Oxyphytosterols as Active Ingredients in Wheat Bran Suppress Human

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Oxyphytosterols as Active Ingredients in Wheat Bran Suppress Human Colon Cancer Cell Growth: Identification, Chemical Synthesis and Biological Evaluation Yingdong Zhu, Dominique Soroka, and Shengmin Sang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf506361r • Publication Date (Web): 06 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

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Oxyphytosterols as Active Ingredients in Wheat Bran Suppress Human Colon Cancer Cell

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Growth: Identification, Chemical Synthesis and Biological Evaluation

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Yingdong Zhu, Dominique Soroka, and Shengmin Sang*

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Center for Excellence in Post-Harvest Technologies, North Carolina Agricultural and Technical

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State University, North Carolina Research Campus, 500 Laureate Way, Kannapolis, NC 28081,

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United States

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Running title: Oxyphytosterols in Wheat Bran

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Corresponding authors:

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Shengmin Sang, Ph.D.

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North Carolina Agricultural and Technical State University

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North Carolina Research Campus

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Tel: 704-250-5710; Fax: 704-250-5709

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Email: [email protected] or [email protected]

1 ACS Paragon Plus Environment


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ABSTRACT

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Consumption of whole grains has been reported to be associated with a lower risk of colorectal

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cancer. Recent studies illustrated that phytochemicals in wheat bran (WB) may protect against

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colorectal cancer. There is a growing interest in the phytosterol contents of foods as either

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intrinsic or added components due to their beneficial health effects. However, little is known

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whether phytosterols in WB contribute the observed chemopreventative activity of the grain. In

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the present study, we directly purified and identified four oxyphytosterols 1-4 from sterol-

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enriched fraction of WB, and also successfully synthesized five sterol oxides 5-8 and 13. Using

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these nine compounds as references, we outlined a comprehensive profile of steroids in WB

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using tandem liquid chromatography mass spectrometry with electrospray ionization (LC-

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ESI/MSn, n = 2-3) techniques for the first time. Among them, three sterol oxides 13, 14 and 18

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are novel compounds, and fourteen compounds 3, 4, 6-11, 13, 14, 16 and 18-20 were reported in

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WB for the first time. Our results on the inhibitory effects of available sterol oxides 1-8 and 13

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against the growth of human colon cancer cells HCT-116 and HT-29 showed that compounds 2-

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8 exerted significant antiproliferative effects, with oxysterol 8 being the most active one in both

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cells. We further demonstrated that four most active sterol oxides 5-8 could induce cell death

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through the apoptosis pathway. Our results showed that phytosterols, particularly

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oxyphytosterols, in WB possess significant antiproliferative properties, and thereby may greatly

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contribute the observed chemoprevention of the whole grain wheat.

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KEYWORDS: Apoptosis, cytotoxicity, chemical synthesis, sterol oxide, wheat bran, colon

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cancer


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INTRODUCTION

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Epidemiological studies have shown that intake of whole-grains or high fiber, particularly

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fiber from cereal grains such as wheat and rye, is associated with a lower risk of several lifestyle-

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related diseases such as type 2 diabetes,1,

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cancers.4,

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discovered a statistically significant 17% reduction in colorectal cancer risk for every three-

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servings-per-day increment of whole-grain intake.6 In 2011, the World Cancer Research

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Fund/American Institute for Cancer Research upgraded that there is “convincing” evidence that

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consumption of dietary fiber protects against colorectal cancer.7 Besides fiber, whole grains are

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rich sources of phytochemicals, minerals, vitamins and other active substances with possible

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health effects.8 Recently, Reddy et al. found that the lipid fraction of wheat bran (WB) strongly

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inhibited colon tumor incidence, multiplicity, and volume in the F344 rat model.9 Our group

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further demonstrated that 2% WB oil significantly inhibited the overall tumorigenesis by 35.7%

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in the Apcmin/+ mouse model.10 More recently, we investigated the anti-cancer ingredients in WB

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oil using a bioassay-guided strategy, and identified alkylresorcinols (ARs) as major active

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components in WB for colon cancer chemoprevention.11 In addition, we also found that

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sphingolipids have a weak activity against the growth of human colon cancer cells.12 However,

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the role of phytochemicals in WB oil accountable for the observed bioactivity of the whole grain

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is not fully understood yet. Whether many other phytochemicals including phytosterols may

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protect against chronic disease remains to be determined.

