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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
New cytotoxic cycloartane triterpene from Cassia italica aerial parts a
Gamal A. Mohamed a
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt Published online: 01 Apr 2014.
To cite this article: Gamal A. Mohamed (2014) New cytotoxic cycloartane triterpene from Cassia italica aerial parts, Natural Product Research: Formerly Natural Product Letters, 28:13, 976-983, DOI: 10.1080/14786419.2014.902820 To link to this article: http://dx.doi.org/10.1080/14786419.2014.902820
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Natural Product Research, 2014 Vol. 28, No. 13, 976–983, http://dx.doi.org/10.1080/14786419.2014.902820
New cytotoxic cycloartane triterpene from Cassia italica aerial parts Gamal A. Mohamed* Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
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(Received 22 January 2014; final version received 4 March 2014) Phytochemical study of the aerial parts of Cassia italica Mill. (family: Fabaceae) growing in Saudi Arabia afforded one new cycloartane triterpene, named (22E)-3-bhydroxycycloart-22-en-24-one (2), together with eight known compounds: b-sitosterol (1), uvaol (3), daucosterol (4), methyl 3,4-dihydroxybenzoate (5), emodin (6), 4hydroxypheny-O-b-D -glucopyranoside (7), aloin (8) and rutin (9). The structure of the isolated compounds was determined by physical, chemical and spectral data (UV, IR, MS, 1D (1H, 13C and DEPT) and 2D (1H – 1H COSY, HSQC and HMBC) NMR), as well as by comparing with authentic samples. Compounds 3 –5 and 7 – 9 were isolated for the first time from the plant. Compound 2 was evaluated for its cytotoxic activity against the L5178Y and PC12 cell lines. The total methanolic extract and compounds 5 –9 exhibited free radical-scavenging activity using DPPH assay. Keywords: Cassia italic; cycloartane; phenolics; anthraquinones; antioxidant; cytotoxicity
1. Introduction Cassia italic (family: Fabaceae) is a perennial shrubby plant known as Eshrinq and grows abundantly in the kingdom of Saudi Arabia. Cassia species such as Cassia italica and Cassia senna are used in Saudi traditional medicine for treatment of constipation, oedema and skin infections (Al-Yahya et al. 1990). Ethno-medically, the decoction of the entire plant or its leaves has been used as laxative, purgative and expectorant (Al-Said 1993). In addition, it is used for rheumatic and intestinal disorders as well as urinary tract infections (Al-Said 1993). The ethanolic extract of the whole plant revealed a wide range of biological activities such as anti-inflammatory, antipyretic, analgesic, anti-neoplastic, antiviral, central nervous system depressant, antioxidant, hepatoprotective, hypoglycaemic and antibacterial (Assane et al. 1994; Ghanadi et al. 2000; Nassr-Allah et al. 2009; Qamar et al. 2011; Sermakkani & Thangapandian 2011; Dabai et al. 2012; Modibbo & Nadro 2012; Masoko et al. 2012; Nadro & Onoagbe 2012). A literature survey on the chemical constituents of C. italica revealed the presence of anthraquinones (Elsayed et al. 1992; Kamzi et al. 1994; Dave & Ledwani 2012; Dabai et al. 2012), flavonoids (Elsayed et al. 1992), sterols (Elsayed et al. 1992), triterpenes (Elsayed et al. 1992), hydrocarbons and fatty acids (Ermakkani & Thangapandian 2012). Reviewing the current literature, there are no previous chemical investigations for the constituents of C. italica growing in Saudi Arabia. Therefore, a phytochemical study of the plant was performed aiming to identify its constituents as well as the biological evaluation of the total methanolic extract (TME) and isolated compounds. This article reports the isolation and characterisation of one new cycloartane triterpene (2) together with eight known compounds (1, 3 –9) from the aerial parts of C. italica (Figure 1). Compounds 3– 5 and 7– 9
*Email: [email protected] q 2014 Taylor & Francis
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30
O 21 18
17 13
1
5
3
RO
28
OH
O
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2 6
5
3
8a
9
9a
2
4 10a
10
4a 4
5
11
O 6
28 16
OH 5
27
7
10
3 OH 3´
8
HO
7
9
O
1´
2
11
CH2OH
4
OH 7
OH
4´
2´
1
4a
3
6 5
H
OH
4
OH
3 5
2 3
5
24
OH
10
O
5´
HO
6´
OH
4
O
OH
3
6
HO
17
15
6
2
5a
1
8 7
6
1a
8a
1 6
OH
O 9
7
1
OH
23
8
O
5 4
21 22
8
10
2
HO OH
OH
3`
14
29
1`
H
11 26 9
3
O
HO HO
18 13
1
2
OH 6`
15
7 30
6
4
HO
1 R= H 4 R= Glucose
12 25
8
10
20
19
27
16
14 9
2
25
23
12 11
19
COOCH3
29
26 24
20
8
7
22
O
1´´
O
OH
9 H3C OH HO
OH 8
O O
HO
1´´´
HO HO
Figure 1. Structures of the isolated compounds.
