Chemical Constituents of Propolis from Vietnamese Trigona minor and

Article pubs.acs.org/jnp

Chemical Constituents of Propolis from Vietnamese Trigona minor and Their Antiausterity Activity against the PANC‑1 Human Pancreatic Cancer Cell Line Hai X. Nguyen,† Mai T. T. Nguyen,†,‡ Nhan T. Nguyen,*,† and Suresh Awale*,§ †

Faculty of Chemistry and ‡Cancer Research Laboratory, VNUHCM−University of Science, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam § Division of Natural Drug Discovery, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan S Supporting Information *

ABSTRACT: The ethanol extract of propolis from the Vietnamese stingless bee Trigona minor possessed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells in nutrient-deprived medium, with a PC50 value of 14.0 μg/mL. Chemical investigation of this extract led to the isolation of 15 cycloartane-type triterpenoids, including five new compounds (1−5), and a lanostane-type triterpenoid. The structures of the new compounds were elucidated on the basis of NMR spectroscopic analysis. Among the isolated compounds, 23-hydroxyisomangiferolic acid B (5) and 27-hydroxyisomangiferolic acid (13) exhibited the most potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions, with PC50 values of 4.3 and 3.7 μM, respectively.

P

diseases. There has been no prior scientific research on the chemical composition and bioactive properties of the propolis from this species, although stingless bee propolis continues to be used as a folk medicine based on empirical knowledge. Tumor cells proliferate uncontrollably and thus require a high demand of glucose and other nutrients to support their growth.20 Most of a tumor’s growth relies on angiogenesis for the supply of nutrients.21,22 However, human pancreatic tumors are known to be inherently hypovascular with an inadequate supply of nutrients to the aggressively proliferating cells.21,22 Nevertheless, pancreatic tumor cells show remarkable tolerance to nutrition starvation and survive in a critically low nutrient condition (referred to as austerity) within the heterogeneous tumor microenvironment.23−25 Therefore, the discovery of anticancer agents that inhibit the tolerance of the cancer cells to nutrient starvation leading to cancer cell death represents a novel “antiausterity” strategy in anticancer drug discovery.23,24 Compounds possessing preferential cytotoxicity in a nutrientdeprived medium (NDM) without toxicity in a nutrient-rich medium (e.g., Dulbecco’s modified Eagle medium, DMEM) are classified as “antiausterity agents”.20,24 As part of a continued antiausterity strategy screening program for the discovery of anticancer agents that preferentially inhibit cancer cells’ viability in nutrition-deprived medium,20,24,26−32 an ethanol extract of

ropolis is a resinous hive product collected by bees from different plant sources and used to repair the hive and create a protective barrier against intruders.1,2 It is also an important chemical weapon of bees against pathogenic microorganisms. The composition and the source of propolis depend on the geographical locations, bee species, foraging distances, and the vegetation area from which the bees collect the flowers, leaf buds, and tree bark.1 For example, the principal sources of European, Nepalese, and Brazilian red and Cuban, Myanmar, Mexican, and Brazilian green propolis were reported as Populus nigra,3 Dalbergia species,4,5 Clusia species,6 Mangifera indica,7 Populus species,8 and Baccharis dracunculifolia,9 respectively. Propolis has been extensively researched due to its versatile pharmacological activities such as anticancer,10 antibacterial,11,12 antifungal,11,13 antiviral,11 immunostimulating,14 anti-inflammatory,15 and antioxidant16 activities. Previous studies on Brazilian red propolis, as well as Myanmar and Mexican propolis, have shown preferential cytotoxicity activity against PANC-1 human pancreatic cancer cells in nutrientdeprived medium.5,7,8,17 Several species of bees are known to produce propolis, including honey bees (Apis mellifera) belonging to the Apini tribe and stingless bees belonging to the Meliponini tribe.18 There are more than 300 reported species in the Meliponini tribe, of which 43 species belonging to two genera (Lisotrigona and Trigona) are found in different regions of Asia.19 In Vietnam, propolis from Trigona minor has been used in traditional medicine as a remedy to improve health and prevent © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 30, 2017

A

DOI: 10.1021/acs.jnatprod.7b00375 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

propolis from the stingless bee T. minor showed preferential cytotoxicity (PC) against PANC-1 cells in NDM, with a PC50 value of 14.0 μg/mL. Chemical investigation of this extract led to the isolation of 15 cycloartane-type triterpenoids, including five new compounds (1−5), and a lanostane-type triterpenoid. We herein report the first chemical investigation of Vietnamese propolis produced by the stingless bee T. minor and its antiausterity activity evaluation against the PANC-1 human pancreatic cancer cell line.

