Antiproliferative Acylated Glycerols from New Zealand Propolis

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Antiproliferative Acylated Glycerols from New Zealand Propolis Stephen Bloor,*,† Owen Catchpole,†,‡ Kevin Mitchell,† Rosemary Webby,† and Paul Davis§ †

Callaghan Innovation, 69 Gracefield Road, PO Box 31310, Lower Hutt 5040, New Zealand Manuka Health NZ Ltd, PO Box 87429, Meadowbank, Auckland 1742, New Zealand § Trinity Bioactives, Lower Hutt 5040, New Zealand ‡

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S Supporting Information *

ABSTRACT: Previous work has shown that a number of phenolic components of NZ propolis possess antiproliferative activity against certain human gastrointestinal cancer cell lines. Here we report on a series of acylglycerols isolated from the nonpolar fraction of propolis resin, which represent further bioactive constituents unrelated to the more usual phenolic compounds generally found in propolis. NZ propolis is sourced from poplar trees, and the acylglycerols have been shown to be present in the leaves and buds of some common poplars. The compounds are a series of monoglycerides containing 3,8-dihydroxy fatty acids, many of which are further acylated with acetic acid residues. The dihydroxy fatty acids are C18 to C24, with the most abundant being C20 and C22. These acylglycerols were found to have strong antiproliferative activity against three human gastrointestinal cell lines, particularly gastric cancer cell line NCI-N87, where one example shows an IC50 of less than 50 μM.

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propolis from three South American countries: Chile, Uruguay, and Argentina.1 In previous work we showed that the antiproliferative activity of New Zealand “poplar”-type propolis against a group of human gastrointestinal cancer cell lines could be ascribed to more than a dozen phenolic compounds.4 We also showed that antiproliferative activity was retained by encapsulation of propolis in cyclodextrins.5 Apart from the well-known phenolic bioactives such as chrysin and CAPE (caffeic acid phenethyl ester) this work identified a number of flavonoids and caffeictype esters with strong activity.4 The major contribution to the total activity of the propolis tincture and resin was attributed to the main active compounds (chrysin, pinocembrin, galangin, 3O-acetylpinobanksin), as these compounds are the most dominant phenolic compounds in New Zealand propolis. Caffeic acid and its esters also collectively contributed to the activity of propolis. 1,1-Dimethylallyl caffeate, benzyl caffeate, and 3-methyl-3-butenyl caffeate all had strong activity and are present in NZ propolis in higher concentration than the known bioactive compound CAPE. Phenolic glycerides have also been reported in poplar-type propolis.6,7 The esters comprise 2acetyl-1,3-diglycerides of ferulic and/or cinnamic acid6 and a 2acetyl-1-feruloyl-3-(3,16)-dihydroxypalmitoylglycerol.7 These compounds had moderate antiproliferative and anti-inflammatory activity in in vitro testing. In the first stage of the previous work we also noted that one of the most antiproliferative fractions was still under investigation. This fraction (S#8) had no identifiable phenolics

ropolis is a heterogeneous material consisting of resin collected by honey bees from the leaf, buds, and bark of certain tree species, which is then admixed with beeswax produced from the hypopharyngeal glands of the worker bees. Propolis is used by bees to defend the hive against invaders and to reduce air flow into the hive to retain heat. For human use, crude propolis is typically extracted with an aqueous ethanolic solvent to separate the more polar resin from the nonpolar wax. The resultant tincture is used to make a variety of products that take advantage of antioxidant and antiinflammatory activity for oral consumption (tablets, capsules, lozenges, throat sprays, tinctures) or antibacterial activity for external application (toothpastes, soaps, skin care products, hair care products). There are around eight or nine distinct varieties of propolis according to the botanical source of the resin.1−3 The main botanical sources of resins and characteristic chemical components are poplars (Europe, North America, southern South America, China) rich in aglycone flavonoids, birch (Russia and Eastern Europe) rich in flavones and flavonols, Baccharis spp. (Brazilian “green” propolis) rich in prenylated derivatives of coumaric acid, Cupressacaea (Mediterranean propolis) rich in labdane-type diterpenoids, and Clusia spp. (central South America and southern Brazil, “red propolis”) rich in propolones.2 New Zealand propolis can be categorized as “European”, i.e., obtained by honey bees mainly from the exudates of poplars. A comparison of the antioxidant activity and total phenol and flavonoid and individual phenol and flavonoid composition for EtOH-extracted propolis samples from 14 countries showed that New Zealand-sourced propolis is similar in composition to propolis from Bulgaria, Uzbekistan, and Hungary and to © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 10, 2018

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DOI: 10.1021/acs.jnatprod.8b00562 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. RP-HPLC chromatogram of New Zealand propolis tincture with detection at 268 and 320 nm and ELSD. Boxed area in the ELSD chromatogram contains the set of acylglycerol peaks.

