Chemical Constituents of the Aerial Parts of Daucus carota subsp. hispidus Growing in Tunisia

Abstract

Three known polyol menthane monoterpenoids, namely, (4R)-1-p-menthen-6,8-diol (1), (4R)-1-p-menthen-4,7-diol (2), and (1R,2R,4R)-p-menthane-1,2,4-triol (3), and 6 known phenolics (4-9), in addition to β-sitosterol 3-O-glucoside (10), were isolated from the aerial parts of Daucus carota subsp. hispidus (Ball) Heywood (Family: Apiaceae) growing in Tunisia. The structures of the isolated compounds were established depending upon the spectroscopic techniques including one and two-dimensional nuclear magnetic resonance (1D, and 2D NMR) and high resolution mass spectroscopy (HRMS). The absolute configuration of the compounds 1 to 3 was determined using experimental circular dichroism (CD) for the first time. Compounds 1 to 3 were reported for the first time from this plant. Compounds 1 to 3 exhibited no antimicrobial and antioxidant activity using superoxide dismutase-like activities. Compounds 2 and 3 exhibited weak activity, while 1 showed negative cytotoxic activity against human mouth squamous carcinoma (HSC-2) and human cervical cells (HeLa) cancer cells.

Keywords

Apiaceae circular dichroism antimicrobial antioxidant cytotoxicity

In recent years, a huge importance is paid to the chemical and biological investigations of indigenous medicinal plants forming a treasure trove of phytoconstituents with extraordinary diverse biological activities and constituting the best source of novel therapeutically effective drugs and biochemicals useful in pharmaceutical and agrochemical industries. The extraction and isolation of targeted nontoxic and potentially antimicrobial secondary metabolites is becoming of paramount importance to fight the microbial resistance to synthetic drugs and to combat food deterioration caused by either bacterial or fungal infections.¹ North Africa is still a promising source of unexplored medicinal plants making the subject of extensive screenings for natural drugs discovery. As an example, Tunisian steppes (Northern Africa) are rich in plants with ethnopharmacological uses; it has more than 500 species of medicinal aromatic plants.² However, many of them remain untapped. Species belonging to Apiaceae family are flowering aromatic plants endowed with high therapeutic benefits including estrogenic, sedative, diuretic, carminative, and antispasmodic properties.³ Daucus is one of the most popular and economically important genera belonging to family Apiaceae. In Tunisia, this genus includes 16 taxa.⁴ Common carrot, Daucus carota L. s. lat. is the richest taxon in subspecies and shows about nine intraspecific taxa.^4,5 Daucus carota is commonly famous in Tunisian traditional medicine for chilblains treatments, the seeds possess carminative and diuretic properties, and they are widely used as appetite stimulants.⁶ It is reported to contain numerous chemical constituents such as flavonoids, coumarins, alkaloids, and anthocyanes.⁷ Besides, terpenoids are cited as the most widespread class of secondary metabolites in D. carota and other Daucus species.⁸ Due to the wide variety of their biological activities, the number of research studies focused on new terpenoids isolation has grown. Within this context and as a part of our ongoing phytochemical and biological investigations of crude extracts from Tunisian medicinal plants, our interest in the discovery of new bioactive constituents prompted us to initiate, and for the first time in this study, the phytochemical and biological investigations of the dichloromethane extract from D. carota subsp. hispidus collected in Tabarka region (Northwestern of Tunisia).

Herein, this work includes the isolation and the structure elucidation of 3 polyol menthane monoterpenoids reported from this plant for the first time,⁶ well-known phenolic compounds and 1 steroid. The compounds 1 to 3 were subjected to in vitro antioxidant using superoxide dismutase (SOD)-like, antimicrobial, and cytotoxic activity against HSC-2 and human cervical cells (HeLa).

The chemical characterization of the chloroform-soluble portion of the aerial parts of Tunisian D. carota subsp. hispidus led to isolation and identification of 3 known menthane monoterpenoids namely (4R)-1-p-menthen-6,8-diol,^1,9,10 1-p-menthen-4,7-diol,^2,11 and (1R,2R,4R)-p-menthane-1,2,4-triol,^3,12 6 known phenolics,^4-9,13,14 and β-sitosterol 3-O-glucoside (10) (Figure 1).

Figure 1

Isolated compounds from Daucus carota subsp. hispidus.