18.3, 19.4, 20.9, 21.1, 22.3), a cyclopropane methylene (δC 29.7), an oxymethylene (δC 58.3), two olefinic carbons (δC 127.6, 151.1), a carboxylic carbon (δC 171.2), an ester carbonyl (δC 171.8), and a ketocarbonyl (δC 216.7). These 1H and 13C NMR spectra resembled those of 27-hydroxymangiferonic acid (9),36 isolated from the same extract, except for the presence of the acetyl group. The location of the acetyl group was deduced to be at C-27 based on the HMBC correlations from the acetyl methyl (δH 2.03) and the H-27 oxymethylene (δH 4.84) to the ester carbonyl carbon (δC 171.8) (Figure 1). The relative configuration of compound 1 was assigned based on the NOESY (Figure 2) analysis and coupling constant data. The NOESY correlations between H-2α/Me-28, Me-28/H-1α, H1α/H-5, H-5/H-7α, H-7α/Me-30, Me-30/H-17, and H-17/ Me-21 suggested the A/B and C/D rings to be trans-fused and H-5 and Me-30 to be in α-axial orientations. The NOESY correlations between H-2β/Me-29, H-2β/H-19a, Me-29/H19b, H-19b/H-8, H-8/Me-18, and Me-18/H-20 suggested the rings B/C to be cis-fused and H-8, Me-18, and Me-29 to be in β-axial orientations. This was further supported by the large trans-diaxial coupling constants of H-2β (J = 14.0) and H-5 (J = 12.2). The NOESY correlations between CH2-23 and CH2-27 suggested an E-configuration of the Δ24(25) double bond. Finally, the absolute configuration of 1 was determined by electronic circular dichroism (ECD) data analysis (Figure 3). Compound 1 showed a negative Cotton effect at 298 nm corresponding to the carbonyl n → π* transition. Application of the octant rule suggests that the polarizable steric bulk resides in the negative octant (Figure 3),44,45 suggesting the absolute configuration of 1 as (5R,8S,9S,10R,13R,14S,17R,20R). Therefore, the structure of compound 1 was concluded as 27acetoxymangiferonic acid. Compound 2 was obtained as a white, amorphous solid. Its molecular formula was determined to be C32H48O6 via the HRESIMS data. The IR absorption bands at 3035, 3500, 1695, 1710, 1740, 2960, and 2860 cm−1 demonstrated the presence of a cyclopropane methylene, a hydroxy, a conjugated acid carbonyl, a ketocarbonyl, and a carbonate carbon. The 1H and 13C NMR data of 2 (Table 1) resembled those of 1 and showed signals of a cyclopropane methylene, four tertiary methyls, a secondary methyl, an oxymethylene, a double bond, a conjugated acid, and a carbonyl, together with the aliphatic methylenes and methines. However, its NMR data showed signals for a methoxy (δH 3.78; δC 55.0) and a carbonate (δC

■

RESULTS AND DISCUSSION Propolis from stingless bees, T. minor, was extracted with EtOH under sonication at room temperature. The extract was suspended in H2O and successively partitioned with n-hexane, CHCl3, and EtOAc to yield n-hexane-, CHCl3-, EtOAc-, and H2O-soluble fractions, respectively. The n-hexane extract showed the most potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells in nutrient-deprived conditions with a PC50 value of 3.6 μg/mL. Further separation and purification of this fraction led to the identification of 16 triterpenoids, including five new compounds (1−5). The known compounds were identified as cycloartenone (6),33 mangiferonic acid (7),34 23-hydroxymangiferonic acid (8),35 27-hydroxymangiferonic acid (9),36 mangiferolic acid (10),34 23-hydroxymangiferolic acid (11),37 27-hydroxymangiferolic acid (12),38 27-hydroxyisomangiferolic acid (13),39 (24E)-3βhydroxycycloart-24-en-26-al (14),40 (23E)-27-nor-3β-hydroxycycloart-23-en-25-one (15),41 and lanosterol (16).42 Compound 1 was isolated as a white, amorphous solid and showed a sodiated molecular ion at m/z 535.3373 [M + Na]+ corresponding to the molecular formula, C32H48O5Na. The IR spectrum of 1 showed absorption bands of a cyclopropane methylene (3035 cm−1), a hydroxy (3450 cm−1), a conjugated acid carbonyl (1690 cm−1), an ester carbonyl (1730 cm−1), and a ketocarbonyl (1715 cm−1) group. The 1H NMR spectrum of 1 (Table 1) displayed a characteristic set of AB doublets of a cyclopropane methylene39,43 at δH 0.57 (J = 4.0 Hz) and 0.78 (J = 4.0 Hz), four tertiary methyl singlets (δH 0.90, 0.99, 1.04, 1.09), a secondary methyl (δH 0.91, d, J = 6.5 Hz), an acetoxy group (δH 2.03, s), an oxymethylene (δH 4.84, s), a conjugated olefinic proton (δH 7.09, t, J = 7.6 Hz), and overlapped signals corresponding to aliphatic methylenes and methines (δH 0.96− 2.70). Its 13C NMR spectrum (Table 1) in combination with DEPT analysis showed the presence of six methyls (δC 18.3, B


C

position

24 25 26 27 28 29 30 OCOCH3-27 OCOCH3-27 OCOOCH3-27

23

20 21 22

17 18 19

12 13 14 15 16

8 9 10 11

7

3 4 5 6

2

1

dd (12.2, 4.1) m m m m m

s s s s

m m m m s d (4.0) d (4.0) m d (6.5) m m m m t (7.6)

2.03 s

4.84 1.04 1.09 0.90

1.31 1.90 1.32 1.60 0.99 0.78 0.57 1.45 0.91 1.56 1.18 2.35 2.22 7.09

δH

m m ddd (14.0, 14.0, 6.4) m

2.05 m 1.18 m 1.65 m

1.71 1.54 0.96 1.37 1.16 1.58

1.85 1.53 2.70 2.30

1a δC

151.1 127.6 171.2 58.3 22.3 20.9 19.4 171.8 21.1

26.1

36.2 18.3 35.3

52.3 18.3 29.7

32.9 45.5 48.9 35.7 28.3

48.0 21.2 26.2 26.8

26.0

216.7 50.4 48.5 21.6

37.6

33.5

4.92 1.04 1.10 0.90

1.32 1.90 1.31 1.61 0.99 0.79 0.58 1.43 0.92 1.58 1.17 2.42 2.26 7.22

δH

dd (12.2, 4.3) m m m m m

m m ddd (13.9, 13.9, 6.4) ddd (13.9, 4.2, 2.5)

s s s s

m m m m s d (4.1) d (4.1) m d (6.5) m m m m t (7.8)