were reported for the first time from the plant. The TME and compounds 5– 9 exhibited free radical-scavenging activity. Compound 2 exhibited cytotoxic activity against the L5178Y and PC12 cancer cell lines. 2. Results and discussion Compound 2 was obtained as colourless needles and gave positive Liebermann –Burchard reaction, suggesting 2 to be a triterpene derivative (Reinhold 1935). Its molecular formula was C30H48O2 as established by the HR-ESI-MS pseudo-molecular ion peak at m/z 441.3642 [M þ H]þ (calc. for C30H49O2, 441.3654), requiring seven degrees of unsaturation. The IR spectrum exhibited absorption bands at 1722 (carbonyl ketone) and 3435 (hydroxyl group) cm21. The 1H NMR spectrum revealed the presence of cyclopropane methylene group at dH 0.55 and 0.32 (each 1H, d, J ¼ 3.6 Hz) (Estrada et al. 2002; Abdel-Monem et al. 2008), four tertiary methyl groups at dH 0.97 (H3-18), 0.95 (H3-28), 0.80 (H3-29) and 0.89 (H3-30), three secondary methyls at dH 1.08 (d, J ¼ 6.4, H3-21), 1.08 (d, J ¼ 6.4 Hz, H3-26) and 0.86 (d, J ¼ 6.4 Hz, H3-27), one oxymethine at dH 3.28 (m, H-3), two trans-coupled olefinic protons at dH 7.53 (1H, dd, J ¼ 15.8, 7.4 Hz, H-22) and 7.71 (1H, d, J ¼ 15.8 Hz, H-23), a hydroxyl group at dH 4.30 (m, 3-OH) and a series of overlapped signals, suggesting the presence of a 9,19-cycloartene-type triterpene with C22ZC23 double bond. The 13C NMR spectrum displayed thirty carbon signals including seven methyls, nine methylenes, eight methines and six quaternary carbons; one of them for ketone moiety at dC 205.5. The 13C NMR spectrum exhibited two olefinic carbons at dC 128.8 (d, C-22) and 148.9 (d, C-23) and an oxymethine at dC78.8 (d, C-3). The presence of oxygen-bearing carbon at C-3 was confirmed by the HMBC spectrum (Figure S6; see supplementary materials), which revealed cross-peaks between H3-28, H3-29, H-5, H-1 and 3-OH group with C-3. The position of the double bond at C22 – C23 was established based on 1H – 1H COSY of H-22 with H-23 and H-20 and further confirmed by the HMBC correlations of H3-21 to C-22 and H-20 and H-25 to C-23. The HMBC cross-peaks of H3-26, H3-27, H-25, H-22 and H-23 to C-24 allowed the ketone moiety to
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be located at C-24. Furthermore, HMBC correlations from H3-18 and H3-30 to C-13 and C-14, H19 to C-5, C-9, and C-10, H-1 to C-2 and C-10, and H3-28 and H3-29 with C-2, C-3, C-4, and C-10 were observed. The relative configuration of H-3 was determined to be a-configuration by comparing the 1H and 13C NMR chemical shifts as well as coupling constant with those of related compounds in the literature (Estrada et al. 2002; Zhang et al. 2005). Thus, from the abovementioned evidences and by comparing the NMR (1H – 1H COSY, HMQC, DEPT and HMBC) spectral data of 2 with those in the literature, the structure of 2 was elucidated as (22E)-3-bhydroxycycloart-22-en-24-one and considered a new natural product. The known compounds (Figure 1) were identified by analysing the spectroscopic data (1D, 2D NMR and MS) and comparing their data with those in the literature as well as cochromatography with authentic samples to be b-sitosterol (1) (Sayedy et al. 2007), uvaol (3) (Mahato & Kundu 1994; Min et al. 2000), daucosterol (4) (Sayedy et al. 2007), methyl 3,4dihydroxybenzoate (5) (Azizuddin et al. 2010), emodin (6) (Guo et al. 2011), 4-hydroxyphenyO-b-D -glucopyranoside (7) (Cepanec & Litvic´ 2008; Prabhu et al. 2011), aloin (8) (Karagianis et al. 2003) and rutin (9) (Malikov & Yuldashev 2002). Acid hydrolysis of compounds 4, 7 and 9 gave b-sitosterol, 4-hydroxyphenol and quercetin as aglycones and glucose and rhamnose as sugar moieties, respectively. 2,20 -Diphenylpicrylhydrazyl (DPPH) radical is widely used as a model system to investigate the scavenging activities of several natural compounds. DPPH is scavenged by the antioxidants through the donation of proton forming the reduced DPPH which can be quantified by its decrease of absorbance. Cassia italica had been known to be a rich source of flavonoids, anthraquinones and phenols, possessing variety of biological activities (Elsayed et al. 1992; Kamzi et al. 1994; Dave & Ledwani 2012; Dabai et al. 2012). The TME and compounds 5 –9 exhibited a concentrationdependent scavenging activity by quenching DPPH radicals (Table 1). A maximum inhibition (86.25% for TME and 74.2% for compound 9) of DPPH free radical was observed at concentrations of 800 mg/mL and 20 mM, respectively. We can, therefore, deduce that the antioxidant activity of C. italica TME as demonstrated in this study could be due to the presence of phenolic compounds. These findings validated the use of this plant in folk medicine for the treatment of some diseases such as skin diseases and urinary tract infections and its great potential to be used as a source of antioxidant phytochemicals. Moreover, rutin (9) exhibited potent DPPH-scavenging activity, which is in accordance with the reported data (Sayed et al. 2006; Zielin´ska et al. 2010). It was reported that 9,19-cyclolanostane triterpene exhibited Table 1. The DPPH radical-scavenging activity results. Sample a
TME TMEa TMEa TMEa TMEa TMEa 5b 6b 7b 8b 9b
Concentration
DPPH (% inhibition)
25 50 100 200 400 800 20 20 20 20 20
38.91 ^ 1.7 56.74 ^ 1.4 67.81 ^ 1.0 76.43 ^ 1.2 82.94 ^ 2.4 86.25 ^ 2.7 70.3 ^ 0.62 42.1 ^ 0.45 21.7 ^ 0.41 36.2 ^ 0.70 74.2 ^ 0.63
Note: Each value represents the mean ^SD, n ¼ 3. a Concentration: mg/mL. b Concentration: mM.
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cytotoxic activity (Kuang et al. 2011). The cytotoxic effect of 2 was tested against L5178Y and PC12 cell lines. It was found that 2 displayed 81% and 69% growth suppression against L5178Y and PC12 cell lines (concentration 22.7 mM) and 73% and 57% (concentration 6.8 mM), with ED50(s) 1.09 and 1.86 mM, respectively.
