New abietane diterpenes from Euphorbia pseudocactus berger (Euphorbiaceae) and their antimicrobial activity
Azza Ramy Abdel-Monem, Enas Hussein Abdelrahman
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
|Date of Submission||27-Jun-2015|
|Date of Decision||22-Jul-2015|
|Date of Web Publication||7-Jul-2016|
Enas Hussein Abdelrahman
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Euphorbia is the largest genus in Euphorbiaceae. Terpenoids were isolated from most species of this genus. Objective: Since no previous study was reported about Euphorbia pseudocactus Berger, we started here a phytochemical investigation on this species to isolate and identify its terpenoid constituents and to estimate the antimicrobial activity of the isolated compounds. Materials and Methods: The n-hexane fraction of the ethanolic extract of E. pseudocactus Berger was chromatographed on silica gel columns, the structures of the isolated compounds (1–5) were identified based on their MS, 1 D, and 2 D NMR spectral data. The antimicrobial activity of the n-hexane fraction and the isolated compounds (1–4) was investigated using diffusion plate method against Gram-positive (Staphylococcus aureus  and Bacillus subtilis ) and Gram-negative (Pseudomonas aeruginosa  and Escherichia coli ) bacteria, yeast (Candida albicans ), and fungi (Aspergillus flavus). Results: Two triterpenes (glut-5-en-3 β-ol  and olean-12,15-diene-3 β-ol ) and three abietane diterpene (3-hydoxy-19-cyclopropenoyloxy-abietane , ent-abieta-9,12,14-triene-12,16-olide , and 12,19-dihydroxy-abieta-5-ene ) were isolated. Compound 1 exhibited no antibacterial activity against the tested bacteria, compound 2 and n-hexane fraction exhibited weak activity, whereas compounds 3 and 4 showed moderate activity. All samples showed no activity against the tested yeast and fungi. Discussion and Conclusion: Five compounds were isolated for the 1st time from E. pseudocactus Berger, three of them (3–5) are new natural compounds. As the major isolated compound (1) exhibited no antimicrobial activity, the observed activity of the n-hexane fraction is mainly due to its diterpenoid constituents.
Keywords: Abietane, antimicrobial, diterpene, Euphorbia pseudocactus, triterpene
|How to cite this article:|
Abdel-Monem AR, Abdelrahman EH. New abietane diterpenes from Euphorbia pseudocactus berger (Euphorbiaceae) and their antimicrobial activity. Phcog Mag 2016;12, Suppl S3:346-9
|How to cite this URL:|
Abdel-Monem AR, Abdelrahman EH. New abietane diterpenes from Euphorbia pseudocactus berger (Euphorbiaceae) and their antimicrobial activity. Phcog Mag [serial online] 2016 [cited 2021 May 10];12, Suppl S3:346-9. Available from: http://www.phcog.com/text.asp?2016/12/46/346/185768
- Two known triterpenes and three new diterpenes were isolated from n-hexane fraction of Euphorbia pseudocactus
- The abietane diterpenoids showed higher antimicrobial activity than n-hexane fraction.
| Introduction|| |
Euphorbiaceae is a large family of flowering plants, composed of over 300 genera. Diverse medicinal properties of Euphorbiaceae members are associated with their wide distribution which is supported by their survival adaptations such as succulence. Genus Euphorbia is the largest genus in this family, comprising about 2000 known species. Traditionally, some species of Euphorbia are useful for the treatment of boils, cuts, and wounds. It is useful for cardiovascular complaints, asthma, cough, and spleen disorders. Certain Euphorbia species have been reported to possess cytotoxic,,,,, antimicrobial,,,,, larvicidal, insecticidal,, anti-inflammatory, hepatoprotective, and antioxidant ,, activities. The diterpenoid constituents, especially those with abietane, tigliane, and ingenane skeletons, are thought to be the main toxicant and bioactive factors.,,
Euphorbia pseudocactus Berger (candelabra spurge) is a multibranched, dwarf-stemmed, candelabra-shaped, succulent herb, 60–120 cm tall. The stems often have distinctive yellow V-shaped markings. It is originating in the subtropical coast of South Africa. It grows in thorny bush-lands and savannah often forming colonies. Herein, we report for the 1st time, the isolation of new abietane terpenoids from this Euphorbia member and the antimicrobial activity of the isolated compounds.