(4R)-1-p-Menthen-6,8-diol (1) was isolated as a white powder with positive optical $[α]_{D}^{25}$ + 46.0 (c 0.01, MeOH). The molecular formula of compound 1 was constructed as C₁₀H₁₈O₂ depending upon high-resolution electron ionization mass spectrometry that showed a molecular ion peak at m /z 170.1316 (calcd. 170.1307) and indicating 2 degrees of unsaturation. The proton nuclear magnetic resonance (¹H NMR) of compound 1 (Table 1) exhibited characteristic signals of oxygenated proton at δ _H 3.86 brs, 1 olefinic proton at δ _H 5.46 brs, protons of 2 methyl groups at δ _H 1.07 brs and δ _H 1.66 s, along with protons of 2 endocyclic methine groups at δ _H 1.25 (m, H_5a) , δ _H 1.85 (m, H_5b) and δ _H 1.66 (m, H_6a), δ _H 2.00 (m, H_6b). The carbon-13 Nuclear magnetic resonance (¹³C NMR) (Table 1) showed 10 carbon resonances that were characterized depending upon distortionless enhanced polarization transfer (DEPT-135) and heteronuclear Single quantum coherence experiments to 3 methyls at δ _C 23.7, 29.5, and 29.7, 2 methylenes at δ _C 30.6, and 36.7, 1 methine at δ _C 42.3, 1 oxygenated methine at δ _C 71.8, 1 olefinic methine at δ _C 128.7, 1 oxygenated quaternary at δ _C 75.3, and 1 quaternary olefinic at δ _C 137.9. The spectroscopic data of compound 1 possessed a polyol monoterpenoid menthene skeleton.^15

-19 The 1D NMR inferred 1 was a substituted-hydroxycyclohexen-ol and close to isolated m-menthane from Chenopodium ambrosioides.¹⁵ The structure of 1 was further suggested depending upon the detailed 2D NMR such as ¹H-¹H correlation spectroscopy (¹H-¹H COSY) and heteronuclear multiple bond correlation (HMBC). The planar cyclohexene skeleton of 1 was further established on the basis of COSY cross-peaks H₂-H_3a, H₂-H_3b, H_3b-H₄, H₄-H_5b, H_5a-H₆, and H_5b-H₆, along with the ¹H-¹³C long-range correlations of H-6 at δ _H 3.86 brs to 2 olefinic carbons C-1 at δ _C 137.9 (J ²) and C-2 at δ _C 128.7 (J ³). Furthermore, the key HMBC correlations displayed between both the methyl proton H₃-7 at δ _H 1.66 s and the proton H-6 with the oxygenated carbon C-6 at δ _C 71.8 (J ³), and 2 olefinic carbons C-1 (J ²) and C-2 (J ³) confirmed the connectivities of the methyl and hydroxyl groups at C-1 and C-6, respectively. The HMBC correlations of the methyl proton H₃-9 at δ _H 1.07 s to C-4 at δ _C 42.3 (J ³), quaternary hydroxylated carbon C-8 at δ _C 75.3 (J ²), and methyl carbon C-10 at δ _C 29.5 alongside the correlations of H-2 at δ _H 5.46 brs to C-4 (J ³) confirmed the location of hydroxyisopropyl attached to C-4 as well as hydroxylated C-8. From the abovementioned data, 1 was confirmed to be 1-p-menthen-6,8-diol. The Nuclear Overhauser Effect Spectroscopy correlations of H-3 (δ _H 2.00 m)/H-4 (δ _H 1.69), H-4/H-5 (δ _H 1.85 m), and H-5/H-6 (δ _H 3.86 brs) established the α orientation of H-6. The R orientation of C-4 was also confirmed depending upon the positive cotton effect at +263 of the circular dichroism (CD) (Figure 2)^20,21 that was supported by the positive optical rotation.^19,22 Based on all the mentioned data, compound 1 was characterized as (4R)-1-p-menthen-6,8-diol reported for the first time from D. carota subsp. hispidus.

Figure 2

Experimental circular dichroism of compounds 1 to 3.

