Bioactivity guided isolation of oxypregnane-oligoglycosides (calotroposides) from the root bark of Calotropis gigantea as potent anticancer agents

Abstract

Bioactivity guided isolation of oxypregnane-oligoglycosides (calotroposides) from the ethanolic extract of root bark of Calotropis gigantea (L.) Dryand. with purple flowers has been performed. Dereplication by NMR and LC-MS analysis confirmed the presence of oxypregnane-oligoglycosides (calotroposides) in the ethyl acetate fraction. One new (7) and six known calotroposides (1–6) were isolated from the most active ethyl acetate fraction of ethanolic extract of Calotropis gigantea and structure elucidation was accomplished by spectroscopic methods (IR, UV, MS and NMR). Isolated calotroposides were investigated for cytotoxic activity against six cancer cell lines (MDA-MB-231, MCF-7, LNCaP, HeLa, K562, and Caco-2) and a non-cancer cell line (HEK-293), where calotroposides 2, 4 and 5 showed highest cytotoxicity (IC₅₀ 8.49 ± 0.41 μM, 6.49 ± 0.15 μM and 9.65 ± 0.44 μM respectively) against MDA-MB-231. Conclusively, calotroposides 2, 4 and 5 have promising anticancer effect against aggressive breast cancer cells and can serve as leads for further structural exploration as anticancer moiety.

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Introduction

Calotropis gigantea (L.) Dryand. (family-Asclepiadaceae) is a glabrous and laticiferous shrub known as “Swallow Wort” or “Milk Weed”, and is distributed mainly in the tropical and subtropical regions of the world.¹ In India, it is used as a traditional medicinal plant with unique medicinal properties. The plant has purgative, alexipharmic and anthelmintic properties and has been reported for the treatment of various diseases i.e. leprosy, ulcers, tumors and diseases of the spleen, liver and abdomen.²

Cardiac glycosides are the most prominent secondary metabolite present in C. gigantea and the plant has been investigated for cardiac glycosides from the latex^1b and leaves.³ Members of this class of compounds have been in clinical use for the treatment of heart failure and atrial arrhythmia. The first generation of glycoside-based anti-cancer drug candidates are under clinical trials.⁴ Second important class of compound, oxypregnane-oligoglycosides also known as calotroposides, have been investigated and isolated from the roots of C. gigantea.⁵ The ethanolic extract of the root of C. gigantea exhibits potent cytotoxic property therefore, the ethanolic extract might be utilized for the development and searching of anti-cancer drug lead molecules.⁶ The steroidal aglycone (pregnanone) also called as calotropone of calotroposides, isolated from the C. gigantea and the cytotoxicity of calotropone were also evaluated and reported against K562 and SGC-7901 cell lines.⁷ Pregnane glycosides, another class of compounds isolated from Caralluma tuberculata are potent cytotoxic agents in the treatment of breast cancer.⁸ Oxypregnane-oligoglycosides (calotroposides) have almost similar structure to the pregnane glycosides. Thus, the cytotoxic activity of ethanolic extract of the root of the C. gigantea and the similarity with the structure of pregnane glycosides drew our interest to isolate the active principles present in the ethanolic extract.

In the present study, we investigated the oxypregnane-oligoglycosides (calotroposides) for anticancer activity and they selectively showed significant anti-breast cancer activity against MDA-MB-231 cancer cell lines.

Results and discussion

Bioactivity guided isolation of calotroposides was carried out from ethanolic extract of the root bark of C. gigantea with purple flower. The ethanolic extract was partitioned into water (F01-CPRB-1), ethyl acetate (F02-CPRB-2) and hexane (F03-CPRB-3) fractions and these fractions were examined for anti-cancer activity as well as subjected to NMR and LC-MS analysis. Ethyl acetate (F02-CPRB-2) fraction was most active against the estrogen receptor independent MDA-MB-231 (breast cancer) cell line. Its nine sub-fractions (F02-1 to F02-9) also retained the bioactivity. One new calotroposides along with six known calotroposides were isolated and characterized from the active (F02-CPRB-2) fraction. All seven pure calotroposides were evaluated against six human cancer cell lines (MDA-MB-231, MCF-7, LNCaP, HeLa, K562, Caco-2) and non-cancer cell line (HEK-293). Three calotroposides 2, 4 and 5, exhibited strong cytotoxicity against MDA-MB-231 cell lines and they were subjected to mechanistic studies.

Profiling of calotroposides

NMR and LC-MS based dereplication, which is the rapid identification of biogenic small molecules present in the mixture, is crucial to the rapid discovery of NCEs. ¹H NMR spectra of water (F01-CPRB-1), ethyl acetate (F02-CPRB-2) and hexane (F03-CPRB-3) fractions of root bark of C. gigantea have been taken and stacked plot showed the selectivity of the calotroposides during fractionation (Fig. 1).

Fig. 1 Stacked plot of ¹H NMR spectra of water (F01-CPRB-1), ethyl acetate (Et-OAc) (F02-CPRB-2) and hexane (F03-CPRB-3) fractions of root bark of C. gigantea.

¹H NMR spectra of ethyl acetate fraction of root bark and aerial parts of C. gigantea indicated the presence of calotroposides and cardiac glycosides respectively, which suggested that the calotroposides are only present in the root barks of C. gigantea (Fig. S1 of the ESI‡). Along with NMR, LC-MS results of ethyl acetate fraction also confirmed the presence of calotroposides in ethyl acetate fraction of C. gigantea as shown in Fig. S2 of the ESI.‡ LC-MS analysis of sub-fraction F02-7 indicated the presence of two isomers detected at m/z 1368 [M + NH₄]⁺ and 1373 [M + Na]⁺ Fig. S3 of the ESI.‡ The purified compounds (1–7) were isolated from ethyl acetate fraction by using Thermo ODS-2, 250 × 10 mm, 5 μm column on waters preparative RP-HPLC.

Structural characterization

The structural characterization of all seven calotroposides (1–7) was accomplished by extensive NMR and Mass analysis as well as by comparison with literature data in case of known compounds.

Compound 1 showed peak at m/z 1043 [M − H]⁻ and 1062 [M + NH₄]⁺ in ESI-MS spectrum, which indicated the molecular weight must be 1044 Da. Which was further confirmed by ESI-HRMS m/z 1067.5525 [M + Na]⁺ and the molecular formula was calculated C₅₆H₈₄O₁₈. IR and UV pattern was same as reported in the literature for this compound and also the ¹H NMR and ¹³C NMR spectra of compound 1 showed signal as reported⁹ and characteristic signals of an oxypregnane-oligoglycosides containing cymarose and oleandrose moieties. Thus the structure was concluded and also reported as 12-O-benzoyldeacetylmetaplexigenin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside.