| Materials and Methods|| |
General experimental procedures
Mass spectra were measured with Thermo scientific, ISQ single quadrupole mass spectrometer (San Jose, CA, USA). NMR analysis was measured on 1 H-NMR (300 MHz),13 C-NMR (75 MHz): Varian Mercury-VX-300 spectrophotometer. Thin layer chromatography (TLC) was performed on precoated silica gel plates using solvent systems S1: n-c6H14: Ethyl acetate (EtOAc) (95:5), S2: N-C6H6: EtOAc (9:1), and S3: n-c6H14: EtOAc (8:2), the spots were detected by spraying with p-anisaldehyde-sulfuric acid spray reagent.
The whole plant of E. pseudocactus Berger was collected from special garden in Abu Rawash-Giza, Egypt, and kindly identified by Dr. Mohamed Elgebaly taxonomy specialist, El-Orman Garden, Giza. A voucher specimen was kept in the herbarium of Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo, Egypt (April 1, 2014).
Extraction and isolation
The fresh whole plant of E. pseudocactus Berger (2 kg) was sliced into small pieces then extracted by maceration in 90% ethyl alcohol (5 L × 3). The combined extracts were evaporated under reduced pressure (≤60°C) to give 110 g of green residue. The residue was fractionated into n-hexane (12.07 g), chloroform (3.9 g), and n-butanol (15.6 g) fractions.
The n-hexane fraction (10 g) was chromatographed on a column of silica gel starting with n-c6H14 then increasing the polarity by stepwise increments of EtOAc till 20% EtOAc. Fractions, 20 ml each, were collected and monitored by TLC, similar fractions were pooled to afford four main fractions. Combined fractions (46–48) were rechromatographed on a silica gel column using n-c6H14: EtOAc (98:2) as eluent to afford compound 1 (1.5 g). Combined fractions (49–55) were rechromatographed on a silica gel column using n-C6H14: EtOAc (98:2) as eluent to afford compounds 2 (100 mg) and 3 (200 mg). Combined fractions (56–64) were rechromatographed on a silica gel column using n-C6H14: EtOAc (95:5) as eluent to afford compound 4 (80 mg). Combined fractions (68–75) were rechromatographed on a silica gel column using n-C6H14: EtOAc (95:5) as eluent to afford compound 5 (40 mg).
White powder; Rf 0.6 (TLC, S1); MS m/z: 426 [M]+;1 H-NMR (300 MHz, CDCl3): δH 5.62 (1H, m, H-6), 3.46 (1H, br s, H-3), 1.16 (3H, s, H-28), 1.14 (3H, s, H-24), 1.10 (3H, s, H-27), 1.04 (3H, s, H-23), 1.01 (3H, s, H-26), 0.99 (3H, s, H-29), 0.95 (3H, s, H-30), 0.85 (3H, s, H-25).13 C-NMR (75 MHz, CDCl3) δC: 141.87 (C-5), 122.24 (C-6), 76.53 (C-3), 49.94 (C-10), 47.67 (C-8), 43.31 (C-18), 41.03 (C-4), 39.53 (C-13), 39.18 (C-22), 38.07 (C-14), 36.25 (C-16), 35.31 (C-19), 35.07 (C-9), 34.84 (C-11), 34.74 (C-29), 33.36 (C-21), 32.61 (C-30), 32.31 (C-15), 32.26 (C-28), 30.59 (C-12), 30.31 (C-17), 29.50 (C-24), 28.45 (C-20), 27.84 (C-2), 25.66 (C-23), 23.87 (C-7), 19.83 (C-26), 18.62 (C-27), 18.43 (C-1), 16.41 (C-25).
