Comparative Phytochemical Screening and Cytotoxic Efficacy of Endemic Cephalaria tuteliana

Abstract

Phytochemical investigation of Cephalaria tuteliana (Caprifoliaceae) endemic to Turkey was carried out for the first time. Thirteen compounds were isolated, including 2 triterpenoid sapogenins, 10 saponins, and 1 iridoid glycoside. This is the first report of the isolation of pomolic acid, tormentic acid, and 3-O-α-l-rhamnopyranosyl-(1→3)-β-d-glucopyranosyl hederagenin, not only from the Cephalaria genus, but also from the Caprifoliaceae family. The structures of the isolated compounds were elucidated by a combination of spectroscopy, including one- and two-dimensional nuclear magnetic resonance, and by comparison with the reported data in the literature. Two triterpene sapogenins, hederagenin and oleanoic acid, the latter being the most typical sapogenin of the oleanane type of triterpene saponins, were evaluated by the MTT method for their cytotoxic effect against A549, HeLa, PANC1, and SHSY5Y cancer cells, and the noncancerous HEK293 cell line. The cytotoxic activity of triterpene aglycones was discussed in detail and a structure-activity relationship was established. C. tuteliana may be considered as a useful source for developing new cytotoxic and immunotherapeutic agents due to its biologically active saponin content.

Cephalaria species, family Caprifoliaceae, are located in Europe, the Eastern Mediterranean, East Asia, and Central and North Africa. Forty species are found in Turkey, 24 of which are endemic. ¹ Some of these have been reported to be used as dye material, food additives, and folk medicine from ancient times. ² The species are rich in flavonoids, ³ iridoids, ⁴ alkaloids, ⁵ and especially saponins. ^6,7 The great diversity of secondary metabolites in Cephalaria species requires studies on their biological activities. It has been reported that the species have cytotoxic, antimicrobial, antifungal, antioxidant, hemolytic, and immunomodulatory properties. ^{8 -11} Recently, our research group investigated some species of Cephalaria and their saponins were shown to have remarkable cytotoxic and immunomodulatory properties. ⁹

Saponins and sapogenins have been studied extensively for their diverse structures and important roles in modern drug development in terms of biological activities, including cytotoxic, antimicrobial, antifungal, and immunomodulatory effects. Studies have demonstrated that they have many positive effects, especially on cancer, stimulation of the immune system, and blood cholesterol levels. ¹²

In this study, the secondary metabolites of Cephalaria tuteliana Kus & Gokturk were examined, for the first time. The structures of the isolated compounds were determined using one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy (¹H, ¹³C, heteronuclear single quantum coherence [HSQC], heteronuclear multiple bond correlation [HMBC], and correlation spectroscopy [COSY]). The sapogenins, pomolic acid (1) ¹³ and tormentic acid (2), ¹⁴ 10 known triterpene saponins, namely elmalienosides A and B (3 and 4), ¹¹ davisianosides A and B (5 and 6), ⁶ α- hederin (7), ¹⁵ 3-O-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl hederagenin 28-O-β-d-glucopyranosyl ester (8), ¹⁶ 3-O-β-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl hederagenin 28-O-β-d-glucopyranosyl ester (9), ¹⁷ dipsacoside B (10), ¹⁸ macranthoidin A (11), ¹⁹ and 3-O-α-l-rhamnopyranosyl-(1→3)-β-d-glucopyranosyl hederagenin (12), ²⁰ and 1 iridoid glycoside, namely laciniatoside I (13) ²¹ were obtained. Among these compounds pomolic acid (1), tormentic acid (2), and 3-O-α-l-rhamnopyranosyl-(1→3)-β-d-glucopyranosyl hederagenin (12) were detected in the genus Cephalaria and the family Caprifoliaceae, for the first time. Thus, these 3 compounds may be of assistance as chemotaxonomic markers to differentiate C. tuteliana from other Cephalaria species.

