Crispene A, B, C and D, four new clerodane type furanoid diterpenes from Tinospora crispa (L.)
Farhad Hossen1, Rubaida Ahasan2, Mohammad Rashedul Haque3, Bilkis Begum1, Choudhury Mahmood Hasan3
1 Department of Clinical Pharmacy and Pharmacology, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh
2 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh
3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000, Bangladesh
|Date of Web Publication||10-Feb-2016|
Choudhury Mahmood Hasan
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Dhaka-1000
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Tinospora crispa (L.) is used to alleviate the symptoms of diabetes mellitus in folk medicine. It is also used for hypertension and to treat malaria, remedy for diarrhea, and as vermifuge. Materials and Methods: Stems of T. crispa were collected, sun dried for several days followed by oven dried for 24 h at a considerably low temperature and then ground into coarse powder. The powdered stems were soaked in methanol at room temperature for 14 days with occasional shaking. The extract was collected by filtration, and the solvent was evaporated under reduced pressure in a rotary evaporator to obtain a solid residue which was then subjected to fractionation using the modified Kupchan partitioning method into n-hexane, CCl4, CHCl3and aqueous soluble fractions. The n-hexane soluble fraction was chromatographed over sephadex (LH-20) and the column was eluted with n-hexane: CH2Cl2:MeOH (2:5:1) followed by CH2Cl2:MeOH (9:1) and MeOH (100%) in order to increase the polarities. The column fractions were then concentrated and subjected to thin layer chromatography screening and the fractions with a satisfactory resolution of compounds were rechromatographed over silica gel to isolate the pure compounds. Results: Four new furanoid diterpenes of clerodane types, Crispene A, B, C, and D (1–4), including one known furanoid diterpene glucoside, borapetoside E (5), were isolated from the stems of T. crispa. The structures of these compounds were elucidated by means of extensive spectroscopic analysis and by comparison of their spectral data with closely related compounds. Conclusion: We have reported four new furanoid diterpenes of clerodane types, including one known furanoid diterpene glucoside. This is the first report of any clerodane diterpene having olefinic bond between C-6 and C-7.
Keywords: Clerodane, Crispene, furanoid diterpene, Menispermaceae, Tinospora crispa
|How to cite this article:|
Hossen F, Ahasan R, Haque MR, Begum B, Hasan CM. Crispene A, B, C and D, four new clerodane type furanoid diterpenes from Tinospora crispa (L.). Phcog Mag 2016;12, Suppl S1:37-41
|How to cite this URL:|
Hossen F, Ahasan R, Haque MR, Begum B, Hasan CM. Crispene A, B, C and D, four new clerodane type furanoid diterpenes from Tinospora crispa (L.). Phcog Mag [serial online] 2016 [cited 2022 Jan 26];12, Suppl S1:37-41. Available from: http://www.phcog.com/text.asp?2016/12/45/37/176116
- Crispene A, B, C, and D, four new furanoid diterpenes of clerodane types from Tinospora crispa
- Crispene C, an unusual furanoid diterpene with olifinic bond between C-6 and C-7
- First report of Crispene D as a free aglycone, though it was earlier reported as an enzymatic hydrolysis product.