5

2

cardiovascular diseases,2,

3

and some types of

A meta-analysis of six prospective studies of whole-grains and colorectal cancer

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Phytosterols are plant components that have a chemical structure similar to cholesterol except

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for the addition of an extra methyl or ethyl group. In plants, more than 200 different types of

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phytosterols have been reported with the most abundant species being β-sitosterol, campesterol



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and stigmasterol.13 Besides their cholesterol-lowering effects,14 other beneficial physiologic

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properties of plant sterols include anti-cancer,15 anti-atherosclerotic16 and anti-inflammatory

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effects.17 It is known that wheat is a rich source of diverse sterols, either in their free, conjugated,

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or oxidized forms. Earlier study led to the identification of four oxyphytosterols including 5,6-

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epoxysitosterols and 7-hydroxysitosterols from wheat flour.18 Iwatsuki et al. isolated and

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identified five free sterols and seven sterol ferulates from WB extract.19 Recently, Prinsen et al.

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reported sterols, sterol glycosides, sterol esters, sterol ferulates, steroid hydrocarbons and steroid

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ketones in WB by using gas chromatography-mass spectrometry techniques.20 However, a

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complete chemical profile and the anti-cancer activities of phytosterols in WB are still unclear.

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In the present study, we purified four oxyphytosterols 1-4 from WB extract and synthesized four

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sterol oxides 5-8, two ferulates 13 and 13a, and two aza-sterols 7a and 8a. Using liquid

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chromatography mass spectrometry (LC/MS), we established the chemical profile of

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phytosterols in WB. Furthermore, the structure-activity relationship (SAR) of sterol oxides

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against the growth of human colon cancer cells was discussed based on both purified and

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synthesized compounds 1-8 and 13.

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MATERIALS AND METHODS

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Materials. Cholesterol, β-sitosterol, stigmasterol and pregnenolone were purchased from

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Sigma (St. Louis, MO) or Thermo Fisher Scientific (Waltham, MA). Anhydrous reactions were

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carried out in oven-dried glassware under a nitrogen atmosphere unless otherwise noted.

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Analytical (250 µm thickness, 2-25 µm particle size) and preparative thin layer chromatography

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(TLC) plates (1000 µm thickness, 2-25 µm particle size) were purchased from Sigma (St. Louis,

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MO) and Sorbent Technologies (Atlanta, GA), respectively. Sephadex LH-20 was purchased

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from Sigma (St. Louis, MO). Medium pressure column chromatography was carried out over


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reverse phase silica gel (60 Å, Sigma, St. Louis, MO), equipped with LabAlliance Series I pump

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and Spectra/chrom fraction collector CF-2. LC/MS-grade solvents and other reagents were

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obtained from Thermo Fisher Scientific (Waltham, MA). HCT-116 and HT-29 human colon

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cancer cells were obtained from American Type Tissue Culture (Manassas, VA). McCoy’s 5A

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medium was purchased from Thermo Fisher Scientific (Waltham, MA). Fetal bovine serum

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(FBS) and penicillin/streptomycin were purchased from Gemini Bio-Products (West Sacramento,

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CA). MTT (3-(4,5-dimethylthiaxol-2-yl)-2,5-diphenyltetrazolium bromide) was procured from

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Calbiochem-Novabiochem (San Diego, CA). Apoptag Plus Peroxydase In Situ Apoptosis

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Detection Kit was purchased from Millipore (Billerica, MA).

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Preparation of Sterol-enriched Fraction from WB. Eight fractions (F1-F8) were obtained

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from WB oil, as described in our previous study.11 Briefly, wheat bran, provided by ConAgra

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Food Ingredients Company, was extracted by 95% ethanol at 40 oC for 24 h to give an ethanol

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extract. After evaporation, the ethanol residue was then suspended in water and partitioned with

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ethyl acetate (EA). After removal of EA in vacuo, the WB oil was obtained. The WB oil was

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subjected to column chromatography (CC) over a normal phase silica gel eluted with a mixture

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of hexane and EA, affording F1-F8, which are stored at -80 oC till analysis. Phytosterols could be

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detected in both F7 and F8. In order to avoid the analytical interference from ARs in F7, we

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enriched sterols from F8. Specifically, part of F8 (6.0 g) was subjected to reverse phase (RP) C18

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silica gel medium pressure CC (30 × 360 mm, 60 Å) eluted with 50% methanol, methanol and

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methanol containing 0.2% formic acid (FA). Sphingolipids remained in acidic methanol eluate.