analysis, as can be seen in some of the peaks “topping out” in both UV and ELSD channels. The workup of more propolis resin through a series of chromatographic steps including RP and normal-phase silica gel chromatography allowed the separation of sufficient material for structural analysis. Compound Identification. The nonpolar sharp and wellresolved peaks observed in the ELSD could be linked to a set of peaks seen in the LCMS runs of the crude propolis and enriched fractions. No UV absorption is associated with these peaks. Mass spectrometry shows prominent peaks in positive mode where [M + H]+ and [M + Na]+ ions were observed. The main components of this set of compounds have molecular weights of 460, 474, 488, and 502 Da. This suggests a homologous series where the components differ by a methylene group. All the mass spectra have common fragmentation ions at m/z 159 and 117. In the negative mode the compounds showed formate adduct ions at [M + 45]+. High mass accuracy MS suggested a molecular formula of C25H48O7 for the 460 Da compound and extra CH2 units for the others in the series. The mixture of compounds was quite complex and proved difficult to separate from the background “polymeric” material. Peracetylation of the crude fractions enriched in these compounds proved to be useful. Not only did this allow separation of the acetylated compounds from the other material but the spectroscopic data were more definitive. The mass spectra of the acetylated mixture showed the compounds had gained three acetyl groups, as indicated by the MW increase by 3 × 42 amu. The compounds of interest were also readily separated by silica gel chromatography to yield sufficient material for NMR spectroscopic analysis. Although the material was still a mixture of the four major compounds, the NMR spectrum was surprisingly well resolved. The spectra showed there were in fact four acetate groups, indicating one of

and required further work to identify the active compounds. This paper reports the results of further work on the active fraction leading to the identification of some novel acylglycerols identified for the first time not only in propolis but in the source resins from poplar trees. The chemistry of the new compounds is reported here as well as the antiproliferative activity of these acylglycerols against a range of human gastrointestinal cell lines.

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RESULTS AND DISCUSSION Initial Separation from Propolis Resin. In earlier work we described the in vitro bioactivity-guided fractionation of “Bio30” propolis using both anti-inflammatory (TNF-α, COX1, COX-2) and anticolon cancer (DLD-1 colon cancer cell viability) assays and determined the phenolic compounds responsible for the activity.4 This work identified a range of phenolic compounds responsible for much of the observed activity. One fraction in particular (fraction S#8) was not pursued at the time, as the activity did not relate to any phenolic-type compounds. Identification of the active compounds in this fraction is now reported in this work. The active fraction of interest was one of the most nonpolar fractions eluted from a reverse-phase (RP) silica chromatographic separation. This fraction showed good levels of antiproliferative activity against a human colon cancer cell line, DLD-1. Further fractionation into four subfractions showed activity to mainly reside in just one of these subfractions. This subfraction showed no UV-absorbing peaks in HPLC; however evaporative light-scattering detection (ELSD) indicated the presence of several peaks in the area of interest. Chromatograms showing the relative retention time of these peaks in a typical propolis tincture sample are shown in Figure 1. In order to observe these peaks in the ELSD, the amount of tincture injected was much higher than for a typical B



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Table 1. 1H NMR and 13C NMR Data (δ) for 1, 2, and 8 (500 MHz) (δ in ppm, J in Hz)a 1b pos.

δC, type

1′ 2′

171.6, C 42.4, CH2

3′ 4′ 5′ 6′ 7′ 8′

67.8, CH 37.1, CH2 25.6, CH2 25.6, CH2 37.7, CH2 70.6, CH

9′ 10′ 11′-17′ 18′ 19′ 20′ 1

37.6, CH2 25.5, CH2 29.3, CH2 31.7, CH2 22.4, CH2 13.42, CH2 63.4, CH2

2 3

69.9, CH 63.2, CH2

acetates CH3 acetate CO

δH (J in Hz)

2c δC

δH (J in Hz)

δC

δH (J in Hz)

HMBC

169.9 39.1

2.57, AB dq (9.6, 7.3)

1′, 3′, 4′

70.2 33.9 25.0 25.0 33.9 74.1

5.18, tt, (7.2, 5.4) 1.6, m 1.25−1.4, m 1.25−1.4, m 1.51, m 4.85, q (7.1)

2′, 4′, 5′ 3′, 5′

3.53, br s

68.2 36.6 25.5 25.7 37.3 71.9

2.45, dd, (15, 8); 2.55 dd, (15, 4) 4.02, br s 1.5−1.6, m 1.25−1.5, m 1.25−1.5, m 1.4−1.5, m 3.6, br s

0.87, t (7.0)

37.6 25.5 29.3 31.9 22.6 14.1

1.4−1.5, m 1.25−1.5, m 1.25, br s 1.25, br s 1.25, br s 0.88, t (7.0)

34.1 25.3 29−30 31.9 22.7 14.1

1.51, m 1.25−1.4, m 1.25, br s 1.25, br s 1.25, br s 0.87, t, (6.5)

4.1−4.3, m

65.1

4.15−4.3, m

62.5

3.82, m 3.59, br s

68 65.1

4.1, m 4.15−4.3, m

69.4 62.2

20.8

2.11, s

20.6, 20.8, 21.0, 21.2

4.29, 4.32. both dd 2, 3 (12.3, 4.4) 5.23 1, 3 4.15, 4.16 both dd (10.6, 2, 3 4.4) 2.09, 2.07, 2.03, 2.03 s

2.39, dd, (15, 8); 2.49, dd (15,4.4) 4.00, br s

172.7 41.6

8c

171.1

10′ (or 6′), 7′, 9′

170.0, 170.3, 170.4, 170.9

a

HMBC correlations are from proton(s) stated to the indicated carbon. bAcetone-d6 solvent. cCDCl3 solvent.

