TRITERPENES FROM THE LEAVES OF Syzygium polycephalum,

S. cumini, AND S. samarangense

C. Y. Ragasa,1* O. B. Torres,1 C.-C. Shen,2 M. K. E. G. Lachica,3

A. B. Sulit,3 D. B. D. L. Chua,3 A. D. M. Ancheta,3 C. J. B. Ismail,3
F. T. E. Bernaldez,3 and D. D. Raga3

1) Chemistry Department and Center for Natural Sciences and Ecological Research, De La Salle University, 2401
Taft Avenue, Manila 1004, Philippines, e-mail: consolacion.ragasa@dlsu.edu.ph;
2) National Research Institute of Chinese Medicine, 155-1, Li-Nong St., Sec. 2, Taipei 112, Taiwan;
3) Department of Biology, School of Science and Engineering, Katipunan Ave., Ateneo de Manila University,
Quezon City 1108, Philippines.

Published in Khimiya Prirodnykh Soedinenii, No. 5, September–October, 2014, pp. 814–815. Original article
submitted January 9, 2013.

Chemistry of Natural Compounds, Vol. 50, No. 5, pp. 942-944, November, 2014

Syzygium polycephalum, locally known as lipote, is an indigenous Philippine tree. It was reported to lower
high blood pressure and high cholesterol level and it exhibits antioxidant activity [1]. A recent study reported that a
decoction of the bark is used for the treatment of dysentery [2]. There is no reported study on the chemical
constituents of S. polycephalum. Syzygium cumini, locally known as duhat, is native to the Philippines and other
Southeast Asian countries. It is marketed for its fruit, which is known for its hypoglycemic property. S. cumini
possesses chemopreventive, radioprotective, and antineoplastic properties and is used in the treatment of diabetes
mellitus, inflammation, ulcers, and diarrhea [3]. S. cumini extract was reported to exhibit antitumor and
antioxidative potential against chemically induced stomach carcinogenesis [3]. Recently, a review of its food and
medicinal uses was presented [4]. Syzygium samarangense, locally known as macopa, is native to the Philippines
and other Southeast Asian countries. An earlier study reported that a mixture of cycloartenyl stearate, lupenyl
stearate, sitosteryl stearate, and 24-methylenecycloartanyl stearate (sample 1) from the air-dried leaves of S.
samarangense exhibited potent analgesic and anti-inflammatory activities at effective doses of 6.25 and 12.5 mg/kg
body weight, respectively. Sample 1 also exhibited negligible toxicity on zebrafish embryonic tissue [5]. Another
study reported the isolation of cytotoxic chalcones and antioxidants from the fruits of S. samarangense [6].

This study was conducted as part of our research on the chemical constituents of trees native to the
Philippines. We earlier reported the chemical constituents of Cinnamomum utile [7], Cinnamomum griffithii [8],
Cinnamomum rupestre, Cinnamomum nanophyllum [9], Ardisia squamulosa [10], and Ficus linearifolia [11]. We
report herein the isolation of ursolic acid (1), oleanolic acid (2), squalene (3), and__β-sitosterol (4) from S.
polycephalum; 1, 4, and betulinic acid (5) from S. cumini; and 1 and lupeol (6) from S. samarangense. To the best of
our knowledge, this is the first report on the isolation of 1–4 from S. polycephalum.

The leaves of S. polycephalum were collected from Makiling Center for Mountain Ecosystems, Los Banos,
Laguna, Philippines in March 2012. A voucher specimen (No. 1) was deposited at the Philippine National
Herbarium, National Museum of the Philippines. The leaves of S. cumini were collected from Arayat, Pampanga,
Philippines in April 2012. A voucher specimen (No. 710) was deposited at the Philippine National Herbarium,
National Museum of the Philippines. The leaves of S. samarangense were collected from Taytay, Rizal, Philippines
in February, 2012. A voucher specimen (No. 624) was deposited at the Philippine National Herbarium, National
Museum of the Philippines. The specimens were authenticated by Wilfredo F. Vendivil of the Philippine National
Herbarium, Museum of the Philippines.

The air-dried leaves (134.5 g) of S. polycephalum were ground in an osterizer, soaked in CH2Cl2 for 3 days,
and then filtered. The filtrate was concentrated under vacuum to afford the crude extract (0.99 g). The air-dried
leaves (400 g) of S. cumini were ground in an osterizer, soaked in CH2Cl2 for 3 days, and then filtered. The filtrate
was concentrated under vacuum to afford the crude extract (23.83 g). The air-dried leaves of S. samarangense (800
g) were ground in an osterizer, soaked in CH2Cl2 for 3 days, and then filtered. The filtrate was concentrated under
vacuum to afford the crude extract (34.4 g). The crude extracts were fractionated by silica gel chromatography using
increasing proportions of acetone in CH2Cl2 (10% increment) as eluents. Fractions were collected and monitored by
thin-layer chromatography (TLC). Fractions with spots of the same Rf values were combined and
rechromatographed in appropriate solvent systems until TLC pure isolates were obtained.

The CH2Cl2 fraction from the chromatography of the crude S. polycephalum leaf extract was
rechromatographed using petroleum ether (5 ×) to afford 3 (7 mg). The 10% to 20% acetone in CH2Cl2 fractions
were combined and rechromatographed (3 ×) using 15% EtOAc in petroleum ether to afford 4 (10 mg) after
washing with petroleum ether. The 30% to 50% acetone in CH2Cl2 fractions were combined and
rechromatographed (4 ×) using Et2O–CH3CN–CH2Cl2 in a 0.5:0.5:9 by volume ratio to afford a mixture of 1 and 2
(9 mg) after washing with petroleum ether.

