32-35Pterospermum truncatolobatum Gagnep. has long been used as a traditional medicine in Vietnam. Its crude extract
showed cytotoxicity against human epidermal carcinoma (KB) cell lines. However, its chemical constituent and biological
activity remains unknown. In the course of our investigation on the Vietnamese medicinal plants, four compounds, taraxerol
(1), betulonic acid (2), -sitosterol (3) and eicosanoic acid (4) were purified from methanolic extract of P. truncatolobatum
by silica gel column chromatography. Their structures were determined by spectral (Mass spectrometry and nuclear
magnetic resonance) analysis and by comparison with the literature reports. Of which, betulonic acid (2) showed moderate
cytotoxicity against all four cancer cell lines, KB, MCF7 (human breast carcinoma), LU (human lung carcinoma), and HepG2
(hepatocellular carcinoma)

Dien, Pham Huu

Linh, Le Thi Khanh

Quang, Dang Ngoc

Uyen, Nguyen Thu

NOPR

Indian Journal of Natural Products and Resources Vol. 13(1), March 2022, pp. 32-35 Cytotoxic constituents from Vietnamese Pterospermum truncatolobatum Gagnep. Le Thi Khanh Linh, Nguyen Thu Uyen, Pham Huu Dien and Dang Ngoc Quang* Faculty of Chemistry, Hanoi National University of Education, 136-Xuan Thuy road, Cau Giay, Hanoi 10000, Vietnam Received 22 October 2020; Revised 16 February 2022 Pterospermum truncatolobatum Gagnep. has long been used as a traditional medicine in Vietnam. Its crude extract showed cytotoxicity against human epidermal carcinoma (KB) cell lines. However, its chemical constituent and biological activity remains unknown. In the course of our investigation on the Vietnamese medicinal plants, four compounds, taraxerol (1), betulonic acid (2), -sitosterol (3) and eicosanoic acid (4) were purified from methanolic extract of P. truncatolobatum by silica gel column chromatography. Their structures were determined by spectral (Mass spectrometry and nuclear magnetic resonance) analysis and by comparison with the literature reports. Of which, betulonic acid (2) showed moderate cytotoxicity against all four cancer cell lines, KB, MCF7 (human breast carcinoma), LU (human lung carcinoma), and HepG2 (hepatocellular carcinoma). Keywords: Betulonic acid, Cancer, Cytotoxic, Medicinal plant, Pterospermum truncatolobatum Gagnep., Taraxerol.  IPC code; Int. cl. (2021.01)- A61K 36/00, A61K 127/00, A61K 135/00, A61P  Introduction The Sterculiaceae is a big plant family in Vietnam, currently comprises 22 genus and 86 species1. They have been used as food, medicine, fiber and wood. Phytochemical investigation on the plants of this family revealed that they contained various secondary metabolites such as alkaloids, phenyl propanoids, flavonoids and terpenoids. Many of them have been found to be effective in treatment of common diseases such as cold, sore throats, cancer, inflammation2-5. The Pterospermum genus has 40 species, mostly distribute in tropical and subtropical Asia6. Chemical investigation of the plants of this genus indicated that some biologically active compounds were characterized such as cytotoxic naphthol from P. yunnanense7, cytotoxic triterpenoids fromP. heterophyllum8, antioxidant phenolic and osteogeniccompounds from P. acerifolium4,9, and