White powder; Rf 0.5 (TLC, S1); MS m/z: 424 [M]+;1H-NMR (300 MHz, CDCl3): δH 5.21 (1H, t, J = Hz, H-12), 4.83 (1H, br s, H-16), 4.62 (1H, br s, H-15), 3.20 (1H, m, H-3), 1.14 (3H, s, H-28), 1.01 (3 H, s, H-24), 0.98 (3H, s, H-27), 0.96 (3H, s, H-26), 0.94 (3H, s, H-25), 0.89 (3H, s, H-30), 0.88 (3H, s, H-29), 0.80 (3H, s, H-23).13 C-NMR (75 MHz, CDCl3) δC: 142.96 (C-13), 129.24 (C-15), 121.97 (C-12), 109.56 (C-16), 79.56 (C-3), 55.43 (C-5), 51.45 (C-9), 47.47 (C-19), 47.34 (C-14), 47.07 (C-18), 39.18 (C-8), 39.00 (C-4), 38.83 (C-1), 37.61 (C-10), 37.04 (C-22), 35.28 (C-21), 33.40 (C-7), 33.55 (C-29), 32.90 (C-20), 28.19 (C-23), 27.75 (C-2), 24.92 (C-27), 23.92 (C-11), 23.76 (C-30), 18.61 (C-6), 18.50 (C-28), 18.22 (C-26), 16.90 (C-24), 16.32 (C-24), 15.62 (C-25).
Yellow oily liquid; Rf 0.4 (TLC, S1); MS m/z: 374 [M]+;1 H-NMR (300 MHz, DMSO): δH 7.69 (1 H, br s, H-3`), 4.64 (1 H, d, J = 4.5 Hz, H-19a), 4.12 (1 H, d, J = 4.5 Hz, H-19b), 2.83 (1 H, br s, H-3), 0.97 (3 H, s, H-20), 0.90 (3 H, s, H-18), 0.85 (6 H, d, J = 7.2 Hz, H-16 and 17).13 C-NMR (75 MHz, DMSO) δC: 166.91 (C-1`), 131.51 (C-3`), 128.59 (C-2`), 74.38 (C-3), 67.37 (C-19), 50.00 (C-9), 49.38 (C-5), 46.79 (C-13), 42.56 (C-4), 38.07 (C-1), 35.85 (C-10), 34.65 (C-8), 34.46 (C-15), 33.05 (C-14), 31.01 (C-7), 30.30 (C-12), 28.33 (C-2), 24.14 (C-11), 23.22 (C-20), 22.34 (C-16, 17), 20.41 (C-6), 13.82 (C-4`), 10.74 (C-18).
Yellow oily liquid; Rf 0.3 (TLC, S2); MS m/z: 298 [M]+;1 H-NMR (300 MHz, DMSO): δH 7.24 (1 H, s, H-11), 7.12 (1 H, m, H-14), 3.89 (1 H, d, J = 2.4 Hz, H-15), 1.16 (3 H, s, CH3-20), 0.83 (6 H, s, CH3-18 and 19), 0.72 (3 H, d, J = 6.3 Hz, CH3-17).13 C-NMR (75 MHz, DMSO): δC 174.25 (C-16), 161.98 (C-12), 147.97 (C-9), 135.60 (C-8), 128.11 (C-14), 127.32 (C-11), 125.68 (C-13), 52.37 (C-15), 50.50 (C-5), 45.12 (C-3), 38.68 (C-1), 36.02 (C-10), 33.62 (C-4), 31.26 (C-7), 29.29 (C-18), 24.46 (17), 21.55 (C-20), 18.34 (C-2 and 6), 13.86 (C-19).
Yellow oily liquid; Rf 0.6 (TLC, S3); MS m/z: 306 [M]+;1 H-NMR (300 MHz, DMSO): δH 5.42 (1H, broad s, H-6), 1.15 (3H, s, H-19), 0.96 (3H, s, H-20), 0.84 (6 H, m, H-16 and 17).13 C-NMR (75 MHz, DMSO): δC 140.74 (C-5), 120.51 (C-6), 74.43 (C-12), 68.77 (C-19), 48.33 (C-13), 47.36 (C-9), 44.74 (C-8), 44.09 (C-4), 42.99 (C-10), 38.08 (C-1), 34.80 (C-11), 31.48 (C-7), 30.18 (C-3), 29.26 (C-14), 29.01 (C-20), 25.82 (C-15), 25.54 (C-17), 18.97 (C-2), 18.02 (C-16), 14.20 (C-18).