| Introduction|| |
Tinospora crispa (L.) (Synonym: Tinospora rumphii Boerl., Menispermum crispum L.; Local name: Gulancha; family - Menispermaceae) is a woody climber with numerous protrusions on the stem and is native to Malesia, Indochina, Indian subcontinent, and China., In traditional medicine, it is used for hypertension, diabetes mellitus, to treat malaria, remedy for diarrhea and as vermifuge. Pharmacological studies on this plant also demonstrated its anti-inflammatory, antioxidant, antimalarial, antiprotozoal, and hypoglycemic activities. Previous chemical investigation on T. crispa have been reported the isolation of borapetol A and B, borapetoside A, B, C, E, and F, tinocrisposide, N-formylanondine, N-formylnornuciferine, N-acetyl nornuciferine, g-sitosterol, picrotein, tinotubride, jatrorhizine, magnoflorine, palmatine, protoberberine, tembolarine, diosmetin, cycloeucalenol, cycloeucalenone and other clerodane type furanoid diterpenes and their glycosides.,,,,,
This paper describes the phytochemical investigation of the stems of T. crispa, which resulted in the isolation and structural elucidation of four new furanoid diterpenes of clerodane types, Crispene A, B, C, and D (1–4), along with a previously isolated and reported furanoid diterpene glucoside, borapetoside E (5) [Figure 1].,
| Materials and Methods|| |
General experimental procedure
Column chromatography was performed on sephadex (LH-20). Optical rotations were measured on an ADP 220 polarimeter (Bellingham Stanley Ltd.,) and concentrations (c) are given in g/100 ml.1 H nuclear magnetic resonance (NMR) and 13 C NMR spectra were acquired at 300 K using Bruker Advance NMR spectrometer at 400 MHz and 100 MHz respectively. Chemical shifts are reported relative to TMS (d = 0.0 ppm). Chemical shifts (d H) are quoted in ppm (parts per million) and referenced to CDCl3 residual chloroform signal 1 H d = 7.26,13 C d = 77.2. Electrospray ionization mass spectroscopy (ESI-MS) data were collected using a Waters Micromass ZQ instrument coupled to a Waters 2695 high-performance liquid chromatography with a Waters 2996 photodiode array. Waters Micromass ZQ parameters used were capillary (kV), 3.38; cone (V), 35; extractor (V), 3.0; source temperature (°C), 100; desolvation temperature (°C), 200; cone flow rate (L/h), 50; and desolvation flow rate (L/h), 250.
Stems of T. crispa were collected from the Tangail district of Bangladesh, in the month of March 2009. The plant was identified by Mr. Sardar Nasir Uddin, Senior Scientific officer, Bangladesh National Herbarium, Dhaka, where a voucher specimen has been deposited (DACB accession number: 35291). The stems were sun dried for several days followed by oven dried for 24 h at a considerably low temperature. The dried stems were then ground into coarse powder using high capacity grinding machine in the Phytochemical Research Laboratory, Faculty of Pharmacy, University of Dhaka.
Extraction and isolation
The powdered stems (600 mg) were soaked in methanol (3 L) at room temperature for 14 days with occasional shaking and the extract was collected by filtration. The solvent was evaporated under reduced pressure in a rotary evaporator to obtain a solid residue (5 g) which was then subjected to fractionation using the modified Kupchan partitioning method  into n-hexane, CCl4, CHCl3, and aqueous soluble fractions. Evaporation of solvent afforded n-hexane (400 mg), CCl4 (1.56 g), CHCl3 (140 mg), and aqueous soluble fractions. The n-hexane soluble fraction was chromatographed over sephadex (LH-20), and the column was eluted with n-hexane: CH2 Cl2:MeOH (2:5:1) followed by CH2 Cl2:MeOH (9:1) and MeOH (100%) in order to increase the polarities. The column fractions were then concentrated and subjected to thin layer chromatography (TLC) screening. The fractions with a satisfactory resolution of compounds were rechromatographed over silica gel separately to obtain the pure compounds 1 (1.4 mg), 2 (1.3 mg), 3 (1.7 mg), 4 (1.6 mg), and 5 (3.8 mg).
| Results and Discussion|| |
The methanol extract of the dried stems of T. crispa was successively partitioned with n-hexane, carbon tetrachloride, and chloroform. The n-hexane fraction was then subjected to column chromatography using sephadex (LH-20) followed by TLC screening and preparative TLC to isolate five pure compounds (1–5) [Figure 1]. The NMR spectra of these compounds suggested that four of them were furanoid diterpenes of clerodane series and another was furanoid diterpene glucoside of the same series.