2.05 m 1.16 m 1.66 m

1.69 1.55 0.95 1.38 1.14 1.59

1.84 1.51 2.70 2.30

2a δC

155.8

153.5 125.8 170.8 61.2 22.3 20.9 19.4

26.0

36.2 18.2 33.6

52.3 18.2 29.7

33.0 45.6 48.9 35.1 28.3

48.0 21.2 26.2 26.9

26.4

216.7 50.4 48.6 21.7

37.6

33.6

m m m m m dd (12.5, 4.7)

s s s s

m m m m s d (4.1) d (4.1) m d (6.4) m m m m t (7.7)

2.06 s

4.84 0.81 0.97 0.90

1.38 1.89 1.28 1.58 0.97 0.56 0.33 1.44 0.91 1.58 1.18 2.40 2.25 7.17

δH m m m m dd (11.1, 4.4)

2.00 m 1.14 m 1.63 m

1.30 1.61 0.78 1.33 1.08 1.52

1.56 1.25 1.75 1.56 3.28

3a

152.6 126.2 169.8 57.8 25.5 14.0 19.3 170.8 20.9

26.0

36.0 18.1 35.1

52.2 18.1 29.9

33.0 45.5 48.9 35.6 28.2

48.0 20.0 26.2 26.5

26.2

78.9 40.5 47.2 21.1

30.4

32.0

δC

Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data for New Compounds 1−5 (δ in ppm, J in Hz)

dd (12.6, 4.3) m m m m m

m m m m s d (3.6) d (3.6) m d (6.3) m m ddd (8.0, 8.0, 2.6)

1.72 0.80 0.83 0.87

s s s s

6.55 d (8.0)

1.25 1.79 1.23 1.55 0.97 0.45 0.30 1.59 0.91 1.57 0.91 4.34

δH m m m m t (2.5)

1.89 m 1.20 m 1.60 m

1.81 1.40 0.71 1.24 1.14 1.47

1.80 0.89 1.75 1.50 3.25

4b δC

145.7 125.3 168.9 12.3 26.1 21.1 18.9

64.4

31.7 18.0 42.8

52.1 17.7 28.9

32.5 44.9 48.5 34.9 27.6

47.1 19.2 26.2 25.8

25.3

74.6 39.5 40.1 20.4

28.5

27.0

m m m m s d (3.6) d (3.6) m d (6.2) m m t (8.2)

1.69 0.79 0.83 0.86

s s s s

6.41 d (7.8)

1.25 1.80 1.23 1.55 0.95 0.44 0.30 1.59 0.90 1.51 0.84 4.30

δH

dd (12.3, 4.0) m m m m m

m m m m brs

1.91 m 1.20 m 1.60 m

1.82 1.40 0.71 1.24 1.14 1.47

1.81 0.89 1.75 1.50 3.24

5b δC

142.6 128.4 170.4 13.0 26.2 21.2 19.0

64.6

31.8 18.1 43.2

52.3 17.9 29.1

32.6 45.0 48.6 35.0 27.7

47.3 19.2 26.3 25.8

25.4

74.7 40.1 40.3 20.6

28.7

27.1

Journal of Natural Products Article



Article

δC

Measured in CDCl3. bMeasured in DMSO-d6. a

position

OCOOCH3-27

Table 1. continued

δH

1a

δC

3.78 s

δH

2a

55.0

δH

3a

δC

δH

4b

δC

δH

5b

δC

155.8) group instead of an acetoxy group (δH 2.03; δC 171.8, 21.1) in 1. In the HMBC spectrum, a correlation between the methoxy protons (δH 3.78) and the carbonate carbonyl carbon (δC 155.8) was observed, which suggested the presence of a methoxycarbonyloxy group. Furthermore, the HMBC correlation between the C-27 oxymethylene protons (δH 4.92) and the carbonate carbonyl (δC 155.8) (Figure 1) suggested its location at C-27. The NOESY data analysis and the coupling constants as well as the negative Cotton effect at 298 nm in the ECD spectrum suggested the same absolute configuration as that of 1. Therefore, the structure of 2 was assigned as (5R,8S,9S,10R,13R,14S,17R,20R)-27-methoxycarbonyloxymangiferonic acid. Compound 3 was isolated as a white, amorphous solid. Its HRESIMS data showed a sodiated molecular ion at m/z 537.3556 [M + Na]+ (calcd for C32H50O5Na, 537.3556), corresponding to the molecular formula C32H50O5. Its IR spectrum showed absorption bands for the cyclopropane methylene and hydroxy, conjugated acid carbonyl, and ester carbonyl groups. The 1H and 13C NMR data of 3 (Table 1) resembled those of 1 except for the presence of an oxymethine group (δH 3.28, dd, J = 11.1, 4.4 Hz; δC 78.9) instead of a carbonyl group (δC 216.7) in 1. In the HMBC spectrum, the H3 oxymethine proton (δH 3.28) showed long-range correlations to C-1 (δC 32.0), C-2 (δC 30.4), C-5 (δC 47.2), C-28 (δC 25.5), and C-29 (δC 14.0) (Figure 1), suggesting the C-3 location of the hydroxy group. The NOESY correlation between H-3 and Me-28 (Figure 2), together with the large 3JH‑3,H‑2β value of 11.1 Hz suggested the hydroxy group at C-3 to be β-equatorial. The absolute configuration of 3 was determined to be the same those of 1, 2, and mangiferolic acid (10) by comparison of their positive specific rotation values. The negative and positive Cotton effects at 236 and 254 nm, respectively, in the ECD spectrum of 3 resembled closely those of 1 and 2. Therefore, the structure of 3 was defined as 27-acetoxymangiferolic acid. Compounds 4 and 5 were both obtained as white, amorphous solids and showed the molecular formula C30H48O4, as deduced from the positive HRESIMS data. The 1 H and 13C NMR spectra of 4 and 5 (Table 1) resembled those of isomangiferolic acid, isolated from M. indica37 and Myanmar propolis,7 except for the presence of an oxymethine group in both 4 (δH 4.34, ddd, J = 8.0, 8.0, 2.6 Hz; δC 64.4) and 5 (δH 4.30, t, J = 8.2 Hz; δC 64.6) and the absence of signals due to one of the aliphatic methylenes in isomangiferolic acid. The C23 location of the oxymethine group was deduced on the basis of the HMBC correlations H-23/C-22, H-23/C-24, H-22/C23, and H-24/C-23 (Figure 1) and the deshielding of C-23. Therefore, compounds 4 and 5 differ only in the configuration at C-23. Owing to limited sample quantities, the C-23 absolute configurations of 4 and 5 were not defined. The relative configurations of 4 and 5 were determined by NOESY correlations and coupling constant data. The NOESY correlations (Figure 2) H-3/H-2β, H-2β/H-19a, H-2β/Me29, and Me-29/H-19b suggested the A ring to be in a chair conformation with an α-axially oriented C-3 hydroxy group. This was further evident from the small coupling constants 3 Jeq‑eq ≈ 3Jeq‑ax of H-3 [(δH‑3 3.25, t, J = 2.5 Hz, 4) and (δH‑3 3.24, brs, 5)]. On the other hand, the NOESY correlations between H-19b/H-8, H-8/Me-18, Me-18/H-20, and H-23/Me27 suggested an E-configuration of the Δ24(25) double bond and ring C to be in a chair and rings B and D to be in a boat conformation. Their positive specific rotations [α]D and the negative Cotton effects at 237 and 239 nm indicated that 4 and D