disubstitution pattern is also supported by MS data analysis (see below). The positive ion mass spectra (LCMS, ESIMS/MS) all show fragment ions at m/z 159 and m/z 117. These are most easily accounted for by a C7H11O4 fragment (three indices of hydrogen deficiency) comprising the C-1−C-5 part of the fatty acid with an acetate group at C-3. The loss of the acetate then gives the other common fragment at 117 amu. The proposed structure for the peracetylated glyceride is shown in Figure 2 with the putative mass spectrometric fragmentation pathway.

the acetate moieties was present in the natural compound. A trisubstituted glycerol was present, and a fatty acid attached at one of the hydroxy groups of the glycerol moiety was also hydroxylated at C-3. The rest of the molecule was composed of the remaining fatty acid chain, which also had a second hydroxy group. The likely chain length of the fatty acid, therefore, was C20, C21, or C22 for the main compounds. Detailed analysis of the NMR data (Table 1) identified the most likely placement of the second hydroxy group on the fatty acid to be at C-8. C-2 of the fatty acid is an isolated methylene group and shows long-range HMBC correlations to C-3 (70 ppm) and C-4 (34 ppm). The H-3 methine proton shows long-range correlations to C-4 (34 ppm) and C-5 (25 ppm). The other methine proton (H-8) shows long-range correlations to C-6 and C-10 (both at 25 ppm) and C-7 and C-9 (both 34 ppm). Also, the C-4, C-7, and C-9 protons show HMBC correlations to the multiple carbons at 25 ppm (C-5, -6, and -10). No correlation is seen between H-4 and any of the carbons with a chemical shift around 29 ppm. The C-4 protons are a clear multiplet, while H-7 and H-9 are overlapped in an isolated multiplet. No direct H−H coupling is seen between H-4 and H-7 or H-9. A long-range correlation is seen between H-7/9 and a carbon with a chemical shift at 29 ppm, which must be C-10. These data fit a pattern of chemical shift order that is consistent with a 3,8-diacetoxy fatty acid. For C-8 the chemical shifts of both the α carbons are at 34 ppm, while the β carbons resonate at 25 ppm. Similarly, C-4 and C-5 resonate at 34 and 25 ppm, respectively. The chemical shift pattern would be somewhat different if the second acetoxy group was placed at C-9 (the C-5 signal would have a higher chemical shift and a probable long-range correlation with H2-4). The 3,8-

Figure 2. Structure of C20 glyceride peracetate (8) and MS degradation to give m/z 159 and m/z 117 ions characteristic of glyceride series.

Further information as to the structural features of the fatty acid moiety were obtained by analysis of the mixture of fatty acids obtained after base hydrolysis of the glyceride mixture. The free fatty acids were liberated by treatment with LiOH and methylated by diazomethane treatment to yield a fatty acid methyl ester (FAME) mixture.8 GCMS analysis of the tetramethylsilyl (TMS) derivatives of the FAME mixture showed a set of 13 acid derivatives (Figure 3). The main peaks represent a homologous series. Peak 4 has a characteristic ion at m/z 383 (corresponding to loss of methyl and TMSOH), and similar ions are seen for peaks 5 (m/z 397), 7 (m/z 411), C



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Figure 3. GCMS chromatogram TIC of TMS derivatives of methylated fatty acids from the glyceride fraction.

Figure 4. RP-LCMS-MRM chromatogram of acylglycerols from propolis (only segment shown).

branched methyl at the terminal end). This fits with evidence from NMR data on the peracetylated mixture, which suggested minor amounts of methyl branching. The LCMS analysis of the crude propolis or partially purified glyceride fractions is discussed next. As shown above, the acylglycerols have characteristic MS patterns and show some common fragment ions, which facilitate analysis using LCMS data. The major compounds are the monoacetates with MWs of 460, 474, 488, and 502 Da, corresponding to C20−C23 fatty acid glycerides with one acetate group. A typical propolis acylglycerol profile is shown in Figure 4. This is a crude propolis resin sample obtained by evaporation to dryness of Manuka Health Bio-30 tincture. The chromatograms are those resulting from multiple reaction monitoring (MRM) transitions specific for each type of compound, and only the relevant portion of the total chromatogram is shown. Generally, the MRMs chosen were those for the molecular ion that yielded an ion at m/z 117 or 159. Peak 1 is the nonacetylated C20 glyceride. Peaks 2, 3, 5, and 6 are the monoacetates of the C20−C23 acylglycerols. Peak 4 is probably the iso-analogue of the C22 glyceride monoacetate, as it has the same MW as the compound in peak 5. Peak 7 represents the

10 (m/z 425), 11 (m/z 439), and 12 (m/z 453), so peaks 4−9 correspond to the C19−C24 dihydroxy fatty acids (methyl ester TMS ethers). Another feature was fragments at m/z 301 and m/z 247 seen for all the peaks. These fragments, especially the fragment with m/z 301, are characteristic of 3,8-dihydroxy acids.9 The other possible dihydroxy acids (3,6; 3,7; or 3,9) would be expected to have different fragmentation patterns based on published data.9 For example, the large peak 5 (C20 dihydroxy) has a mass spectrum [m/z (rel intensity), 397(43), 301(100), 271(76), 247(37), 73(71)] that is similar to that described for methyl 3,8-bis[trimethylsilyloxy]eicosanoate [literature values: 487(1), 397(5), 333(2), 301(26), 271(24), 247(3), 243(1), 211(4), 175(7), 143(3), 129(13), 73(100)]. Thus, the dihydroxy acids in the glyceride fraction are the C19−C24 3,8-dihydroxy fatty acids. Note that the relative peak size indicates that the C20 (peak 5) and C22 (peak 10) chain lengths are probably the dominant compounds. Smaller minor peaks with highly similar MS fragmentation to the larger peaks were also seen (some difference in relative abundance of low m/z fragments). Peak 9 is similar to 10 and peak 12 to 13. It is probable these minor peaks are the iso-versions of the C22 and C24 acids (i.e., a D