Estimation of microbiological activity
The antibacterial and antifungal activities were carried out in the Microbiology Division of Microanalytical Center of Cairo University, using the diffusion plate method.,,
One mL of each sample (100 mg/mL) is placed on a dish (9 cm diameter) containing a solid bacterial medium (nutrient agar broth) or fungal medium (Dox's medium) which has been heavily seeded with the spore suspensions of the tested organism. After incubation (24 h for bacteria and 5 days for fungi), the diameter of the clear zone of inhibition surrounding the sample is taken as measure of the inhibitory power of the sample against the particular tested organism. Compounds were tested against Gram+ve [Bacillus subtilis (6051) and Staphylococcus aureus (12600)], Gram-ve (Pseudomonas aeruginosa (10145) and Escherichia coli (11775)) bacteria, yeast [Candida albicans (7102)] and fungi [Aspergillus flavus]. Ampicillin and amphotericin B were used as references for bacteria and fungi, respectively. The results are depicted in [Table 1].
|Table 1: Antimicrobial activity of Euphorbia pseudocactus Berger (n-hexane fraction and isolated compounds)|
Click here to view
| Results and Discussion|| |
Two triterpenes and three diterpenes [Figure 1] were isolated from the n-hexane fraction of the ethanolic extract of E. pseudocactus Berger by successive fractionation on silica gel columns. Mass and 13 C-NMR spectral data of compounds 1 and 2 suggesting a triterpene nucleus with a molecular formula C30H50O and C30H48O, respectively. The presence of correlation in the HMBC spectrum of compound 1 between an olefinic quaternary carbon at δC 141.87 and carbon of a tertiary alcohol at δC 76.53 with two singlet at δH 1.14 (CH3-24) and 1.04 (CH3-23) confirm the presence of a double bond at C-5. By comparing the chemical shift of compound 1 with the reported data, it was identified as glut-5-en-3 β-ol (glutinol). NMR spectrum of compound 2 showed signals of three olefinic protons, two equivalent protons at δH 4.83 (br s) and 4.62 (br s) corresponding to H-15 and 16, and a third one at δH 5.21 (t) assigned for H-12. Their corresponding carbons according to HMQC displayed at δC 129.24, 109.56, and 121.97, respectively, and another quaternary olefinic carbon appeared at δC 142.96. HMBC spectrum showed a correlation between carbon of a tertiary alcohol at δC 79.56 and the signals of 23 and 24 CH3. By comparing our data with the reported one,, compound 2 was identified as olean-12,15-diene-3 β-ol. Mass and 13 C-NMR spectral data of compounds 3, 4, and 5 indicate a diterpene skeleton with a molecular formula C24H38O3, C20H26O2, and C20H34O2, respectively. NMR spectrum of compound 3 showed a signal of an oxygenated carbon at δC 67.37 has two protons at δH 4.12 and 4.64, these protons showed a correlation with carbonyl carbon at δC 166.91. This was assigned for the acyl group at C-19. Two olefinic carbons appeared at δC 131.51 and 128.59, and a downfield shifted olefinic proton appeared at δH 7.69, indicating the presence of a conjugated olefinic bond with the carbonyl carbon. Another oxygenated carbon appeared at δC 74.38, its proton displayed at δH 2.83 and showed a correlation with a methyl signal (CH3-20), this revealed the presence of hydroxyl group at C-3. From the given data, compound 3 was identified as 3-hydoxy-19-cyclopropenoyloxy-abietane. NMR spectrum of compound 4 showed signals of six olefinic carbons at δC 161.98, 147.97, 135.60, 128.11, 127.32, and 125.68 and two olefinic protons at δH 7.24 and 7.12, which revealed the presence of an aromatic system at ring C. The olefinic carbon at 161.98 showed a correlation with a proton at δH 3.89 (d, J = 2.4 Hz, H-15), this proton present on carbon displayed at δC 52.37 according to HMQC. Furthermore, a signal of carbonyl carbon appeared at δC 174.25, this confirmed the presence of C-12,16 olide group. Thus, compound 4 was identified as ent-abieta-9, 12, 14-triene-12,16-olide. NMR spectrum of compound 5 displayed an olefinic proton at δH 5.42 (br s) and two olefinic carbons at δC 140.74 and 120.56. Furthermore, signals of two oxygenated carbons displayed at δC 74.43 and 68.77, while their corresponding protons overlapped with the solvent peak. A methyl signal at δH 1.15 (s) showed a correlation in HMBC with the olefinic carbon at δC 140.74 and the oxygenated one at δC 68.77, this revealed the presence of an olefinic bond at C-5 and a hydroxyl group at C-19. From the given data and by comparison with similar structures,, compound 5 was identified as 12,19-dihydroxy-abieta-5-ene.