Article

Figure 1. Connectivities (bold lines) deduced by the COSY spectrum and significant HMBC correlations (solid arrows) observed for 1−5.

Figure 2. Key NOESY (solid arrows) correlations of compounds 1−5.

characteristic constituents of M. indica,31,37,39,46 while the others, except 15 and 16, were also found in this plant.31,34,37,39,46 Moreover, M. indica is predominantly available in the Ben Tre Province of Vietnam, where the propolis used in this study has been collected.31 Therefore, the predominant plant source of this propolis sample is suggested to be M. indica. Interestingly, the propolis of the Myanmar honey bee, A. mellifera, was also found to contain cycloartane-type triterpenoids.7 All the isolated compounds were tested for their preferential cytotoxic activity against a PANC-1 cell line in NDM by employing an antiausterity strategy. The data are presented as preferential cytotoxicity (PC50) values, which represent 50% cancer cell death in NDM without apparent toxicity in DMEM (Table 2). Among the compounds tested, the new 23hydroxyisomangiferolic acid B (5) and 27-hydroxyisomangiferolic acid (13) displayed the strongest preferential cytotoxicity, with PC50 values of 4.3 and 3.7 μM, respectively, which are comparable to that of arctigenin, a positive control (PC50, 0.8 μM). The presence of a hydroxy group at C-23 or C-27 seems to enhance activity, i.e., 12 ≥ 11 > 10; 8 > 9 ≥ 7. The presence

Figure 3. ECD spectra and octant projection of 1 and 2.

5 were in the same family as isomangiferolic acid. Therefore, the structures of 4 and 5 were concluded to be 23hydroxyisomangiferolic acid A and B, respectively. Fifteen cycloartane-type triterpenoids from the EtOHsoluble extract of propolis of the Vietnamese stingless bee T. minor were also isolated. Compounds 7, 9, and 10 are major constituents of this extract, and they have been reported as E



Article

noncytotoxic concentration in DMEM and allowed to form colonies for 10 days. As shown in Figure 5, 5 significantly inhibited colony formation in a concentration-dependent manner. Therefore, 23-hydroxyisomangiferolic acid B (5) may serve as an anticancer lead for the development of drugs against pancreatic cancer.

Table 2. Preferential Cytotoxicity of Compounds 1−16 against the PANC-1 Cells compound