Article

(MPA). In each case a diester was formed with the chiral reagent attached to the hydroxy groups at C-3 and C-8. The products were examined by 1H NMR. Only the signals for H-2, H-3, and H-8 are sufficiently well separated for detailed analysis. The changes in the shifts of these derivatives [Δδ(S−R), Table 2] can then be compared with relevant examples from

diacetate of the C20 glyceride. The general pattern of compounds is somewhat similar to that seen above for the GCMS analysis of the methylated free fatty acids. At this stage the location of the fatty acid attachment to the glycerol moiety was not resolved, the site of acetylation was uncertain, and none of the individual glyceride compounds had been purified. Using a combination of preparative RPHPLC and size exclusion chromatography several of the individual acylglycerols were obtained in purity suitable for NMR spectroscopic analysis. The NMR data for two of these compounds, the major non-acetylated compound 1 and the major isolated monoacetate 2, are presented in Table 1. The NMR spectra of the individual purified monoacetates 2, 3, and 5 were difficult to distinguish from each other, as the minor differences were buried in the aliphatic part of the NMR spectrum. The position of the acetate group in the monoacetate 2 could be assigned by comparison of the NMR data (Table 1) for the non-acetylated C20 compound, 1, the monoacetylated C20 compound, 2, and the peracetate, 8. In particular, only the C-3′ protons of the glycerol unit in the monoacetate 2 show chemical shifts consistent with acetylation (3.59 in 1 vs 4.15−4.30 in 2). An HMBC correlation between the C-3′ protons and the acetate carbonyl was also observed. This shows the monoacetate compounds have the glycerol acylated at the 3 and the acetyl group at the 1 position; that is, compound 2 is 1-O-acetyl-(3′,8′-dihydroxyeicosanoyl) glycerol. The LCMS fragmentation pattern initially suggested acetylation at the C-3 of the fatty acid, as this explains the fragment ion at m/z 159. It is possible that this fragment ion arises from trans-acetylation before loss of the glycerol moiety. A sufficient quantity of the diacetate could not be isolated for defining the location of the second acetate group in compound 7. Based upon analysis of propolis samples using LCMS and a standard of the major purified acylglycerol, 2, the acylglycerols are present in a typical propolis tincture at approximately 0.2% (w/w) of the weight of dewaxed propolis solids. A search of the literature using the molecular formulas and structures determined for the acylglycerols in propolis showed only one other example of this specific type of compound, a series of acylglycerols isolated from the leaf exudate of Paulownia sp.9 Thirty acylglycerols were isolated from Paulownia, and many were acetylated. The acetates were variously located on either the fatty acid or the glycerol moiety, resulting in the large number of analogues. However, on hydrolysis, only C18 and C20 mono- and dihydroxy fatty acids were seen. In the New Zealand propolis it is likely a similar range of degrees of acetylation is present, but the fatty acids are C20, C21, and C22 hydroxy fatty acids. This is somewhat unusual, as generally fatty acids are synthesized from two carbon units, and hence the resultant series would normally be C18, C20, C22, etc. Also, the hydroxy fatty acids in compounds from Paulownia are positioned at the C-2 position of the glycerol moiety. Stereochemistry. Further work was required to determine the configuration of the oxygenated stereogenic centers of the 3,8-dihydroxy fatty acids. As the length of the fatty acid chain has no effect upon the chemical shift of the protons at C-2, -3, and -8, the crude glyceride mixture was used for this work. The crude mixture was hydrolyzed using LiOH as described previously. The free fatty acid was converted to the methyl ester using diazomethane. Samples of the methyl ester were then derivatized using R- or S-α-methoxyphenylacetic acid

Table 2. 1H NMR Chemical Shift Differences between (R)and (S)-MPA Esters for 3,8-Dihydroxy Fatty Acid Methyl Esters from Propolis Compared with a Similar Experiment with Byrsonic Acid10 and Oncidinol (Using Mosher’s Acid)8 hydrogen

δ dihydroxy FAME

δ di R ester FAME

δ di S ester FAME

Δ δ(S− R)

Δ δ(S− R) byrsonic

Δ δ(S− R) oncidinol

Ha-C(2) Hb-C(2) H-3 H-8

2.5 2.41 4.01 3.57

2.6 2.5 5.22 4.76

2.43 2.35 5.15 4.86

−0.17 −0.15 −0.07 +0.10

−0.11 −0.13 −0.08 +0.07 (H-7)