Table 1

¹H (500 MHz) and ¹³C (125 MHz) NMR of 1 to 3.^{a, b, c, d}

No.	1		2		3
No.	¹H	¹³C; type	¹H	¹³C; type	¹H	¹³C; type
1	-	137.9; s	-	136.7; s	-	71.6; s
2	5.46 brs	128.7; d	5.54 brs	119.6; d	3.53 s	74.9; d
3	1.66 m 2.00 m	30.6; t	1.91 m 2.16 m	33.7; t	1.73 dt (5.0, 10.0) 1.90 m	33.8; t
4	1.69 m	42.3; d	-	71.7; s	-	75.3; s
5	1.25 m 1.85 m	36.7; t	1.58 m 1.68 m	30.6; t	2.02 dd (5.0, 10.0)	29.2; t
6	3.86 brs	71.8; d	1.99 m 2.19 m	22.3; t	1.45 m 1.83 td (10.0, 5.0)	29.6; t
7	1.66 s	23.7; q	3.93 s	65.7; t	1.33 s	27.7; q
8	-	75.3; s	1.63 m	36.7; d	1.59 m	38.5; d
9	1.07 s	29.7; q	0.94 d (10.0)	15.9; q	0.94 d (10.0)	16.6; q
10	1.07 s	29.5; q	0.91 d (10.0)	15.8; q	0.92 d (10.0)	16.7; q

^a J (MHz).

^bMultiplicity of carbons was determined by DEPT-135 and HMQC experiments.

^cs, quaternary; d, methine; t, methylene; q, methyl.

^dCompounds 1 and 2 were measured in CD₃OD, while 3 was measured in CDCl₃.

Compound 2 was isolated as amorphous white powder with a positive optical rotation $[α]_{D}^{25}$ +52.3 (c 0.01, MeOH). The molecular formula of compound was confirmed to be C₁₀H₁₈O₂ by HRCIMS that exhibited a molecular ion peak at m /z 170.1306 (calcd. 170.1307) implying 2 degrees of unsaturation. The ¹H and ¹³C NMR (Table 1) of 2 was very close to those of 1 with the presence of oxygenated exo-methylene at δ _H 3.93 s, and δ _C 65.7, a tertiary hydroxyl group at δ_C 71.7 as well as the disappearance of 1 methyl and 1 oxygenated methane. Additionally, the presence of 2 methyls belongs to isopropyl moiety which were strongly shown in ¹H NMR at δ _H 0.94 d (J = 10.0, H-9) and δ _H0.91 d (J = 10.0, H-10.0) and in ¹³C NMR at δ _C 15.9 (C-9) and δ _C 15.8 (C-10). Comparison of ¹H and ¹³C NMR data of compound 2 with those previously published in the literature¹¹ suggested that 2 is 1-p-menthen-4,7-diol. This was approved by the significant long-range correlations H₇-C₁, H₇-C₂, H₇-C₆, H₇-C₄, and H₁₀-C₄. The R orientation of C-4 was confirmed by the positive cotton effect at +255 of the CD (Figure 2)^20,21 that was supported by the positive optical rotation.^19,22 Subsequently, compound 2 was established as (4R)-1-p-menthen-4,7-diol reported for the first time in D. carota subsp. hispidus.

Compound 3 was determined as C₁₀H₂₀O₃ according to the HRCIMS molecular ion peak at m/z 188.1411 (calcd. 188.1412) deducing only 1 degree of unsaturation. The ¹H and ¹³C NMR (Table 1) of 3 were very close to 1 except for the disappearance of the olefinic protons and carbons along with the appearance of oxygenated proton at δ _H 3.53 s (H-2) and at δ _C 74.9 (C-2) and 2 oxygenated quaternary carbon signals at δ _C 71.6 (C-1) and δ _C 75.3 (C-4). Additionally, the ¹H NMR exhibited upfield shift of 1 methyl (Me-7) by 0.33 ppm to be at δ _H 1.33 s and downfield shift in ¹³C NMR by 4 ppm to be at δ _C 27.7. Thus, ¹H and ¹³C data of 3 compared with those of the literature suggested the structure of p-menthane-1,2,4-triol. The HMBC correlations of H₃-9 and H₃-10 to C-8 at δ _C 38.5 (J ²), the quaternary oxygenated carbon C-4 at δ _C 75.3 (J ³) as well as HMBC correlations of H-2 at δ _H 3.53 s and H-8 at δ _H 1.59 m to C-4 confirmed the location of hydroxyl group in C-4. β-OH orientation in C-1 and C-2 along was deduced from the NOE correlation of H-2 at δ _H 3.53 s with Me-7 at δ _H 1.33 s. The positive cotton effect at +244 of the CD (Figure 2) deduced the R orientation of C-4^20,21 that can be more supported with the positive optical rotation in methanolic solution +53.3(c 0.01, MeOH).^19,22 From all these mentioned data, compound 3 was constructed as (1R,2R,4R)-p-menthane-1,2,4-triol reported for the first time from D. carota subsp. hispidus.

