Taraxer-14-en-3β-ol, an Anti-Inflammatory Compound from Sterculia foetida L.

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Taraxer-14-en-3β-ol (1) was shown to be the active ingredient in the leaves of Sterculia foetida L. The alcohol 1, its acetate and ketone showed anti-inflammatory activity against TPA induced mouse ear oedema with inhibition ratios of 60.0, 58.57 and 40.57 at 0.5 mg/ear, respectively. The percentage inhibition of inflammation increased with dose for each compound.

Sterculia foetida L. leaf extract exhibited significant anti-inflammatory and CNS depressant activity [1]. The presence of taraxerol, β-sitosterol, n-octacosanol and glucuronyl derivatives of procyanidin, scutellarein and luteolin is reported [1]. To locate the hitherto unknown active ingredient, the extract was purified by column chromatography and isolated compounds were screened. Local anti-inflammatory activity was determined using the TPA-induced ear inflammation model in Swiss Albino mice. The results show dose-dependent and significant local anti-inflammatory activity of compound 1 against oedema provoked by local application of TPA. Comparison of the physical and spectral data of 1 with the literature confirmed it to be taraxer-14-en-3β-ol. Screening of its acetate 2 and ketone 3 (Fig. [1]) indicated their activities to be dose-dependent and the ketone to be the least active, possibly due to the absence of free/protected hydroxyl group at C-3. The activity of alcohol 1 was superior to that of the acetate 2. This could be due to the presence of a free hydroxy group at C-3. This trend is illustrated by the inhibition ratios at 0.5 mg/ear. The data also indicate the increased inhibition of inflammation with increase in dose in each case (Table [1]). However, it was slightly less in comparison with that of indomethacin. The phorbol ester provides a suitable model for evaluation of both topical and systemic anti-inflammatory agents. The majority of its activities appear to involve or be dependent on arachidonic acid release and metabolism and interaction with protein kinase C [2].

Materials and Methods

Melting points are uncorrected. IR spectra were recorded in CCl₄ solutions on a Shimadzu IR 470 spectrophotometer. Optical rotations were measured with a Perkin-Elmer spectropolarimeter. NMR spectra were recorded on Bruker AM250 (¹H, 250.13 MHz, ¹³C, 62.9 MHz) and Bruker DRX500 spectrometers (¹H, 500.13 MHz, ¹³C, 125.7 MHz) using TMS as an internal standard (chemical shifts in δ, ppm).

Leaves of Sterculia foetida L. were collected from Pune and were authenticated by matching with the voucher specimen AHMA: 581 available with the Agharkar Herbarium at Maharashtra Association (AHMA) at Agharkar Research Institute.

Shade-dried leaves (1100 g) were extracted with EtOH (3 L). The alcohol extract was evaporated under vacuum to yield total crude extract as a dark viscous residue (35 g, 3.18 %). Part of this (10 g) was adsorbed on silica gel (60 - 120 mesh, 15 g) and loaded on an MPLC column of TLC silica gel G (2.6 × 46 cm, 1 : 15 g). The column was eluted using hexane (1 L), hexane/EtOAc (19 : 1, 1.8 L), hexane/EtOAc (9 : 1, 0.4 L), hexane/EtOAc (17 : 3, 0.4 L), hexane/EtOAc (1 : 1, 0.4 L), EtOAc (0.4 L), EtOH (0.4 L) at 4.8 mL/min, t_R of 1: between 1 - 200 mL 2: between 200 - 400 mL. Similarly eluted were fractions 3 - 24 with an elution volume 200 mL each. Fractions 6 - 10 eluted with hexane/EtOAc (19 : 1) were combined (0.450 g) and recrystallised with hexane yielding compound 1 (0.125 g). TLC single spot, (10 % EtOAc in hexane, R_f: 0.45); m. p. 278 °C; [α]_D ²⁰: + 0.2° (CHCl₃) lit.[3], m. p. 278 - 280 °C, [α]_D ²⁰: + 2°; IR (CCl₄): ν = 3600, 1540, 1460, 1380, 1030, 1000 cm^-1; ¹H-NMR (250.133 Hz, deuterated acetone): proton at C-7 four peaks 6.5916, 6.5790, 6.5615, 6.5473 of equal intensities, methyl protons 2.1297, 1.9809, 1.9632, 1.9459, 1.9333, 1.8556, 1.8017 (7 sets of methyl protons); elemental analysis found: C 84.35; H 11.96; calculated for C₃₀H₅₀O: C 84.44; H 11.74 %.

Monoacetate 2, prepared by treating taraxer-14-en-3β-ol (1, 150 mg), with acetic anhydride was obtained as a colourless solid (125 mg); m. p. 297 - 299 °C; [α]_D ²⁰: + 11.73° (CHCl₃), (lit. [4] m. p. 302 °C); IR (CCl₄): ν = 1720, 1440, 1360, 1240, 1030, 1000 cm^-1; elemental analysis found: C 76.47; H 11.64; calculated for C₃₂H₅₂O₂ (2H₂O): C 76.25; H 11.20 %.