−0.11 −0.12 −0.06 +0.08 (H-6)

the literature. There are three examples where direct comparisons are valid; these are the floral oil compounds byrsonic acid10 and oncidinol8 and the acylglycerols from Paulownia.9 In the case of byrsonic acid, hydrolysis produced a 3,7dihydroxy fatty acid. Preparation of the diesters using chiral Mosher’s acid [methoxy(trifluoromethyl)phenylacetic acid] produced changes (Table 2) that permitted assignment of the acid as (3R,7R)-3,7-dihydroxydocosanoic acid. Similarly, for oncidinol, the acid was assigned as a (3R,6R)-dihydroxy acid based on the results shown in Table 2 using Mosher’s acid. In both examples, the results are similar to those seen for the propolis acylglyceride sample in Table 2. The Δδ(S−R) values are negative for H-2 and H-3 and positive for the remote hydroxy position. Paulownia has a range of dihydroxy fatty acids acylated to glycerol including 3,8-dihydroxy fatty acids as seen in the propolis acylglyceride fraction, enabling a direct comparison of the same hydroxylation pattern. Chiral derivatives were prepared from these compounds.9 However, the chiral reagent used was a naphthalene derivative that is not commercially available. However, the same general shielding/deshielding effects should be observed. In the work described for the Paulownia acids the individual 3- and 8-monoesters were prepared, and the chemical shift differences are shown in Table 3. The authors concluded a 3R, 8R absolute configuration based on these results. Along with preparing these esters they also prepared Mosher’s acid derivatives using the 3-acetyl versions of the acylglycerols; that is, only the C-8 position was derivatized with the chiral reagent. The Δδ(S−R) values seen Table 3. 1H NMR Chemical Shift Differences between (R)and (S)-Naphthylmethoxyacetic Acid Esters for Selected Paulownia Compounds (3,8-Dihydroxy Fatty Acids)9

E

hydrogen

3-mono ester Δδ(S−R)

8-mono ester Δδ(S−R)

Ha-C(2) Hb-C(2) H-3 H-8

+0.11 +0.1 +0.1 −0.23

+0.18 +0.17 +0.25



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the same dihydroxy fatty acid acylglycerols seen in propolis were present in many of the more commonly planted New Zealand poplar cultivars analyzed. The acylglycerols were found on leaves, twigs, and buds of many poplars and appeared to be generally associated with the resin on these plant parts. It is anticipated that the individual acylglycerols could be isolated from poplar exudates in a similar manner to that used for propolis. Poplar cultivars such as “Tasman”, “Selwyn”, and “Fraser” are typical examples of the widely planted Populus × euramericana. The dihydroxy fatty acid acylglycerols were generally found to be absent in some of the other cultivars such as the P. maximowiczii × nigra clones and the Chinese poplar species P. yunnanensis. Acylglycerols were absent in hybrids not containing a cross with P. deltoides. These types of glycerides were also absent in samples of willow leaf and buds, while in Paulownia plant parts sampled in New Zealand low levels of different types of acylglycerols were present, showing that these plant species are not the source of the acylglycerols in New Zealand propolis. Figure 6 shows an LCMS-MRM chromatogram for acylglycerols from P. euramericana cultivar “Fraser” leaf extract, which qualitatively shows a similar pattern to the corresponding propolis glyceride fraction in Figure 4. Although the Paulownia compounds are structurally the closest to the propolis acylglycerols, there are a number of reports of other acylglycerols from other plant exudates.11−13 In each case the fatty acid was reported as attached at the 2position. Although acyl migration may readily occur during extraction and workup, it would appear unlikely that the propolis acylglycerols have undergone acyl migration to the more stable C-1 analogues since the source of poplar exudates shows the same pattern of compounds. In poplar, the resins are reportedly produced by stipules, which start producing resin at the bud stage,14 whereas the acylglycerols mentioned above arise from glandular trichome exudates.11−13 Antiproliferative Activity. The relative antiproliferative bioactivities of the two major monoacetates, 2 and 3, as well as the acylglycerol-rich concentrate fraction were examined against three human gastrointestinal cancer cell lines to confirm that the isolated compounds were the source of the antiproliferative activity observed in earlier work.4 Samples of 1 and the deacetylated versions of 2 and 3 were also tested (these were isolated from preparative HPLC and derived from hydrolysis during workup). The initial bioassay-guided fractionation work was performed using DLD-1 colon adenocarcinoma cells. These compounds were also tested for activity versus two other cell lines, KYSE-30 (esophageal squamous cancer) and NCI-N87 (gastric carcinoma), and compared with the positive controls glycerol monopalmitate15,16 and the anticancer drug 5-fluorouracil. The results are shown in Table 5 and confirm the high level of activity in these assays for the acylglycerols. The level of activity is similar to or greater than that which we reported for other propolis compounds, such as pinocembrin and the various caffeates.4 However, unlike the phenolic compounds, which are less active against the gastric carcinoma NCI-N87 cell line, the acylglycerols are highly inhibitory of proliferation and cytotoxic at 110 μM.

for H-2 and H-3 with the Mosher’s reagent were negative, showing that the naphthyl reagent produces the opposite Δδ(S−R) values to the Mosher’s esters for the same stereogenic center. As the reagent we used for the chiral derivatization of the propolis glyceride dihydroxy fatty acid is similar to Mosher’s acid (a trifluoromethyl group at the αposition vs a hydrogen), we can confidently assign the propolis glyceride dihydroxy fatty acid as (3R,8R)-3,8-dihydroxyeicosanoic acid (or heneicosanoic/dodecanoic acid for the other major fatty acids) as shown in Figure 5.

Figure 5. Compounds 1 (R = H, n = 10), 2 (R = Ac, n = 10), 3 (R = Ac, n = 11), and 4 (R = Ac, n = 12).