In the light of our studies on the cytotoxic and immunomodulatory activities of triterpenic compounds, ⁹ the cytotoxic activities of the n-butanol extract, compounds 1 - 2, and two common aglycones, oleanoic acid and hederagenin, were examined by the MTT method against cancerous A549, HeLa, PANC1, and SHSY5Y cells, and the noncancerous HEK293 cell line. The cytotoxicity results for 1 - 2, hederagenin and oleanoic acid, which are the most typical sapogenins of Cephalaria species, were compared to determine structure-activity relationships (Figure 1). As seen in Table 1, while pomolic acid and tormentic acid did not show any cytotoxicity, hederagenin and oleanoic acid were moderately active. Hederagenin showed cytotoxic activity against the cancerous A-549, HeLa, PANC1, and SHSY5Y cell lines, as well as the noncancerous HEK-293 cells, with half-maximal inhibitory concentration (IC₅₀) values of 44.5, 30.8, 31.4, 23.0, and 37.0 µM, respectively. Oleanoic acid displayed cytotoxicity against A-549, HeLa, mPANC1, and SHSY5Y cells, with IC₅₀ values of 31.6, 68.9, 44.5 and 18.9, respectively. However, oleanoic acid did not exhibit any cytotoxicity to the noncancerous HEK-293 cell line. Thus, it can be concluded that an extra hydroxyl group (-OH) and different locations of methyl (-CH₃) groups on the aglycone may not result in higher cytotoxicity (Figure 1). Contrary to the view that the extra -OH groups increase the cytotoxic activity due to increase in water solubility, the cytotoxicity of compounds 1 and 2 was less than that of oleanoic acid. It seems that there is no contribution to the cytotoxicity of the extra –OH group in the first ring of tormentic acid. In addition to this, it is clear that the methyl groups at C-29 and C-30 play an important role in the cytotoxicity of triterpene aglycones. Hederagenin was more potent by assay than oleanoic acid, except against the A549 cell line, suggesting that the presence of 23-OH enhances the activity of oleanoic acid.

Figure 1

The viability of cancerous A549, SHSY5Y, HeLa, and PANC1 cells, and noncancerous HEK293 cells after treatment with the n-butanol extract, and compounds 1 - 2 from Cephalaria tuteliana, and aglycones hederagenin and oleanoic acid. Cell viability was determined by MTT assay, normalized to a control (100% viability).

Table 1

The Cytotoxic Activities of n-Butanol Extract, Compounds 1 - 2 and Aglycones Hederagenin and Oleanoic Acid.

Cell lines µM
Compounds	A549	HeLa	PANC1	SHSY5Y	HEK293
1	-	-	-	-	-
2	-	-	-	-	-
Hederagenin	44.5 ± 0.9	30.8 ± 1.5	31.4 ± 1.5	23.0 ± 0.5	37.0 ± 0.9
Oleanoic acid	31.6 ± 1.0	68.9 ± 1.1	44.5 ± 2.5	18.9 ± 1.16	>50
n-Butanol extract	-	-	-	-	-
Doxorubicin	14.0 ± 0.6	10.4 ± 0.3	15.9 ± 2.0	4.7 ± 0.04	25.85 ± 4.0

-, not active.

We previously showed that elmalienosides A and B (3 and 4) ¹¹ produced significant hemolysis and induced a remarkable interleukin 1β secretion; thus, they may be good candidates as immunomodulatory components. Besides that, davisianoside B (6) ⁶ was also evaluated for its cytotoxic activities against A549 and HeLa cancerous cells via the MTT assay. ⁹ Compound 6 had cytotoxic effects that were higher than that of the standard drug doxorubicin under in vitro conditions. Additionally, the toxic effect of 6 was less than that of doxorubicin against the non-cancerous kidney cell line HEK-293.

The results of this investigation show that C. tuteliana has potential immunomodulatory and cytotoxic activities indicating medicinal applications due to the innate immune response and cytotoxic properties of these saponins. Thus, the n-butanol extract of C. tuteliana, which contains these saponins, may be a good source for additive materials for different pharmaceutical products.

Experimental

General

Vacuum liquid chromatography (VLC), medium-pressure liquid chromatography (MPLC), thin-layer chromatography (TLC), and column chromatography (CC) were used. VLC using Lichroprep RP18 (Merck 9303, 25-40 µm) was utilized for initial fractionation. In the second MPLC step, Buchi brand pumps (C-605) and glass columns (15/460, 26/920, 49/230) were used. Silica gel 60 (0.063-0.200, Merck 7734) was employed as an adsorbent for MPLC and CC applications. TLC plates (F₂₅₄ Merck 5554 silica gel) were used to monitor the components, to determine their purity, and to help identify them. The UV active components were observed at wavelengths of 254 and 366 nm. Components were visualized by spraying with 20% H₂SO₄ solution and heating at 120°C.

The structures of the purified compounds were determined using spectroscopic methods. Values of J were recorded in Hertz; dimethyl sulfoxide d ₆ (DMSO-d ₆) was used as solvent, and tetramethylsilane as the internal standard using Varian AS 400 MHz and 600 MHz spectrometers for 1D- (¹³C and ¹H) and 2D-NMR (HMBC, HMQC, COSY) analysis.

Plant Material

Cephalaria tuteliana Kus & Gokturk (Caprifoliaceae) ²² was collected from Istanbul, Kirac, Bahcesehir in July 2012 at about 90 m (N 41 04 358, E 28 41 195) by H. Sumbul and R.S. Gokturk (RS Gokturk 7526). A voucher specimen has been deposited at the Herbarium of the Research and Application Centre of Akdeniz University in Antalya-Turkey.