cytotoxicphenolic compounds from P. lanceifolium10. So far,there was no report on the chemical constituents andbiological activity of the plant Pterospermumtruncatolobatum Gagnep. When screening thebiological activity of the Vietnamese medicinal plant,we found that the n-hexane, dichloromethane, ethylacetate and n-butanol extracts of a rare plant,P. truncatolobatum showed moderate cytotoxic activityagainst KB cell lines, with the IC50 values of 14.57,54.09, 7.6, and 182.5 g/mL, respectively. Thus, phytochemical study of the extracts of the leaves and stems of this plant was carried out. This paper describes the isolation, structural elucidation and cytotoxicity of four compounds from the n-hexane and ethyl acetate extracts of P. truncatolobatum. Materials and Methods  Plant sample collection and identification The P. truncatolobatum was collected in March 2018 in Langson province, north of Vietnam and identified by Nghiem Duc Trong, Department of Botany, Hanoi Pharmacy University. Voucher specimen (LKL-1801) has been deposited at the Faculty of Chemistry, Hanoi University of Education, Vietnam. General Nuclear magnetic resonance spectroscopy (NMR) was recorded on a Bruker AMX-500 (500 MHz for 1H-NMR spectrum and 125 MHz for 13C-NMR spectrum) with an internal standard TMS. The chemical shifts () are expressed in parts per million (ppm). HR-MS was recorded on a SCIEX X500 QTOF system. TLC was performed on silica gel plates (Kiesegel 60 F254, Merck). The spots of TLC were detected under UV lights (254, 302, and 366 nm) and by spraying with 10% H2SO4 in methanol, followed by heating at 120 °C. Column chromatography was carried out on silica gel 60 (0.2-0.5 mm, 0.04-0.063 mm, Merck). ————— *Correspondent authorEmail: quangdn@hnue.edu.vnLINH et al.: CYTOTOXIC CONSTITUENTS FROM PTEROSPERMUM TRUNCATOLOBATUM GAGNEP.   33 Extraction and Isolation The fresh leaves and stems of P. truncatolobatum (4.0 kg) were dried and extracted with methanol. All solvent was removed to give the crude extract (131 g), which was partitioned between n-hexane, CH2Cl2, EtOAc, n-butanol and water. The n-hexane extract (10.8 g) was subjected to a silica gel column chromatography, using n-hexane/EtOAc gradient to give 11 sub-fractions. Sub-fraction 7 (0.71 g) was recrystallized to give compound 3 (17 mg). Compounds 1 (37 mg) and compound 4 (32 mg) were isolated from sub-fraction 8 (0.87 g) by silica gel column chromatography, eluting with n-hexane/EtOAc (6/1) and followed by recrystallization. The EtOAc extract (10 g) was chromatographed on a silica gel column, using n-hexane/EtOAc from 6/1 to 1/1 to give 8 sub-fractions. Sub-fraction 2 (225 mg) was purified by silica gel column, eluting with n-hexane/EtOAc (4/1) to afford compound 2 (18 mg) as white crystals.   Spectral data of isolated compounds Taraxerol (1): 1H NMR: H 5.53 (dd, J = 3.5, 8.5 Hz, H-15), 3.19 (dd, J = 4.5, 11.0 Hz, H-3), 2.04  (m, H-19), 1.93 (m, H-16), 1.66 (H-2, H-7), 1.64  (m, H-2, H-16), 1.63 (m, H-1, H-11), 1.61 (m, H-6a), 1.51 (m, H-6b), 1.49 (m, H-11), 1.46 (m, H-18), 1.45 (m, H-7), 1.38 (m, H-22a), 1.36 (m, H-19), 1.31 (m, H-12), 1.26 (m, H-21), 1.09 (s, H-27), 1.03 (m, H-12), 0.99 (m, H-22b), 0.98 (m, H-1, H-9), 0.96 (s, H-23), 0.95 (s, H-29), 0.93 (s, H-24), 0.91 (s, H-26, H-30), 0.87 (m, H-5), 0.80 (s, H-28), 0.77 (s, H-25). 