Compound 2 showed peak at m/z 1187 [M − H]⁻, 1206 [M + NH₄]⁺ and 1211 [M + Na]⁺ in ESI-MS spectrum, confirmed the molecular weight of compound 1188 Da and determined the molecular formula C₆₃H₉₆O₂₁ by ESI-HRMS observed peak at m/z 1211.6285 [M + Na]⁺. IR and UV pattern were similar as compound 1. Compared with the literature chemical shift values of ¹H NMR and ¹³C NMR signals, compound 2 showed signal as reported for characteristic signals of an oxypregnane-oligoglycosides containing cymarose and oleandrose moieties. The compound was identified as 12-O-benzoyllineolon-3-O-β-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside.⁵

Compound 3 showed peak at m/z 1203 [M − H]⁻, 1263 [M + CH₃COO]⁻, 1222 [M + NH₄]⁺ and 1227 [M + Na]⁺ in ESI-MS spectrum, suggested that the molecular weight must be 1204 Da, which is 16 Da more than compound 2. ESI-HRMS was found at m/z 1227.6273 [M + Na]⁺ and the molecular formula was calculated C₆₃H₉₆O₂₂. IR and UV pattern were similar as compound 1 and comparable with reported values and also the ¹H NMR and ¹³C NMR data of compound 3 showed characteristic signals of an oxypregnane-oligoglycosides containing cymarose and oleandrose moieties. The compound was identified as 12-O-benzoyldeacetyl-metaplexigenin-3-O-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside.⁵

Compound 4 showed peak at m/z 1187 [M − H]⁻, 1206 [M + NH₄]⁺ and 1211 [M + Na]⁺, confirmed the molecular weight 1188 Da. ESI-HRMS m/z 1211.6335 [M + Na]⁺ observed and determined the molecular formula C₆₃H₉₆O₂₁. IR and UV pattern were similar as compound 1 and compared with the literature and also the ¹H NMR and ¹³C NMR spectra of compound 4 showed signal same as reported for characteristic signals of an oxypregnane-oligoglycosides containing cymarose and oleandrose moieties. The compound was established as 12-O-benzoyllineolon-3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside.⁹

Compound 5 showed peak at m/z 1203 [M − H]⁻, 1263 [M + CH₃COO]⁻, 1222 [M + NH₄]⁺ and 1227 [M + Na]⁺, suggested that the molecular weight must be 1204 Da, which is 16 Da more than compound 4. ESI-HRMS at m/z 1227.6290 [M + Na]⁺ showed the molecular formula C₆₃H₉₆O₂₂. IR and UV pattern were similar as compound 1 and comparable with reported values. ¹H NMR and ¹³C NMR data of compound 5 showed characteristic signals of an oxypregnane-oligoglycosides containing cymarose and oleandrose moieties. This compound was identified as 12-O-benzoyldeacetylmetaplexigenin-3-O-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranoside.⁹

When we initiated this work, LC-MS data indicated the presence of two new isomers detected at m/z 1368 [M + NH₄]⁺ and 1373 [M + Na]⁺ Fig. S3 of the ESI.‡ During the structural characterization by utilizing various 1D and 2D NMR analysis along with mass analysis, we have found that somebody recently reported one compound having same mass. After detailed analysis and comparison with data of reported compound, we have identified one of our isolated compound (6) similar as reported.¹⁰ We were then quite interested to find out the structure of other new isomer to see the difference in structure and by extensive 2D NMR analysis we have characterised this as compound 7.

Compound 6 showed peak at m/z 1349 [M − H]⁻, 1409 [M + CH₃COO]⁻, 1368 [M + NH₄]⁺ and 1373 [M + Na]⁺, which confirmed the molecular weight 1350 Da. The molecular formula was calculated C₆₉H₁₀₆O₂₆ by ESI-HRMS and observed peak at m/z 1368.7307 [M + NH₄]⁺ and 1373.6911 [M + Na]⁺. Absorption band due to a hydroxyl group (3436.37 cm⁻¹), =C–H (3022.25 cm⁻¹) and carbonyl (1705 cm⁻¹) in IR spectrum and UV pattern showed characteristic absorption at 232.0 nm and 273 nm. The structure of the isolated compound 6 was elucidated by extensive 2D-NMR experiments (COSY, TOCSY, NOESY, HSQC and HMBC) and mass spectral analysis. Six anomeric proton were observed in the ¹H-NMR spectrum at δ 4.44 (1H, dd, J = 9.7, 1.2 Hz), δ 4.67 (1H, dd, J = 9.4, 1.5 Hz), δ 4.74 (1H, dd, J = 9.4, 1.5 Hz), δ 4.76 (1H, dd, J = 9.3, 1.2 Hz) and at δ 4.83 (1H, both dd, J = 9.7, 1.8 Hz) and one anomeric signal at δ 4.91 (1H, dd, J = 9.0, 1.0 Hz) for glucopyranose and this indicated that all the monosaccharide moieties and all the glycosidic linkage are in β orientation as suggested from the coupling constant. Signals at δ 7.43 ppm (2H, t, J = 7.5 Hz), δ 7.54 ppm (1H, t, J = 7.5 Hz), δ 7.95 ppm (2H, d, J = 7.5 Hz) suggested the presence of benzoyl groups. HMBC correlations of CH₃-21 and CH₂-16 to C-20 fixed the position of carbonyl at δ 209.5 ppm. Important HMBC correlations of H-2′, H-6′ and H-12 to C-7′ (δ 165.3 ppm) determined the linkage between 12-O-benzoyllineolon to pregnanone (aglycone) moiety. HMBC correlation of CH₃-18 to C-13 and C-17 and HMBC correlations of CH₃-19 to C-10, C-9, and C-5 established the skeleton of pregnanone (aglycone) moiety. Anomeric proton of β-D-cymarose showing HMBC correlation with C-3 of ring A of pregnanone (aglycone) moiety and proton H-3, showed HMBC correlation with C-1′ of β-D-cymarose at first position of sugar chain. nOe and HMBC correlations suggested the conformations of aglycone moiety (Fig. 2).

Fig. 2 Important nOe and HMBC correlations of the aglycone moiety of calotroposides.