PC50, μMa

compound

PC50, μMa

1 2 3 4 5 6 7 8 arctigeninb

77.5 32.9 22.6 16.1 4.3 >100 >100 16.8 0.8

9 10 11 12 13 14 15 16 paclitaxelc

93.8 >100 64.4 47.6 3.7 19.7 16.4 68.7 >100

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a JASCO DIP-140 digital polarimeter (JASCO International Co., Ltd., Japan). ECD measurements were carried out on a JASCO J-805 spectropolarimeter. IR spectra were measured with a Shimadzu IR-408 spectrophotometer (Shimadzu Pte., Ltd., Singapore). NMR spectra were recorded on a Bruker Avance III 500 spectrometer (Bruker BioSpin AG, Bangkok, Thailand) with TMS as an internal standard, and chemical shifts are expressed in δ values. HRESIMS data were acquired on a Bruker micrOTOF-QII mass spectrometer (Bruker Singapore Pte., Ltd., Singapore). Silica gel 60, 40−63 μm (230−400 mesh ASTM), for column chromatography was purchased from Scharlau (Scharlau, Barcelona, Spain). Analytical and preparative TLC were carried out on precoated Kieselgel 60F254 or RP-18 plates (0.25 or 0.5 mm thickness) from Merck (Merck KGaA, Darmstadt, Germany). Biological Material. Stingless bee propolis (Trigona minor) was collected in the Ben Tre Province, Vietnam, in July 2011, and was identified by Dr. Trung Quang Le, Bee Research and Development Centre, Ha Noi, Vietnam. A voucher specimen (MCE0042) was deposited at the Division of Medicinal Chemistry, Faculty of Chemistry, VNUHCM−University of Science. Extraction and Isolation. Stingless bee propolis (2 kg) was extracted with EtOH under sonication (5 × 1 L, 12 h each) at room temperature to yield an EtOH-soluble fraction (1 kg). This extract was suspended in H2O (1 L) and successively partitioned with n-hexane (5 × 1 L), CHCl3 (5 × 1 L), and EtOAc (5 × 1 L) to give n-hexane (860 g), CHCl3 (15 g), EtOAc (25 g), and H2O (100 g) soluble extracts, respectively. Part of the n-hexane-soluble fraction (220 g) was subjected to silica gel column chromatography (9.5 × 150 cm), eluted with acetone/n-hexane gradient mixtures (0−100%), to yield 14 fractions (fr.1, 36.7 g; fr.2, 47.3 g; fr.3, 34.2 g; fr.4, 33.9 g; fr.5, 14.8 g; fr.6, 8.1 g; fr.7, 6.1 g; fr.8, 6.6 g; fr.9, 6.5 g; fr.10, 6.3 g; fr.11, 5.6 g; fr.12, 7.9 g; fr.13, 3.6 g; fr.14, 2.3 g). Fraction 3 (10.0 g) was dissolved in MeOH/CHCl3 (50:50) and left overnight to give crystals of 7 (500.0 mg). The mother liquor (9.0 g) was chromatographed over silica gel column chromatography with an acetone/n-hexane gradient system, to yield seven fractions (fr.3.1, 1.2 g; fr.3.2, 2.4 g; fr.3.3, 0.6 g; fr.3.4, 0.2 g; fr.3.5, 1.2 g; fr.3.6, 1.1 g; fr.3.7, 1.3 g). Subfraction 3.3 was chromatographed over a silica gel column with EtOAc/n-hexane gradient mixtures (0−50%) and further purified by preparative TLC with CHCl3 (100%), to give 6 (5.0 mg) and 16 (6.0 mg). Subfraction 3.4 was purified with MeOH/CHCl3 gradient mixtures (0−20%), to obtain 14 (5.0 mg) and 15 (6.0 mg). Fraction 5 (14.8 g) was also recrystallized with MeOH/CHCl3 (50:50), to give 10 (1.2 g). Fraction 9 (6.5 g) was passed over a silica gel column, eluted with acetone/nhexane mixtures, to afford seven subfractions (fr.9.1, 110 mg; fr.9.2, 1.1 g; fr.9.3, 780 mg; fr.9.4, 2.0 g; fr.9.5, 330 mg; fr.9.6, 780 mg; fr.9.7, 1.4 g). Subfraction 9.3 was chromatographed over a silica gel column with EtOAc/n-hexane gradient mixtures (0−50%), to afford five subfractions, fr.9.3.1−5. Subfraction 9.3.2 (97 mg) was separated by preparative TLC with MeOH/CHCl3 (2:98), to give 3 (4.2 mg) and 8 (6.8 mg). Subfraction 9.4 was subjected to silica gel column chromatography with MeOH/CHCl3 gradient mixtures (0−50%), to afford seven subfractions, fr.9.4.1−7. Fr.9.4.4 (68 mg) was purified by preparative TLC with acetone−n-hexane (30:70), to give 4 (2.5 mg) and 5 (2.0 mg), while fr.9.4.7 (160.0 mg) was recrystallized with MeOH/CHCl3 (50:50), to give 11 (46.0 mg). Fraction 10 (5.0 g) was chromatographed via silica gel column chromatography, eluted with a MeOH/CHCl3 gradient system, to give seven subfractions (fr.10.1, 300 mg; fr.10.2, 700 mg; fr.10.3, 50 mg; fr.10.4, 860 mg; fr.10.5, 1.3 g; fr.10.6, 1.2 g; fr.10.7, 480 mg). Subfraction 10.4 was chromatographed

a

Concentration at which 50% of cells were killed preferentially in NDM. bPositive control. cNegative control.

of an α-hydroxy substituent at C-3 tends to enhance the activity significantly (5 > 4 ≈ 8 > 11; 13 > 12 > 9). 23Hydroxyisomangiferolic acid B (5; PC50, 4.3 μM) and 27hydroxyisomangiferolic acid (13; PC50, 3.7 μM) both met these criteria and showed significant preferential cytotoxic activity. 23-Hydroxyisomangiferolic acid B (5) was further evaluated for its effect on the cell morphology of PANC-1 cells in NDM. The microscopic images were analyzed under phase-contrast and fluorescence mode using an ethidium bromide/acridine orange (EB/AO) reagent. AO is a cell-permeable dye and emits a green fluorescence in live cells. EB is permeable in dead cells only and emits a red fluorescence. As shown in Figure 4, the

Figure 4. Fluorescent [ethidium bromide (EB)/acridine orange (AO)] and phase-contrast overlay images of PANC-1 cells at 24 h. (a) Control, live cells stained only with AO emitted bright green fluorescence, (b) treatment with 23-hydroxyisomangiferolic acid B (5) (5 μM) led to total death of PANC-1 cells within 24 h; cells stained with EB emitted red fluorescence, (c) merged fluorescent and phasecontrast image of PANC-1 cells in the control and (d) treated with 5 (5 μM) showing morphological alteration.

cells in the control after 24 h were alive and stained with AO, giving a green fluorescence. However, when treated with 5 μM 23-hydroxyisomangiferolic acid B (5) for 24 h, the PANC-1 cells changed morphologically and gave an exclusive red fluorescence, indicating dead cells. To further evaluate the effect of 5, a colony formation assay on PANC-1 cells was performed. In this assay, PANC-1 cells were treated with 5 at a F