Antimicrobial Activity Assay

Compounds 1 to 3 did not show antimicrobial activity against the used strains of microorganisms.

Superoxide Dismutase-Like Activity Assay

Compounds 1 to 3 did not exhibit antioxidant activity using SOD assay as Vitamin C was used as a positive control (IC₅₀: 62.5 µM).

Cytotoxic Activity Against HeLa and HSC-2 Cancer Cells

Monoterpenoids 1 to 3 were in vitro evaluated against the cancer cell lines, HSC-2 mouth squamous cell carcinoma, and HeLa (Supplemental Figure S28) using a modified 3- [4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.²¹ Compounds 2 and 3 exhibited weak activity with higher responses of HSC2 than HeLa [2 > 3 (at 100 μM)] but compound 1 exhibited no activity against 2 cancer cells.

Experimental

General Experimental Procedures

Optical rotations were recorded on a JASCO P-2300 polarimeter (Tokyo, Japan). ¹H (500 MHz), and ¹³C NMR (125 MHz) spectra were recorded on a Bruker 500 NMR spectrometer (United States). The chemical shifts were given in δ (ppm), and the coupling constants were reported in Hz. High resolution mass spectroscopy spectra were obtained on a JEOL JMS-700 instrument (Tokyo, Japan). Circular dichroism was displayed using CD spectra: JASCO 810 spectropolarimeter. Column chromatography (CC) was carried out on normal-phase silica gel column, Silica gel 60 (Merck, 230-400 mesh; Merck, Darmstadt, Germany). Precoated silica gel plates (Merck, Kieselgel 60 F₂₅₄, 0.25 mm; Merck, Darmstadt, Germany) and precoated RP-18 F_254S plates (Merck, Darmstadt, Germany) were used for thin layer chromatography analysis; and detection was achieved by spraying with (1:9) H₂SO₄-MeOH followed by heating. High-performance liquid chromatography (HPLC) was performed on a Jasco PU-980 pump intelligent HPLC pump equipped with a Jasco UV-970 intelligent UV/VIS detector at 210 nm. A semipreparative reversed-phase column (Supelco C18 column 250 × 10 mm, 5 µm) was used for HPLC. Isolera one flash chromatography (Biotage, Suite C Charlotte, NC, United States) was used for flash chromatography and purification.

Plant Material

Aerial parts of D. carota subsp. hispidus were collected in Tabarka region (Northwestern of Tunisia) on September 2016 and identified by one of the authors (R.E.M.). A voucher specimen (AP/D-c-hsp; 00127/2016) was deposited in local herbarium at the Department of Botany and Plant Biology, Faculty of Pharmacy of Monastir, Tunisia.

Extraction and Isolation

Daucus carota subsp. hispidus aerial parts (2200 g) were air-dried under room temperature until constant weight to be coarsely powdered. The pulverized plant sample was macerated exhaustively in methanol until complete extraction to afford 140 g of crude methanol extract obtained after filtration and solvent evaporation under vacuum. The methanol extract thus obtained was suspended in water and partitioned between dichloromethane, ethyl acetate, and butanol to yield 78, 4, and 21 g of crude extracts successively. The dichloromethane extract was subjected to first chromatographic separation over a normal-phase silica gel stationary phase eluted with n-hexane gradually increased with EtOAc and MeOH to afford 20 fractions (F1-F20). Fraction F18 (1.5 g) was further chromatographed over a normal-phase silica gel CC eluted with a solvent gradient of increasing polarity (CH₂Cl₂/MeOH) starting from pure CH₂Cl₂ to yield 10 subfractions (G1-G10). The subfraction G8 (100 mg) was subjected to RP-18 HPLC (MeOH-H₂O, 5.5:4.5) and afforded compounds 1 (6.3 mg) and 2 (5.6 mg). The subfraction G6 subjected to Isolera one flash chromatography using a mixture of hexane/EtOAc (3:7) as eluent, afforded compound 3 (14.3 mg). The other known compounds (4-9) were isolated and purified using different chromatographic techniques.