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3. Experimental 3.1. General experimental procedures Melting points were carried out using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). The UV spectrum was determined using the Perkin Elmer double beam spectrophotometer Model 550S, attached to a Hitachi recorder Model 561, using 1 cm quartz cell. Optical rotation was measured on a JASCO digital polarimeter. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). HR-ESI-MS was recorded on an LTQ Orbitrap (ThermoFinnigan, Bremen, Germany). EI-MS was recorded on a Finnigan MAT TSQ 7000 mass spectrometer. ESI-MS and LC – MS spectra were obtained with an LCQ DECA mass spectrometer (Thermo Finnigan, Bremen, Germany) coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). 1D and 2D NMR spectra (chemical shifts in ppm and coupling constants in Hz) were recorded on Bruker BioSpin GmbH 400 MHz Ultrashield spectrometer using DMSO-d6, CDCl3 and CD3OD as solvents, with TMS as the internal reference. Solvents were distilled before spectroscopic measurements. Column chromatographic separations were performed on SiO2 60 (0.04 – 0.063 mm, Merck) and Sephadex LH20 (0.25 – 0.1 mm, Merck). TLC was performed on pre-coated TLC plates with SiO2 60 F254 (0.2 mm, Merck). The solvent systems used for TLC analyses were CHCl3:MeOH (97:3, S1), CHCl3:MeOH (90:10, S2), CHCl3:MeOH (85:15, S3) and n-BuOH:HOAc:H2O (4:1:2, S4). The compounds were detected by UV absorption at lmax 255 and 366 nm followed by spraying with anisaldehyde/H2SO4 reagent and heating at 1108C for 1 – 2 min. 3.2. Plant material C. italica aerial parts were collected in November 2011 from the campus of King Abdulaziz University and were authenticated by Dr. Abdulaziz. A. Fayed, Prof. of Plant Taxonomy, Faculty of Science, Assiut University, Assiut, Egypt, as well as morphological features and database present in the library (Collenette 1999; Migahid 2001). A voucher specimen has been deposited at the Department of Natural products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia (Registration code AXG2011). 3.3. Extraction and isolation The air-dried powdered aerial parts of C. italica (500 g) were extracted with 70% MeOH (6 £ 2.5 L) at room temperature. The combined methanol extract was concentrated under reduced pressure to afford a dark brown residue (31.0 g). The latter was suspended in distilled H2O (200 mL) and successively partitioned between n-hexane and EtOAc to yield n-hexane (4.9 g), EtOAc (9.2 g) and aqueous (10.1 g) fractions, respectively. From each, total, n-hexane and EtOAc fractions, 5.0, 1.0, and 1.0 g, respectively, were stored for biological studies. The nhexane fraction (3.5 g) was subjected to VLC on SiO2 column eluted with n-hexane:EtOAc gradient to yield three sub-fractions. Sub-fractions B (890 mg, n-hexane:EtOAc, 50:50) and C (950 mg, EtOAc, 100%) were chromatographed separately on SiO2 (60 g £ 50 cm £ 1 cm)
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eluted with n-hexane:EtOAc gradient to afford compounds 1 (42 mg, sub-fraction B), 2 (5.4 mg, sub-fraction C) and 3 (8.5 mg, sub-fraction C). The EtOAc fraction (7.0 g) was subjected to VLC on SiO2 column chromatography using CHCl3:MeOH gradient to afford six sub-fractions. Sub-fraction B (827 mg, CHCl3:MeOH, 90:10) was chromatographed over SiO2 column chromatography using CHCl3:MeOH gradient elution to give 4 (62 mg). SiO2 column chromatography of sub-fraction C (659 mg, CHCl3:MeOH, 75:25) using CHCl3:MeOH gradient gave 5 (17 mg) and 6 (11 mg). Sub-fraction D (490 mg, CHCl3:MeOH, 50:50) was treated similar to sub-fraction C to afford 7 (13 mg). A Sephadex LH-20 column chromatography (40 g £ 50 cm £ 1 cm) of sub-fraction E (970 mg) using MeOH as an eluent yielded impure 8 and 9, which were further purified by repeated chromatography on SiO2 column (40 g £ 50 cm £ 1 cm) using CHCl3:MeOH gradient to afford 8 (17 mg) and 9 (32 mg). 3.4. Spectral data (22E)-3-b-hydroxycycloart-22-en-24-one (1): Colourless needles (5.4 mg); Rf: 0.69, silica gel 60 F254 (S1); m.p. 181– 1828C; [a ]D þ 31.5 (c 0.5, CHCl3); UV (CHCl3): lmax (log 1) 254 nm (2.44) nm; IR (KBr) nmax 3435, 2930, 1722, 1645, 870, 723 cm21; 1H NMR (400 MHz, CDCl3): dH 1.62 (1H, m, H-1A), 1.27 (1H, m, H-1B), 1.59 (1H, m, H-2A), 1.72 (1H, m, H-2B), 3.28 (1H, m, H-3), 1.50 (1H, m, H-5), 1.61 (2H, m, H-6), 1.75 (1H, m, H-7A), 1.22 (1H, m, H-7B), 1.28 (1H, m, H-8), 1.97 (1H, m, H-11A), 1.05 (1H, m, H-11B), 2.48 (1H, m, H-12A), 2.39 (1H, m, H12B), 1.36 (1H, m, H-15A), 1.25 (1H, m, H-15B), 1.28 (2H, m, H-16), 1.59 (1H, m, H-17), 0.97 (3H, s, H-18), 0.55 (1H, d, J ¼ 3.6 Hz, H-19A), 0.32 (1H, d, J ¼ 3.6 Hz, H-19B), 2.61 (1H, m, H-20), 1.08 (3H, d, J ¼ 6.4 Hz, H-21), 7.53 (1H, dd, J ¼ 15.8, 7.4 Hz, H-22), 7.71 (1H, d, J ¼ 15.8 Hz, H-23), 2.37 (1H, m, H-25), 1.08 (3H, d, J ¼ 6.4 Hz, H-26), 0.86 (3H, d, J ¼ 6.4 Hz, H-27), 0.95 (3H, s, H-28), 0.80 (3H, s, H-29), 0.89 (3H, s, H-30), 4.30 (1H, m, 3-OH); 13C NMR (100 MHz, CDCl3): dC 32.9 (t, C-1), 32.0 (t, C-2), 78.8 (d, C-3), 40.5 (s, C-4), 48.0 (d, C-5), 21.1 (t, C-6), 30.1 (t, C-7), 47.1 (d, C-8), 19.9 (s, C-9), 36.6 (s, C-10), 26.0 (t, C-11), 37.5 (t, C-12), 45.3 (s, C-13), 48.8 (s, C-14), 35.7 (t, C-15), 26.5 (t, C-16), 52.7 (d, C-17), 18.0 (q, C-18), 30.3 (t, C-19), 40.8 (d, C-20), 18.1 (q, C-21), 128.8 (d, C-22), 148.9 (d, C-23), 205.5 (s, C-24), 35.5 (d, C-25), 18.3 (q, C-26), 19.8 (q, C-27), 25.4 (q, C-28), 14.0 (q, C-29), 18.4 (q, C-30); HR-ESI-MS m/z [M þ H]þ441.3642 (calcd for C30H49O2, 441.3654). 3.5. Antioxidant activity The antioxidant activity was evaluated using DPPH assay as previously outlined (Burits & Bucar 2000; Kumarasamy et al. 2004; Raziq et al. 2011; Mohamed et al. 2013a). One millilitre of the TME (25, 50, 100, 200, 400, and 800 mg/mL) and compounds 5– 9 (20 mM) were mixed with 1 mL of DPPH (4 mg was dissolved in 50 mL HPLC MeOH to obtain a concentration of 80 mg/mL) and allowed to stand for half an hour for any reaction to occur; 20 mM concentrations of tested compounds were used as previously published (Mohamed et al. 2013a). The UV absorbance was recorded at 517 nm and compared with DPPH in MeOH (blank). The experiment was performed in triplicate. The average absorption was recorded for each concentration. The same procedure was followed for the standard propyl gallate (a known synthetic antioxidant) set as 100% antioxidant activity. The % free radical-scavenging activity was calculated using the following formula: absorbance with the sample antioxidant activity ¼ 100 £ 1 2 absorbance of the blank