The 1 H NMR spectra of 1–4, which are summarized in [Table 1] and [Table 2], suggested some common structural features: The presence of an olefinic bond (C-3 and C-4), a furan ring (C-13–C-16), a carbonyl group (C-18), and two angular methyl groups (C-19 and C-20). The spectra also suggested the presence of two sp3-hybridized quaternary carbons (C-5 and C-9), two sp3-hybridized methines (C-8 and C-10) and an olefinic proton (C-3), which confirmed the absence of proton at C-4. The one proton broad signals at δ 6.72–7.01 and no cross connection between C-3 proton and any olefinic protons in correlation spectroscopy (COSY) experiment exclude the possibility of the double bond at C-1 and C-2. The spectra of 1, 2, and 4 showed signals attributed to four sp3-hybridized methylenes (C-1, C-2, C-7, and C-11) and two oxygenated methine groups (C-6 and C-12). Whereas, the spectrum of 3 indicated three sp3-hybridized methylenes (C-1, C-2, and C-11) and one oxygenated methine group (C-12).
|Table 2: Comparison of 1H NMR spectral data of compounds 4 and 5 with the published data|
Click here to view
The ESI-MS of compound 1 showed a pseudo molecular ion [M + Na]+ peak at m/z 365.0 corresponding to the molecular formula C20H22O5 and compound 2 showed a pseudo molecular ion [M + H]+ peak at m/z 361.10 relevant to C20H24O6. The 1 H NMR spectra of 1 and 2 [Table 1] displayed a one proton double doublet at δ 5.51 (J = 11.6, 4.8 Hz) and δ 5.33 (J = 12.0, 4.0 Hz), which were assigned to C-12. The difference in chemical shifts of C-12 (δ 5.51 and δ 5.33) is may be due to the dissimilarity in the fusion of B/C rings (cis-fused in 1 and trans-fused in 2). The downfield chemical shifts of the protons at C-12 were also indicative of the presence of a lactone ring at C-12 and C-17. Strong cross peaks were observed between C-12 proton with the C-11β and C-11α protons in the COSY spectra. The one proton doublet of doublets (dd) at δ 2.42 (J 4.0, 3.6 Hz) in 1 and δ 2.48 (J = 12.6, 2.2 Hz) in 2 at C-8 were indicative of the presence of neighboring carbonyl group at C-17. The proton at C-6 of compound 1 showed a downfield dd at δ 4.62 (1H, J = 10.6, 6.6 Hz) which revealed the presence of another lactone ring between C-6 and C-18. The large coupling (10.6 Hz) is indicative of axial orientation of H-6. On the other, the proton at C-6 of compound 2 showed the corresponding downfield dd at δ 4.46 (1H, J = 10.6, 7.4 Hz), which indicated the presence of a free hydroxyl group (C-6). Again, the large coupling (10.6 Hz) confirms that the oxymethine proton at C-6 is axial. Based on these results and from a comparison with the reported data of closely related compounds,,,,, the structure of 1 and 2 were characterized as shown. Compound 1 and 2 are found to be new diterpenes and given the trivial names Crispene A and Crispene B, respectively.
The relative configuration at various centers in 1 and 2 were derived from extensive dreiding model study and comparing the published 1 H NMR data of similar compounds. In the literatures, most of the bicyclic furanoid diterpenes of clerodane types isolated from Tinospora genus, the H-10 and C-19 methyl are in α orientation, i.e. A/B rings are cis-fused. Again, C-20 methyl and H-12 are in β and α configuration, respectively. In compound 1 and 2, the H-10 appears as broad doublet (6.0 Hz and 6.8 Hz) indicating that this proton is in axial (α) position (with respect to B ring) and the C-19 methyl is equatorial (α). As the H-10 is axial and α, from the biogenetic point the methyl group at C-9 must be in axial and β orientation. The H-8 proton in compound 1 appears as dd at δ 2.42 (J = 4.0, 3.6 Hz) showing its equatorial (β) orientation. Since both H-8 and C-20 methyl are in β orientation, B/C rings are cis-fused. The oxymethine proton at C-12 appears as dd at δ 5.51 (J = 11.6, 4.8 Hz) and the large coupling (11.6 Hz) confirms it to be axial (α). In compound 2, the H-8 proton appears as dd at δ 2.48 (J = 12.6, 2.2 Hz) and the large coupling of 12.6 Hz showed it to be in axial (α) position. Hence, the B/C rings are trans-fused. C-12 proton appears as dd at δ 5.33 (J = 12.0, 4.0 Hz), the large coupling of 12.0 Hz indicative of axial orientation and α as the C ring in half-chair conformation. In both 1 and 2, the relative deshielding of C-7 equatorial proton at δ 2.80 and 2.58, respectively, support earlier findings  that C (8)-C (17)-O-C (12) are on the same plane.
