Two New Triterpenes from the Husks of Xanthoceras sorbifolia

• Abstract
• Materials and Methods
• References

Abstract

Two new triterpenes, 22-angeloyl-21-epoxyangeloylbarringtogenol C (1) and 21,22-diangeloyl-24-hydroxy-R₁-barrigenol (2), and the known 21,22-diangeloyl-R₁-barrigenol (3) were isolated from the husks of Xanthoceras sorbifolia Bunge. Their structures were elucidated based on detailed NMR analysis. Compounds 1 and 2 inhibited significantly the growth of cancer cell lines (A375-S2, HeLa) with IC₅₀ values ranging from 2.9 to 76.3 μmol/L.

Some coumarins [1], together with triterpenes, saponins [2], [3], [4], [5] and flavonoids [6], have been isolated from the seeds and wood of Xanthoceras sorbifolia Bunge (Sapindaceae), which is used in traditional medicine for the treatment of rheumatism and enuresis of children in China. The mentally stimulating activity of the fruit extract has also been reported in the literature [7]. Aiming at exploring the diverse activities of the other parts of this plant, we examined the extract of the husks and report here the structural elucidation of two new triterpenes, 1 and 2, as well as their cytotoxicity.[]

Compound 1 was obtained as an amorphous powder. The molecular formula of C₄₀H₆₂O₈ was deduced from ESI-MS (m/z = 693.3 [M + Na]⁺) and ¹³C-NMR data. The ¹H-NMR spectrum (Table [1]) showed the characteristic signals of an olean-12-ene moiety and two signals at δ = 6.66, 6.33 (each 1H, d, J = 10.1 Hz), which were assigned to H-21 and H-22 based on their correlations with C-29 and C-30 or with C-28, respectively, in the HMBC spectrum. Furthermore, the signals of an angeloyl functionality were observed: an olefinic proton at δ = 6.04 (1H, q, J = 6.2 Hz), and two vinylic methyls at δ = 2.14 (3H, d, J = 6.2 Hz), 1.91 (3H, s). The remaining prominent signals at δ = 1.64 (3H, s), 1.39 (3H, d, J = 5.3 Hz) and 3.08 (1H, q, J = 5.3 Hz) were not assigned. The ¹³C-NMR and DEPT (135°) spectra (Table [1]) revealed the signals of an angeloyl moiety and 30 typical carbon signals. They corresponded well to those of barringtogenol C found in reference [8]. The other signals observed at δ = 169.8, 60.2, 59.9, 20.0 and 14.0, combined with the signals not assigned in the ¹H-NMR spectrum, suggested the presence of a 2,3-epoxy-2-methylbutyryl moiety by comparison to a related, reported compound [9]. As an NOE between δ = 3.08 (H-3′) and δ = 1.64 (H-5′) was observed in the NOESY spectrum of 1, the moiety above was deduced to be epoxyangeloyl. Based on HSQC, HMBC and NOESY measurements, the signals of the angeloyl and epoxyangeloyl groups were assigned. Furthermore, the positions of these two acyl groups were determined with the correlations between δ = 6.66 (H-21) and δ = 169.8 (C-1′), and δ = 6.33 (H-22) and δ = 167.6 (C-1″), respectively, in the HMBC spectrum. Consequently, the structure of 1 was concluded to be 22-angeloyl-21-epoxyangeloylbarringtogenol C.

Compound 2, obtained as white needles, had the molecular formula C₄₀H₆₂O₉ based on ESI-MS and NMR data (Table [1]). The ESI-MS of 2 showed the [M + Na]⁺ ion at m/z = 709.5 and fragment ions from the stepwise cleavage of angeloyl moieties at m/z = 609.4 and 509.3. This was confirmed with the signals of the two angeloyl groups in the ¹³C-NMR spectrum. The substitution position was determined by the significant downfield shifts of C-21 (δ = 78.6) and C-22 (δ = 73.4) as well as by the crosspeaks between H-21 (δ = 6.72) and C-1′ (δ = 167.7) and H-22 (δ = 6.34) and C-1″ (δ = 168.1), respectively, observed in the HMBC spectrum. Comparison of the NMR data of 2 to those of the known compound 21,22-diangeloyl-R₁-barrigenol (3) showed that the signals of their B, C, D, and E rings and of the acyl groups were identical, except for the disappearance of the signal of one methyl moiety. Simultaneously, a pair of protons at δ = 3.69, 4.51 (each 1H, d, J = 10.5 Hz) and a signal of carbon bearing oxygen at δ = 64.7 revealed the presence of another hydroxymethyl group besides the one located at C-28. The long-range correlations between the protons at δ = 3.69, 4.51 and the carbon signals at δ = 56.2 (C-5) and 80.1 (C-3) in the HMBC spectrum and the NOE between δ = 3.65 (H-3) and 1.53 (Me-23) observed in the NOESY spectrum confirmed that Me-24 on the skeleton was substituted by a hydroxyl group. Therefore, 2 was assigned as 21,22-diangeloyl-24-hydroxy-R₁-barrigenol.