Antimicrobial Activity Assay

The isolated monoterpenoids 1 to 3 were evaluated for antimicrobial activity against Bacillus subtilis NBRC 13719 (B. subtilis), Staphylococcus aureus NBRC 100910 (S. aureus), Escherichia coli NBRC 102203 (E. coli), Candida albicans NBRC 1594 (C. albicans), and Trichophyton rubrum NBRC 5807 (T. rubrum) as described by Yoshikawa et al. Ketoconazol (LKT Laboratories, Inc., United States) was used as a reference reagent for the bacterial strains. The antimicrobial activity was performed using microdilution assays as described before.²³

Superoxide Dismutase-Like Activity Assay

The monoterpenoids 1 to 3 were evaluated for SOD-like activity according to the method of Ukeda et al²⁴ using a SOD Assay Kit-WST (Dojindo Lab., Kumamoto, Japan).

Cytotoxicity Assay

Cell Culture

HSC-2 and HeLa were obtained from Japanese Collection of Research Bioresources Cell Bank. Both cells were cultured in minimum essential medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin at 37°C in a humidified atmosphere of 5% CO₂.

Cell Viability Assay

Cytotoxic activity of the isolated monoterpenoids 1 to 3 was performed using a modified MTT assay as described before.²⁵ A 100 µL aliquot of the cells suspension (5 × 10⁴ cells/mL) was seeded into each well of the 96-well microplate and incubated at 37°C in a 5% CO₂ for 18 to 24 hours. After incubation, culture medium was discarded and a 100 μL of culture medium containing the tested compounds was incubated at 37°C in a 5% CO₂ for 48 hours. After incubation, the culture medium was discarded and a 100 µL of culture medium containing 0.5 mg/mL MTT was added into each well and incubated at 37°C in a 5% CO₂ for 4 hours. After incubation, the culture medium was discarded and the cells were lysed by the addition of 100 µL into each well. The absorbance of the converted dye was measured at a wavelength of 570 nm with background subtraction at 620 nm using microplate reader Model 680 (BioRad).

Statistical Analysis

Statistical analysis was conducted using one-way analysis of variance. P < 0.05 was considered to indicate the statistical significance.

(4R)-1-p-Menthen-6,8-Diol (1)

$[α]_{D}$ : +46.0 (c 0.01, MeOH).

Rf: 0.43 (n-hexane-EtOAc, 3:2).

¹H NMR (500 MHz) and ¹³C NMR (125 MHz, CD₃OD): Table 1

CI-MS (EI): m/z (%) =170 [M] (10), 152 (85), 109 (100), 79 (75), 59 (93), 43 (38).

HRCI-MS: m/z 170.1316 [M], [calcd for C₁₀H₁₈O₂: 170.1307].

4R-1-p-Menthen-4,7-Diol (2)

$[α]_{D}$ : +52.3 (c 0.01, MeOH).

Rf: 0.46 (n-hexane-EtOAc, 3:2).

¹H NMR (500 MHz) and ¹³C NMR (125 MHz, CD₃OD): Table 1

HRCI-MS (CI, positive mode): m/z (%) =169 [M-1] (15), 152 (80), 135 (88), 127 (57), 109 (52), 81 (70), 79 (100), 43 (28), HRCI-MS (CI, positive mode): m/z 170.1306 [M], (calcd for C10H18O2: 170.1307).

(1R,2R,4R)-p-Menthane-1,2,4-Triol (3)

$[α]_{D}$ : +53.3 (c 0.01, MeOH).

Rf: 0.58 (n-hexane-EtOAc, 3:2).

¹H NMR (500 MHz) and ¹³C NMR (125 MHz, CD₃OD): Table 1

CI-MS (CI, positive mode): m/z (%) =188 [M] (11), 171 (50), 153 (100), 135 (100), 127 (100), 111 (100), 99 (100), 81 (82), 71(92), 43 (100).

HRCI-MS: m/z 188.1411[M], [calcd for C₁₀H₂₀O₃: 188.1412]

Supplemental Material

Supplementary material - Supplemental material for Chemical Constituents of the Aerial Parts of Daucus carota subsp. hispidus Growing in Tunisia

Supplemental material, Supplementary material, for Chemical Constituents of the Aerial Parts of Daucus carota subsp. hispidus Growing in Tunisia by Saoussen Hammami, Abdelsamed I. Elshamy, Ridha El Mokni, Ali Snene, Kanako Iseki, Hatem Dhaouadi, Yasuko Okamoto, Midori Suenaga, Masaaki Noji, Akemi Umeyama, and Yoshinori Asakawa in Natural Product Communications

Footnotes

Acknowledgments

Dr Elshamy gratefully acknowledges the Takeda Science Foundation, Japan for the financial support.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was also supported by Tokushima Bunri University, Japan, University of Monastir, Tunisia, and National Research Centre Centre, Egypt.

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