HSQC spectrum of H-6 (δ 5.37 ppm) to C-6 (δ 117.5 ppm) showed a characteristic peak of olefinic proton. This gave COSY correlation to H-7. It also gave HMBC correlation to C-5 at (δ 140.7 ppm). COSY and TOCSY established the spin system CH₂-1/CH₂-2/H-3/CH₂-4 in ring A, H-6/CH₂-7 in ring B, H-8/H-9/CH₂-11/H-12 in ring C, CH₂-15/CH₂-16 in ring D, and H-2′ to H-6′ in benzoyl group. COSY and TOCSY correlations established the spin system for sugar moieties. These experimental values were well in accordance with the recently reported in literature. The compound was identified as 12-O-benzoylisolineolon-3-O-β-D-cymaropyranosyl-(1-4)-β-D-cymaropyranosyl-(1-4)-β-D-oleandropyranosyl-(1-4)-β-D-oleandropyranosyl-(1-4)-β-D-cymaropyranosyl-(1-4)-β-D-glucopyranoside.¹⁰

Compound 7 showed peak at m/z 1349 [M − H]⁻, 1368 [M + NH₄]⁺ and 1373 [M + Na]⁺, which indicated that the molecular weight of compound must be 1350 Da. From the ESI-HRMS observed peak at m/z 1368.7304 [M + NH₄]⁺ and 1373.6860 [M + Na]⁺ and the molecular formula calculated was C₆₉H₁₀₆O₂₆. Almost all 2D NMR spectra were similar to compound 6.

Only NOESY spectrum showed somewhat different correlations. nOe correlation gave a strong evidence of presence of β-D-oleandrose at 5^th position of the glycone portion. One distinguished nOe correlation between H-3′′′′′ and H-1′′′′′ was observed in the compound 7 which indicated that the OCH₃-3′′′′′ was in equatorial position and such correlation is absent in compound 6. It indicated that in compound 6, β-D-cymarose is present while in case of compound 7 β-D-oleandrose sugar is present at 5^th position (Fig. 3). These NMR data established the structure of compound 7 and has been elucidated as 12-O-benzoylisolineolon-3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-glucopyranoside.

Fig. 3 Expanded NOESY spectra showing the difference between two isomers calotroposide 6 and calotroposide 7.

Structures of all purified calotroposides (1–7) have been depicted in the Fig. 4.

Fig. 4 Structures of isolated calotroposides 1–7.

Anti-cancer activity

Three different fractions from the ethanolic extract of C. gigantea were tested against MDA-MB-231, MCF-7 and HEK-293 cell lines. Only ethyl acetate (CPRB-2) fraction showed activity against MDA-MB-231 cells with IC₅₀ of 48.50 ± 2.56 μg mL⁻¹. Water (CPRB-1) and hexane (CPRB-3) fractions were found inactive against all the three cell lines and ethyl acetate (CPRB-2) fraction found inactive against MCF-7 cells considering IC₅₀ more than 100 μg mL⁻¹ as inactive. Further, the nine sub-fractions of ethyl acetate fraction (CPRB-2) were evaluated for cytotoxicity (Fig. 5).

Fig. 5 Anti-cancer activity of (a) three fractions (F-01-CPRB-1), (F-02-CPRB-2) and (F-03-CPRB-3) and (b) nine sub-fractions (F-02-1 to F-02-9) of (F-02-CPRB-2) fraction.

Similar to the fraction CPRB-2, all the sub-factions showed significant cytotoxicity against MDA-MB-231 cancer cell line only. The seven pure isolated compounds (1–7) were examined for cytotoxicity. Anti-cancer activity of these compounds (1–7) against a panel of cell lines in terms of their IC₅₀ has been shown in Table 1.

Table 1 Anti-cancer activity of calotroposides against various cancer cell lines in terms of IC₅₀ (μM) values. IC₅₀ is presented as mean ± SEM calculated from three (n = 3) independent experiments

Comp.	MDA-MB-231	MCF-7	LNCaP	HeLa	K562	Caco-2	HEK-293
1	16.52 ± 0.73	>100	>100	>100	>100	>100	>100
2	08.49 ± 0.41	>100	>100	>100	29.96 ± 1.42	9.42 ± 0.18	>100
3	12.50 ± 0.27	>100	>100	>100	>100	>100	>100
4	06.49 ± 0.15	>100	>100	>100	26.66 ± 1.62	>100	>100
5	09.65 ± 0.44	>100	>100	>100	>100	>100	>100
6	23.26 ± 0.47	>100	>100	>100	>100	>100	>100
7	13.75 ± 0.79	>100	>100	>100	>100	>100	>100
Tam citrate	09.83 ± 0.02	08.77 ± 0.02	16.36 ± 0.01	12.00 ± 0.09	—	10.2 ± 0.09	21.5 ± 0.03
Cisplatin	—	—	—	—	31.45 ± 0.17	—	—
Doxo	01.55 ± 0.14	02.40 ± 0.23	—	—	—	—	—

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All the pure compounds (1–7) were found to be cytotoxic against MDA-MB-231 cell lines, whereas only some compounds were active against other cancer cell lines. None of the compounds showed significant cytotoxicity against MCF-7, LNCaP, HeLa as well as non-cancer HEK-293 cells (as IC₅₀ more than 100 μM). Out of all the active compounds against MDA-MB-231 cells, compound 2 (8.49 ± 0.41 μM), 4 (6.49 ± 0.15 μM) and 5 (9.65 ± 0.44 μM) exhibited lowest IC₅₀ amongst the compounds. In case of leukemia cell lines K562, only compound 2 and 4 showed cytotoxicity with IC₅₀ 29.96 ± 1.42 and 26.66 ± 1.62 μM respectively. Among all the compounds only compound 2 showed cytotoxic effects against Caco-2 cell line with IC₅₀ value 9.42 ± 0.18 μM.

Effect of active compounds on MDA-MB-231 cell morphology

Effect of compounds 2, 4 and 5 on the cellular morphology of MDA-MB-231 cell lines was evaluated at IC₅₀ and sub-IC₅₀ concentrations by phase contrast microscopy. Fig. 6 shows the morphological changes in MDA-MB-231 cell lines induced by calotroposides. Changes in the cellular morphology were clearly observed in the treatment groups. Cell number reduction and cellular shrinkages were evident in all treated groups as compared to vehicle control.

Fig. 6 Effect of compounds 2, 4 and 5 on cellular morphology of MDA-MB-231 cell lines after 24 h treatment at their respective IC₅₀ and sub-IC₅₀ concentrations. Cells were observed and photographed by phase-contrast microscopy (X10). Control, doxorubicin IC₅₀, 2-sub-IC₅₀, 2-IC₅₀, 4-sub-IC₅₀, 4-IC₅₀, 5-sub-IC₅₀, 5-IC₅₀.