Article

Figure 5. Effect of 23-hydroxyisomangiferolic acid B (5) on colony formation in PANC-1 cells. The figure is a representative of three replications. **p < 0.01 when compared with the untreated control group. 265 mg/L CaCl2·2H2O, 0.1 mg/L Fe(NO3)3·9H2O, 400 mg/L KCl, 200 mg/L MgSO4·7H2O, 6400 mg/L NaCl, 700 mg/L NaHCO3, 125 mg/L NaH2PO4, 15 mg/L phenol red, 25 mM/L HEPES buffer (pH 7.4), and MEM vitamin solution (Life Technologies, Inc., Rockville, MD, USA); the final pH was adjusted to 7.4 with 10% NaHCO3. Arctigenin, the positive control, was isolated from the seed of Arctium lappa.24 After 24 h of incubation with each test compound in DMEM and NDM, the cells were washed twice with PBS and the medium replaced with 100 μL of DMEM containing a 10% WST-8 cell counting kit solution. After 3 h of incubation, the absorbance at 450 nm was measured (PerkinElmer EnSpire multimode plate reader). Cell viability was calculated from the mean values of data from three wells by using the following equation:

over a silica gel column with MeOH/CHCl3 gradient mixtures (0− 50%), to yield two subfractions, fr.10.4.1 and 10.4.2. Subfraction 10.4.2 (50 mg) was separated by preparative TLC with 2-propanol/CH2Cl2/ n-hexane (2:8:90), to afford 1 (5.2 mg) and 2 (5.0 mg). Subfraction 10.5 was passed over a silica gel column with MeOH/CHCl3 gradient mixtures (0−50%), to give two subfractions, fr.10.5.1 and 10.5.2. Fr.10.5.2 (650 mg) was recrystallized with MeOH/CHCl3 (50:50), to give 9 (300 mg). Fraction 11 (5.6 g) was separated over a silica gel column with a MeOH/CHCl3 gradient system, to yield five subfractions (fr.11.1, 80 mg; fr.11.2, 330 mg; fr.11.3, 60 mg; fr.11.4, 190 mg; fr.11.5, 4.6 g). Subfraction 11.5 was chromatographed over a silica gel column with MeOH/CHCl3 gradient mixtures (0−50%), to afford five subfractions, fr.11.5.1−5. Subfraction 11.5.2 (380 mg) was successively separated by column chromatography with EtOAc/nhexane gradient mixtures (0−50%) and preparative TLC with MeOH/ CHCl3 (1:99), to afford 12 (15.0 mg) and 13 (4.0 mg). Compound 1: white, amorphous powder; [α]25 D +16 (c 1, DMSO); IR νmax (CHCl3) 3450, 3035, 2960, 2860, 1730, 1715, 1690, 1625, 1450 cm−1; ECD (c 2.0 × 10−4 M, EtOH) [θ]231 −1314, [θ]246 +648, [θ]298 −1416; 1H and 13C NMR (CDCl3, 500 MHz), see Table 1; HRESIMS m/z 535.3373 [M + Na]+ (calcd for C32H48O5Na, 535.3399). Compound 2: white, amorphous powder; [α]25 D +26 (c 1, DMSO); IR νmax (CHCl3) 3500, 3035, 2960, 2860, 1740, 1710, 1695, 1620, 1450 cm−1; ECD (c 1.8 × 10−4 M, EtOH) [θ]231 −1855, [θ]248 +944, [θ]300 −1627; 1H and 13C NMR (CDCl3, 500 MHz), see Table 1; HRESIMS m/z 551.3346 [M + Na]+ (calcd for C32H48O6Na, 551.3349). Compound 3: white, amorphous powder; [α]25 D +31 (c 1, DMSO); IR νmax (CHCl3) 3400, 3030, 2950, 2870, 1730, 1690, 1625, 1450 cm−1; ECD (c 1.9 × 10−4 M, EtOH) [θ]215 −1573, [θ]236 −2347, [θ]254 +484; 1H and 13C NMR (CDCl3, 500 MHz), see Table 1; HRESIMS m/z 537.3556 [M + Na]+ (calcd for C32H50O5Na, 537.3556). Compound 4: white, amorphous powder; [α]25 D +18 (c 1, DMSO); IR νmax (CHCl3) 3400, 3030, 2950, 2870, 1680, 1630, 1450 cm−1; ECD (c 2.1 × 10−4 M, EtOH) [θ]213 −2386, [θ]237 −1798; 1H and 13C NMR (DMSO-d6, 500 MHz), see Table 1; HRESIMS m/z 495.3420 [M + Na]+ (calcd for C30H48O4Na, 495.3450). Compound 5: white, amorphous powder; [α]25 D +37 (c 1, DMSO); IR νmax (CHCl3) 3400, 3030, 2950, 2870, 1680, 1630, 1450 cm−1; ECD (c 2.1 × 10−4 M, EtOH) [θ]214 −1957, [θ]239 −2300; 1H and 13C NMR (DMSO-d6, 500 MHz), see Table 1; HRESIMS m/z 495.3417 [M + Na]+ (calcd for C30H48O4Na, 495.3450). Preferential Cytotoxicity Assay against PANC-1 Cells. The PANC-1 human pancreatic cancer cell line (RBRC-RCB2095) was purchased from the Riken BRC cell bank and maintained in standard DMEM with 10% fetal bovine serum (FBS) supplemented at and stored at 37 °C under a humidified atmosphere of 5% CO2 and 95% air. Briefly, human pancreatic cancer cells were seeded in 96-well plates (1.5 × 104/well) and incubated in fresh DMEM at 37 °C under 5% CO2 and 95% air for 24 h. After the cells were washed twice with phosphate-buffered saline (PBS), the medium was changed to serially diluted test samples in both DMEM and NDM24 with a control and blank in each test plate. The composition of the NDM was as follows:

