Cytotoxic Triterpenoids from the Fruits of Zizyphus jujuba

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The following eleven triterpenoic acids were isolated from the fruits of Zizyphus jujuba (Rhamnaceae): colubrinic acid, alphitolic acid, 3-O-cis-p-coumaroylalphitolic acid (3), 3-O-trans-p-coumaroylalphitolic acid (4), 3-O-cis-p-coumaroylmaslinic acid, 3-O-trans-p-coumaroylmaslinic acid, betulinic acid (7), oleanolic acid, betulonic acid (9), oleanonic acid and zizyberenalic acid. The in vitro cytotoxicities of the triterpenoic acids against K562, B16(F-10), SK-MEL-2, PC-3, LOX-IMVI, and A549 tumor cell lines were investigated by the sulforhodamin B (SRB) method. Among these compounds, the lupane-type triterpenes, such as compounds 3, 4, 7, and 9, showed high cytotoxic activities. In particular, the cytotoxic activities of 3-O-p-coumaroylalphitolic acids (compounds 3 and 4) were better than those of non-coumaroic triterpenenoids (compounds 7 and 9). These results suggest that the coumaroyl moiety at the C-3 position of the lupane-type triterpene may play an important role in enhancing cytotoxic activity.

In our search for natural products with anti-tumor effects from plants, we identified the EtOAc-soluble fraction of the MeOH extract of the fruits Zizyphus jujuba to show high cytotoxic activity against B16(F-10) tumor cell lines with ED₅₀ values of less than 10 μg/mL. Z. jujuba is a thorny rhamnaceous plant widely distributed in Europe and Southeastern Asia. The seed, known as Suan Tsao Zen in China, is a famous Chinese medicine used for the treating of insomnia [1]. Until recently, several triterpenes, cyclopeptide alkaloids and flavonoids have been isolated from the seed, fruit and root of this plant [2], [3], [4], [5], [6], [7]. In this paper, we report the cytotoxic activity of seven triterpenoic acids and four phenolic carboxylic acid esters of triterpenoic acid isolated from fruits of Z. jujuba against six tumor cell lines.

Repeated column chromatography of the EtOAc-soluble fraction of the MeOH extract of Z. jujuba on silica gel followed by gel filtration on Sep-Pak C18 cartridge and preparative HPLC, led to the isolation of eleven triterpenoic acids (Fig. [1]) identified as colubrinic acid (1) [8], alphitolic acid (2), 3-O-cis-p-coumaroylalphitolic acid (3), 3-O-trans-p-coumaroylalphitolic acid (4) [9], 3-O-cis-p-coumaroylmaslinic acid (5), 3-O-trans-p-coumaroylmaslinic acid (6), betulinic acid (7), oleanolic acid (8), betulonic acid (9), oleanonic acid (10) [5] and zizyberenalic acid (11) [8], the structures of which were elucidated on the basis of ESI-MS and ¹H-NMR data, and by comparison of melting points and optical rotation with reference data. In particular, compounds 1 and 11 have been isolated from the root of Paliurus hemsleyanus [8]; they have now been isolated from Z. jujuba for the first time.

All compounds (1 - 11) were tested for their cytotoxic activity against K562, B16(F-10), SK-MEL-2, PC-3, LOX-IMVI and A549 tumor cell lines (Table [1]). Among them, the oleanane compounds - 5, 6, 8, and 10 - had no significant cytotoxic activity against any of the cancer cell lines tested. However, 3-O-cis-p-coumaroylalphitolic acid (3), 3-O-trans-p-coumaroylalphitolic acid (4), betulinic acid (7), and betulonic acid (9) as lupane compounds exhibited cytotoxic activity. In particular, 3-O-cis/trans-p-coumaroylalphitolic acids (3, 4) showed significant cytotoxic activities. In a previous report, it was stated that cytotoxic activities of coumaroylkaempferols such as platanoside and tiliroside depend on the number of substituted coumaroyl moieties [10]. These results suggest that the p-coumaroyl moiety at the C-3 position of the lupane skeleton may play an important role in enhancing cytotoxic activity. The mechanism of action of these coumaroylalphitolic acids is still being investigated in our laboratories.

Materials and Methods

Plant material: Fruits of Zizyphus jujuba was purchased from oriental medicine markets in Kumsan, Korea. A voucher specimen (KRIBB 1013) was deposited in the herbarium of the Korea Research Institute of Bioscience and Biotechnology (KRIBB).