Effect of compounds on cell cycle of MDA-MB-231

The effect of compounds 2, 4 and 5 on cell cycle distribution of MDA-MB-231 cell lines was investigated and results are displayed in Fig. 7. There was a significant (p < 0.001) arrest of cell cycle progression in G₀/G₁ phase in all treatment groups in comparison to vehicle control. Compound 4 treated group showed highest G₀/G₁ arrest (79.40 ± 1.21%) at IC₅₀ concentration. In doxorubicin (positive control) treated group, significant (p < 0.001) G₂/M arrest was observed as compared to vehicle control.

Fig. 7 Effect of compounds on cell cycle distribution of MDA-MB-231 cells (a) graph depicting cell cycle distribution treated with compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ concentrations for 24 h (b) histogram showing the comparative cell cycle distribution treated with compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ concentrations for 24 h. ***P < 0.001.

Cell apoptosis induced by treatment of calotroposides

To investigate the mechanism of cell death, MDA-MB-231 cells were treated with calotroposides and subjected to Annexin-V/PI apoptotic assay. The results are shown in Fig. 8. A dose dependent significant (P < 0.05) increase in total apoptotic populations was observed in the treatment groups as compared to vehicle control. Compound 4 showed highest total apoptotic cell population at both IC₅₀ (11.93 ± 0.95%) and sub-IC₅₀ (6.57 ± 0.09%) amongst all calotroposides.

Fig. 8 Effect of calotroposides on apoptosis of MDA-MB-231 cells (a) graph depicting total apoptosis of MDA-MB-231 cells treated with compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ concentrations for 24 h (b) histogram showing the comparative total apoptosis of MDA-MB-231 cells treated with compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ concentrations for 24 h. ***P < 0.001, **P < 0.01, *P < 0.05.

Effect of compounds on mitochondrial membrane potential of MDA-MB-231 cells

Change in the MMP induced by treatment of compounds was investigated in MDA-MB-231 cell lines. Results are shown in Fig. 9. A dose dependent rise in green: red ratio was observed in MDA-MB-231 cells treated with compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ which was significantly high (p < 0.05) as compared to vehicle control. The reduction MMP was more in compound 4 treated cells as indicated by highest (1.06 ± 0.12) green: red ratio at IC₅₀ concentration. Compounds 2 and 5 also exhibited significant (p < 0.05) rise in green: red ratio indicating that there was loss of MMP on treatment of calotroposides.

Fig. 9 Change in MMP of MDA-MB-231 cell lines treated with compounds 2, 4 and 5 (a) graphs exhibiting the change in red green fluorescence by compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ concentrations for 24 h (b) histogram exhibiting comparative change in MMP depicted as green: red ratio by compounds 2, 4 and 5. ***P < 0.001, **P < 0.01, *P < 0.05.

Calotroposides induced production of reactive oxygen species in MDA-MB-231 cells

The involvement of intracellular ROS in the cell death process induced by calotroposides was investigated in MDA-MB-231 cells and the results are depicted in Fig. 10. After 24 h of treatment, there was a significant increase (p < 0.001) in ROS level of all groups treated with compounds 2, 4 and 5 and compound 4 (175.2 ± 5.38) treated group (at IC₅₀) exhibited highest increase in ROS level in comparison to vehicle control. The order of increase in ROS was highest with compound 4 followed by 2 and 5 treated groups at both the IC₅₀ and sub-IC₅₀ concentrations.

Fig. 10 Effect of compounds 2, 4 and 5 on ROS production in MDA-MB-231 cell lines (a) graphs exhibiting the mean ROS generation by compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ concentrations for 24 h with H₂O₂ as positive control (b) histogram exhibiting comparative mean ROS generation by compounds 2, 4, 5 and H₂O₂ with statistical significance. ***P < 0.001.

Effect of active compounds 2, 4 and 5 on expression of apoptotic marker proteins

On the basis of above findings, it is evident that compounds 2, 4 and 5 mediated cell death is due to apoptosis. The alterations in molecular signaling during treatment induced apoptosis are studied by western blotting and results are showed in Fig. 11. Compound 4 treated MDA-MB-231 cells exhibited downregulation of Bcl2 and upregulation of Bax protein in both sub-IC₅₀ and IC₅₀ concentrations. Tumor suppressor protein p21 was also found to be increased in compound 4 treated cells. Expression level of p-Akt and Akt was decreased, and at the same time NFκB was also decreased as compared to vehicle control in the treatment groups.

Fig. 11 Effect of compound 4 treatment at IC₅₀ and sub-IC₅₀ concentrations for 24 h on the expression of apoptosis related proteins in MDA-MB-231 cells. Representative western blots showing expression levels of (a) Bcl2 family proteins Bax and Bcl2, oncoprotein p-Akt and Akt, and (b) NFκB and tumour suppressor, p21.

Effect of lead compounds on migration of MDA-MB-231 cells

Wound healing assay was employed for the purpose of investigating the wound healing potential of MDA-MB-231 cells treated with compounds 2, 4 and 5 at their respective ½IC₅₀ and ¼IC₅₀ concentrations. Fig. 12 illustrated the effect of calotroposides on cell's ability to migrate across gap created in cell monolayer. It was observed that all the three compounds 2, 4 and 5 inhibited cell migration in comparison to the vehicle control. Compounds 2 and 4 were more effective than compound 5 in inhibiting cell migration at both concentrations.

Fig. 12 Effect of compounds 2, 4 and 5 on cell migration of MDA-MB-231 cell lines at their respective ½IC₅₀ and ¼IC₅₀ concentrations in both 0 h and 24 h time points. Cells were observed and photographed by phase-contrast microscope (X10).