The known compound 3 was identified as 21,22-diangeloyl-R₁-barrigenol by comparison of its physical and NMR data to those reported [2] [10].

Using the MTT method, compounds 1 - 3 were tested for their cytotoxicity in vitro against human tumor cell lines (A375-S2, HeLa). Compound 2 exhibited the strongest activity with IC₅₀ values of 4.1 and 2.9 μmol/L while compound 3 was nearly inactive (Table [2]). The results indicate that the hydroxyl group positioned at C-24 is essential for biological activity. Recently, the metabolites of aescin produced by bacteria in the gut, which have structures similar to that of compound 2, have been reported [11]. Therefore, the significant cytotoxic activity of 2 measured in our assay indicated that aescin, used as an anti-inflammatory medicine in patients, has potential antitumor activity.

Materials and Methods

Melting points were determined on a Yanaco MP-S₃ apparatus without correction. ESI-MS was performed on an Agilent 1100 ion trap spectrometer. Optical rotations were measured with a Perkin-Elmer 241 polarimeter. NMR spectra were recorded on a Bruker ARX-300 or Bruker AV-600 spectrometer with TMS as an internal standard.

The plant material was collected in Shenyang, Liaoning Province, P. R. China, in October 1997. A voucher specimen (No. 0 154 620) was deposited in the Herbarium Department of the Institute of Applied Ecology, Chinese Academy of Sciences.

The cytotoxic effects on human malignant A375-S2 and human cervical cancer HeLa cells were measured with the MTT assay as described [12]. A375-S2 and HeLa cells were seeded at a density of 1 × 10⁴ cells/well in 96-well plates. After 12-h incubation, the cells were incubated with various concentrations of the tested compounds and taxol (reference compound) for 24 h. Cell growth was measured with the MTT assay on a plate reader. The percentage of cell inhibition was calculated as follows: cell death (%) = [A₄₉₀(control) - A₄₉₀(test)]/A₄₉₀(control) × 100.The 50 % inhibitory concentrations (IC₅₀) of the compounds were determined by the LOGIT method.

The dried husks of X. sorbifolia Bunge (15.0 kg) were extracted with 70 % aqueous ethanol (120 L × 3). After evaporation of the ethanol under vacuum, the extract was suspended in H₂O and partitioned with CHCl₃ (5 L × 3) and n-BuOH (5 L × 5) successively. The CHCl₃ extract (100 g) was subjected to column chromatography (CC) over silica gel (500 g, 200 - 300 mesh), eluted with petroleum ether (PE, 60 - 90 °C) and acetone resulting in fractions C-1 - C-15 (100 : 0 to 100 : 50, 5 L each). Fr. C-7 [PE-acetone (100 : 8), 5000 mL, 3 g] was further subjected to CC on silica gel (30 g, 200 - 300 mesh), eluted with PE-EtOAc (10 : 1, 10 mL each) to give 1 (50 - 100 mL, 10 mg) and 3 (200 - 300 mL, 970 mg). Fr. C-11 [PE-acetone (100 : 24), 5000 mL, 1.5 g] was purified by PTLC on silica gel with CHCl₃-MeOH (15 : 1) as the developing solvent to afford 2 (R_f = 0.4, 17 mg).

22-Angeloyl-21-epoxyangeloylbarringtogenol C (1): white amorphous powder; m. p. 255 - 257 °C; [α]_D ²⁰: + 24° (c 0.02, MeOH); positive ESI-MS: m/z = 693.3 [M + Na]⁺, 577.2 [M + Na - epoxyangeloxyl]⁺, 477.0 [M + Na - epoxyangeloxyl - angeloxyl]⁺; DEPT, ¹H- and ¹³C-NMR data are listed in Table [1].

21,22-Diangeloyl-24-hydroxy-R1-barrigenol (2): white needles; m. p. 259 - 261 °C; [α]_D ²⁰: + 35°(c 0.10, MeOH); positive ESI-MS: m/z = 709.5 [M + Na]⁺, 609.4 [M + Na - angeloxyl]⁺, 509.3 [M + Na - 2 × angeloxyl]⁺; DEPT, ¹H- and ¹³C-NMR data are listed in Table [1].