Cell viability (%) = [Abs(test sample) − Abs(blank)/Abs(control) − Abs(blank)] × 100% Morphological Assessment of Cancer Cells. PANC-1 cells were seeded in 60 mm dishes (1 × 106) and incubated in fresh DMEM at 37 °C under 5% CO2 and 95% air for 24 h. After the cells were washed twice with PBS, the medium was changed to NDM (control) or 23-hydroxyisomangiferolic acid B (5, 5 μM) in NDM (treated). After a 24 h incubation, 2 μL of EB/AO reagent was added to each test well and incubated for 5 min, and morphology captured using an EVOSFL cell imaging system (40× objective) under fluorescent and phase-contrast modes. Colony Formation Assay. PANC-1 cells were plated in 12-well plates at a density of 500 cells/well in DMEM (1 mL/well) and incubated at 37 °C under humidified 5% CO2 for 24 h for the cell attachment. The cells were treated with 23-hydroxyisomangiferolic acid B (5) at noncytotoxic concentrations of 12.5, 25, and 50 μM in DMEM and allowed to grow for 10 days. After 10 days, cells were washed with PBS, fixed with 4% formaldehyde, and stained in crystal violet for 10 min. Each experiment was repeated three times. Colony area measurement was carried out by the ImageJ plugin “Colony Area”.

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00375.

■

Copies of spectroscopic data for 1−5 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail (N. T. Nguyen): [email protected]. Tel: +84-907426-331. Fax: +84-838-353-659. *E-mail (S. Awale): [email protected]. Tel: +81-76434-7640. Fax: +81-76-434-7640. G



Article

ORCID

(27) Nguyen, H. X.; Nguyen, M. T. T.; Nguyen, T. A.; Nguyen, N. Y. T.; Phan, D. A. T.; Thi, P. H.; Nguyen, T. H. P.; Dang, P. H.; Nguyen, N. T.; Ueda, J. Y.; Awale, S. Fitoterapia 2013, 91, 148−153. (28) Nguyen, M. T. T.; Nguyen, N. T.; Nguyen, K. D. H.; Dau, H. T. T.; Nguyen, H. X.; Dang, P. H.; Le, T. M.; Nguyen Phan, T. H.; Tran, A. H.; Nguyen, B. D.; Ueda, J.-Y.; Awale, S. Planta Med. 2014, 80, 193−200. (29) Nguyen, H. X.; Nguyen, N. T.; Dang, P. H.; Thi Ho, P.; Nguyen, M. T. T.; Van Can, M.; Dibwe, D. F.; Ueda, J. Y.; Awale, S. Phytochemistry 2016, 122, 286−293. (30) Nguyen, H. X.; Nguyen, N. T.; Dang, P. H.; Thi, P. H.; Nguyen, M. T. T.; Can, M. V.; Dibwe, D. F.; Ueda, J. Y.; Matsumoto, K.; Awale, S. Nat. Prod. Commun. 2016, 11, 723−724. (31) Nguyen, H. X.; Do, T. N. V.; Le, T. H.; Nguyen, M. T. T.; Nguyen, N. T.; Esumi, H.; Awale, S. J. Nat. Prod. 2016, 79, 2053− 2059. (32) Nguyen, N. T.; Nguyen, M. T. T.; Nguyen, H. X.; Dang, P. H.; Dibwe, D. F.; Esumi, H.; Awale, S. J. Nat. Prod. 2017, 80, 141−148. (33) Gandhe, S.; Lakavath, S.; Palatheeya, S.; Schuehly, W.; Amancha, K.; Kiran Reddy Nallamaddi, R.; Palepu, A.; Thakur, Y.; Rao Adavi Rao Belvotagi, V.; Kumar Bobbala, R.; Narasimha Appa Rao Achanta, V.; Kunert, O. Chem. Biodiversity 2013, 10, 1613−1622. (34) Escobedo-Martínez, C.; Concepción Lozada, M.; HernándezOrtega, S.; Villarreal, M. L.; Gnecco, D.; Enríquez, R. G.; Reynolds, W. Magn. Reson. Chem. 2012, 50, 52−57. (35) Elfita, E.; Muharni, M.; Latief, M.; Darwati, D.; Widiyantoro, A.; Supriyatna, S.; Bahti, H. H.; Dachriyanus, D.; Cos, P.; Maes, L. Phytochemistry 2009, 70, 907−912. (36) Anjaneyulu, V.; Babu, J. S.; Krishna, M. M. Acta Cienc. Indica, Chem. 1992, 18, 173−176. (37) Anjaneyulu, V.; Satyanarayana, P.; Viswanadham, K. N.; Jyothi, V. G.; Rao, K. N.; Radhika, P. Phytochemistry 1999, 50, 1229−1236. (38) Sharma, S. K.; Ali, M. J. Indian Chem. Soc. 1995, 72, 339−342. (39) Anjaneyulu, V.; Ravi, K.; Prasad, K. H.; Connolly, J. D. Phytochemistry 1989, 28, 1471−1477. (40) Madureira, A. M.; Gyemant, N.; Ascenso, J. R.; Abreu, P. M.; Molnar, J.; Ferreira, M. J. U. J. Nat. Prod. 2006, 69, 950−953. (41) Zhang, H. J.; Tan, G. T.; Hoang, V. D.; Hung, N. V.; Cuong, N. M.; Soejarto, D. D.; Pezzuto, J. M.; Fong, H. H. S. J. Nat. Prod. 2003, 66, 263−268. (42) Emmons, G. T.; Wilson, W. K.; Schroepfer, G. J., Jr. Magn. Reson. Chem. 1989, 27, 1012−1024. (43) Herz, W.; Watanabe, K.; Kulanthaivel, P.; Blount, J. F. Phytochemistry 1985, 24, 2645−2654. (44) Moffitt, W.; Woodward, R.; Moscowitz, A.; Klyne, W.; Djerassi, C. J. Am. Chem. Soc. 1961, 83, 4013−4018. (45) Snatzke, G. Angew. Chem., Int. Ed. Engl. 1968, 7, 14−25. (46) Anjaneyulu, V.; Harischandra Prasad, K.; Ravi, K.; Connolly, J. D. Phytochemistry 1985, 24, 2359−2367.