Search for new drugs and new chemical entities is a continuous process and plant drugs are of prime importance because of their vast biodiversity as well as adequate therapeutic potential. Numerous plant extracts, active fractions and pure phytomolecules are explored for anticancer potential and many of them are proven to be promising in cure and prevention of cancer.¹¹ In the present exploration we investigated anticancer prospective of C. gigantea a medium-sized shrub, that has been reported for purgative, alexipharmic, and anthelmintic properties and is traditionally employed for treatment for leprosy, leucoderma, ulcers as well as tumors.^12,13 Water, ethyl acetate and hexane fractions of ethanolic extract of root bark of C. gigantea were evaluated for cytotoxicity and it was found that ethyl acetate fraction (CPRB-2) was showing best cytotoxicity against MDA-MB-231 cell lines, however no such activity was observed against MCF-7. In HEK-293, the normal human kidney cell lines, all the extracts were found to be non-toxic. These results indicated that CPRB-2 might exhibit promising effect in its enriched fractions as well as pure form. Based on these findings nine sub-fractions were prepared and evaluated for cytotoxicity. All the sub-fractions exhibited cytotoxic effect against MDA-MB-231 cells and further bioactivity guided isolation of purified compounds were done from these organic fractions. A series of seven calotroposides were isolated and subjected to anti-proliferative properties against six different cancer cell lines of various origins. Among compounds 2, 4 and 5 showed best cytotoxicity against MDA-MB-231 cell lines as evident from lowest IC₅₀. Based on the screening results, compounds 2, 4 and 5 were selected as lead calotroposides and were further investigated for their anti-proliferative activity. These calotroposides were also investigated for anticancer effect in other cancer cell lines, but no promising cytotoxicity was seen. C. gigantea (extracts/fractions/purified compounds) has been investigated for anticancer activity against various cancer cell lines such as myelogenous leukemia K562, human gastric cancer SGC-7901.⁷ Literature also reveals the anticancer potential of calotroposides from Calotropis procera in some cancer cell lines.¹⁰ However no such report are available on the anti-breast cancer activity of calotroposides from C. gigantea.

The cytotoxicity exhibited by the compounds 2, 4 and 5 prompted us to investigate further on other biological parameters to ensure anticancer potential. Cellular morphology of the MDA-MB-231 cells treated with selected calotroposides has shown some evidence of compounds induced cell death. MDA-MB-231 is an aggressive cancer cell line, which exhibits migratory properties. In the wound healing studies, it was seen that the selected calotroposides displayed inhibitory effect on the cellular relocation across the wound. These findings are in support of the cytotoxic effect of the compounds.

The largely known pharmacological mechanism of action of cardiac glycosides is through inhibition of the Na⁺, K⁺-ATPase.¹⁴ Also, cardiac glycosides have been reported as β-adrenergic agonists;¹⁵ showed to upregulate BDNF peptide downstream of melanocortin receptors;¹⁶ showed to inhibit cancer cell by targeting DNA-topoisomerase II cleavable complexes similar to etoposide.¹⁷ Recently, pregnane glycosides were also reported to interfere with steroidogenic enzymes.¹⁸ As steroid and growth factors play critical role in cancer proliferation, therefore inhibition steroidogenesis could be a possible mode of action against cancer. Same report has also showed some degree of pregnane glycosides binding with glucocorticoid receptor.¹⁸ Overall, anti-cancer activity of cardiac glycosides are widely reported, but the mechanism of action of oxypregnane-oligoglycosides involved in specific inhibition of cancer is still not clear.

We further proceeded for specific in vitro assays to evaluate the anticancer potential of lead calotroposides. Cell cycle analysis showed G₀/G₁ phase arrest of the cells in all treatment groups. In order to know the type of cell death caused by the calotroposides, Annexin V/PI apoptosis assay was performed. It has been observed that dose dependent increase in apoptotic population was found in the treatment groups. MMP studies illustrated that MMP was disrupted in all the treatment groups. Moreover, the ROS generation experiment followed similar trend and it has been noted that calotroposides caused increased ROS generation in all the treated groups. The results are in accordance with the previously reported literature where the anticancer potential of various phytomolecules are investigated and validated through similar experiments.^13,19 Release of apoptogenic factors is very critical in the process of apoptosis. The Bcl2 family members play key role in alteration of mitochondrial outer membrane permeability. The ratio of pro-apoptotic protein Bax and anti-apoptotic protein Bcl2 determines cells fate to undergo apoptosis or not.²⁰ Selected calotroposides altered the expression levels of Bax and Bcl2 proteins in MDA-MB-231, which might be the reason for mitochondrial membrane destabilizations as evident from the decrease in MMP. The upregulation of tumour suppressor protein p21 was observed in calotroposides treated group, which is known to be involved in regulation of cell cycle progression.²¹ Activated oncoprotein Akt is responsible for cell survival and suppression of apoptosis and it is found to be overexpressed in majority of cancers by growth factor mediated cell survival pathway.²² The calotroposides treatment downregulated the expression of p-Akt and total Akt in MDA-MB-231 cells, suggesting that apoptosis might be mediated by inactivation of Akt. Furthermore Akt signalling is responsible for regulation of SKP2 protein which is a critical factor in ubiquitin mediated degradation of cell cycle regulatory protein p21.²³ NFκB is a transcription factor which promotes cancer progression and oncogenesis through inhibition of apoptosis and cellular differentiation, as well as promotion of cell proliferation and migration.²⁴ Selected calotroposides decreased the expression NFκB in MDA-MB-231 cells, which could be correlated with our experimental findings of wound healing and Annexin-V/PI apoptotic assay. Previous reports revealed that Akt phosphorylates key regulatory kinase IKK, which brings about degradation of inhibitory protein IκB, leading to activation of NFκB.²⁵

Our results are in agreement with the previously reported research work where it was observed that various compounds form Calotropis species exhibited anticancer activity.^10,26 However we found that, anti-breast cancer activity of purified compounds 2, 4 and 5 from C. gigantea was not yet reported. These findings based on experimental evidences led to our understanding that isolated oxypregnane-oligoglycosides have promising cytotoxic effects on aggressive breast cancer model, MDA-MB-231 cell lines and can be further explored for cancer prevention and treatment.

Conclusions

C. gigantea is traditionally used as a folk medicine and it has been the rich subject for phytochemical as well as bioactive investigations. Its chemical composition is not completely known and bioactivities of all constituents have not been thoroughly investigated yet. Several oxypregnane-oligoglycosides (calotroposides) has been isolated and characterized from the roots of C. gigantea. In our present study, one new (7) and six known calotroposides (1–6) were isolated from the root bark of C. gigantea (purple). The bioactivity of these isolated calotroposides was evaluated for the first time for anti-cancer activity against various cancer cells lines and normal human cell line. Compounds 2, 4 and 5 exhibited strong cytotoxicity amongst all isolated compounds. Active calotroposides caused apoptotic cell death and arrest cells in G₀/G₁ phase, which was also supported by disruption in MMP and increased ROS. Alterations in the expression levels of apoptosis related proteins further confirmed anti-cancer potential of the active calotroposides. These promising in vitro activities may of interest to future calotroposides structure-based drug design and development, particularly against aggressive triple negative breast cancer (TNBC). However, more detailed evaluation including in vivo activity of pure calotroposides compounds is needed for further progress in this area.