The assignment of the (3R, 8R) absolute configuration is also consistent with known similar compounds, suggesting these compounds are probably derived from a similar biosynthetic pathway. Ideally individual monoesters should also be prepared for the compounds under study. This would allow for any effects due to the overlapping effects of the two aromatic ester moieties. However, in this case it was difficult to just form monoesters, as the reaction tended to go to completion quite quickly and a 1:1 mixture of the acid and ester also produced mostly diester. As there are suitable examples of other similar diester compounds, attempts to prepare monoesters were discontinued. Plant Source of Acylglycerols. The source of the acylglycerol compounds in NZ propolis is from the exudates of some species and hybrids of poplar trees. Analysis of leaf and twig exudates using LCMS shows the presence of the same acylglycerols in the tree resin extracts at about the same relative quantity as seen in propolis resin. Table 4 presents the results of analysis of leaf and twig extracts of a range of common poplar trees found in New Zealand. The analysis showed that Table 4. Qualitative 3,8-Dihydroxy Fatty Acid Acylglycerol Content in New Zealand Poplar Cultivarsa plant part cultivar Kulu Oxford Pecam Selwyn Tasman Luisa Avanzo Fraser Yunnan Geyles Italica

poplar description P. ciliata Wall. ex Royle P. maximowiczii Henry × berolinensis Dipp. P. maximowiczii × nigra P. × euramericana Guinier (= × canadensis Moench) P. × euramericana Guinier (= × canadensis Moench) P. × euramericana Guinier (= × canadensis Moench) P. × euramericana Guinier (= × canadensis Moench) P. yunnanensis Dode P. maximowiczii × nigra P. nigra L.

twig

leaf

np np np +++

np np np +++

++

++

+++

+++

+++

++

np np np

np np np

■

a

EXPERIMENTAL SECTION

General Experimental Procedures. Propolis resin was obtained from Manuka Health NZ Ltd. as bulk samples and as Bio-30 tincture (commercially available). R- and S-Methoxy-phenyl acetic acid, N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide, and N,N-dimethyla-

Levels estimated from LCMS data on plant extracts: np = acylglycerols not present, ++ levels of acylglycerols somewhat lower than seen in propolis, +++ acylglycerol levels similar to those in propolis. F



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Figure 6. RP-LCMS chromatogram (MRM) showing acylglycerols in P. euramericana “Fraser” leaf extract.

Table 5. Antiproliferative Activity of Propolis Acylglycerols against Human Cell Lines DLD-1, KYSE-30, and NCI-N87 (% Inhibition of Proliferation versus Cells-Only Control)a cell line (% inhibition of proliferation, 110, 55, or 22 μM) DLD-1 compound

110

acylglycerol concentrate acylglycerol 1 acylglycerol 1b acylglycerol 1c acylglycerol 2 acylglycerol 3 MGP (glycerol monopalmitate)

82 80 76 nd 94 91 72

55

13

NCI-N87 22

110 83 100 100 100 100 100 100

nd

55

86

KYSE-30 22

39

110 44 52 29 82 52 29 75

55

22

50

9

a

Acylglycerols 1b and 1c are equivalent to compounds 2 and 3 without the acetyl group. bPositive control: 5-fluorouracil at 450 nM gave 21, 8, and 17% inhibition against DLD-1, NCI-N87, and KYSE-30, respectively. cnd = not determined. Synergi C12 column (4 μm, RP Max 80 Å, 250 × 30 mm). Injection volumes between 0.5 and 1.5 mL and a flow rate of 20 mL/min were employed. Solvents were 80% aqueous MeOH (containing 0.1% trifluoroacetic acid) and EtOAc/MeOH (4:1 v/v). The initial eluent composition consisted of 80% aqueous MeOH. The solvent composition was held for 5 min at the initial conditions before the EtOAc/MeOH solvent concentration was increased linearly to 100% over 35 min. The chromatography was carried out at room temperature (18−20 °C). Fractions were collected manually with online detection carried out at 210, 268, and 327 nm and also by evaporative light scattering detection. The main components of this fraction were quite nonpolar, eluting late in the chromatogram, and showed minimal UV absorption at the wavelengths typically used for analysis on other propolis fractions containing phenolics, i.e., 268 and 327 nm. However, ELSD revealed a complex mixture of components. Owing to the destructive nature of ELSD, preparative HPLC fractions were collected manually with online detection carried out at 210 nm. Based on the similarity of analytical profiles, the collected fractions were pooled into four fractions. These pooled fractions were subjected to the bioassay using DLD-1 cells as described earlier.4 The most active fraction was S#8 90% F3, which completely inhibited proliferation and was cytotoxic at the concentration tested (200 μg/ mL). Analysis of this active fraction by HPLC-ELSD and LCMS showed that a series of sharp peaks with characteristic mass spectra were concentrated in this fraction. Purification of Acylglycerols. A large-scale extract was prepared from 1.1 kg of dewaxed propolis resin using EtOAc (3 L) as the

