SUPPLEMENTARY MATERIAL
Cytotoxic agents for KB and SiHa cells from n-hexane fraction of Cissampelos pareira and its chemical composition†
Manju Balaa,b, Kunal Pratapc, Praveen Kumar Vermaa,b, Yogendra Padwadc*and Bikram Singha,b*
a Academy of Scientific & Innovative Research, India; b Natural Plant Products Division, CSIR-Institute of Himalayan Bioresource, Palampur, Himachal Pradesh, 176061, India; c Regulatory Research Centre, Department of Biotechnology, CSIR-Institute of Himalayan Bioresource, Palampur, Himachal Pradesh, 176061, India.
* Corresponding author:
Bikram Singh, Academy of Scientific & Innovative Research, India; Natural Plant Products Division, CSIR-Institute of Himalayan Bioresource, Palampur, Himachal Pradesh, 176061, India; c Regulatory Research Centre, Department of Biotechnology, CSIR-Institute of Himalayan Bioresource, Palampur, Himachal Pradesh, 176061, India.
Tel.: 91-1894-230426; Fax: 91-1894-230433
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Yogendra Padwad, Regulatory Research Centre, Department of Biotechnology, CSIR-Institute of Himalayan Bioresource, Palampur, Himachal Pradesh, 176061, India.
Tel.: 91-1894-230742-43 (ext. 473)
E- mail:
Abstract: The present study aimed to investigate the chemical composition and biological activity of n-hexane fraction of Cissampelos pareira roots. Eleven constituents were characterized by gas chromatography-mass spectrometry analysis and five molecules, namely, oleanolic acid (1), oleic acid (2), ethyl oleate (3), 1-docosene (4) and tetracosane (5) were isolated using column chromatography. The in vitro study of extract and isolated molecules against KB (human oral squamous carcinoma) and SiHa (Human Cervical Cancer) cell lines revealed the oleanolic (1) and oleic acid (2) as potent cytotoxic molecules with potential anticancer activity. The IC50 values of n-hexane extract (CPHF), oleanolic (1) and oleic acid (2) were >300, 56.08 and 70.7 µg/mL (µM), respectively, against KB cell lines and >300, 47.24 and 80.2 µg/mL (µM), respectively, against SiHa cell lines.
Keywords: Cissampelos pareira; Anti-cancer activity; Gas chromatography-mass spectrometry; n-Hexane fraction.
Table of Contents
1. General information
2. Experimental section
3. 13C NMR values for compounds 1-5
4. Table S2 for chemical composition of n-hexane fraction from C. pareira.
5. Table S3. In vitro cytotoxicity and IC50 values of compounds (1-3) and n-hexane extract against SiHa & KB cancer cells.
6. NMR spectra of isolated compounds
References
1. General information
Column chromatography was carried out with 60-120 mesh silica gel. Silica (60-120 mesh) and TLC silica gel 60 F254 plates were purchased from Merck India Ltd. 1H NMR and 13C NMR experiments were performed on Bruker Avance-600 spectrometer. Chemical shifts are reported in parts per million (ppm) downfield from an internal standard. The GC-MS analysis was carried out on a Shimadzu (QP 2010) series GC-MS (Tokyo, Japan) equipped with a FID, AOC-20i auto-sampler coupled, and a DB-5 capillary column, (30 m x 0.25 mm i.d., 0.25 µm). The initial temperature of column was 40 °C held for 5min and was programmed to 230 °C at 4 °C/min, then held for 5 min at 230 °C; the sample injection volume was 2 µl in dichloromethane. Helium was used as carrier gas at a flow rate of 1.1 ml min-1on split mode (1:50).
2. Experimental
Chemicals
Silica gel (60-120) mesh was purchased from Merck India Ltd. Different solvents (analytical grade) and all other chemicals were also purchased from Merck India Ltd. Mumbai.
Plant material
Plant material was collected from Palampur, Himachal Pradesh, India during June 2012. The plant was identified from the CSIR-IHBT herbarium, Palampur, India, where a voucher specimen (# PLP-16567) has been deposited.