Materials and methods

Plant materials

The root bark of C. gigantea with purple flower was collected from Jabalpur, Madhya Pradesh (India) in Dec. 2011. The herbarium of the same has been deposited in the CSIR-CDRI Herbarium with voucher specimen number 24561.

Chemicals and biochemicals for biological activity

Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute medium (RPMI), fetal bovine serum (FBS), antibiotic solution (penicillin/streptomycin, 0.1% v/v), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and ribonuclease A, JC-1 stain, propidium iodide and Annexin V-FITC apoptosis detection kit, phosphate buffered saline, p-formaldehyde, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA) were purchased from Sigma Aldrich, St. Louis, MO, USA. All antibodies used in western blot analysis were purchased form CST, USA. Autoclaved Milli-Q water (Millipore, Milli-Q plus 185, Bedford, MA, US) was used for cell culture studies.

Cell lines

All the cell lines were originally procured from ATCC (Manassas, VA, USA) and maintained in the laboratory. MCF-7, MDA-MB-231 (breast cancer cell lines), HeLa (cervical cancer line) and HEK-293 (normal human epithelial kidney cell line) were maintained in DMEM supplemented with 10% FBS, K562 (leukemia cell line), LNCaP (prostate cancer cell line), were maintained in RPMI supplemented with 10% FBS, Caco-2 (colorectal cancer cell line), were maintained in DMEM supplemented with 15% FBS. Antibiotic/antimycotic solution (1%) was added to culture media and cells were maintained in a CO₂ incubator (5% CO₂) at 37 °C and 95% relative humidity following the standard protocols.

Experimental

General experimental procedures. Melting points was taken on precision micro melting point apparatus and are uncorrected. UV spectra were taken with Shimadzu UV spectrophotometer (UV-1800). Infrared spectra were obtained on a FT-IR spectrophotometer. ESIMS, LCMS data were recorded on the LCQ (Thermo scientific) ion trap and AQUITY TQD mass spectrometers. HRMS were performed on a Q-TOF mass spectrometer. Analytical and preparative RP-HPLC was performed on Waters HPLC using PDA detector. ODS-2 column was used for the method development in analytical and isolation in preparative RP-HPLC. LR grade solvents were used for column chromatography and spectral grade solvents were used for spectroscopic measurements. ¹H, ¹³C, DEPT 135, DEPT 90, COSY, DQF-COSY, HSQC, Edited-HSQC, HMBC and NOESY spectra were recorded on Bruker Av II 400 and 300 MHz spectrometers with the residual solvent signal or TMS as internal reference. Chemical shifts are given in parts per million (ppm) downfield from TMS, and coupling constants are measured in Hz. Silica gel (Merck, 60–120 and 100–200 mesh size) were used for column chromatography (CC) at normal pressure. Compounds were separated on a precoated aluminium plate (Merck, silica gel 60 F254).

Extraction and isolation. Powdered air-dried root bark of C. gigantea (1.1 kg) was repeatedly extracted 3 times with 95% ethanol (3 × 1000 ml) for 72 h. The ethanolic extract was filtered under vacuum and concentrated under reduced pressure using rotavapor at 40 °C. The weight of extract was 138.00 g and coded as CPRB.

The ethanolic extract (CPRB) was partitioned between aqueous and n-hexane (1 : 1), got separated by separating funnel and hexane phase was concentrated under reduced pressure and gave hexane fraction (F03-CPRB-3) (39 g). Ethyl acetate was added into aqueous phase and got it partitioned. This procedure led to the separation into two phases, aqueous and ethyl acetate phases. Both liquid phase concentrated and resulted into water fraction (F01-CPRB-1) (46 g) and ethyl acetate fraction (F02-CPRB-2) (53 g). The ethyl acetate fraction (F02-CPRB-2) was loaded (53 g) on silica gel 60–120 (0.120–0.250 mm) mesh (1 kg) for column (100 cm × 12 cm) chromatography packed in CH₂Cl₂. Stepwise elution was carried out using CH₂Cl₂/MeOH gradient solvent system (100 : 0, 98 : 2, 96 : 4, 94 : 6, 92 : 8, 90 : 10, 85 : 15, 80 : 20, 70 : 30, 60 : 40, 50 : 50 and 0 : 100). TLC pattern was carried out, visualised under UV lamp (254 nm) and similar sub-fractions were collected and concentrated. At the same time all 12 sub-fractions were subjected to LC-MS analysis. Out of 12 sub-fractions [F02-1 (11 g), F02-2 (3 g), F02-3 (5 g), F02-4 (2 g), F02-5 (5 g), F02-6 (4 g), F02-7 (7.5 g), F02-8 (5.5 g), F02-9 (3 g), F02-10 (2 g), F02-11 (2.5 g), F02-12 (10 mg)], 9 sub-fractions contained calotroposides, which was indicated by the dereplication of the ethyl acetate fraction as well as the sub-fractions of the ethyl acetate fraction by LC-MS and MS-MS analysis. Sub-fraction F02-1 was further subjected to column chromatography and yields five fraction (F02-1.1-and F02-1.5) using hexane/ethyl acetate gradient solvent systems (silica-gel 60–120 mesh size, 50 : 50 to 0 : 100). Fractions were submitted for MS-MS analysis and found that sub-fraction F02-1.4 (2 g) contains compound 1 which was further purified by preparative RP-HPLC (ODS-2, H₂O/ACN (50 : 50 to 40 : 60), with flow rate 3.5 mL min⁻¹) and yielded 12 mg. Sub-fraction F02-3 was subjected to column chromatography over silica gel 100–200 mesh size with CH₂Cl₂/MeOH (96.5-3.5–94 : 6) as the eluent to give four sub-factions (F02-3.1 to F02-3.4) among these, sub-fractions F02-3.2 (500 mg) was subjected to LC-MS analysis which indicated the presence of two isomers having ESI-MS m/z 1206 [M + NH₄]⁺ and two isomers having ESI-MS m/z 1222 [M + NH₄]⁺ and this sub-fraction was then applied to preparative RP-HPLC eluted with (H₂O/ACN (62 : 38 to 55 : 45), with flow rate 3.0 mL min⁻¹) yielded compounds 2 (17 mg), 3 (11 mg), 4 (15 mg) and 5 (18 mg). LC-MS analysis of sub-fraction F02-7 was indicated the presence of two isomers having ESI-MS m/z 1368 [M + NH₄]⁺ and 1373 [M + Na]⁺ and then subjected to preparative RP-HPLC with gradient solvent system (H₂O/ACN (70 : 30 to 60 : 40), with flow rate 3.0 mL min⁻¹) gave pure compound 6 (3.5 mg) and 7 (3 mg). The procedure of bioactivity guided isolation has also been shown in a flow chart Fig. S4 of the ESI‡ and LC gradient elution method utilized for the separation of compounds in different fractions and sub-fractions in LC-MS is depicted in Table S1 of the ESI.‡