13C NMR: C 158.1 (C-14), 116.9 (C-15), 79.0 (C-3), 55.6 (C-5), 49.3 (C-18), 48.8 (C-9), 41.3 (C-19), 39.0 (C-8), 38.8 (C-4), 38.0 (C-1), 37.8 (C-10, C-13, C-17), 36.7  (C-16), 35.8 (C-12), 35.1 (C-7), 33.7 (C-21), 33.4  (C-29), 33.1 (C-22), 29.9 (C-26), 29.8 (C-28), 28.8  (C-20), 28.0 (C-23), 27.2 (C-2), 25.9 (C-27), 21.3  (C-30), 18.8 (C-6), 17.5 (C-11), 15.5 (C-25), 15.4  (C-24).  Crystal data for 1: Program used to solve structure ShelXT11. Refinement: on Least Squares minimization. Crystal size: 0.4 × 0.4 × 0.3 mm3. C30H50O, MW 426, monoclinic, P212121, a = 13.5489(7) Å, b = 6.2054(3) Å, c = 30.2721(15) Å,  = 90.00o,  = 94.620(3)°,   = 90.00o, V = 2536.9(2) Å3, Z = 4, MoK radiation,   = 0.71073 Å,  = 0.064 mm-1, 6087 reflections, 298 parameters; R = 0.0833, Rw = 0.1911, S = 1.307.  Betulonic acid (2): 1H NMR: H 4.74 (d, J = 1.5 Hz, H-29a), 4.62 (d, J = 1.5 Hz, H-29b), 3.01 (m, H-19), 2.50 (m, H-2), 2.42 (m, H-2), 1.99 (m, H-15, H-22), 1.70 (s, H-30), 1.7 (m, H-12), 1.64 (m, H-18), 1.52 (m, H-6), 1.50 (m, H-21), 1.48 (m, H-22), 1.46 (m, H-16), 1.45 (m, H-11a), 1.44 (m, H-7), 1.42 (m, H-15), 1.38 (m, H-1, H-9), 1.34 (m, H-5, H-11b), 1.07 (s, H-23), 1.06 (m, H-12), 1.02 (s, H-24), 1.00 (s, H-27), 0.99  (s, H-26), 0.93 (s, H-25). 13C NMR: C 218.1 (C-3), 181.2 (C-28), 150.3 (C-20), 109.7 (C-29), 56.3 (C-17), 55.0 (C-5), 49.9 (C-9), 49.2 (C-18), 47.3 (C-4), 46.9 (C-19), 42.5 (C-14), 40.7 (C-8), 39.6 (C-1), 38.5 ( C-13), 37.0 (C-22), 36.3 (C-10), 34.1 (C-2), 33.6 (C-7), 32.1 (C-16), 30.6 (C-15), 29.7 (C-21), 26.7 (C-23), 25.5 (C-12), 21.4 (C-11), 21.0 (C-24), 19.6 (C-6), 19.4 (C-30), 15.9 (C-25), 15.8 (C-26), 14.6 (C-27). +IDA-TOF-MS: m/z 477.3332 [M+Na]+, calcd. for C30H46O3Na: 477.3345. -sitosterol (3): 1H NMR: H 5.36 (s, H-6), 3.52 (m, H-3), 1.01 (s, H-19), 0.92 (d, J = 6.5 Hz, H-21), 0.85 (t, J = 7.2 Hz, H-29), 0.84 (d, J = 6.5 Hz, H-26), 0.82 (d, J = 6.0 Hz, H-27), 0.68 (s, H-18). 13C NMR: C 140.8 (C-5), 121.7 (C-6), 71.8 (C-3), 56.8 (C-14), 56.1 (C-17), 50.2 (C- 9), 45.9 (C-24), 42.4 (C-4), 42.3 (C-13), 39.8 (C-12), 37.3 (C-1), 36.5 (C-10), 36.2  (C-20), 34.0 (C-22), 31.9 (C-2), 31.9 (C-8), 31.9  (C-7), 29.2 (C-25), 28.3 (C-16), 26.2 (C-15, C-23), 23.1 (C-28), 21.1 (C-11), 19.8 (C-26), 19.4 (C-27), 19.1 (C-21), 19.1 (C-18), 12.0 (C-29), 11.9 (C-19). Eicosanoic acid (4): 1H NMR: H 2.34 (t, H-2), 1.63 (m, H-3), 1.26 (m, H-4  H-19), 0.87 (m, H-20). 13C NMR: C 179.9 (C-1), 34.0 (C-2), 31.9 (C-18), 29.6  (C-6  C16), 29.4 (C-17), 29.1 (C-4), 24.7 (C-3), 22.7 (C-19), 14.1 (C-20).  Bioassay  Cytotoxic assay was carried out as described in the literature12.  Results and Discussion The 1H NMR spectrum of compound 1 showed the presence of one olefinic proton at 5.53 ppm, one carbinol at 3.19 ppm together with eight singlet methyls at 1.09 (3H), 0.96 (3H), 0.95 (3H), 0.93 (3H), 0.91 (6H), 0.80 (3H) and 0.77 (3H) ppm. In addition, its 13C NMR spectrum displayed 30 carbon signals, including two olefinic carbons (158.1 and 116.9 ppm), one oxygen-bearing carbon (79.0 ppm) and eight singlet methyls as written in section Spectral Data of Isolated Compounds. This spectral data suggested that this compound is a triterpenoid with a hydroxyl group