Mai T. T. Nguyen: 0000-0001-8006-4028 Nhan T. Nguyen: 0000-0001-5142-4573 Suresh Awale: 0000-0002-5299-193X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This research was supported by a grant from the Vietnam National University, Ho Chi Minh City (No. B2015-18-02), to N.T.N. Biological evaluation was supported by the JSPS Kakenhi to S.A.

■

REFERENCES

(1) Bankova, V. S.; Castro, S. L. d.; Marcucci, M. C. Apidologie 2000, 31, 3−15. (2) Ghisalberti, E. L. Bee World 1979, 60, 59−84. (3) Bankova, V. Evid. Based Complement. Alternat. Med. 2005, 2, 29− 32. (4) Awale, S.; Shrestha, S. P.; Tezuka, Y.; Ueda, J. Y.; Matsushige, K.; Kadota, S. J. Nat. Prod. 2005, 68, 858−864. (5) Awale, S.; Li, F.; Onozuka, H.; Esumi, H.; Tezuka, Y.; Kadota, S. Bioorg. Med. Chem. 2008, 16, 181−189. (6) Hernández, I. M.; Fernandez, M. C.; Cuesta-Rubio, O.; Piccinelli, A. L.; Rastrelli, L. J. Nat. Prod. 2005, 68, 931−934. (7) Li, F.; Awale, S.; Zhang, H.; Tezuka, Y.; Esumi, H.; Kadota, S. J. Nat. Prod. 2009, 72, 1283−1287. (8) Li, F.; Awale, S.; Tezuka, Y.; Esumi, H.; Kadota, S. J. Nat. Prod. 2010, 73, 623−627. (9) Teixeira, É. W.; Negri, G.; Meira, R. M. S. A.; Message, D.; Salatino, A. Evid. Based Complement. Alternat. Med. 2005, 2, 85−92. (10) Chen, C. N.; Weng, M. S.; Wu, C. L.; Lin, J. K. Evid. Based Complement. Alternat. Med. 2004, 1, 175−185. (11) Kujumgiev, A.; Tsvetkova, I.; Serkedjieva, Y.; Bankova, V.; Christov, R.; Popov, S. J. Ethnopharmacol. 1999, 64, 235−240. (12) Scazzocchio, F.; D’Auria, F. D.; Alessandrini, D.; Pantanella, F. Microbiol. Res. 2006, 161, 327−333. (13) Silici, S.; Koc, N. A.; Ayangil, D.; Cankaya, S. J. Pharmacol. Sci. 2005, 99, 39−44. (14) Orsatti, C.; Missima, F.; Pagliarone, A.; Bachiega, T.; Búfalo, M.; Araújo, J.; Sforcin, J. Phytother. Res. 2010, 24, 1141−1146. (15) Paulino, N.; Teixeira, C.; Martins, R.; Scremin, A.; Dirsch, V. M.; Vollmar, A. M.; Abreu, S. R.; de Castro, S. L.; Marcucci, M. C. Planta Med. 2006, 72, 899−906. (16) Kumazawa, S.; Ueda, R.; Hamasaka, T.; Fukumoto, S.; Fujimoto, T.; Nakayama, T. J. Agric. Food Chem. 2007, 55, 7722−7725. (17) Li, F.; He, Y.-M.; Awale, S.; Kadota, S.; Tezuka, Y. Chem. Pharm. Bull. 2011, 59, 1194−1196. (18) Bankova, V.; Popova, M. Pharmacogn. Rev. 2007, 1, 88−92. (19) Chinh, T. X.; Sommeijer, M. J. Apidologie 2005, 36, 493−503. (20) Awale, S.; Nakashima, E. M. N.; Kalauni, S. K.; Tezuka, Y.; Kurashima, Y.; Lu, J.; Esumi, H.; Kadota, S. Bioorg. Med. Chem. Lett. 2006, 16, 581−583. (21) Sakamoto, H.; Kitano, M.; Suetomi, Y.; Maekawa, K.; Takeyama, Y.; Kudo, M. Ultrasound Med. Biol. 2008, 34, 525−532. (22) Feig, C.; Gopinathan, A.; Neesse, A.; Chan, D. S.; Cook, N.; Tuveson, D. A. Clin. Cancer Res. 2012, 18, 4266−4276. (23) Izuishi, K.; Kato, K.; Ogura, T.; Kinoshita, T.; Esumi, H. Cancer Res. 2000, 60, 6201−6207. (24) Awale, S.; Lu, J.; Kalauni, S. K.; Kurashima, Y.; Tezuka, Y.; Kadota, S.; Esumi, H. Cancer Res. 2006, 66, 1751−1757. (25) Kim, S. E.; Park, H.-J.; Jeong, H. K.; Kim, M.-J.; Kim, M.; Bae, O.-N.; Baek, S.-H. Biochem. Biophys. Res. Commun. 2015, 463, 205− 210. (26) Win, N. N.; Awale, S.; Esumi, H.; Tezuka, Y.; Kadota, S. J. Nat. Prod. 2007, 70, 1582−1587. H


Chemical Constituents of Propolis from Vietnamese Trigona minor and

Recommend Documents