MTT assay. Anti-cancer activity of water fraction (F01-CPRB-1), ethyl acetate fraction (F02-CPRB-2), hexane fraction (F03-CPRB-3), sub-fractions (F02-1, F02-2, F02-3, F02-4, F02-5, F02-6, F02-7, F02-8, F02-9) and isolated seven calotroposides (1–7) obtained from C. gigantea was carried out using MTT assay.²⁷ Cells at the density of 1 × 10⁴ cells per well were seeded in 100 μL of respective growth medium (as per the specific cell lines), with 10% FBS in 96-well micro culture plates. The well plates were incubated at 37 °C in a humidified CO₂ incubator until cells achieved their normal morphology. Cells were treated with variable concentrations of extracts/fractions/pure compounds which were diluted in growth medium without phenol red, supplemented with 2% FBS for 24 h, with respective vehicle control. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma chemicals, USA, 5 mg mL⁻¹ in PBS) 10 μL was added to each well and plates were incubated for 2 h in CO₂ incubator. Supernatant was removed and formazan crystals were solubilized in 100 μL DMSO. Absorbance was taken at 540 nm in spectrophotometer (MicroQuant, BioTek) and cell inhibition was calculated. The IC₅₀ values were calculated by Graph Pad Prism (5.0).

Cellular morphological analysis. The test compounds induced morphological changes in MDA-MB-231 cells were visualized by phase contrast microscopy.²⁸ The cells were plated at a density of 1 × 10⁴ cells per well in 24-well plates. After overnight incubation, cells were treated with IC₅₀ and sub-IC₅₀ concentrations of compounds 2, 4, 5, positive control doxorubicin (IC₅₀) and vehicle control for 24 h. The induced morphological changes were observed under phase contrast microscope.

Cell cycle analysis. Cell cycle analysis was done employing flow cytometry by propidium iodide fluorescent marker in MDA-MB-231 cell lines.²⁹ Cells were seeded in 6 well plates in the density of 1 × 10⁶ cells per well, treated with IC₅₀ and sub-IC₅₀ concentrations of compounds 2, 4, 5, doxorubicin (IC₅₀) and vehicle control for 24 h and harvested by mild trypsinization. Collected cells were washed with PBS and re-suspended in chilled 70% ethanol for 1 h. After fixation cells were washed 2 times with PBS, treated with RNase A (10 mg mL⁻¹), and then stained with PI (30 μg mL⁻¹; Sigma, USA) for 30 min at room temperature in dark. The DNA content of cells was measured using FACS Calibur flow cytometer (Becton-Dickinson, SanJose, CA, USA) equipped with ModFit LT 2.0 and analyzed with CellQuest software.

Cell apoptosis analysis. Apoptotic cell populations were detected employing Annexin-V apoptosis assay kit as per manufacturer's instructions.³⁰ Cells were seeded in the density of 1 × 10⁶ cells per well in 6 well plates and compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ were given for 24 h along with vehicle control and positive control (doxorubicin; IC₅₀). After 24 h incubation cells were trypsinized, washed with PBS and re-suspended in 1× binding buffer. For double staining, 2 μL Annexin V-FITC (from 50 μg mL⁻¹ stock) and 4 μL of PI (from 100 μg mL⁻¹ stock) were added and cell suspension was incubated in dark for 30 min. Further cell suspension was centrifuged, pellet was re-suspended in 300 μL of binding buffer and analysed in flow cytometer (FACSCalibur, BD Biosciences). Total apoptosis was calculated and plotted against treatment groups.

Measurement of mitochondrial membrane potential (MMP). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenz imidazolylcarbocyanine iodide (JC-1) was used to monitor mitochondrial membrane potential (MMP) changes (ΔΨ_M) in MDA-MB-231 cells.³¹ Cells were seeded in a density of 1 × 10⁶ cells per well in 6 well plates and treatments of compounds 2, 4 and 5 were given at IC₅₀ and sub-IC₅₀ concentrations along with vehicle control and positive control (doxorubicin; IC₅₀) and incubated at 37 °C in CO₂ incubator for 24 h. After incubation cell monolayers were washed with PBS, trypsinized and pelleted. Cell pellets were stained with culture medium containing 5 μg mL⁻¹ JC-1 dye for 15 min, and then analysed in FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA, USA) equipped with ModFit LT 2.0 and analysed with Cell Quest software.

Reactive oxygen species (ROS) detection. Treatment induced reactive oxygen species generation in MDA-MB-231 cells were measured by H₂DCFDA staining.³² Cells were seeded in 6 well plates at the rate of 1 × 10⁶ cells per well and allowed to attain morphology. Compounds 2, 4 and 5 at IC₅₀ and sub-IC₅₀ concentration, positive control (doxorubicin; IC₅₀) and vehicle control were given for 24 h. Cells were harvested, washed with PBS and incubated in medium containing 10 μg mL⁻¹ of H₂DCFDA for 30 min in dark. Cell suspensions were centrifuged and pellets were re-suspended in PBS. Increase in green fluorescence was monitored using FACS Calibur flow cytometer (Becton-Dickinson, San Jose, CA, USA).

Western blot analysis. Expression patterns of proteins involved in cell proliferation and apoptosis were evaluated by western blotting.²⁰ Cells were seeded in T-25 flasks, and treated with sub-IC₅₀ and IC₅₀ of compound 4 and IC₅₀ of doxorubicin as reference standard. Cell monolayers were washed and harvested in 1 mL of cold PBS, and cell suspension was centrifuged (800 rpm for 12 min at 4 °C). Cell pellets were lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% nonidet-P40, 0.5% sodium deoxycholate, 0.1% SDS, and 1 mM sodium orthovanadate) supplemented with phosphatase and protease inhibitor cocktail. Total protein was estimated in cell lysates employing Bradford assay. Protein samples were separated by SDS-PAGE and then transferred to PVDF membrane. Membranes were blocked in 1% BSA and then incubated with primary antibody viz. β-actin, Bax, Bcl2, p21, Akt, p-Akt, NF-κB (CST, Danvers, MA, USA). Membranes were washed, incubated with secondary antibodies and developed using ECL reagent and visualized in chemidoc (GE Healthcare Lifesciences).