Isolation procedure: The fruits of Z. jujuba (1 kg) were extracted with MeOH (3 L) at room temperature (7 days × 3) to yield an extract of 400 g. The MeOH extract was suspended in H₂O (1 L) and was then shaken with EtOAc (1 L × 2, each time, 75 g), and BuOH saturated with H₂O (1 L × 3, each time, 200 g), successively. A portion of the EtOAc-soluble fraction (3 g) was divided into ten fractions (Fr. 1 - Fr. 10, 40 mL each) by gel filtration on Sep-Pak C18 Cartridges (5 G) eluted with acetonitrile 0 - 100 % (each fraction was increased by 10 % with acetonitrile). The fractions 7 and 8 (500 mg) were separated by preparative HPLC on YMC-Pak ODS-AQ column (300 × 10 mm I.D.) eluted with aqueous acetonitrile (75 %, 1.5 mL/min) to give fifteen subfractions (Fr. 1′ - Fr.15′, each 10 mL). These sub-fractions were further purified by preparative HPLC eluting with 75 % aqueous acetonitrile to afford compounds 1 - 11 (Fig. [1]). Yields: 1 (tR: 14.6 min, 3 mg, from Fr. 2′), 2 (tR: 16.6 min, 11 mg, from Fr. 2′), 3 (tR: 19.8 min, 8 mg, from Fr. 3′), 4 (tR: 20.4 min, 7 mg, from Fr. 3′), 5 (tR: 21.2 min, 2 mg, from Fr. 3′), 6 (tR: 22.4 min, 1 mg, from Fr. 3′), 7 (tR: 22.8 min, 17 mg, from Fr. 4′, 5′), 8 (tR: 24.3 min, 3 mg, from Fr. 4′, 5′), 9 (tR: 26.9 min, 9 mg, from Fr. 6′), 10 (tR: 27.7 min, 7 mg, from Fr. 6′, 7′), 11 (tR: 29.1 min, 7 mg, from Fr. 8′).

Colubrinic acid (1): white needles (in MeOH), m. p. 262 - 264 °C, ESI-MS: m/z = 469.5 [M - H]^-, 493.6 [M + Na]⁺, [α]_D ¹⁸: -17.0° (c 0.5, pyridine).

Alphitolic acid (2): colorless needles (in CHCl₃-MeOH), m. p. 275 - 278 °C, ESI-MS: m/z = 471.4 [M - H]^-, 495.6 [M + Na]⁺. [α]_D ¹⁹: -4.0° (c 1.0, pyridine).

3-O-(cis-p-Coumaroyl)-alphitolic acid (3): white powder, m. p. 208 - 210 °C, ESI-MS: m/z = 617.6 [M - H]^-. [α]_D ²⁰: + 0.9° (c 1.0, pyridine).

3-O-(trans-p-Coumaroyl)-alphitolic acid (4): white powder, m. p. 279 - 280 °C, ESI-MS: m/z = 617.6 [M - H]^-, 641.7 [M + Na]⁺. [α]_D ²⁵: +44.1° (c 0.8, pyridine).

3-O-(cis-p-Coumaroyl)-maslinic acid (5): white powder, m. p. 278 - 282 °C, ESI-MS: m/z = 617.6 [M - H]^-, 641.7 [M + Na]⁺. [α]_D ²⁴: + 9.1° (c 1.1, pyridine).

3-O-(trans-p-Coumaroyl)-maslinic acid (6): white powder, m. p. 190 - 194 °C, ESI-MS: m/z = 617.6 [M - H]^-, 641.7 [M + Na]⁺. [α]_D ²⁰: + 0.9° (c 1.0, pyridine).

Betulinic acid (7): white needles (in CHCl₃-MeOH), m. p. 275 - 278 °C, ESI-MS: m/z = 455.6 [M - H]^-, 479.1 [M + Na]⁺, [α]_D ²⁰: + 7.8° (c 0.9, pyridine). The IR and NMR spectra were superimposable with those of an authentic sample.

Oleanolic acid (8): white needles (in MeOH), 306 - 308 °C, ESI-MS: m/z = 455.5 [M - H]^-, 479.3 [M + Na]⁺, [α]_D ²⁰: + 75.2° (c 1.0, CHCl₃). The IR and NMR spectra were superimposable with those of an authentic sample.

Betulonic acid (9): white powder, m. p. 253 - 255 °C, ESI-MS: m/z = 453.5 [M - H]^-, 477.5 [M + Na]⁺, [α]_D ²⁰: + 7.0° (c 1.0, pyridine). Identified by direct comparison (¹H-NMR) with an authentic sample, which was synthesized from betulinic acid by Jones oxidation.