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3.6. Cytotoxicity test The cytotoxicity of 2 against L5178Y (mouse lymphoma cells) and PC12 (brain tumour cells of the rats) at concentrations of 6.8 and 22.7 mM was determined using the microculture tetrazolium assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)), and compared with that of untreated controls. Of the test samples, stock solutions in ethanol 96% (v/v) were prepared. Exponentially growing cells were harvested, counted and diluted appropriately. Of the cell suspension, 50 mL containing 3750 cells were pipetted into 96-well microtitre plates. Subsequently, 50 mL of a solution of the test samples containing the appropriate concentration was added to each well. The small amount of ethanol present in the wells did not affect the experiments. The test plates were incubated at 378C with 5% CO2 for 72 h. A solution of MTT was prepared at 5 mg/mL in phosphate-buffered saline (PBS; 1.5 mM KH2PO4, 6.5 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl; pH 7.4) and from this solution, 20 mL was pipetted into each well. The yellow MTT penetrates the healthy living cells and in the presence of mitochondrial dehydrogenases, MTT is transformed to its blue formazan complex. After an incubation period of 4 h at 378C in a humidified incubator with 5% CO2, the medium was centrifuged (15 min, 208C, 210 £ g) with 200 mL DMSO, the cells were lysed to liberate the formed formazan product. After thorough mixing, the absorbance was measured at 520 nm using a scanning microtitre-well spectrophotometer. The colour intensity is correlated with the number of healthy living cells (Mohamed et al. 2013b). 3.7. Acid hydrolysis The solution of the isolated glycosides (4, 7 and 9) (5 mg of each in 10 mL MeOH) was treated with 5% H2SO4 (1.5 mL) and refluxed on a boiling water bath for 3 h. The aglycone was extracted with EtOAc and concentrated under reduced pressure. It was further purified on Sephadex LH-20 column using MeOH as an eluent and identified by co-TLC with an authentic sample. The sugars in the aqueous layer were identified by co-paper chromatography with authentic materials using solvent system (S4) (Sayedy et al. 2007). 4. Conclusion In conclusion, in this study, nine compounds were isolated and elucidated from C. italica Mill. Compound 2 is a new natural compound. Compounds 3– 5 and 7– 9 were reported for the first time from the plant. The TME and compounds 5 and 9 exhibited high antioxidant activity. Compound 2 displayed cytotoxic activity against the L5178Y and PC12 cell lines. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S6. Acknowledgements I would like to express my deep thanks to Prof. Dr W.E.G. Mu¨ller (Institute fu¨r Physiologische Chemie, Duesbergweg 6, D-55099 Mainz, Germany) for cytotoxicity testing.
References Abdel-Monem AR, Abdel-Sattar E, Harraz FM, Petereit F. 2008. Chemical investigation of Euphorbia schimperi C. Presl Rec Nat Prod. 2:39– 45. Al-Said MS. 1993. Traditional medicinal plants of Saudi Arabia. AMJ Chinese Med. 2:1–12. Al-Yahya MA, Al-Meshal IA, Mossa JS, Al-Badr AA, Tariq M. 1990. Saudi plants. A phytochemical and biological approach. Riyadh: King Saud University; p. 349.
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Assane M, Nydyema R, Bassene E, Sere A, Gaye O. 1994. Purgative activity of Cassia italica. Dakar Med. 39:125–128. Azizuddin, Makhmoor T, Choudhary MI. 2010. Radicals scavenging potential of compounds isolated from Vitex agnus castus. Turkisch J Chem. 34:119–126. Burits M, Bucar F. 2000. Antioxidant activity of Nigella sativa essential oil. Phytother Res. 14:323–328. Cepanec I, Litvic´ M. 2008. Simple and efficient synthesis of arbutin. ARKIVOC. ii:19–24. Collenette S. 1999. Wild flowers of Saudi Arabia. King of Saudi Arabia: National Commission for Wild life Conservation and Development (NCWCD) & Sheila Collenette, King Fahd National Library; p. 523. Dabai YU, Kawo AH, Aliyu RM. 2012. Phytochemical screening and antibacterial activity of the leaf and root extracts of S italic. Afr J Pharm Pharmacol. 6:914–918. Dave H, Ledwani L. 2012. A review on anthraquinones isolated from Cassia species and their applications. Indian J Nat Prod Resour. 3:291– 319. Elsayed NH, Abu-Dooh AM, Elkhrisy EAM, Mabry TJ. 1992. Flavonoids of Cassia italica. Phytochemistry. 31:2187. Ermakkani M, Thangapandian V. 2012. GC–MS analysis of Cassia italica leaf methanol extract. Asian J Pharm Clin Res. 5:90–94. Estrada S, Acevedo L, Rodriguez M, Toscano RA, Mata R. 2002. New triterpenoids from the orchids Scaphyglottis livida and Nidema boothii. Nat Prod Lett. 16:81–86. Ghanadi AR, Ghasemi Dehkordi NA, Sohrabi MJ. 2000. Isolation and identification of flavonoids from Iranian Senna. J Res Med Sci. 5:54–55. Guo S, Feng B, Zhu R, Ma J, Wang W. 2011. Preparative isolation of three anthraquinones from Rumex japonicus by high-speed counter-current chromatography. Molecules. 16:1201–1210. Kamzi MH, Abdul-Malik, Saira-Hameed, Akhtar N, Ali SN. 1994. An anthraquinone derivative from Cassia italica. Phytochemistry. 36:761–763. Karagianis G, Viljoen A, Waterman PG. 2003. Identification of major metabolites in Aloe littoralis by high-performance liquid chromatography nuclear magnetic resonance spectroscopy. Phytochem Anal. 14:275–280. Kumarasamy Y, Byres M, Cox PJ, Delazar A, Jaspars M, Nahar L, Shoeb M, Sarker SD. 2004. Isolation, structure elucidation, and biological activity of flavone 6-C-glycosides from Alliaria petiolata. Chem Nat Compd. 40:122–128. Kuang H, Su Y, Yang B, Xia Y, Wang Q, Wang Z, Yu Z. 2011. Three new cycloartenol triterpenoid saponins from the roots of Cimicifuga simplex Wormsk. Molecules. 16:4348–4357. Mahato SB, Kundu AP. 1994. 13C NMR spectra of pentacyclic triterpenoids – a compilation and some salient features. Phytochemistry. 37:1517–1575. Malikov VM, Yuldashev MP. 2002. Phenolic compounds of plants of the Scutellaria genus. Distribution, structure, and properties. Chem Nat Compd. 