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The neutral methanol fraction was further loaded on Sephadex LH-20 CC eluted with 50%

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ethanol and ethanol. The ethanol eluates were evaporated in vacuo, giving the sterol-enriched

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fraction (2.0 g).



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Purification of Oxyphytosterols 1-4 from Sterol-enriched Fraction and Their NMR

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Data. Repeated purification of the sterol-enriched fraction (1.0 g) by RP C18 silica gel medium

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pressure CC eluted with methanol, giving stigmast-4-en-6β-ol-3-one (1) (4 mg), stigmast-4,22-

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dien-6β-ol-3-one (2) (6 mg), stigmast-5-en-3β-ol-7-one (3) (8 mg), and stigmast-5,22-dien-3β-ol-

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7-one (4) (5 mg).

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Stigmast-4-en-6β-ol-3-one (1) as a white solid: 1H NMR (700 MHz, Py-d5) δ 1.64 (1H, m,

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H-1a), 1.92 (1H, m, H-1b), 2.45 (1H, m, H-2a), 2.60 (1H, m, H-2b), 6.07 (1H, s, H-4), 4.57 (1H,

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m, H-6), 1.28 (1H, m, H-7a), 2.21 (1H, m, H-7b), 2.30 (1H, m, H-8), 0.91 (1H, m, H-9), 1.48

115

(2H, m, H-11), 1.17 (1H, m, H-12a), 2.04 (1H, m, H-12b), 1.02 (1H, m, H-14), 1.16 (1H, m, H-

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15a), 1.65 (1H, m, H-15b), 1.27 (1H, m, H-16a), 1.88 (1H, m, H-16b), 1.17 (1H, m, H-17), 0.74

117

(3H, s, H-18), 1.57 (3H, s, H-19), 1.43 (1H, m, H-20), 1.02 (3H, d, J = 6.6 Hz, H-21), 1.14 (1H,

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m, H-22a), 1.43 (1H, m, H-22b), 1.28 (2H, m, H-23), 1.02 (1H, m, H-24), 1.71 (1H, m, H-25),

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0.89 (3H, d, J = 6.5 Hz, H-26), 0.91 (3H, d, J = 6.5 Hz, H-27), 1.32 (2H, m, H-28), and 0.92 (3H,

120

t, J = 6.6 Hz, H-29); 13C NMR (175 MHz, Py-d5) δ 37.9 (t, C-1), 35.4 (t, C-2), 200.1 (s, C-3),

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126.3 (d, C-4), 170.3 (s, C-5), 70.3 (d, C-6), 40.1 (t, C-7), 30.7 (d, C-8), 54.5 (d, C-9), 38.9 (s, C-

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10), 21.7 (t, C-11), 40.4 (t, C-12), 43.2 (s, C-13), 56.7 (d, C-14), 25.0 (t, C-15), 29.0 (t, C-16),

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56.6 (d, C-17), 12.5 (q, C-18), 20.0 (q, C-19), 36.8 (d, C-20), 19.4 (q, C-21), 34.6 (t, C-22), 26.9

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(t, C-23), 46.5 (d, C-24), 30.0 (d, C-25), 19.6 (q, C-26), 20.4 (q, C-27), 23.9 (t, C-28), and 12.6

125

(q, C-29); positive ESIMS, m/z 429 [M + H]+.

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Stigmast-4,22-dien-6β-ol-3-one (2) as a white solid: 1H NMR (700 MHz, Py-d5) δ 6.07 (1H,

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s, H-6), 5.24 (1H, dd, J = 15.2, 8.8 Hz, H-22), 5.09 (1H, dd, J = 15.2, 8.8 Hz, H-23), 4.56 (1H, m,

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H-6), 2.61 (1H, m), 2.46 (1H, m), 2.28-2.18 (2H, m), 2.09 (1H, m), 2.01 (1H, m), 1.93 (1H, m),

129

1.78 (1H, m), 1.68-1.41 (5H, m), 1.37-1.0 (10H, m), 1.57 (3H, s, H-19), 1.12 (3H, d, J = 6.6 Hz, 6 ACS Paragon Plus Environment