ESI-MS of compound 3 gave a mol. wt. 417.20 [M + H]+ suggestive of C23H28O7. The 1 H NMR spectrum of 3 [Table 1] displayed a one proton double doublet at δ 5.94 (J = 8.0, 7.6 Hz), which was assigned at C-12. The downfield shift (δ 5.94) of this proton was also indicative of the presence of an acetoxy group (OAc) at C-12, which was confirmed by the methyl signal at δ 1.56 (3H, s). In the mass spectrum, a presence of a strong fragment at m/z 358 produced by the loss of 59 also supports the presence of OAc group. The relative shielding of the acetoxymethyl is quite rare but not uncommon.,, The location of the OAc group at C-12 was also augmented by the cross-linking observed in the COSY between the oxymethine proton (δ 5.94) with C-11 protons at δ 2.28 and 1.92. Two downfield doublets at δ 6.38 (1H, J = 10.4 Hz) and δ 6.56 (1H, J = 10.4 Hz) revealed the presence of two olefinic protons and COSY showed that these protons only couple with each other, hence this double bond must be at C-6 and C-7. It was also substantiated by the presence of a 1 H singlet of C-8 (β and equatorial) proton at δ 2.09. It is interesting to note that in the related diterpenes, the Hβ-8 proton appears as broad singlet, even when there are two protons at C-7., The placement of oxymethine proton at C-12 is determined on the basis of its close similarity in the chemical shift value (δ 5.94) with those reported for the same proton (H-12) in amritoside A and B. In addition, the nuclear overhauser effect experiment showed cross peaks between Hβ-8 and CH3 OCO − indicated that the orientation of H-12 is α [Figure 2]. The location of another CH3 OCO − (δ 3.73, 3H, s) was placed at C-18 on common biogenetic ground. From these spectral data, the structure of 3 was elucidated as shown. To our knowledge, there is no record of any clerodane diterpene having olefinic bond between C-6 and C-7. This new compound is given the trivial name Crispene C.
Compound 4 has the molecular formula C21H26O6 as determined by ESI-MS at m/z 375.20 [M + H]+. The 1 H NMR spectrum of 4 [Table 1] and [Table 2] suggested the presence of all the common structural features of 5 except the data for glucose moiety at C-12. Compound 5 exhibited a one proton dd at δ 5.27 (J = 9.6, 2.8 Hz) which was assigned to the C-12 proton and revealed the presence of glycosidic linkage. On the other, compound 4 exhibited the corresponding signal at δ 5.13 (1H, bd, J = 8.4 Hz) which indicated the presence of free hydroxyl group at C-12. The signal at δ 3.73 (3H, s) was due to the ester methyl group at C-18. On the basis of these results and from a comparison with the published data,, compound 4 was identified as the aglycone of 5. Though it was earlier reported as an enzymatic hydrolysis product of borapetoside E (5),, this is the first record of 4 as free aglycone and the trivial name Crispene D has been proposed.
Compound 5 was characterized as borapetoside E by comparing the spectral data [Table 1] and [Table 2] with the published data of this compound., This diterpene glucoside was first reported from Tinospora tuberculata and later on isolated and the structure was revised as 5.