Cell Culture
Two human cancer cell lines, KB (human squamous oral carcinoma, ATCC No. CCL-17) and SiHa (human cervical cancer, ATCC No. HTB-35) were obtained from Guru Nanak Dev University, Amritsar and national animal cell culture repository at National Center for Cell Science, Pune, respectively. The cells were maintained in complete Dulbecco’s Modified Eagle Medium (Invitrogen life technologies) with 10% fetal bovine serum (Invitrogen life technologies) and 1% antibiotic antimycotic (Invitrogen life technologies) in a humidified 5% CO2 incubator at 37 °C. About 5x106 cells were re-suspended in complete medium.
Preparation of n-hexane extract
The air dried powdered roots of C. pareira (1.3 kg) were extracted thrice with ethanol-water (80:20, 7 Lit.) at room temperature by percolation method. The combined percolations were concentrated under reduced pressure to obtain dried brown extract (345.0 g) which was dissolved in H2O and fractionated, sequentially, with n-hexane, CHCl3 and n-BuOH. Each fraction was dried to yield n-hexane (110.0 g), CHCl3 (45.0 g), n-BuOH (75.0 g) fractions and water extract (90.0 g).
Preparation of methyl esters
The n-hexane fraction (500 mg) was derivatized by methanol and sulphuric acid under nitrogen atmosphere as described (Christie 1990) and methyl esters obtained were evaluated by gas chromatography-mass spectrometry (GC–MS).
Gas chromatography-mass spectrometry (GC-MS) analysis
The GC-MS analysis was carried out on a Shimadzu (QP 2010) series GC-MS (Tokyo, Japan) equipped with a FID, AOC-20i auto sampler coupled, and a DB-5 capillary column, (30 m x 0.25 mm i.d., 0.25 μm). The initial temperature of column was 40 °C held for 5 min and was programmed to 230 °C at 4 °C/min, then held for 5 min at 230 °C; the sample injection volume was 2 μL in High Performance Liquid chromatography (HPLC) grade dichloromethane. Helium was used as carrier gas at a flow rate of 1.1 mL min-1 on split mode (1:50). A standard solution of n-alkanes (C8-C23) was used to obtain the retention indices. Individual components were identified by retention indices (RI) with those reported in literature and also by comparison of their mass spectra (MS) with NIST data base and Adams libraries (Adams 2004; NIST/EPA/NIH 1998).
Isolation of compounds
The n-hexane (5.0 g) fraction was subjected to column chromatography over silica gel (60–120 mesh) using a gradient elution with mixture of n-hexane: CHCl3. The elution of packed column with pure n-hexane yielded the oleic acid (2, 87.0 mg), 1-docoscene (4, 27.0 mg) and tetracosane (5, 19.0 mg). On increasing the polarity with 9:1 mixture of n-hexane and chloroform, ethyl oleate (3, 39.0 mg) was isolated and oleanolic acid (1, 65.0 mg) was obtained as a white powder at ratio of 6:4 of n-hexane and chloroform.
1H NMR and 13C NMR experiments were performed on Bruker Avance-600 spectrometer. Chemical shifts were reported in parts per million (ppm) downfield from an internal standard. The NMR values (Table S1) were also confirmed by comparing with literature data [Bhatt 2011; http://lipidlibrary.aocs.org/nmr].
Oleanolic acid (1): Obtained as white solid (65.0 mg); 1H NMR (CD3OD, 600 MHz) d 0.63 (s, 3H, CH3), 0.67 (s, 3H, CH3),0.74 (s, 3H, CH3), 0.81 (s, 3H,CH3), 0.84 (s, 3H, CH3), 1.01 (s, 3H,CH3), 1.14 (s, 3H, CH3), 3.16-3.17 (m, 1H), 5.10 (s, 1H) and 13C NMR (CD3OD, 150 Hz) is given in Table S1.
Oleic acid (2): Obtained as yellowish oil (87.0 mg); 1H (CDCl3, 600 MHz) d 0.87 (t, 3H, J = 6.6, 6.6 Hz), 1.25-1.30 (m, 22H), 1.60-1.64 (m, 2H), 1.96-2.06 (m, 2H), 2.32-2.35 (m, 2H), 5.31-5.37 (m, 2H); 13C NMR (CDCl3, 150 MHz) is given in Table S1 and EI-MS m/z 282 [M]+ 264, 222, 151, 138, 112, 97, 83, 69, 55, 41, 27.