38:358–406. Masoko P, Gololo SS, Mokgotho MP, Eloff JN, Howard RL, Mampuru LJ. 2012. Evaluation of the antioxidant, antibacterial and antiproliferative activities of the acetone extract of the roots of Senna italica (Fabaceae). Afr J Trad CAM. 7:138–148. Migahid AM. 2001. Flora of Saudi Arabia, University Libraries. Vol. 2, 3rd ed. Riyadh, Saudi Arabia: King Saud University; p. 10–11. Min BS, Kim YH, Lee SM, Jung HJ, Lee JS, Na MK, Lee CO, Lee JP, Bae K. 2000. Cytotoxic triterpenes from Crataegus pinnatifida. Arch Pharm Res. 23:155–158. Modibbo AA, Nadro MS. 2012. Hepatoprotetive effects of Cassia italica leaf extracts on carbon tetrachloride-induced jaundice. Asian J Biochem Pharm Res. 2:382–387. Mohamed GA, Ibrahim SRM, Al-Musayeib NM, Ross SA. 2013. New anti-inflammatory flavonoids from Cadaba glandulosa Forssk. Arch Pharm Res. doi:10.1007/s12272-013-0305-1 Mohamed GA, Ibrahim SRM, Ross SA. 2013. New ceramides and isoflavone from the Egyptian Iris germanica L. rhizomes. Phytochem. Lett. 6:340–344. Nadro MS, Onoagbe IO. 2012. Anti-hyperlipidaemic and antioxidant effect of aqueous and ethanolic extracts of Cassia italica leaves in streptozotocin-induced diabetes in rats. J Med Plants Res. 6:4675–4681. Nassr-Allah AA, Aboul-Enein AM, Aboul-Enein KM, Lightfoot DA, Cocchetto A, ElShemy HA. 2009. Anti-cancer and anti-oxidant activity of some Egyptian medicinal plants. J Med Plants Res. 3:799–808. Prabhu K, Karar PK, Hemalatha S, Ponnudurai K. 2011. Isolation and spectral identification of arbutin from the roots of Viburnum erubescens Wall. EX DC. Int J Res Ayurveda Pharm. 2:889–892. Qamar F, Afroz S, Feroz Z, Siddiqui S, Ara A. 2011. Evaluation of hypoglycemic effect of Cassia italica. J Basic Appl Sci. 7:61–64. Raziq N, Muhammad N, Chishti KA, Saeed M, Rahman S, Khan H. 2011. Correlation of the antioxidant capacity with the phenolic contents of Hypericum monogynum and Hypericum perforatum. Afr J Pharm Pharmacol. 5:1872– 1876. Reinhold JG. 1935. Liebermann–Burchard reaction velocities of sterols: I. Differences between free and ester cholesterol applied to the determination of cholesterol esters, II. A test for the presence of coprostenol in plasma. Am J Med Sci. 189:302 –303.
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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
New cytotoxic cycloartane triterpene from Cassia italica aerial parts a
Gamal A. Mohamed a
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt Published online: 01 Apr 2014.
To cite this article: Gamal A. Mohamed (2014) New cytotoxic cycloartane triterpene from Cassia italica aerial parts, Natural Product Research: Formerly Natural Product Letters, 28:13, 976-983, DOI: 10.1080/14786419.2014.902820 To link to this article: http://dx.doi.org/10.1080/14786419.2014.902820
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Natural Product Research, 2014 Vol. 28, No. 13, 976–983, http://dx.doi.org/10.1080/14786419.2014.902820
New cytotoxic cycloartane triterpene from Cassia italica aerial parts Gamal A. Mohamed* Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
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(Received 22 January 2014; final version received 4 March 2014) Phytochemical study of the aerial parts of Cassia italica Mill. (family: Fabaceae) growing in Saudi Arabia afforded one new cycloartane triterpene, named (22E)-3-bhydroxycycloart-22-en-24-one (2), together with eight known compounds: b-sitosterol (1), uvaol (3), daucosterol (4), methyl 3,4-dihydroxybenzoate (5), emodin (6), 4hydroxypheny-O-b-D -glucopyranoside (7), aloin (8) and rutin (9). The structure of the isolated compounds was determined by physical, chemical and spectral data (UV, IR, MS, 1D (1H, 13C and DEPT) and 2D (1H – 1H COSY, HSQC and HMBC) NMR), as well as by comparing with authentic samples. Compounds 3 –5 and 7 – 9 were isolated for the first time from the plant. Compound 2 was evaluated for its cytotoxic activity against the L5178Y and PC12 cell lines. The total methanolic extract and compounds 5 –9 exhibited free radical-scavenging activity using DPPH assay. Keywords: Cassia italic; cycloartane; phenolics; anthraquinones; antioxidant; cytotoxicity
1. Introduction Cassia italic (family: Fabaceae) is a perennial shrubby plant known as Eshrinq and grows abundantly in the kingdom of Saudi Arabia. Cassia species such as Cassia italica and Cassia senna are used in Saudi traditional medicine for treatment of constipation, oedema and skin infections (Al-Yahya et al. 1990). Ethno-medically, the decoction of the entire plant or its leaves has been used as laxative, purgative and expectorant (Al-Said 1993). In addition, it is used for rheumatic and intestinal disorders as well as urinary tract infections (Al-Said 1993). The ethanolic extract of the whole plant revealed a wide range of biological activities such as anti-inflammatory, antipyretic, analgesic, anti-neoplastic, antiviral, central nervous system depressant, antioxidant, hepatoprotective, hypoglycaemic and antibacterial (Assane et al. 1994; Ghanadi et al. 2000; Nassr-Allah et al. 2009; Qamar et al. 2011; Sermakkani & Thangapandian 2011; Dabai et al. 2012; Modibbo & Nadro 2012; Masoko et al. 2012; Nadro & Onoagbe 2012). A literature survey on the chemical constituents of C. italica revealed the presence of anthraquinones (Elsayed et al. 1992; Kamzi et al. 1994; Dave & Ledwani 2012; Dabai et al. 2012), flavonoids (Elsayed et al. 1992), sterols (Elsayed et al. 1992), triterpenes (Elsayed et al. 1992), hydrocarbons and fatty acids (Ermakkani & Thangapandian 2012). Reviewing the current literature, there are no previous chemical investigations for the constituents of C. italica growing in Saudi Arabia. Therefore, a phytochemical study of the plant was performed aiming to identify its constituents as well as the biological evaluation of the total methanolic extract (TME) and isolated compounds. This article reports the isolation and characterisation of one new cycloartane triterpene (2) together with eight known compounds (1, 3 –9) from the aerial parts of C. italica (Figure 1). Compounds 3– 5 and 7– 9
*Email: [email protected] q 2014 Taylor & Francis
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30
O 21 18
17 13
1
5
3
RO
28
OH
O
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2 6
5
3
8a
9
9a
2
4 10a
10
4a 4
5
11
O 6
28 16
OH 5
27
7
10
3 OH 3´
8
HO
7
9
O
1´
2
11
CH2OH
4
OH 7
OH
4´
2´
1
4a
3
6 5
H
OH
4
OH
3 5
2 3
5
24
OH
10
O
5´
HO
6´
OH
4
O
OH
3
6
HO
17
15
6
2
5a
1
8 7
6
1a
8a
1 6
OH
O 9
7
1
OH
23
8
O
5 4
21 22
8
10
2
HO OH
OH
3`
14
29
1`
H
11 26 9
3
O
HO HO
18 13
1
2
OH 6`
15
7 30
6
4
HO
1 R= H 4 R= Glucose
12 25
8
10
20
19
27
16
14 9
2
25
23
12 11
19
COOCH3
29
26 24
20
8
7
22
O
1´´
O
OH
9 H3C OH HO
OH 8
O O
HO
1´´´
HO HO
Figure 1. Structures of the isolated compounds.