minopyridine were purchased from Sigma-Aldrich (St. Louis, MO, USA). NMR spectra were recorded using a Bruker Avance III 500 MHz NMR spectrometer (Bruker, Karlsruhe, Germany). LCMS analysis was performed using a Shimadzu LCMS system with a Nexera UPLC and an 8040 triple quad MS (Shimadzu, Kyoto, Japan). The system was operated using an ESI source in both negative and positive modes. The other conditions used were nebulizer gas flow of 3 L/min, desolvation line temperature of 250 °C, heating block temperature of 400 °C, and drying gas flow of 15 L/min. A Waters QTOF MS was also employed for direct infusion ESIMS analysis of some samples (Waters, Milford, MA, USA). Preparative HPLC was performed using a Gilson 321 pump and PDA detector (Gilson, Middleton, WI, USA). HPLC analysis was performed using a Waters H-class UPLC system equipped with PDA and ELSD detectors (Waters, Milford, MA, USA). The solvent program used for propolis analysis (e.g., as shown in Figure 1) is similar to that described below for LCMS analysis of acylglycerols. The IR instrument used was a Bruker Tensor with ATR insert (Bruker, Karlsruhe, Germany). GCMS analysis was performed with a Shimadzu QP2010 Ultra instrument equipped with a Restek RTX5 ms 30 m column using a temperature program of 170−320 °C, with a split ratio of 20 (Shimadzu, Kyoto, Japan). Bioactivity-Guided Isolation of Propolis Fraction. The 90% aqueous EtOH elution fraction (propolis fraction S#8) produced in the work reported earlier4 was further fractionated using preparative HPLC as follows. The propolis fraction S#8 was dissolved in neat EtOH and chromatographed by preparative HPLC on a Phenomenex G



Article

acetic acid); HRMS (QTOF) m/z 609.3618 (calcd for C31H54O10Na, 609.3615). LCMS Analysis of Acylglycerols. Chromatography was performed using a Supelco Ascentis Express C18 column (150 × 3 mm, 2.7 μm, 90 Å) with a H2O (A):MeCN (B) gradient at a flow rate of 0.22 mL/min. The water contained 0.1% formic acid. The solvent gradient time program was as follows: initial 20% B, hold for 3 min, then linear increase to 30% at 8 min, 40% at 37 min, 60% at 46 min, 80% at 52 min, 100% at 8 min, and hold for 4 min before returning to starting conditions. A shorter gradient was employed for more rapid analysis. The Shimadzu 8040 MS detector was operated using a electrospray ion source, and acylglycerols were analyzed by MRM in positive mode. Transitions were m/z 461 to m/z 117 for the main monoacetate, with other transitions to the same daughter ion being m/z 475, 489, and 503 for other monoacetates, 503, 517, and 531 for diacetates, and 410 and 419 for non-acetylated glycerides. The other conditions used were nebulizer gas flow of 3 L/min, desolvation line temperature of 250 °C, heating block temperature of 400 °C, and drying gas flow of 15 L/min. MRM dwell times and collision energies were all set at 20 ms and −35 V. Chiral Ester Derivatization. The method used was that described by Freire et al.17 A 10 mg amount of the free fatty acid methyl ester mixture was dissolved in approximately 0.8 mL of CH2Cl2 (dry) in a 5 mL reaction vial. The solution was stirred at room temperature (RT), and 3 equiv (22.3 mg) of R-methoxyphenylacetic acid, 2 equiv (10 μL) of N-(3-(dimethylamino)propyl)-N′ethylcarbodiimide, and a catalytic amount of N,N-dimethylaminopyridine were added. The reaction was worked up by washing the reaction solution with water, 1 M HCl, NaHCO3, and water. The organic layer was dried under vacuum and purified using a small silica gel column to obtain the diester product (approximately 8 mg). The S-diester was obtained in a similar fashion. Samples for 1H NMR analysis were dissolved in CDCl3 and run at 500 MHz. Determination of Plant Sources of Acylglycerols. Samples of poplar buds, twigs, and leaf material were collected from the curated Plant and Food Research national poplar collection at Aokautere, near Palmerston North, New Zealand, from September 2014 to February 2015. Most samples were from coppiced trees, as these were easily accessed. Leaf and twig samples were weighed and extracted by dipping in absolute EtOH at RT for approximately 2 min. The samples were extracted as is without maceration or slicing. The extracts were dried and weighed, and samples of each prepared for analysis by making up solutions in EtOH at 5 mg/mL. Samples of leaf and flower material were collected from a Paulownia tomentosa tree in Lower Hutt, NZ (Tirohanga Rd., GPS −41.201586, 174.90840). The plant material was extracted by dipping the plant material into ethyl acetate to extract the waxy outer layers only. Antiproliferative (Cell Viability) Assays: DLD-1, KYSE-30, and NCI-N87 Gastrointestinal Cancer Assays. Human gastrointestinal cancer cells DLD-1 (colorectal adenocarcinoma cells [ATCC CCl-221, obtained from ATCC]); NCI-N87 (gastric carcinoma cells [ATCC CRL-5822, obtained from ATCC]), KYSE30 (esophageal squamous cell carcinoma [ECACC, obtained from Sigma-Aldrich]) were revived from cryostorage and cultured in the presence of the test and reference samples. The culture conditions for the cells were those described by the supplier of the cells, and the assay methodology was based on previously reported procedures.4 Briefly, working solutions of the test compounds and positive controls were prepared by dissolving the test fractions in 15% EtOH/Hank’s balanced saline solution to a concentration of 2 mg/mL solids. The final concentration of each sample in the test well was 110, 55, or 22 μM. The 5-fluorouracil positive control test well concentrations were 450, 150, and 50 nM. After incubation an MTT cell viability assay was performed on the cultures to determine the effect of the samples on the inhibition of cell proliferation. Results were expressed as the percentage proliferation of cells cultured in the presence of the sample in comparison to the cells-only control. Six replicates were used for both test and control samples.