Oleanonic acid (10): white powder, m. p. 226 - 229 °C, ESI-MS: m/z = 453.6 [M - H]^-, 477.5 [M + Na]⁺, [α]_D ²⁰: +101° (c 1.63, CHCl₃). Identified by direct comparison (¹H-NMR) with an authentic sample, which was synthesized from oleanolic acid by Jones oxidation.

Zizyberenalic acid (11): white powder; m. p. 214 - 216 °C, ESI-MS: m/z = 451.5 [M - H]^-, 475.5 [M + Na]⁺, [α]_D ¹⁸: +24° (c 0.5, MeOH).

Cytotoxicity activity: The in vitro tumor cells assay was carried out by a sulforhodamin B (SRB) method [12]. For the SRB method, 100 μL of the cell suspension (4 - 50000 cells/mL) in the culture medium were inoculated to each well of 96-well microtiter plates. One day after plating, a time zero control plate was made. Compounds were directly treated on plates, and the cells were incubated for 48 h in a humidified 5 % CO₂ atmosphere at 37 °C. The cells were fixed with 50 μL of 10 % trichloroacetic acid (TCA) solution for 1 h at 4 °C and the plates were washed 5 times with tap-water and air-dried. 50 μL of SRB solution (0.4 % in 1 % acetic acid) were added and staining was done at room temperature for 30 min. The residual dye was washed out with 1 % acetic acid and the plates were air-dried. To each well, 100 μL of Tris buffer solution (1 mM, pH 10.5) were added. The optical density (OD) of each well was measured with a microtiter plate reader at 540 nm. The growth inhibition was calculated according to the previously described method [12]. Briefly, the value (T_s - T_o), in which the OD at time-zero (T_o) is subtracted from that of a treated well (T_s), was divided by calculated value of untreated control (T_c). The 50 % growth inhibition value (ED₅₀) was calculated by the Probit method [13].

Acknowledgements

We are grateful to KBSI for ¹H- and ¹³C-NMR spectral measurements.

References

• 1 Namba T. The Encyclopedia of Wakan-Yaku with Color Picture. Vol. I Osaka, Japan; Hoikusha Co 1993: 142- 4
  Search in Google Scholar
• 2 Woo W S, Kang S S, Shim S H, Wagner H, Chari V M, Seligmann O, Obermeier G. The structure of spinosin (2″-O-β-glucosylswertisin) from Zizyphus vulgaris var. spinosus .  Phytochemistry. 1979;  18 353-5
  CrossrefPubMedSearch in Google Scholar
• 3 Woo W S, Kang S S, Shim S H, Wagner H, Seligmann O, Chari V M. Acylated flavone-C-glycosides from the seeds of Zizyphus jujuba .  Phytochemistry. 1980;  19 2791-3
  CrossrefPubMedSearch in Google Scholar
• 4 Naglaa M N. Phytoconstituents of Zizyphus spina-christi L. fruits and their antimicrobial activity.  Food Chemistry. 2002;  76 77-81
  CrossrefPubMedSearch in Google Scholar
• 5 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishioka I. Studies on the constituents of Zizyphi Fructus. II. Structure of new p-coumaroylates of maslinic acid.  Chem Pharm Bull. 1978;  26 3075-9
  PubMedSearch in Google Scholar
• 6 Tripathi M, Pandey M B, Jha R N, Pandey V B, Tripathi P N, Singh J P. Cyclopeptide alkaloids from Zizyphus jujuba .  Fitoterapia. 2001;  72 507-10
  CrossrefPubMedSearch in Google Scholar
• 7 Lee S S, Lin B F, Karin C L. Three triterpene esters from Zizyphus jujuba .  Phytochemistry. 1996;  43 847-51
  CrossrefPubMedSearch in Google Scholar
• 8 Lee S S, Shy S N, Karin C S. Triterpenes from Paliurus hemsleyanus .  Phytochemistry. 1997;  46 549-54
  CrossrefPubMedSearch in Google Scholar
• 9 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishioka I. Studies on the constituents of Zizyphi Fructus. I. Structure of three new p-coumaroylates of alphitolic acid.  Chem Pharm Bull. 1978;  26 1798-802
  PubMedSearch in Google Scholar
• 10 Kostas D, Costas D, Sofia M, Marios M, Theodoros T, Dimitrios K. Cytotoxic activity of kaempferol glycosides against human leukemic cell lines in vitro .  Pharmacological Research. 2000;  41 89-88
  PubMedSearch in Google Scholar
• 11 Darrick S, Kim H L, John M P, Emily P. Synthesis of betulinic acid derivatives with activity against human melanoma.  Bioorg Med Chem Lett. 1998;  8 1707-12
  CrossrefPubMedSearch in Google Scholar
• 12 Skehan P, Streng R, Scudiero D, Monks A, McMahon J, Vistica D. New colorimetric cytotoxicity assay for anticancer-drug screening.  Journal of National Cancer Institute. 1990;  82 1007-13
  PubMedSearch in Google Scholar
• 13 Wu L, Smythe A M, Stinson S F, Mullendore L A, Monks A, Scudiero D A. Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening.  Cancer Research. 1992;  52 3029-34
  PubMedSearch in Google Scholar