were reported for the first time from the plant. The TME and compounds 5– 9 exhibited free radical-scavenging activity. Compound 2 exhibited cytotoxic activity against the L5178Y and PC12 cancer cell lines. 2. Results and discussion Compound 2 was obtained as colourless needles and gave positive Liebermann –Burchard reaction, suggesting 2 to be a triterpene derivative (Reinhold 1935). Its molecular formula was C30H48O2 as established by the HR-ESI-MS pseudo-molecular ion peak at m/z 441.3642 [M þ H]þ (calc. for C30H49O2, 441.3654), requiring seven degrees of unsaturation. The IR spectrum exhibited absorption bands at 1722 (carbonyl ketone) and 3435 (hydroxyl group) cm21. The 1H NMR spectrum revealed the presence of cyclopropane methylene group at dH 0.55 and 0.32 (each 1H, d, J ¼ 3.6 Hz) (Estrada et al. 2002; Abdel-Monem et al. 2008), four tertiary methyl groups at dH 0.97 (H3-18), 0.95 (H3-28), 0.80 (H3-29) and 0.89 (H3-30), three secondary methyls at dH 1.08 (d, J ¼ 6.4, H3-21), 1.08 (d, J ¼ 6.4 Hz, H3-26) and 0.86 (d, J ¼ 6.4 Hz, H3-27), one oxymethine at dH 3.28 (m, H-3), two trans-coupled olefinic protons at dH 7.53 (1H, dd, J ¼ 15.8, 7.4 Hz, H-22) and 7.71 (1H, d, J ¼ 15.8 Hz, H-23), a hydroxyl group at dH 4.30 (m, 3-OH) and a series of overlapped signals, suggesting the presence of a 9,19-cycloartene-type triterpene with C22ZC23 double bond. The 13C NMR spectrum displayed thirty carbon signals including seven methyls, nine methylenes, eight methines and six quaternary carbons; one of them for ketone moiety at dC 205.5. The 13C NMR spectrum exhibited two olefinic carbons at dC 128.8 (d, C-22) and 148.9 (d, C-23) and an oxymethine at dC78.8 (d, C-3). The presence of oxygen-bearing carbon at C-3 was confirmed by the HMBC spectrum (Figure S6; see supplementary materials), which revealed cross-peaks between H3-28, H3-29, H-5, H-1 and 3-OH group with C-3. The position of the double bond at C22 – C23 was established based on 1H – 1H COSY of H-22 with H-23 and H-20 and further confirmed by the HMBC correlations of H3-21 to C-22 and H-20 and H-25 to C-23. The HMBC cross-peaks of H3-26, H3-27, H-25, H-22 and H-23 to C-24 allowed the ketone moiety to
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be located at C-24. Furthermore, HMBC correlations from H3-18 and H3-30 to C-13 and C-14, H19 to C-5, C-9, and C-10, H-1 to C-2 and C-10, and H3-28 and H3-29 with C-2, C-3, C-4, and C-10 were observed. The relative configuration of H-3 was determined to be a-configuration by comparing the 1H and 13C NMR chemical shifts as well as coupling constant with those of related compounds in the literature (Estrada et al. 2002; Zhang et al. 2005). Thus, from the abovementioned evidences and by comparing the NMR (1H – 1H COSY, HMQC, DEPT and HMBC) spectral data of 2 with those in the literature, the structure of 2 was elucidated as (22E)-3-bhydroxycycloart-22-en-24-one and considered a new natural product. The known compounds (Figure 1) were identified by analysing the spectroscopic data (1D, 2D NMR and MS) and comparing their data with those in the literature as well as cochromatography with authentic samples to be b-sitosterol (1) (Sayedy et al. 2007), uvaol (3) (Mahato & Kundu 1994; Min et al. 2000), daucosterol (4) (Sayedy et al. 2007), methyl 3,4dihydroxybenzoate (5) (Azizuddin et al. 2010), emodin (6) (Guo et al. 2011), 4-hydroxyphenyO-b-D -glucopyranoside (7) (Cepanec & Litvic´ 2008; Prabhu et al. 2011), aloin (8) (Karagianis et al. 2003) and rutin (9) (Malikov & Yuldashev 2002). Acid hydrolysis of compounds 4, 7 and 9 gave b-sitosterol, 4-hydroxyphenol and quercetin as aglycones and glucose and rhamnose as sugar moieties, respectively. 2,20 -Diphenylpicrylhydrazyl (DPPH) radical is widely used as a model system to investigate the scavenging activities of several natural compounds. DPPH is scavenged by the antioxidants through the donation of proton forming the reduced DPPH which can be quantified by its decrease of absorbance. Cassia italica had been known to be a rich source of flavonoids, anthraquinones and phenols, possessing variety of biological activities (Elsayed et al. 1992; Kamzi et al. 1994; Dave & Ledwani 2012; Dabai et al. 2012). The TME and compounds 5 –9 exhibited a concentrationdependent scavenging activity by quenching DPPH radicals (Table 1). A maximum inhibition (86.25% for TME and 74.2% for compound 9) of DPPH free radical was observed at concentrations of 800 mg/mL and 20 mM, respectively. We can, therefore, deduce that the antioxidant activity of C. italica TME as demonstrated in this study could be due to the presence of phenolic compounds. These findings validated the use of this plant in folk medicine for the treatment of some diseases such as skin diseases and urinary tract infections and its great potential to be used as a source of antioxidant phytochemicals. Moreover, rutin (9) exhibited potent DPPH-scavenging activity, which is in accordance with the reported data (Sayed et al. 2006; Zielin´ska et al. 2010). It was reported that 9,19-cyclolanostane triterpene exhibited Table 1. The DPPH radical-scavenging activity results. Sample a
TME TMEa TMEa TMEa TMEa TMEa 5b 6b 7b 8b 9b
Concentration
DPPH (% inhibition)
25 50 100 200 400 800 20 20 20 20 20
38.91 ^ 1.7 56.74 ^ 1.4 67.81 ^ 1.0 76.43 ^ 1.2 82.94 ^ 2.4 86.25 ^ 2.7 70.3 ^ 0.62 42.1 ^ 0.45 21.7 ^ 0.41 36.2 ^ 0.70 74.2 ^ 0.63
Note: Each value represents the mean ^SD, n ¼ 3. a Concentration: mg/mL. b Concentration: mM.