|Figure 1: Structures of the isolated compounds (1-5) from Euphorbia pseudocactus Berger|
Click here to view
Compounds 1–4 and the n-hexane fraction were tested for their antimicrobial activity, the results showed that compounds 3 and 4 exhibited moderate activity against the tested Gram-positive and Gram negative bacteria while compound 2 and n-hexane fraction exhibited weak activity. Compound 1 exhibited no activity against the tested bacteria. All the tested samples showed no activity against the tested yeast and fungi.
| Conclusion|| |
Two triterpenes (1 and 2) and three abietane diterpenes (3–5) were isolated for the 1st time from E. pseudocactus Berger. The three diterpenes were isolated for the 1st time in nature. Compounds 3 and 4 exhibited moderate antibacterial activity, whereas the major isolated compound (1) showed no activity. Thus, the observed antibacterial activity of the n-hexane fraction could be attributed mainly to its diterpenoid content.
The authors are grateful for Dr. Mohamed Elgebaly, taxonomy specialist, El Orman Garden, Giza, for identification of the plant sample.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Webster GL. Classification of the Euphorbiaceae
. Ann Mo Bot Gard 1994;81:3-32.
Jassbi AR. Chemistry and biological activity of secondary metabolites in Euphorbia
from Iran. Phytochemistry 2006;67:1977-84.
Manandhar NP. Plants and People of Nepal. Portland, Oregon, USA: Timber Press; 2002. p. 599.
Watanabe T, Rajbhandari KR, Malla KJ, Yahara S. A Handbook of Medicinal Plants of Nepal. Japan: Ayur Seed Life Environmental Institute; 2005. p. 262.
Joshi SG. Medicinal Plants. New Delhi, India: Oxford and IBH Publishing; 2006. p. 491.
Raja SR, Dinesh MG, Jayal ET. An in vitro
study of cytotoxic activity of Euphorbia hirta
on HEP2 cells of human epithelioma of larynx. Int J Pharm Pharm Sci 2011;3:101-3.
Özlem SA, Tülay AC. Antioxidant, cytotoxic and apoptotic activities of extracts from medicinal plant Euphorbia platyphyllos
L. J Med Plants Res 2013;7:1293-304.
Rauf A, Khan A, Uddin N, Roohullah. Cytotoxic study of aerial parts of Euphorbia Milli
and Euphorbia pulcherrima
. Topclass J Herb Med 2013;2:266-9.
Mahmoud A, Mustafa G, Farough F, Ebrahim E. Cytotoxic activities of Euphorbia kopetdaghi
against OVCAR-3 and EJ-138 cell lines. J Herb Med Pharmacol 2015;4:49-52.
Aisha A, Raja AS, Muhammad AR, Muhammad S. Antioxidant, antimicrobial, antitumor, and cytotoxic activities of an important medicinal plant (Euphorbia royleana
) from Pakistan. J Food Drug Anal 2015;23:109-15.
Abubakar EM. Antibacterial activity of crude extracts of Euphorbia hirta
against some bacteria associated with enteric infections. J Med Plants Res 2009;3:498-505.