at C-3 and a double bond13. The relative configuration of 3-OH was determined as  -equatorial by the proton splitting pattern of H-3 in its 1H NMR spectrum with big coupling constants  INDIAN J NAT PROD RESOUR, MARCH 2022   34 (J = 4.5, 11.0 Hz). In addition, the NMR spectral data of compound 1 were identical with those of taraxerol13. Furthermore, a suitable crystal of compound 1 was obtained and its ORTEP drawing (Fig. 1) was described, confirming the structure of taraxerol as shown in Fig. 2.  The HR-MS of compound 2 has a quasi-molecular ion peak at m/z 477.3332 [M+Na]+, corresponding to the molecular formula of C30H46O3Na with 7 degrees of unsaturation. Analysis of its 1H NMR spectrum revealed the presence of one exo-methylene at 4.74 and 4.62 ppm, six singlet methyls at 1.70, 1.07, 1.02, 1.00, 0.99, 0.93 ppm. The 13C NMR spectrum of compound 2 displayed 30 carbon signals, including one conjugated ketone (218.1 ppm), one carboxylic acid (181.2 ppm), one exo-methylene (109.7 and 150.3 ppm). Then, the structure of compound 2 was deduced by the interpretation of its 2D NMR spectra (HSQC, HMBC, ROESY). The ketone group was located at C-3 based on the HMBC correlations between H-1, H-2, H-23, H-24 and C-3. In addition, the carboxylic group was at C-28 since H-18 was coupled to C-28 in its HMBC spectrum. Furthermore, exo-methylene was bonded at C-20 since H-29 was correlated with C-19 and C-30 in its HMBC spectrum. Finally, H-5 and H-18 were -oriented since H-5 was coupled to H-24; H-18 was coupled to H-27 in its ROESY spectrum. Consequently, compound 2 was found to be betulonic acid, which was previously isolated from Hopea odorata Roxb.14.   Compounds 3 and 4 were characterized as  -sitosterol (3)15 and eicosanoic acid (4)16 by comparing their spectral data with those of published papers. Previously, betulonic acid (2) has shown anti-inflammatory, anti-melanoma, anti-protozoal, and anti-viral activities14,17,18. In this study, all four extracts of this plant showed cytotoxicity against KB cell lines, and then we tested the activity of compound 2 toward four cancer cells, using ellipticine as a standard. The result was illustrated in Table 1. Accordingly, compound 2 had non-selective activity against all four cancer cells, especially it could inhibit moderately Hep-G2 and KB cell lines with its IC50 values of 20.33 and 16.0 g/mL, respectively.  Conclusion  Four secondary metabolites from the extracts of Vietnamese medicinal plant, Pterospermum truncatolobatum were successfully purified and structural determined as taraxerol (1), betulonic acid (2), -sitosterol (3) and eicosanoic acid (4). The moderate cytotoxic activity of betulonic acid (2) against all four cancer cell lines suggested the possible application of P. truncatolobatum for cancer treatment.  Conflict of interest The authors declare there is no conflict of interest.   References  1 Nguyen T B, List of Vietnamese plant, (Agricultural Publishing House, Hanoi), 2003, 536–554. 2 Al Muqarrabun L M R and Ahmat N, Medicinal uses, phytochemistry and pharmacology of family Sterculiaceae: A review, Eur J Med Chem, 2015, 92, 514–520.    