References

• 1 Chen Y J, Takeda T, Ogihara Y. Studies on the constituents of Xanthoceras sorbifolia Bunge I.  Shoyakugaku Zasshi. 1984;  38 203-6
  PubMedSearch in Google Scholar
• 2 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge II. Major sapogenol and a prosapogenin from the fruits of Xanthoceras sorbifolia Bunge.  Chem Pharm Bull. 1984;  32 3378-83
  PubMedSearch in Google Scholar
• 3 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge III. Minor prosapogenins from the fruits of Xanthoceras sorbifolia Bunge.  Chem Pharm Bull. 1985;  33 127-34
  PubMedSearch in Google Scholar
• 4 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge IV. Structures of the minor prosapogenins.  Chem Pharm Bull. 1985;  33 1043-8
  PubMedSearch in Google Scholar
• 5 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge V. Major saponins from the fruits of Xanthoceras sorbifolia Bunge.  Chem Pharm Bull. 1985;  33 1387-94
  PubMedSearch in Google Scholar
• 6 Ma C M, Nakamura N, Hattori M, Kakuda H, Qiao J C, Yu H L. Inhibitory effects on HIV-1 protease of constituents from the wood of Xanthoceras sorbifolia .  J Nat Prod. 2000;  63 238-42
  CrossrefPubMedSearch in Google Scholar
• 7 Li X B, Wang H D, Guo Y. Xanthoceras sorbifolia fruit extracts for enhancing mental activity. CN: 1 084 876 Oct 5th, 1994
  Search in Google Scholar
• 8 Greca M D, Fiorentino A, Monaco P, Previtera L. Polyoxygenated oleanane triterpenes from Hydrocotyle ranunculoides .  Phytochemistry. 1994;  35 201-4
  PubMedSearch in Google Scholar
• 9 Ahmad V U, Fizza K, Sultana A. Isolation of two sesquiterpenes from Pluchea arguta .  Phytochemistry. 1989;  28 3081-3
  CrossrefPubMedSearch in Google Scholar
• 10 Ahmad V U, Fatima I, Fatima A. The sapogenins from Dodonaea viscosa .  Fitoterapia. 1987;  58 361-2
  PubMedSearch in Google Scholar
• 11 Chen J Y, Wu L J, Teng H L, Liu K. Study on aescin metabolism in intestinal flora in vitro .  Chin Tradit Herb Drugs. 2003;  34 970-3
  PubMedSearch in Google Scholar
• 12 Zhang Y, Wu L J, Tashiro S, Onodera S, Ikejima T. Evodiamine induced tumor cell death through different pathways: apoptosis and necrosis.  Acta Pharmacol Sin. 2004;  25 83-9
  PubMedSearch in Google Scholar

Prof. Xian Li

Shenyang Pharmaceutical University

Box 49

Shenyang 110016

People’s Republic of China

Phone: +86-24-2398-6475

Email: Proflixian@163.com

References

• 1 Chen Y J, Takeda T, Ogihara Y. Studies on the constituents of Xanthoceras sorbifolia Bunge I.  Shoyakugaku Zasshi. 1984;  38 203-6
  PubMedSearch in Google Scholar
• 2 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge II. Major sapogenol and a prosapogenin from the fruits of Xanthoceras sorbifolia Bunge.  Chem Pharm Bull. 1984;  32 3378-83
  PubMedSearch in Google Scholar
• 3 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge III. Minor prosapogenins from the fruits of Xanthoceras sorbifolia Bunge.  Chem Pharm Bull. 1985;  33 127-34
  PubMedSearch in Google Scholar
• 4 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge IV. Structures of the minor prosapogenins.  Chem Pharm Bull. 1985;  33 1043-8
  PubMedSearch in Google Scholar
• 5 Chen Y J, Takeda T, Ogihara Y, Iitaka Y. Studies on the constituents of Xanthoceras sorbifolia Bunge V. Major saponins from the fruits of Xanthoceras sorbifolia Bunge.  Chem Pharm Bull. 1985;  33 1387-94
  PubMedSearch in Google Scholar
• 6 Ma C M, Nakamura N, Hattori M, Kakuda H, Qiao J C, Yu H L. Inhibitory effects on HIV-1 protease of constituents from the wood of Xanthoceras sorbifolia .  J Nat Prod. 2000;  63 238-42
  CrossrefPubMedSearch in Google Scholar
• 7 Li X B, Wang H D, Guo Y. Xanthoceras sorbifolia fruit extracts for enhancing mental activity. CN: 1 084 876 Oct 5th, 1994
  Search in Google Scholar
• 8 Greca M D, Fiorentino A, Monaco P, Previtera L. Polyoxygenated oleanane triterpenes from Hydrocotyle ranunculoides .  Phytochemistry. 1994;  35 201-4
  PubMedSearch in Google Scholar
• 9 Ahmad V U, Fizza K, Sultana A. Isolation of two sesquiterpenes from Pluchea arguta .  Phytochemistry. 1989;  28 3081-3
  CrossrefPubMedSearch in Google Scholar
• 10 Ahmad V U, Fatima I, Fatima A. The sapogenins from Dodonaea viscosa .  Fitoterapia. 1987;  58 361-2
  PubMedSearch in Google Scholar
• 11 Chen J Y, Wu L J, Teng H L, Liu K. Study on aescin metabolism in intestinal flora in vitro .  Chin Tradit Herb Drugs. 2003;  34 970-3
  PubMedSearch in Google Scholar
• 12 Zhang Y, Wu L J, Tashiro S, Onodera S, Ikejima T. Evodiamine induced tumor cell death through different pathways: apoptosis and necrosis.  Acta Pharmacol Sin. 2004;  25 83-9
  PubMedSearch in Google Scholar

Prof. Xian Li

Shenyang Pharmaceutical University

Box 49

Shenyang 110016

People’s Republic of China

Phone: +86-24-2398-6475

Email: Proflixian@163.com