Rao B, Karthik L, Elumalai EK, Srinivasan K, Gaurav K. Antibacterial and antifungal activity of Euphorbia hirta
L. leaves: A comparative study. J Pharm Res 2010;3:548-9.
Rajeh MA, Zuraini Z, Sasidharan S, Latha LY, Amutha S. Assessment of Euphorbia hirta
L. leaf, flower, stem and root extracts for their antibacterial and antifungal activity and brine shrimp lethality. Molecules 2010;15:6008-18.
Kader J, Noor HM, Radzi SM, Abdul Wahab NA. Antibacterial activities and phytochemical screening of the acetone extract from Euphorbia hirta
. Int J Med Plants Res 2013;2:209-14.
Gayathri A, Ramesh KV. Antifungal activity of Euphorbia hirta
L. inflorescence extract against Aspergillus flavus
– A mode of action study. Int J Curr Microbiol Appl Sci 2013;2:31-7.
Samidurai K, Mathew N. Bioassay guided fractionation and GC-MS analysis of Euphorbia lactea
extract for mosquito larvicidal activity. Int J Pharm Pharm Sci 2014;6:344-7.
Sandhyarani S, Kumar PK. Insecticidal activity of ethanol extract of leaves of Euphorbia nivulia
. Int J Pharmcol Scr Methods 2014;4:102-4.
Singh SK, Prabha T, Kavitha B, Chouhan HS, Bharti SK. Anti-inflammatory and hepatoprotective activities of ethanolic extract of Euphorbia thymifolia
Linn. Pharmacologyonline 2009;1:986-94.
Garipelli N, Runja C, Potnuri N, Pigili RK. Anti-inflammatory and antioxidant activities of ethanolic extract of Euphorbia thymifolia
Linn. whole plant. Int J Pharm Pharm Sci 2014;4:516-9.
Ahmad SF, Attia SM, Bakheet SA, Ashour AE, Zoheir KM, Abd-Allah AR. Anti-inflammatory effect of Euphorbia hirta
in an adjuvant-induced arthritic murine model. Immunol Invest 2014;43:197-211.
Qi-Cheng W, Yu-Ping T, An-Wei D, Fen-Qiang Y, Li Z, Jin-Ao D.13
C-NMR data of three important diterpenes isolated from Euphorbia
species. Molecules 2009;14:4454-75.
Norihisa K, Nobuhiro A, Tatsuya A, Koetsu T, Ken-ichi K. Pharmacological prospects of oxygenated abietane-type diterpenoids from Taxodium distichum
Cones. Adv Biol Chem 2014;4:109-15.
González MA. Aromatic abietane diterpenoids: Their biological activity and synthesis. Nat Prod Rep 2015;32:684-704.
LLIFLE – Encyclopedia of Living Forms. CC-BY-SA Creative Commons Attribution License. Available from: http://www.llifle.com
. [Last updated on 2005 Nov 14; Last cited on 2015 Oct 11].
Muanz DN, Kim BW, Euler KL, Williams L. Antibacterial and antifungal activities of nine medicinal plants from Zaire. Int J Pharmacogn 1994;32:337-45.
Harborne JB, Williams CA. A survey of antifungal compounds from higher plants, 1982-1993. Phytochemistry 1994;37:19-42.
Elham SD, Nabila AK, Ahmad MF. Synthesis and antimicrobial evaluation of some new pyridine based heterocycles. Heterocycles 2010;81:2247-56.
Good JL, Akisha T. Analysis of Sterols. 1st
ed. Blackie Academic and Professional Press, Chapman Hall; 1997.
Mahato SB, Kundu AP. 13CNMR spectra of pentacyclic triterpenoids. A compilation and some salient features. Phytochemistry 1994;37:1517-75.
Lai AR, Cambie RC, Rutledge PS, Woodgate PD. Ent-pimarane and ent-abietane diterpenes from Euphorbia fidjiana
. Phytochemistry 1990;29:2239-46.
| Authors|| |
Enas Hussein Abdelrahman Assoc.Prof. Pharmacognosy Department Cairo University, Egypt, My researches about natural products chemistry and Phytotherapy.