Fig. 1 — The ORTEP drawing of compound 1   Fig. 2 — Structures of compounds 1-3 Table 1 — Cytotoxic activity of compound 2 (IC50, g/mL) Cancer cells Compound 2 Ellipticine Hep-G2 20.33±1.67 0.36±0.03 Lu 67.15±3.65 0.32±0.03 KB 16.0±0.95 0.24±0.02 MCF7 37.47±1.52 0.51±0.03  LINH et al.: CYTOTOXIC CONSTITUENTS FROM PTEROSPERMUM TRUNCATOLOBATUM GAGNEP.   35 3 Chen W, Tang W, Lou L and Zhao W, Pregnane, coumarin and lupane derivatives and cytotoxic constituents from Helicteres angustifolia, Phytochemistry, 2006, 67,  1041–1047.  4 Dixit P, Khan M P, Swarnkar G, Chattopadhyay N and Maury R, Osteogenic constituents from Pterospermum acerifolium Willd. Flowers, Bioorg Med Chem Lett, 2011, 21, 4617–4621. 5 Li S, Shi Y, Shang X Y, Cui B S, Yuan Y, et al., Triterpenoids from the roots of Pterospermum heterophyllum Hance, J Asian Nat Prod Res, 2009, 11, 652–657. 6 Feng G M, Flora Reipublicae Popularis Sinicae, vol 49, (Beijing, Science Press), 1984, 172–179. 7 Li H, Huang G, Liu B, Liu Y, Zhan R, et al., A new naphthol from the twigs and leaves of Pterospermum yunnanense, Nat Prod Res, 2014, 28(19), 1539–1543. 8 Li S, Shi Y, Shang X Y, Cui B S, Yuan Y, et al., Triterpenoids from the roots of Pterospermum heterophyllum Hance, J Asian Nat Prod Res, 2009, 11, 652–657. 9 Saboo S, Tapadiya R, Khadabadi S S and Deokate U A, In vitro antioxidant activity and total phenolic, flavonoid contents of the crude extracts of Pterospermum acerifolium wild leaves (Sterculiaceae), J Chem Pharm Res, 2010, 2, 417–423. 10 Pal L C, Prateeksha, Singh B N, Pande V and Rao C V, Phenolics-Enriched fraction of Pterospermum lanceifolium Roxb. efficiently reverses the Hepatocellular carcinoma in NDEA-Induced HCC Rats, Nutr Cancer, 2021, 21, 1–16. 11 Sheldrick G M, SHELXT–Integrated space-group and crystal-structure determination, Acta  Cryst, 2015, A71, 3–8. 12 Scudiero D A, Shoemaker R H, Paull K D, Monks A and Tierney S, Evaluation of a soluable tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines, Cancer Res, 1988, 48, 4827–4833. 13 Paul B D, Rao G S and Kapadia G J, Isolation of myricadiol, myricitrin, taraxerol, and taraxerone from Myrica cerifera L. Root bark, J Pharm Sci, 1974, 63, 958–959. 14 Satiraphan M, Pamonsinlapatham P, Sotanaphun U, Sittisombut C, Raynaud F, et al., Lupane trierpenes from the leaves of the tropical rain forest tree Hopea odorata Roxb. and their cytotoxic activities, Biochem Systemat Ecol, 2012, 44, 407–412. 15 Chaturvedula V S P and Prakash I, Isolation of Stigmasterol and -Sitosterol from the dichloromethane extract of Rubus suavissimus, Int Curr Pharm J, 2012, 1, 239–242. 16 Wishart D S, Knox C, Guo A C, Eisner R, Young N, et al., HMDB: A knowledgebase for the human metabolome, Nucleic Acids Res, 2009, 37, D603–D610. 17 Yogeeswari P and Sriram D, Betulinic acid and its derivatives: A review on their biological properties, Curr Med Chem, 2005, 12, 657–666. 18 Domínguez-Carmona D B, Escalante-Erosa F, García-Sosa K, Ruiz-Pinell G, Gutierrez-Yapu D, et al., Antiprotozoal activity of betulinic acid derivatives, Phytomedicine, 2010, 17, 379–382.  

Cytotoxic constituents from Vietnamese Pterospermum truncatolobatum Gagnep.

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Cytotoxic constituents from Vietnamese Pterospermum truncatolobatum Gagnep.

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