):970-976.), boiled dill seeds are effective in reducing labor duration.

Because they have some components such as limonene and tannin, dill seeds may increase the contractions of the uterus and may positively affect the delivery process (Hekmatzadeh et al., 2014Hekmatzadeh SF, Bazarganipour F, Malekzadehc J, Goodarzi F, Aramesh S. A randomized clinical trial of the efficacy of applying a simple protocol of boiled Anethum Graveolens seeds on pain intensity and duration of labor stages. Complementary Ther Med. 2014;22(6):970-976.). Their study had compatible results with their previous study. there is no connection between dill and pain labor intensity, and Hekmatzedh et al. (2011). According to them there is no connection between dill and pain and intensity in labor.

The fact that dill caused a decreased fertility index and diestrus phase prolongation, but dill leaf aqueous did not induce these changes was reported by Monsefi et al. (2014Monsefi M, Lohrasbi P, Abpaikar Z, Bakhtiari S. Anti-implantation and anti-fertility potentials of Anethum graveolens L. extracts in rats. Toxicol Environ Chem. 2014;96(9):1402-1413.). They reported that dill seed is useful as a medicinal herb with its infertility potential. In this study, we investigated the in vitro cytotoxic and antiangiogenic effects of dill seed oil on rat adipose tissue endothelial cells.

In this study, there was 38.2% limonene, 1.5% cis-isodihydrocarvone, 56.4% carvone and 0.8% dillapiol in the content of dill seed oil (Table I). Stammati et al. (1999Stammati A, Bonsi P, Zucco F, Moezelaar R, Alakomi HL, von Wright A. Toxicity of selected plant volatiles in microbial and mammalian short term assays. Food Chem Toxicol . 1999;37(8):813-823.) reported that with different concentrations of (S) (+) carvone, inhibition of cell viability and colony-forming ability of Hep-2 cells were found to be dose-dependent. In addition, carvone led to the fragmentation of nuclei which is typical for a condensed apoptotic phenotype in Hep-2 cells. In the angiogenic process, the proliferation and survival of endothelial cells are significant (Loutrari et al., 2004Loutrari H, Hatziapostolou M, Skouridou V, Papadimitriou E, Roussos C, Kolisis FN, et al. Perillyl alcohol is an angiogenesis inhibitor. J Pharmacol Exp Ther. 2004;311(2):568-575.). Lazutka et al. (2001Lazutka JR, Mierauskienė J, Slapšytė G, Dedonytė V. Genotoxicity of dill (Anethum graveolens L.) and pine (Pinus sylvestris L.) essential oils in human lymphocytes and Drosophila melanogaster. Food Chem Toxicol. 2001;39(5):485-492.) reported that essential oil from dill seed has cytotoxic and genotoxic effects on human lymphocytes. According to the results of our studies, the inhibitory effect of dill seed oil on RATEC proliferation depended on dose and time. This result resembles Stammati’s and Lazutka’s works (Stammati et al., 1999Stammati A, Bonsi P, Zucco F, Moezelaar R, Alakomi HL, von Wright A. Toxicity of selected plant volatiles in microbial and mammalian short term assays. Food Chem Toxicol . 1999;37(8):813-823.; Lazutka et al., 2001Lazutka JR, Mierauskienė J, Slapšytė G, Dedonytė V. Genotoxicity of dill (Anethum graveolens L.) and pine (Pinus sylvestris L.) essential oils in human lymphocytes and Drosophila melanogaster. Food Chem Toxicol. 2001;39(5):485-492.).

According to the report of Monsefi et al. (2006Monsefi M, Ghasemi M, Bahaoddini A. The effects of anethum graveolens L. on female reproductive system of rats. DARU. 2006(a);14:131-135.a) dill can be used by women who have irregular menstrual cycles as a regulatory agent or it can be an antifertility agent. Degradation of the basement membrane of existing blood vessels, migration and proliferation of endothelial cells, and organization of endothelial cells into capillary tubes are the processes that can be considered the basic steps of the angiogenesis process (Klagsbrun, Moses 1999Klagsbrun M, Moses MA. Molecular angiogenesis. Chem Biol. 1999;6(8):217-224.). Meanwhile, the ability of endothelial cells to form capillary tubes is a specialized function of this cell type. The result of vascular tube formation is a finely tuned balance between proliferation, migration and differentiation (Soeda et al., 2000Soeda S, Kozako T, Iwata K, Shimeno H. Oversulfated fucoidan inhibits the basic fibroblast growth factor-induced tube formation by human umbilical vein endothelial cells: its possible mechanism of action. Biochim Biophys Acta. 2000;1497(1):127-134.).

In this study, there was a model in which the culture of RATECs on Matrigel causes the formation of tube-like structures, similar to capillary blood vessels characteristic of angiogenesis. Our data suggest that dill seed oil can be a strong angiogenic inhibitor able to decrease tube formation of rat adipose tissue endothelial cells. Furthermore, the results of the cytoskeletal study were similar: F-actin in RATECs was obviously and rapidly disorganized after exposure to dill seed oil.

In summary, the conclusions from the experimental data are as follows: Dill seed oil displayed potent anti-angiogenic activity in vitro, inhibited the proliferation of rat adipose tissue endothelial cells (RATECs), depolymerized F-actin stress fibers, and disrupted endothelial tube formation. These findings indicate that dill seed oil is a new anti-angiogenic agent, and its development as a new contraceptive is possible.

ACKNOWLEDGMENTS

The authors would like to thank Anadolu University, Faculty of Science, Biology Department for their interest and for providing support for the study.

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Publication Dates

  • Publication in this collection
    08 May 2023
  • Date of issue
    2023

History

  • Received
    02 Apr 2020
  • Accepted
    07 Mar 2022
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
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