The 10% to 30% acetone in CH2Cl2 fractions from the chromatography of the crude S. cumini leaf extract
were combined and rechromatographed (5 ×) using 15% EtOAc in petroleum ether to afford 4 (18 mg) after
washing with petroleum ether. The 40% to 60% acetone in CH2Cl2 fractions were combined and rechromatographed
(6 ×) using Et2O–CH3CN–CH2Cl2 in a 0.5:0.5:9 by volume ratio to afford 1 and 5 (12 mg) after washing with
petroleum ether.

The 20% to 30% acetone in CH2Cl2 fractions from the chromatography of the crude S. samarangense leaf
extract were combined and rechromatographed (3 ×) using 15% EtOAc in petroleum ether to afford 6 (17 mg) after
washing with petroleum ether. The 40% to 60% acetone in CH2Cl2 fractions were combined and rechromatographed
(6 ×) using diethyl ether–acetonitrile–CH2Cl2 in a 0.5:0.5:9 by volume ratio to afford 1 (25 mg) after washing with
petroleum ether.

Chromatographic separation of the dichloromethane extracts of the leaves of Syzygium polycephalum

afforded ursolic acid (1), oleanolic acid (2), squalene (3), and β-sitosterol (4); Syzygium cumini yielded 1, 4, and
betulinic acid (5); Syzygium samarangense afforded 1 and lupeol (6). The structures of 1–6 were identified by NMR
spectroscopy and confirmed by comparison of their 13C NMR data with those reported in the literature for ursolic
acid [12], oleanolic acid [12], squalene [13], β-sitosterol [14], betulinic acid [15], and lupeol [12], respectively.

Ursolic acid and betulinic acid were previously reported as constituents of the chloroform extract of S.
cumini [16]. Ursolic acid was also reported as a constituent of S. samarangense [17]. A review on the anti-
tumorigenic and chemopreventive activity of ursolic acid is provided in [18]. Betulinic acid induces apoptotic cell
death in cancer cells by triggering the mitochondrial pathway of apoptosis, while normal cells are relatively resistant
to it [19]. A review on betulinic acid for cancer treatment and prevention is available [20]. Oleanolic acid exhibits
potent anti-tumor activity against many tumor cell lines [21]. Thus, the three Syzygium species afforded triterpenes
with reported anticancer properties, which may contribute to the cytotoxic, anti-tumorigenic, and anticancer
activities of these three Syzygium species.

REFERENCES
1. H. B. Florido and F. F. Cortiguerra, Res. Inform. Ser. Ecosyst., 15, 6 (2003).
2. K. Roosita, C. M. Ku, C. M. Shanto, M. Sekiyama, Y. Fachrunozi, and K Ohtsuka, J. Ethnopharmacol.,
115, 71 (2008).
3. P. K. Goyal, P. Verma, P. Sharma, J. Parmar, and A. Agarwal, Asian J. Cancer Rev., 11, 753 (2010).
4. S. Swanni, N. Thakeor, M. Patil, and P. Halalankar, Food Nutr. Sci., 3, 1100 (2012).
5. D. Raga, C. L. C. Cheng, K. C. I. C. Lee, W. Z. P. Olaziman, V. J. A. De Guzman, C.-C. Shen, F. C. Franco,
and C. Y. Ragasa, Z. Naturfosch., 66c, 235 (2011).
6. M. J. Simirgiotis, S. Adachi, S. To, and H. Yang, Food Chem., 107, 813 (2008).
7. D. L. Espineli, E. M. G. Agoo, C.-C. Shen, and C. Y. Ragasa, Chem. Nat. Compd., 49, 769 (2013).
8. C. Y. Ragasa, E. M. G. Agoo, C.-C. Shen, and D. L. Espineli, Chem. Nat. Compd., 49, 127 (2013).
9. C. Y. Ragasa, E. M. G. Agoo, C.-C. Shen, and D. L. Espineli, Chem. Nat. Compd., 49, 757 (2013).
10. C. Y. Ragasa, D. L. Espineli, D. D. Raga, A. A. Herrera, and C.-C. Shen, Chem. Nat. Compd., 49, 388 (2013).
11. C. Y. Ragasa, O. B. Torres, C.-C. Shen, L. O. Bernardo, E. H. Mandia, K. A. De Castro-Cruz, and P.-W. Tsai,
Chem. Nat. Compd., 50, 172 (2014).
12. S. B. Mahato and A. P. Kondo, Phytochemistry, 37, 1517 (1994).
13. J. M. Brown and D. R. M. Martens, Tetrahedron, 33, 931 (1977).
14. J. M. C. Cayme and C. Y. Ragasa, Kimika, 20, 5 (2004).
15. C. Y. Ragasa, A. B. Alimboyoguen, and C.-C. Shen, Philipp. Scient., 46, 78 (2009).
16. T. Boonruad and N. Chansuwanich, Bull. Dept. Med. Sci., 42, 109 (2000).
17. R. Srivastava, A. K. Shaw, and D. K. Kulsireshtha, Phytochemistry, 38, 687 (1995).
18. L. Novotory, A. Vachalkova, and D. Briggs, Neoplasma, 48, 241 (2001).
19. S. Fulda, Int. J. Mol. Sci., 9, 1096 (2008).
20. L. Tripathi, P. Kumar, and R. Singh, Curr. Bioact. Compds., 5, 160 (2009).
21. R. Zhou, Z. Zhang, L. Zhao, C. Jia, S. Xu, Q. Mai, M. Lu, M. Huang, L. Wang, X. Wong, D. Jin, and X. Bai,
J. Orthop. Res., 29, 846 (2011).