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H-21), 0.94 (3H, t, J = 6.5 Hz, H-29), 0.92 (3H, d, J = 6.5 Hz, H-27), 0.89 (3H, d, J = 6.5 Hz, H-

131

26), and 0.76 (3H, s, H-18); 13C NMR (175 MHz, Py-d5) δ 200.1 (s, C-3), 170.3 (s, C-5),139.2

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(d, C-22), 130.0 (d, C-23), 126.3 (d, C-4), 73.0 (d, C-6), 56.7, 56.5, 54.6, 51.9, 43.0, 41.2, 40.2,

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40.1, 38.8, 37.9, 35.1, 32.6, 30.7, 29.7, 26.1, 25.0, 21.9, 21.7, 21.6, 20.0, 19.6, 13.0 (q, C-29),

134

and 12.7 (q, C-18); positive ESIMS, m/z 427 [M + H]+.

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7-Keto-β-sitosterol (3) as a white solid: 1H NMR (600 MHz, CD3OD+CDCl3) δ 5.64 (1H, s,

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H-6), 3.56 (1H, m, H-3), 2.48-2.45 (1H, m), 2.40-2.32 (2H, m), 2.29-2.25 (1H, m), 2.06-2.03 (1H,

137

m), 1.98-1.95 (1H, m), 1.90-1.88 (2H, m), 1.69-1.46 (6H, m), 1.37-1.00 (13H, m), 1.21 (3H, s,

138

H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.85 (3H, t, J = 7.3 Hz, H-29), 0.83 (3H, d, J = 6.8 Hz, H-

139

27), 0.81 (3H, d, J = 6.8 Hz, H-26), and 0.70 (3H, s, H-18);

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CD3OD+CDCl3) δ 204.5 (s, C-7), 168.7 (s, C-5), 126.2 (d, C-6), 70.9 (d, C-3), 55.8 (d, C-17),

141

51.1 (d, C-14), 51.1 (d, C-9), 46.9, 46.4, 44.1, 42.5, 39.7, 39.4, 37.3, 37.1, 34.9, 31.6, 30.1, 29.4,

142

27.2, 27.0, 23.9, 22.1, 20.2 (q, C-27), 19.5 (q, C-26), 19.4 (q, C-21), 17.7 (q, C-19), 12.4 (q, C-

143

29), and 12.3 (q, C-18); positive ESIMS, m/z 429 [M + H]+.

13

C NMR (150 MHz,

144

7-Keto-stigmasterol (4) as a white solid: 1H NMR (600 MHz, CD3OD+CDCl3) δ 5.63 (1H, s,

145

H-6), 5.15 (1H, dd, J = 15.1, 8.7 Hz, H-23), 5.01 (1H, dd, J = 15.1, 8.7 Hz, H-22), 3.56 (1H, m,

146

H-3), 2.48-2.45 (1H, m), 2.40-2.34 (1H, m), 2.31-2.24 (2H, m), 2.07-1.99 (2H, m), 1.97-1.94

147

(1H, m), 1.90-1.87 (1H, m), 1.75-1.71 (1H, m), 1.62-1.38 (7H, m), 1.34-1.10 (7H, m), 1.20 (3H,

148

s, H-19), 1.01 (3H, d, J = 6.7 Hz, H-21), 0.83 (3H, d, J = 6.4 Hz, H-27), 0.79 (3H, t, J = 7.3 Hz,

149

H-29), 0.78 (3H, d, J = 6.4 Hz, H-26), and 0.70 (3H, s, H-18);

150

CD3OD+CDCl3) δ 204.4 (s, C-7), 168.5 (s, C-5), 139.0 (d, C-22), 130.3 (d, C-23), 126.1 (d, C-6),

151

70.6 (d, C-3), 55.6 (d, C-17), 52.2, 51.0, 50.9, 46.2, 43.8, 42.3, 41.1, 39.4, 39.3, 37.2, 32.7, 31.4,

13

C NMR (150 MHz,



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29.8, 27.1, 26.1, 21.9, 21.8 (q, C-27), 21.4 (q, C-26), 19.4 (q, C-21), 17.7 (q, C-19), 12.6 (q, C-

153

29), and 12.6 (q, C-18); positive ESIMS, m/z 427 [M + H]+.

154

General Procedure A for the Synthesis of Sterol Acetates. A mixture of sterols (1.0 eq),

155

acetic anhydride (Ac2O) (10.0 eq) and pyridine (Py) (50.0 eq) was incubated at 37 oC overnight.