Ethyl oleate (3): Obtained as yellowish oil (39.0 mg); 1H (CDCl3, 600 MHz) d 0.88 (t, 3H, J = 6.6, 6.6 Hz), 1.26-1.30 (m, 25H), 1.61 (m, 2H), 2.01 (m, 2H), 2.27-2.30 (m, 2H), 4.11-4.14 (m, 2H), 5.31-5.37 (m, 2H); 13C NMR (CDCl3, 150 MHz,) is given in Table S1 and EI-MS m/z 310 [M]+ 281, 264, 246, 235, 222, 207, 180, 152, 137, 123, 111, 101, 97, 88, 83, 69, 55, 41.
1-Docosene (4): Obtained as colourless oil (27.0 mg); 1H (CDCl3, 600 MHz) d 0.85-0.90 (m, 3H), 1.31-1.39 (m, 36H), 2.02-2.06 (m, 2H), 4.92-4.94 (m, 1H), 4.98-5.01 (m, 1H), 5.79-5.82 (m, 1H); 13C NMR (CDCl3, 150 MHz) is given in Table S1; EI-MS m/z 308 [M]+ 280, 266, 252, 238, 224, 210, 181, 152, 139, 125, 111, 97, 83, 69, 55, 41.
Tetracosane (5): Obtained as colourless oil (19.0 mg); 1H (CDCl3, 600 MHz) d 0.85-0.90 (m, 6H), 1.31-1.39 (m, 44H) and 13C NMR (150 MHz, CDCl3) is given in Table S1.
Cytotoxicity assay
Upon reaching 70% confluence, the cells were trypsinized for 5 min at 37 °C and washed twice with phosphate-buffered saline (PBS) by centrifugation and incubated at a density of 20,000 cells/well in 96-well plates in 100 μL of complete medium. After 24 h cells were treated with several dilutions (10, 25, 50 and 100 μL/mL) of pure molecules and crude extract (50, 100, 200 and 300 μg/mL) in 100 μL of complete medium. The cells supplemented with medium only were taken as negative control for the experiment whereas cells treated with vinblastin (1 μM) were used as positive control. Plates were then incubated at 37 °C for 24, 48 and 72 h respectively in CO2 incubator. After each timeline, 50 μL of 50% trichloroacetic acid (Merck) was added to the wells for fixing the cells and plates were further incubated at 4 °C for 1 h. Plates were washed five to six times with distilled water and then air-dried. For mapping the cellular protein concentration, cells were stained with SRB dye. SRB solution, 100 μL (1% in acetic acid) was added to the wells and incubated for 30 min at room temperature. Plates were again washed for five to six times with 1% acetic acid (Merck) and air dried. Then, 100 μL of 10 mM tris base (Sigma) was added to the wells. The absorbance was measured using microplate reader (BioTeK Synergy H1 Hybrid Reader) at wavelength of 540 nm (Vanicha & Kanyawim 2006). The percentage growth inhibition was calculated using following formula:
% Cell Inhibition = 100-{(As-Ab)/(Ac-Ab)} x 100
Where, As is the absorbance value of sample compound; Ab is the absorbance value of blank; and Ac is the absorbance value of control (Carmichael 1987).
Statistical analysis
Data represented here is the result of three independent experiments. Values shown in the tables showing % growth inhibition ± standard deviation was calculated in Microsoft excel. Level of significance (p<0.001) was calculated by unpaired t-test using Graphpad Quickcalcs online software.
3. 13C NMR spectral data for compounds (1-5)
Table S1. 13C-NMR (150 MHz) spectral data of 1-5.