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cytotoxic activity (Kuang et al. 2011). The cytotoxic effect of 2 was tested against L5178Y and PC12 cell lines. It was found that 2 displayed 81% and 69% growth suppression against L5178Y and PC12 cell lines (concentration 22.7 mM) and 73% and 57% (concentration 6.8 mM), with ED50(s) 1.09 and 1.86 mM, respectively.
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3. Experimental 3.1. General experimental procedures Melting points were carried out using an Electrothermal 9100 Digital Melting Point apparatus (Electrothermal Engineering Ltd, Essex, England). The UV spectrum was determined using the Perkin Elmer double beam spectrophotometer Model 550S, attached to a Hitachi recorder Model 561, using 1 cm quartz cell. Optical rotation was measured on a JASCO digital polarimeter. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). HR-ESI-MS was recorded on an LTQ Orbitrap (ThermoFinnigan, Bremen, Germany). EI-MS was recorded on a Finnigan MAT TSQ 7000 mass spectrometer. ESI-MS and LC – MS spectra were obtained with an LCQ DECA mass spectrometer (Thermo Finnigan, Bremen, Germany) coupled to an Agilent 1100 HPLC system equipped with a photodiode array detector. The IR spectra were measured on a Shimadzu Infrared-400 spectrophotometer (Kyoto, Japan). 1D and 2D NMR spectra (chemical shifts in ppm and coupling constants in Hz) were recorded on Bruker BioSpin GmbH 400 MHz Ultrashield spectrometer using DMSO-d6, CDCl3 and CD3OD as solvents, with TMS as the internal reference. Solvents were distilled before spectroscopic measurements. Column chromatographic separations were performed on SiO2 60 (0.04 – 0.063 mm, Merck) and Sephadex LH20 (0.25 – 0.1 mm, Merck). TLC was performed on pre-coated TLC plates with SiO2 60 F254 (0.2 mm, Merck). The solvent systems used for TLC analyses were CHCl3:MeOH (97:3, S1), CHCl3:MeOH (90:10, S2), CHCl3:MeOH (85:15, S3) and n-BuOH:HOAc:H2O (4:1:2, S4). The compounds were detected by UV absorption at lmax 255 and 366 nm followed by spraying with anisaldehyde/H2SO4 reagent and heating at 1108C for 1 – 2 min. 3.2. Plant material C. italica aerial parts were collected in November 2011 from the campus of King Abdulaziz University and were authenticated by Dr. Abdulaziz. A. Fayed, Prof. of Plant Taxonomy, Faculty of Science, Assiut University, Assiut, Egypt, as well as morphological features and database present in the library (Collenette 1999; Migahid 2001). A voucher specimen has been deposited at the Department of Natural products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia (Registration code AXG2011). 3.3. Extraction and isolation The air-dried powdered aerial parts of C. italica (500 g) were extracted with 70% MeOH (6 £ 2.5 L) at room temperature. The combined methanol extract was concentrated under reduced pressure to afford a dark brown residue (31.0 g). The latter was suspended in distilled H2O (200 mL) and successively partitioned between n-hexane and EtOAc to yield n-hexane (4.9 g), EtOAc (9.2 g) and aqueous (10.1 g) fractions, respectively. From each, total, n-hexane and EtOAc fractions, 5.0, 1.0, and 1.0 g, respectively, were stored for biological studies. The nhexane fraction (3.5 g) was subjected to VLC on SiO2 column eluted with n-hexane:EtOAc gradient to yield three sub-fractions. Sub-fractions B (890 mg, n-hexane:EtOAc, 50:50) and C (950 mg, EtOAc, 100%) were chromatographed separately on SiO2 (60 g £ 50 cm £ 1 cm)
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eluted with n-hexane:EtOAc gradient to afford compounds 1 (42 mg, sub-fraction B), 2 (5.4 mg, sub-fraction C) and 3 (8.5 mg, sub-fraction C). The EtOAc fraction (7.0 g) was subjected to VLC on SiO2 column chromatography using CHCl3:MeOH gradient to afford six sub-fractions. Sub-fraction B (827 mg, CHCl3:MeOH, 90:10) was chromatographed over SiO2 column chromatography using CHCl3:MeOH gradient elution to give 4 (62 mg). SiO2 column chromatography of sub-fraction C (659 mg, CHCl3:MeOH, 75:25) using CHCl3:MeOH gradient gave 5 (17 mg) and 6 (11 mg). Sub-fraction D (490 mg, CHCl3:MeOH, 50:50) was treated similar to sub-fraction C to afford 7 (13 mg). A Sephadex LH-20 column chromatography (40 g £ 50 cm £ 1 cm) of sub-fraction E (970 mg) using MeOH as an eluent yielded impure 8 and 9, which were further purified by repeated chromatography on SiO2 column (40 g £ 50 cm £ 1 cm) using CHCl3:MeOH gradient to afford 8 (17 mg) and 9 (32 mg). 