Crispene A (1)
Amorphous powder.1 H-NMR (CDCl3, 400 MHz): [Table 1]. ESI-MS (positive-ion mode) m/z: 365.0 [M + Na]+ (Calculated for C20H22O5 Na: 365.40), 350, 329, 288, 255, 155, 151, 102, 100, 91, 89.
Crispene B (2)
Amorphous powder.1 H-NMR (CDCl3, 400 MHz): [Table 1]. ESI-MS (positive-ion mode) m/z: 361.10 [M + H]+ (Calculated for C20H25O6: 361.41), 289, 238, 214, 158, 141, 116, 101, 100, 90, 89.
Crispene C (3)
Amorphous powder.1 H-NMR (CDCl3, 400 MHz): [Table 1]. ESI-MS (positive-ion mode) m/z: 417.20 [M + H]+ (Calculated for C23H29O7: 417.48), 379, 358, 347, 288, 255, 198, 142, 123, 100, 91, 89, 84.
Crispene D (4)
Colorless gum.1 H-NMR (CDCl3, 400 MHz): [Table 1] and [Table 2]. ESI-MS (positive-ion mode) m/z: 375.20 [M + H]+ (Calculated for C21H27O6: 375.44), 357, 339, 289, 214, 158, 141, 130, 119, 102, 89.
Borapetoside E (5)
Amorphous powder,: 47.1 (c 0.34, CDCl3).1 H-NMR (CDCl3, 400 MHz): [Table 1] and [Table 2].13 C-NMR (CDCl3, 100 MHz) δ:16.46 (C-1, CH2), 24.17 (C-2, CH2), 142.37 (C-3, CH), 134.30 (C-4, C), 39.30 (C-5, C), 82.87 (C-6, CH), 29.45 (C-7, CH2), 46.55 (C-8, CH), 39.26 (C-9, C), 45.57 (C-10, CH), 46.62 (C-11, CH2), 69.0 (C-12, CH), 125.86 (C-13, C), 108.93 (C-14, CH), 143.66 (C-15, CH), 140.20 (C-16, CH), 178.39 (C-17, C), 166.8 (C-18, C), 27.15 (C-19, CH3), 21.69 (C-20, CH3), 51.69 (CH3 COO-), 98.99 (C-1', CH), 73.86 (C-2', CH), 76.40 (C-3', CH), 70.73 (C-4', CH), 75.08 (C-5', CH), 62.55 (C-6', CH2). ESI-MS (positive-ion mode) m/z: 559.14 [M + Na]+ (Calculated for C27H36O11 Na: 559.58), 357, 356, 204, 152, 119, 80.
| Conclusion|| |
We have isolated and characterized four new furanoid diterpenes of clerodane types, Crispene A, B, C and D (1–4), including one known furanoid diterpene glucoside, borapetoside E (5), from the stems of T. crispa (L.). To the best of our knowledge, there is no record of any clerodane diterpene, like Crispene C (3), having olefinic bond between C-6 and C-7.
We thank Mr. Sardar Nasir Uddin, Senior Scientific officer, Bangladesh National Herbarium for identifying the plant material and the Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, United Kingdom for providing ESI-MS determinations and NMR spectra.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Ling KH, Kian CT, Hoon TC. A Guide to Medicinal Plants: An Illustrated, Scientific and Medicinal Approach. Singapore: World Scientific Publishing Co. Pte. Ltd.; 2009.
Khare CP, editor. Indian Medicinal Plants. An Illustrated Dictionary. New York: Springer Science; 2007.
Cavin A, Hostettmann K, Dyatmyko W, Potterat O. Antioxidant and lipophilic constituents of Tinospora crispa
. Planta Med 1998;64:393-6.
Yusuf UK, Noor H. Flavone O-glycosides from Tinospora crispa
. Fitoterapia 1995;66:280.
Kongkathip N, Dhumma-upakorn P, Kongkathip B, Chawananoraset K, Sangchomkaeo P, Hatthakitpanichakul S. Study on cardiac contractility of cycloeucalenol and cycloeucalenone isolated from Tinospora crispa
. J Ethnopharmacol 2002;83:95-9.