Position / dC (ppm)1 / 2 / 3 / 4 / 5
1 / 27.8a / 180.2 / 173.7 / 114.0 / 14.1a
2 / 25.0a / 34.0 / 34.2a / 139.2 / 22.7a
3 / 78.3 / 27.2 / 24.8 / 33.8 / 31.9a
4 / 33.5 / 29.0 / 29.6 / 31.9 / 29.6a
5 / 45.8 / 29.1 / 27.0 / 28.9 / 29.6a
6 / 18.1a / 29.2 / 27.0 / 29.1 / 29.6a
7 / 29.3 / 29.3 / 28.9 / 29.3 / 29.6a
8 / 39.1 / 29.4 / 23.1 / 29.5 / 29.6a
9 / 46.2 / 31.9a / 31.7a / 29.6a / 29.6a
10 / 30.2 / 129.9 / 129.8 / 29.6a / 29.6a
11 / 25.0a / 130.0 / 129.6 / 29.6a / 29.6a
12 / 122.2 / 31.9a / 31.7a / 29.7a / 29.6a
13 / 143.8 / 29.5 / 28.9 / 29.7a / -
14 / 41.5 / 29.6 / 28.9 / 29.7a / -
15 / 32.1 / 29.6 / 29.1 / 29.7a / -
16 / 27.8a / 29.7 / 29.2 / 29.7a / -
17 / 41.3 / 22.5 / 22.4 / 29.7a / -
18 / 32.4 / 14.0 / 14.0 / 29.7a / -
19 / 38.4 / - / - / 29.7a / -
20 / 26.4 / - / - / 29.7a / -
21 / 32.6 / - / - / 22.7 / -
22 / 23.1 / - / - / 14.1 / -
23 / 14.4 / - / - / - / -
24 / 14.8 / - / - / - / -
25 / 16.3 / - / - / - / -
26 / 18.1a / - / - / - / -
27 / 22.5 / - / - / - / -
28 / 180.3 / - / - / - / -
29 / 27.3 / - / - / - / -
30 / 27.4 / - / - / - / 14.1a
1’ / - / - / 59.9 / - / 22.7a
2’ / - / - / 13.8 / - / 31.9a
3’ / - / - / - / - / 29.6a
4’ / - / - / - / - / 29.6a
5’ / - / - / - / - / 29.6a
6’ / - / - / - / - / 29.6a
7’ / - / - / - / - / 29.6a
8’ / - / - / - / - / 29.6a
9’ / - / - / - / - / 29.6a
10’ / - / - / - / - / 29.6a
11’ / - / - / - / - / 29.6a
12’ / - / - / - / - / 29.6a
a Overlapped signals
4. Table S2. Chemical composition of n-hexane fraction from C. pareira.
S. No. / Compounds / Concentration (%) / RI / Mass fragments1 / Methyl tetradecanoate
(Methyl myristate) / 0.09 / 1893 / 242 [M]+, 211, 199, 157, 143, 129, 101, 87, 74, 69, 57, 41
2 / Methyl pentadecanoate / 0.14 / 2016 / 256 [M]+, 225, 213, 199, 171, 157, 143, 129, 101, 87, 74, 69, 41
3 / Methyl-9-hexadecenoate
(Methyl palmitoleate) / 0.48 / 2104 / 268 [M]+, 236, 194, 152, 137, 123, 97, 87, 74, 69, 55, 41
4 / Methyl hexadecanoate
(Methyl palmitate) / 22.27 / 2142 / 270 [M]+, 239, 227, 199, 185, 171, 143, 129, 101, 87, 74, 57, 41
5 / 14-Methyl hexadecanoate / 0.28 / 2258 / 284 [M]+, 253, 241, 227, 213, 199, 185, 157, 143, 125, 101, 87, 74, 57, 41, 27
6 / Methyl-9,12-octatadecadienoate
(Methyl linoleate) / 11.75 / 2343 / 294 [M]+, 263, 164, 150, 136, 123, 109, 95, 81, 67, 55, 41, 27
7 / Methyl-9-octadecenoate
( Methyloleate) / 47.26 / 2356 / 296 [M]+, 264, 222, 180, 166, 152, 137, 123, 97, 83, 74, 69, 55, 41
8 / Methyl octadecenoate
(Methyl stearate) / 6.93 / 2381 / 298 [M]+, 267, 255, 199, 143, 129, 101, 87, 74, 57, 43, 41
9 / 3-Octyloxiraneoate / 0.28 / 2456 / 294, 284, 263, 157, 150, 135, 125,109, 95, 81, 67, 55, 41, 39
10 / 11-Methyl eicosanoate / 0.24 / 2566 / 292, 250, 208, 152, 135, 125, 98, 97, 83, 69, 55, 41
11 / Methyl eicosanoate
(Methyl arachate) / 0.96 / 2583 / 326 [M]+, 283, 241, 227, 199, 185, 143, 129, 101, 87, 74, 57, 43, 41
5. Table S3. In vitro cytotoxicity showing percent growth inhibition of compounds (1-3) and n-hexane extract against SiHa & KB cancer cells.