extraction solvent. The propolis resin was coarsely chopped and soaked in EtOAc at room temperature with mechanical stirring for 2 h. The resulting solution was filtered but not concentrated. Around 20% of this extract solution was chromatographed using silica gel, after first absorbing the extract onto a portion of silica gel (50 g) and evaporating the EtOAc using a rotary evaporator. The preabsorbed solid was applied to the top of a large silica gel column (100 × 45 mm) and eluted with 1:1 hexanes/Et2O (v/v), and 8 × 150 mL fractions were collected. Then the column was eluted with Et2O to collect a further four 150 mL fractions before beginning elution with EtOAc. The four EtOAc fractions #13−#16 (F13−F16) were much darker in color than the earlier fractions. A final fraction F17 was collected by elution with acetone. Silica TLC (visualization using phosphomolybdic acid in EtOH followed by heat) and LC-MS were used to track progression of the purification process. The acylglycerolrich fractions from silica gel chromatography (fractions 13−17, wt 1.6 g) were combined and subjected to RP column chromatography (Machery-Nagel Chromabond C18 70 mL 10 g SPE type column) using mixtures of water and EtOH without acid to yield a final acylglycerol-rich fraction. This process was repeated with the remaining EtOAc extract as required. A portion of the combined fraction was used for preparative HPLC followed by Sephadex LH-20 size exclusion chromatography to obtain two monoacetates, 2 and 3 (90 and 74 mg, C20 and C21 dihydroxy fatty acid monoacetates, respectively), and three non-acetylated compounds, 1, 7, and 8 (45, 16, and 23 mg; C20, C21, and C22 dihydroxy fatty acid monoglycerides, respectively). The separation progress was tracked using HPLC with ELSD, and the purity of the individual compounds isolated was assessed to be >95% by HPLC, LC-MS, and NMR. Preparative HPLC was performed using a Phenomenex Synergi 4 μm-RP Max 80 Å 250 × 30 mm C12 column (gradient elution using water with 0.1% formic acid and MeCN), and fractions were analyzed offline using TLC and LC-MS. Data are reported for 1 and 2 only. Other compounds show almost identical NMR data. Compound 1 (C20 non-acetylated): cream solid; [α]20D −2 (c 0.2, acetone); FTIR (ATR) νmax 3350, 2915, 2848, 1728, 1455 cm−1; NMR data see Table 1; HRMS m/z 441.3201 (calcd for C23H46O6Na, 441.3193). Compound 2 (C20 monoacetate): cream solid; [α]20D −0.1 (c 0.3, CHCl3); FTIR (ATR) νmax 3345, 2914, 2849, 1722, 1380, 1264 cm−1; NMR data see Table 1; HRMS (QTOF) m/z 483.3296 (calcd for C25H48O7Na, 483.3298). Hydrolysis of Acylglycerol Fraction. Hydrolysis of a sample of the acylglycerol-rich fraction was performed as described in Reis et al.10 Approximately 100 mg of the acylglycerol-rich fraction was mixed with 4 mL of THF/MeOH/H2O (3:1:1) and 55 mg of LiOH. This was stirred at 0 °C for 30 min and then at room temperature for 48 h. The reaction mixture was acidified with HOAc (to pH ca. 2.8) and extracted with CHCl3. The dried extract was esterified with diazomethane and subjected to silica gel chromatography using hexanes/EtOAc mixtures. A fraction (40 mg) comprising one main spot on silica TLC (Rf ca. 0.3 with 1:1 hexanes/EtOAc, visualization was by phosphomolybdic acid in EtOH dip followed by heat) was collected. Preparation of Peracetate. Peracetylation of the acylglycerolrich fraction was carried out by dissolving the partially purified acylglycerol fraction (approximately 150 mg) in Ac2O (20 mL) and adding a small amount of N,N-dimethylaminopyridine. After stirring at room temperature overnight the reaction mixture was worked up by adding MeOH and toluene (approximately 20 mL of each) and evaporated to dryness using a rotary evaporator. The residue was chromatographed on silica gel using hexanes/EtOAc mixtures to obtain a purified peracetate mixture (8, approximately 80 mg). Peracetate (8): oil; [α]20D 0 (c 0.2, CHCl3); FTIR(ATR) νmax 2924, 2853, 1737, 1370, 1217 cm−1; 1H NMR data see Table 1; QTOF MS (positive ion m/z) 665.6, 651.5, 637.5, 623.5, 609.5, MS/ MS on 665.6 (m/z) 605.6 (loss of acetic acid), 545.5 (loss of further acetic acid), 507.5 (loss of glycerol residue), 447.5 (loss of further H



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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00562. NMR data for compounds 1, 2, and peracetate, 8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Stephen Bloor: 0000-0002-1936-5857 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Mr. T. Jones (Plant and Food Research, NZ) is thanked for access to poplar collection, colleagues Dr. M. Vyssotski and Dr. K. Lagutin for advice on lipid analysis and absolute configuration, and Manuka Health New Zealand Ltd for providing the samples of “Bio30” propolis and ongoing support in propolis research. This work was also supported by funding from the New Zealand Government through Callaghan Innovation.

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REFERENCES

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Antiproliferative Acylated Glycerols from New Zealand Propolis

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