Pachaly P, Adnan AZ, Will G. NMR-Assignments of N-Acylaporphine Alkaloids from Tinospora crispa
. Planta Med 1992;58:184-7.
Pathak AK, Jain DC, Sharma RP. Chemistry and biological activities of the genera Tinospora
. Int J Pharmacogn 1995;33:277-87.
Choudhary MI, Ismail M, Shaari K, Abbaskhan A, Sattar SA, Lajis NH, et al.
cis-Clerodane-type furanoditerpenoids from Tinospora crispa
. J Nat Prod 2010;73:541-7.
Fukuda N, Yonemitsu M, Kimura T. Studies on the constituents of the stems of Tinospora tuberculata
, IV. Isolation and structure elucidation of the five new furanoid diterpene glycosides borapetoside C-G. Liebigs Ann Chem 1993;1993:491-5.
Martin TS, Ohtani K, Kasai R, Yamasaki K. Furanoid diterpene glucosides from Tinospora rumphii
. Phytochemistry 1996;42:153-8.
Vanwagenen BC, Larsen R, Cardellina JH, Randazzo D, Lidert ZC, Swithenbank C. Ulosantoin, a potent insecticide from the sponge Ulosa ruetzleri
. J Org Chem 1993;58:335-7.
Fukuda N, Yonemitsu M, Kimura T, Hachiyama S, Miyahara K, Kawasaki T. Studies on the constituents of the stems of Tinospora tuberculata
BEUMEE. II. New diterpenoids, borapetoside A and borapetol A. Chem Pharm Bull 1985;33:4438-44.
Fukuda N, Yonemitsu M, Kimura T. Studies on the constituents of the stems of Tinospora tuberculata
Beumee. III. New diterpenoids, borapetoside B and borapetol B. Chem Pharm Bull 1986;34:2868-72.
Waterman PG, Reza-ul-Zalil, Hasan CM, Jabbar A. Columbin from the Root of Momordica cochinchinensis
: High-field NMR Studies. Planta Med 1985;51:181-2.
Maurya R, Manhas LR, Gupta P, Mishra PK, Singh G, Yadav PP. Amritosides A, B, C and D: Clerodane furano diterpene glucosides from Tinospora cordifolia
. Phytochemistry 2004;65:2051-5.
Fuji K, Tanaka K, Li B, Shingu T, Sun H, Taga T. Novel diterpenoids from Taxus chinensis
. J Nat Prod 1993;56:1520-31.
Baek NI, Chung MS, Shamon L, Kardono LB, Tsauri S, Padmawinata K, et al.
Selligueain A, a novel highly sweet proanthocyanidin from the rhizomes of Selliguea feei
. J Nat Prod 1993;56:1532-8.
Li W, Wei K, Fu H, Koike K. Structure and absolute configuration of clerodane diterpene glycosides and a rearranged cadinane sesquiterpene glycoside from the stems of Tinospora sinensis
. J Nat Prod 2007;70:1971-6.
Gangan VD, Pradhan P, Sipahimalani AT, Banerji A. Cordifolisides A, B, C: Norditerpene furan glycosides from Tinospora cordifolia
. Phytochemistry 1994;37:781-6.
| Authors|| |
Dr. Choudhury Mahmood Hasan, is a Professor of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Dhaka, Bangladesh. His research focuses on the chemical and biological studies of local medicinal plants with emphasis on structure elucidation of the secondary metabolites by spectroscopic techniques (UV, IR, NMR, MS etc.). He is the main/ co-author of 232 peer-reviewed papers published in different international and local scientific journals. Dr. Hasan is a member of American Chemical Society (ACS), American Society of Pharmacognosy (ASP), Royal Society of Chemistry (RCS) and a fellow of Bangladesh Academy of Sciences (BAS).
[Figure 1], [Figure 2]
[Table 1], [Table 2]