Taraxer-14-en-3-one (3, 71 mg) was obtained by Jone’s oxidation of 1 (150 mg); m. p. 235 - 237 °C; [α]_D ²⁰: + 14.47° (CHCl₃), (lit. [5] m. p. 238 - 239 °C); IR (CCl₄): ν = 1720, 1460, 1380, 1000 cm^-1; elemental analysis found: C 85.14; H 11.31; calculated for C₃₀H₄₈O: C 84.91; H 11.32 %.

Animal experiments were conducted as per guidelines suggested by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) under Ministry of Statistics and Programme Implementation, Government of India.

Male Swiss Albino mice were obtained from the National Institute of Virology, Pune. They were inbred in the animal house facilities at Agharkar Research Institute, for several generations for the last 19 years. They were housed in polypropylene cages in an air-conditioned area at 25 ± 2 °C with 10 to 14 h light and dark cycle and were given Amrut brand balanced animal feed and water ad-libitum. A group of at least six animals was used for individual treatments.

Ear oedema was induced [6] on the right ear by topical application of TPA (2.5 μg in 20 μL of acetone). The left ear (control) received the vehicle (acetone). The compound was applied at 0.25, 0.5 and 1 mg/ear, respectively, immediately after application of TPA. Indomethacin in a dose of 0.5 mg/ear was used as a positive control. The thickness of ear was measured before and at 4 h after application of TPA using a micrometer (Digitrix mark II, Japan).

The oedema was expressed as mean ± SEM from these values and these results were analysed by Student’s t-test. Based on this the percentage inhibition of inflammation and inhibition ratio at 0.5 mg/ear were calculated to assess the anti-inflammatory activity (Table [1]).

Acknowledgements

S. W. A. B. thanks the Royal Society for an Industry Fellowship and the Royal Society of Chemistry Journal Grant for visiting India.

References

• 1 Mujumdar A M, Naik D G, Waghole R J, Kulkarni D K, Kumbhojkar M S. Pharmacological studies on Sterculia foetida leaves.  Pharm Biol. 2000;  38 13-7
  PubMedSearch in Google Scholar
• 2 Young J M, De Young L M. Pharmacological methods in the control of inflammation. In: Spector J, Back N, editors New York; Alan R. Liss, Inc 1989: pp 215-31
  Search in Google Scholar
• 3 Brooks C JW. Observations on taraxerol (skimmiol). J Chem Soc 1955: 1674-7
  Search in Google Scholar
• 4 Paul B D, Rao G S. Kapadia GJ. Isolation of myricadiol, myricitrin, taraxerol and taraxerone from Myrica cerifera L. root bark.  J Pharmaceutical Sci. 1974;  63 958-9
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• 5 Bates R B, Jacobsen N E, Setzer W N, Stessman C C. NMR assignments and conformation of taraxerenes.  Magnetic Resonance in Chemistry. 1998;  36 539-41
  CrossrefPubMedSearch in Google Scholar
• 6 De Young L, Kheifts J, Young J. Edema and cell in filtration in the phorbol treated mouse ear are temporally separate and can be differentially modulated by pharmacological agents.  Agents and Actions. 1989;  26 335-41
  CrossrefPubMedSearch in Google Scholar

Dr. D. G. Naik

Chemistry Group

Agharkar Research Institute

Pune-411 004

India

Phone: +91-020-25653680

Fax: +91-020-25651542

Email: dgnpune@yahoo.co.in

References

• 1 Mujumdar A M, Naik D G, Waghole R J, Kulkarni D K, Kumbhojkar M S. Pharmacological studies on Sterculia foetida leaves.  Pharm Biol. 2000;  38 13-7
  PubMedSearch in Google Scholar
• 2 Young J M, De Young L M. Pharmacological methods in the control of inflammation. In: Spector J, Back N, editors New York; Alan R. Liss, Inc 1989: pp 215-31
  Search in Google Scholar
• 3 Brooks C JW. Observations on taraxerol (skimmiol). J Chem Soc 1955: 1674-7
  Search in Google Scholar
• 4 Paul B D, Rao G S. Kapadia GJ. Isolation of myricadiol, myricitrin, taraxerol and taraxerone from Myrica cerifera L. root bark.  J Pharmaceutical Sci. 1974;  63 958-9
  PubMedSearch in Google Scholar
• 5 Bates R B, Jacobsen N E, Setzer W N, Stessman C C. NMR assignments and conformation of taraxerenes.  Magnetic Resonance in Chemistry. 1998;  36 539-41
  CrossrefPubMedSearch in Google Scholar
• 6 De Young L, Kheifts J, Young J. Edema and cell in filtration in the phorbol treated mouse ear are temporally separate and can be differentially modulated by pharmacological agents.  Agents and Actions. 1989;  26 335-41
  CrossrefPubMedSearch in Google Scholar

Dr. D. G. Naik

Chemistry Group

Agharkar Research Institute

Pune-411 004

India

Phone: +91-020-25653680

Fax: +91-020-25651542

Email: dgnpune@yahoo.co.in