3.4. Spectral data (22E)-3-b-hydroxycycloart-22-en-24-one (1): Colourless needles (5.4 mg); Rf: 0.69, silica gel 60 F254 (S1); m.p. 181– 1828C; [a ]D þ 31.5 (c 0.5, CHCl3); UV (CHCl3): lmax (log 1) 254 nm (2.44) nm; IR (KBr) nmax 3435, 2930, 1722, 1645, 870, 723 cm21; 1H NMR (400 MHz, CDCl3): dH 1.62 (1H, m, H-1A), 1.27 (1H, m, H-1B), 1.59 (1H, m, H-2A), 1.72 (1H, m, H-2B), 3.28 (1H, m, H-3), 1.50 (1H, m, H-5), 1.61 (2H, m, H-6), 1.75 (1H, m, H-7A), 1.22 (1H, m, H-7B), 1.28 (1H, m, H-8), 1.97 (1H, m, H-11A), 1.05 (1H, m, H-11B), 2.48 (1H, m, H-12A), 2.39 (1H, m, H12B), 1.36 (1H, m, H-15A), 1.25 (1H, m, H-15B), 1.28 (2H, m, H-16), 1.59 (1H, m, H-17), 0.97 (3H, s, H-18), 0.55 (1H, d, J ¼ 3.6 Hz, H-19A), 0.32 (1H, d, J ¼ 3.6 Hz, H-19B), 2.61 (1H, m, H-20), 1.08 (3H, d, J ¼ 6.4 Hz, H-21), 7.53 (1H, dd, J ¼ 15.8, 7.4 Hz, H-22), 7.71 (1H, d, J ¼ 15.8 Hz, H-23), 2.37 (1H, m, H-25), 1.08 (3H, d, J ¼ 6.4 Hz, H-26), 0.86 (3H, d, J ¼ 6.4 Hz, H-27), 0.95 (3H, s, H-28), 0.80 (3H, s, H-29), 0.89 (3H, s, H-30), 4.30 (1H, m, 3-OH); 13C NMR (100 MHz, CDCl3): dC 32.9 (t, C-1), 32.0 (t, C-2), 78.8 (d, C-3), 40.5 (s, C-4), 48.0 (d, C-5), 21.1 (t, C-6), 30.1 (t, C-7), 47.1 (d, C-8), 19.9 (s, C-9), 36.6 (s, C-10), 26.0 (t, C-11), 37.5 (t, C-12), 45.3 (s, C-13), 48.8 (s, C-14), 35.7 (t, C-15), 26.5 (t, C-16), 52.7 (d, C-17), 18.0 (q, C-18), 30.3 (t, C-19), 40.8 (d, C-20), 18.1 (q, C-21), 128.8 (d, C-22), 148.9 (d, C-23), 205.5 (s, C-24), 35.5 (d, C-25), 18.3 (q, C-26), 19.8 (q, C-27), 25.4 (q, C-28), 14.0 (q, C-29), 18.4 (q, C-30); HR-ESI-MS m/z [M þ H]þ441.3642 (calcd for C30H49O2, 441.3654). 3.5. Antioxidant activity The antioxidant activity was evaluated using DPPH assay as previously outlined (Burits & Bucar 2000; Kumarasamy et al. 2004; Raziq et al. 2011; Mohamed et al. 2013a). One millilitre of the TME (25, 50, 100, 200, 400, and 800 mg/mL) and compounds 5– 9 (20 mM) were mixed with 1 mL of DPPH (4 mg was dissolved in 50 mL HPLC MeOH to obtain a concentration of 80 mg/mL) and allowed to stand for half an hour for any reaction to occur; 20 mM concentrations of tested compounds were used as previously published (Mohamed et al. 2013a). The UV absorbance was recorded at 517 nm and compared with DPPH in MeOH (blank). The experiment was performed in triplicate. The average absorption was recorded for each concentration. The same procedure was followed for the standard propyl gallate (a known synthetic antioxidant) set as 100% antioxidant activity. The % free radical-scavenging activity was calculated using the following formula: absorbance with the sample antioxidant activity ¼ 100 £ 1 2 absorbance of the blank
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3.6. Cytotoxicity test The cytotoxicity of 2 against L5178Y (mouse lymphoma cells) and PC12 (brain tumour cells of the rats) at concentrations of 6.8 and 22.7 mM was determined using the microculture tetrazolium assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)), and compared with that of untreated controls. Of the test samples, stock solutions in ethanol 96% (v/v) were prepared. Exponentially growing cells were harvested, counted and diluted appropriately. Of the cell suspension, 50 mL containing 3750 cells were pipetted into 96-well microtitre plates. Subsequently, 50 mL of a solution of the test samples containing the appropriate concentration was added to each well. The small amount of ethanol present in the wells did not affect the experiments. The test plates were incubated at 378C with 5% CO2 for 72 h. A solution of MTT was prepared at 5 mg/mL in phosphate-buffered saline (PBS; 1.5 mM KH2PO4, 6.5 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl; pH 7.4) and from this solution, 20 mL was pipetted into each well. The yellow MTT penetrates the healthy living cells and in the presence of mitochondrial dehydrogenases, MTT is transformed to its blue formazan complex. After an incubation period of 4 h at 378C in a humidified incubator with 5% CO2, the medium was centrifuged (15 min, 208C, 210 £ g) with 200 mL DMSO, the cells were lysed to liberate the formed formazan product. After thorough mixing, the absorbance was measured at 520 nm using a scanning microtitre-well spectrophotometer. The colour intensity is correlated with the number of healthy living cells (Mohamed et al. 2013b). 3.7. Acid hydrolysis The solution of the isolated glycosides (4, 7 and 9) (5 mg of each in 10 mL MeOH) was treated with 5% H2SO4 (1.5 mL) and refluxed on a boiling water bath for 3 h. The aglycone was extracted with EtOAc and concentrated under reduced pressure. It was further purified on Sephadex LH-20 column using MeOH as an eluent and identified by co-TLC with an authentic sample. The sugars in the aqueous layer were identified by co-paper chromatography with authentic materials using solvent system (S4) (Sayedy et al. 2007). 4. Conclusion In conclusion, in this study, nine compounds were isolated and elucidated from C. italica Mill. Compound 2 is a new natural compound. Compounds 3– 5 and 7– 9 were reported for the first time from the plant. The TME and compounds 5 and 9 exhibited high antioxidant activity. Compound 2 displayed cytotoxic activity against the L5178Y and PC12 cell lines. Supplementary material Supplementary material relating to this article is available online, alongside Figures S1 – S6. Acknowledgements I would like to express my deep thanks to Prof. Dr W.E.G. Mu¨ller (Institute fu¨r Physiologische Chemie, Duesbergweg 6, D-55099 Mainz, Germany) for cytotoxicity testing.
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