Extraction, Isolation, and Purification

C. tuteliana plant was dried in dark and dry conditions. The ground plant material (1.6 kg) was extracted with MeOH (5 L × 4) at room temperature to yield 388 g of concentrated material. In the second step, n-BuOH/H₂O (1:1) extraction (each 100 mL × 7) was performed to separate the possible biologically active components. The n-BuOH fraction was extracted with n-hexane (100 mL × 20) to separate nonpolar and oily substances. The dried n-BuOH phase (72 g) was separated by VLC (adsorbent: RP silica gel and solvent system: MeOH: H₂O gradient from 0% to 100% MeOH via 10% increase for each step). Fractions 80:20 and 90:10 (MeOH:H₂O) were combined (25 g) and applied to a silica gel column and eluted with CHCl₃:MeOH:H₂O (from 90:10:0.5 to 61:32:7); 30 subfractions were obtained. Compounds 1 (150 mg), 2 (58 mg), 4 (175 mg), and 7 (38 mg) were obtained directly from this column. Compounds 12 (40 mg), 8 (106 mg), 6 (63 mg), and 9 (108 mg) were obtained from fractions 21, 22, 23, and 25, respectively, by further silica gel open column chromatography eluting with CHCl₃:MeOH:H₂O (from 90:10:1 to 61:32:7 + 5% MeOH). The other VLC fractions 60:40 and 70:30 (MeOH:H₂O) were combined (9 g) and MPLC was applied to this fraction. Fractionation was performed using a silica gel column (26 × 920 mm column, solvent system CHCl₃: MeOH: H₂O, 90:10:0-61:32:7, flow rate 25 mL/min and maximum pressure: 40 bar). Thirteen subfractions were obtained; 8 to 10 were combined (2.8 g) and subjected to further silica gel CC using CHCl₃:MeOH:H₂O (80:20:2-65:35:10). Compounds 3 (60 mg), 5 (47 mg), 10 (110 mg), and 11 (26 mg) were obtained directly from this column. Compound 13 (120 mg) was obtained by another MPLC application (adsorbent: silica gel, column: 26 × 920 mm, flow rate 25 mL/min and solvent system 90:10:1-61:32:7 / CHCl₃:MeOH:H₂O, maximum pressure:40 bar) of 50:50 (MeOH: H₂O) VLC fraction.

In Vitro Cytotoxic Activity Assays

A549, Hela, PANC1, and SHSY5Y cancer cell lines and a normal cell line HEK293 were used for testing cytotoxicity. ⁹ All cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA.). The cell lines were maintained in Dulbecco’s modified Eagle’s medium F12, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (Gibco, NY, USA). The cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. The cells were subcultured twice a week, and cells in the exponential growth phase were used in the experiments.

Cytotoxicity of the n-butanol extract, compounds 1 - 2, hederagenin and oleanoic acid was determined using a modified MTT assay. ²³ The assay principle is based on the cleavage of MTT that forms formazan crystals by cellular succinate dehydrogenases in viable cells. DMSO is added to the wells to dissolve the formazan crystals. Briefly, all cell lines were cultivated for 24 hours in 96-well microplates with an initial cell number of 1 × 10⁵ cells/mL in a humidified atmosphere with 5% CO₂, at 37°C. Then, the cultured cells were treated with different concentrations of compounds (0.5, 5, and 50 µg/mL) followed by incubation for 48 hours at 37°C. Doxorubicin (Sigma, St. Lois, MO, USA) was used as a positive control. The optical density of the dissolved material was measured at 570 nm with UV-Vis spectrophotometer (Thermo Multiskan Spectrum).

The viability (%) was determined by the following formula:

$$\begin{gathered} \mathrm{\%}\mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}=\left[\right(\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d}\mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l}\mathrm{s}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{c}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d})-(\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\left)\right] \\ /\left[\right(\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l})-(\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k}\left)\right]\times 100 \end{gathered}$$

The mean IC₅₀ is the concentration of the agent that reduces cell growth by 50% under the experimental conditions and it is the average from at least 3 independent measurements that will be reproducible and statistically significant. The IC₅₀ values were reported at ±95% confidence intervals. This analysis was performed with Graph Pad Prism 5 (San Diego, CA, USA).

Supplemental Material

Supplemental material - Supplemental material for Comparative Phytochemical Screening and Cytotoxic Efficacy of Endemic Cephalaria tuteliana

Supplemental material, Supplemental material, for Comparative Phytochemical Screening and Cytotoxic Efficacy of Endemic Cephalaria tuteliana by Merve Dagli, Nazli Boke Sarikahya, Ayse Nalbantsoy and Suheyla Kirmizigul in Natural Product Communications