Wound healing assay. MDA-MB-231 cells were seeded in 12 well plates at a density of 1 × 10⁶ cells per well and grown to 90% confluence. In each well of the plate a 1 mm wide scratch was made using 200 μL pipette tip. Cells were washed with PBS and incubated with ½IC₅₀ and ¼IC₅₀ concentrations of compounds 2, 4 and 5 along with vehicle control and doxorubicin (½IC₅₀) as positive control for 24 h in humidified incubator at 5% CO₂ and 37 °C temperature. Cell migration across the wound was observed under phase contrast microscope, and the micro-photographs were captured as well as analysed.³³
Statistical analysis. All the data were presented as mean ± SEM. Data are subjected to ANOVA (one way and two way) by GraphPad Prism 5 and p < 0.05 was considered as statistically significant.
Calotroposide K (6). (12-O-Benzoylisolineolon-3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-glucopyranoside): brown viscous residue, [α]_D + 9.7° (c = 1.1, in MeOH at 22 °C). IR (CHCl₃) cm⁻¹ 3400, 3018, 1636, 1215, 1083. UV (MeOH) nm (log ε) 230 (4.56), 273 (3.15), 280 (3.01). mp 196–200 °C. ¹H-NMR (CDCl₃) δ (ppm) 1.24, 1.88 (1H both m, CH₂-1), 1.90, 2.00 (1H both m, CH₂-2), 3.56 (1H, m, H-3), 2.22, 2.20 (1H both m, CH₂-4), 5.33 (1H, brs, H-6), 2.01, 2.22 (1H both m, CH₂-7), 1.63 (1H, m, H-9), 2.02, 1.52 (1H both m, CH₂-11), 4.84 (1H, m, H-12), 2.12, 1.90 (1H both m, CH₂-15), 2.29, 2.18 (1H both m, CH₂-16), 3.25 (H, m, H-17), 1.54 (3H, s, CH₃-18), 1.13 (3H, s, CH₃-19), 2.05 (3H, s, CH₃-21) (pregnanone moiety), 7.95 (2H, d, J = 7.5 Hz, H-2′,6′), 7.44 (2H, t, J = 7.5 Hz, H-3′,5′), 7.55 (1H, t, J = 7.5 Hz, H-4′) (benzoyl moiety), 4.83, 4.67, 4.44, 4.76, 4.74, 4.91 (1H each, dd, J₁ = 9.70 Hz, J₂ = 1.51 Hz) (anomeric protons), 1.62–1.78 (2H × 6 = 12H, C-2 protons of sugars), 3.80–3.89 (6H, C-3 protons of sugars), 3.21–3.28 (6H, C-4 protons of sugars), 1.26–1.36 (6H, C-5 protons of sugars), 1.60–1.61, 1.20–1.27 (15H, 5 × CH₃ C-6 protons of sugars), 3.37–3.43 (18H, 6 × CH₃ C-3-OMe protons of sugars). ¹³C NMR (CDCl₃) (Table S2 of the ESI‡), ESI-MS m/z 1368 [M + NH₄]⁺ and 1373 [M + Na]⁺, ESI-HRMS m/z 1373.6904 [M + Na]⁺ (calculated), 1373.6911 (found), molecular formula: C₆₉H₁₀₆O₂₆.
Calotroposide O (7). 12-O-Benzoylisolineolon-3-O-β-D-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-oleandropyranosyl-(1→4)-β-D-glucopyranoside: brown viscous residue, [α]_D + 16.7° (c = 1.1, in MeOH at 22 °C). IR (CHCl₃) cm⁻¹ 3436, 3022, 1637, 1401, 1215, 1063. UV (MeOH) nm (log ε) 230 (4.56), 273 (3.15), 280 (3.01). mp 185–190 °C. ¹H-NMR (CDCl₃) δ (ppm) 1.10, 1.88 (1H both m, CH₂-1), 1.90, 2.02 (1H both m, CH₂-2), 3.57 (1H, m, H-3), 2.23, 2.40 (1H both m, CH₂-4), 5.37 (1H, brs, H-6), 2.05 (2H, m, CH₂-7), 1.61 (1H, m, H-9), 2.01, 1.53 (1H both m, CH₂-11), 4.84 (1H, m, H-12), 2.12, 1.88 (1H both m, CH₂-15), 2.09, 2.18 (1H both m, CH₂-16), 3.27 (H, m, H-17), 1.54 (3H, s, CH₃-18), 1.12 (3H, s, CH₃-19), 2.04 (3H, s, CH₃-21) (pregnanone moiety), 7.95 (2H, d, J = 7.6 Hz, H-2′,6′), 7.44 (2H, t, J = 7.5 Hz, H-3′,5′), 7.55 (1H, t, J = 7.5 Hz, H-4′) (benzoyl moiety), 4.84, 4.66, 4.44, 4.74, 4.75, 4.92 (1H each, dd, J₁ = 9.70 Hz, J₂ = 1.51 Hz) (anomeric protons) 1.58–1.79 (2H × 6 = 12H, C-2 protons of sugars), 3.76–3.89 (6H, C-3 protons of sugars), 3.16–3.28 (6H, C-4 protons of sugars), 1.26–1.36 (6H, C-5 protons of sugars), 1.15–1.40 (15H, 5 × CH₃ C-6 protons of sugars) 3.34, 3.36, 3.38, 3.37, 3.39 (18H, 6 × CH₃ C-3-OMe protons of sugars). ¹³C NMR (CDCl₃) (Table S2 of the ESI‡), ESI-MS m/z 1368 [M + NH₄]⁺ and 1373 [M + Na]⁺, ESI-HRMS m/z 1368.7298 [M + NH₄]⁺ (calculated), 1368.7304 (found), molecular formula: C₆₉H₁₀₆O_26.

Conflict of interest

Authors declare no conflict of interest.

Acknowledgements

R. M., S. D. and T. J. are thankful to UGC and CSIR, New Delhi, for fellowships. We thank Mr R. K. Purushottam for his help in HPLC analysis. We sincerely thank SAIF, CSIR-CDRI, for the analytical facilities. Authors acknowledge CSIR network project BSC0106 for financial support.

References

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Footnotes

† This manuscript is CDRI communication no. 9366.

‡ Electronic supplementary information (ESI) available: Additional information including spectral data is provided. See DOI: 10.1039/c6ra23600f

§ Authors contributed equally.

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