Dr. Yung Hee Kho

Korea Research Institute of Bioscience and Biotechnology

P.O. Box 115

Yusong

Daejon 305-600

Korea

Fax: +82-42-860-4595

Email: yhkho@kribb.re.kr

References

• 1 Namba T. The Encyclopedia of Wakan-Yaku with Color Picture. Vol. I Osaka, Japan; Hoikusha Co 1993: 142- 4
  Search in Google Scholar
• 2 Woo W S, Kang S S, Shim S H, Wagner H, Chari V M, Seligmann O, Obermeier G. The structure of spinosin (2″-O-β-glucosylswertisin) from Zizyphus vulgaris var. spinosus .  Phytochemistry. 1979;  18 353-5
  CrossrefPubMedSearch in Google Scholar
• 3 Woo W S, Kang S S, Shim S H, Wagner H, Seligmann O, Chari V M. Acylated flavone-C-glycosides from the seeds of Zizyphus jujuba .  Phytochemistry. 1980;  19 2791-3
  CrossrefPubMedSearch in Google Scholar
• 4 Naglaa M N. Phytoconstituents of Zizyphus spina-christi L. fruits and their antimicrobial activity.  Food Chemistry. 2002;  76 77-81
  CrossrefPubMedSearch in Google Scholar
• 5 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishioka I. Studies on the constituents of Zizyphi Fructus. II. Structure of new p-coumaroylates of maslinic acid.  Chem Pharm Bull. 1978;  26 3075-9
  PubMedSearch in Google Scholar
• 6 Tripathi M, Pandey M B, Jha R N, Pandey V B, Tripathi P N, Singh J P. Cyclopeptide alkaloids from Zizyphus jujuba .  Fitoterapia. 2001;  72 507-10
  CrossrefPubMedSearch in Google Scholar
• 7 Lee S S, Lin B F, Karin C L. Three triterpene esters from Zizyphus jujuba .  Phytochemistry. 1996;  43 847-51
  CrossrefPubMedSearch in Google Scholar
• 8 Lee S S, Shy S N, Karin C S. Triterpenes from Paliurus hemsleyanus .  Phytochemistry. 1997;  46 549-54
  CrossrefPubMedSearch in Google Scholar
• 9 Yagi A, Okamura N, Haraguchi Y, Noda K, Nishioka I. Studies on the constituents of Zizyphi Fructus. I. Structure of three new p-coumaroylates of alphitolic acid.  Chem Pharm Bull. 1978;  26 1798-802
  PubMedSearch in Google Scholar
• 10 Kostas D, Costas D, Sofia M, Marios M, Theodoros T, Dimitrios K. Cytotoxic activity of kaempferol glycosides against human leukemic cell lines in vitro .  Pharmacological Research. 2000;  41 89-88
  PubMedSearch in Google Scholar
• 11 Darrick S, Kim H L, John M P, Emily P. Synthesis of betulinic acid derivatives with activity against human melanoma.  Bioorg Med Chem Lett. 1998;  8 1707-12
  CrossrefPubMedSearch in Google Scholar
• 12 Skehan P, Streng R, Scudiero D, Monks A, McMahon J, Vistica D. New colorimetric cytotoxicity assay for anticancer-drug screening.  Journal of National Cancer Institute. 1990;  82 1007-13
  PubMedSearch in Google Scholar
• 13 Wu L, Smythe A M, Stinson S F, Mullendore L A, Monks A, Scudiero D A. Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening.  Cancer Research. 1992;  52 3029-34
  PubMedSearch in Google Scholar

Dr. Yung Hee Kho

Korea Research Institute of Bioscience and Biotechnology

P.O. Box 115

Yusong

Daejon 305-600

Korea

Fax: +82-42-860-4595

Email: yhkho@kribb.re.kr