156

The solvent was removed in vacuo. The residue was reconstituted in ethyl acetate (EA), washed

157

with water, dried over Na2SO4, and filtered. The filtration was evaporated off, and the residue

158

was subjected to a short pad of Celite, eluting with a mixture of hexane/EA (20:1), to give sterol

159

acetates.

160

General Procedure B for the Synthesis of 7-Ketosterol Acetates. Chromium trioxide

161

(CrO3) (15.0 eq) was suspended in dry dichloromethane (DCM) and stirred for 30 min at -20 oC.

162

3,5-Dimethylpyrazole (15.0 eq) was added in one portion and the reaction mixture was stirred for

163

30 min at -20 oC. Sterol acetates (1.0 eq) were added and the mixture was stirred at -20 oC and

164

allowed to warm up to room temperature (rt) overnight. EA was then added and the brown

165

suspension was filtered through Celite. The filtration was concentrated, and the residue was

166

purified by CC or pre-TLC to give 7-ketosterol acetates.

167

General Procedure C for the Synthesis of 7-Ketosterols. LiOH (4.0 eq) was added at rt to

168

a stirred solution of 7-ketosterol acetates (1.0 eq) in THF/MeOH/H2O (3:1:1). The resulting

169

mixture was stirred at rt for 2 h. The organic solvents were evaporated and the residue was

170

diluted with water, and extracted with EA. The organic layer was washed with water and brine,

171

dried over Na2SO4 and concentrated in vacuo. The residue was purified by pre-TLC to give 7-

172

ketosterols.


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General Procedure D for the Synthesis of 7β-Hydroxysterols. A suspension of 7-

174

ketosterols (1.0 eq) and cerium chloride heptahydrate (4.0 eq) in methanol was stirred at 0 oC for

175

10 min. Sodium borohydride (2.5 eq) was added to the suspension and the mixture was stirred at

176

rt for 4 h–18 h. The reaction solution was partitioned between water and EA. The organic layer

177

was washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure.

178

The residue was purified by pre-TLC to give 7β-hydrolxysterols.

179

General Procedure E for the Synthesis of 3-O-(4-Acetoxyferuloyl)-oxysterols. Trans-4-

180

acetoxy ferulic acid (1.1 eq) and 7-oxysterols (1.0 eq) were dissolved in dry DCM at 0 oC. DCC

181

(3.0 eq) and DMAP (2.5 eq) were added, and the mixture was stirred at rt for 6 h. The solid

182

formed was filtered off, and the solution was washed with water, 10% HOAc and again with

183

water. The organic extracts were dried over Na2SO4 and concentrated in vacuo. The residue was

184

purified by pre-TLC to give 3-O-(4-acetoxyferuloyl)-oxysterols.

185

General Procedure F for the Synthesis of Oxysterol Ferulates. 3-O-(4-Acetoxyferuloyl)-

186

oxysterols (1.0 eq) was dissolved in a mixture of DCM/MeOH (2:1, 3 mL). K2CO3 (0.2 eq) was

187

added, and the mixture was stirred at rt for 5 h. The reaction was quenched by adding a saturated

188

aqueous NH4Cl solution. The organic layers were separated, washed with water and brine, dried

189

over Na2SO4 and concentrated under reduced pressure. The residue was purified by pre-TLC

190

(H/E = 3:1) to give oxysterol ferulates.

191

Synthesis of Sterol Oxides 3-8.

192

β-Sitosterol acetate (22). Procedure A was followed by using β-sitosterol (414 mg, 1.0

193

mmol), Ac2O (0.94 mL, 10.0 mmol) and Py (4.07 mL, 50.0 mmol), giving the title compound

194

(456 mg, yield: 100%) as a white solid: positive ESIMS, m/z 457 [M + H]+. 9 ACS Paragon Plus Environment


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Stigmasterol acetate (23). Procedure A was followed by using stigmasterol (412 mg, 1.0

196


197

(454 mg, yield: 100%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.39 (1H, d, J = 4.7 Hz,

198


199

H-3), 2.35-2.31 (2H, m), 2.10-1.95 (4H, m), 2.05 (3H, s, OAc-3), 1.90-1.85 (2H, m), 1.76-1.68

200

(1H, m), 1.62-1.39 (10H, m), 1.32-1.12 (5H, m), 1.11-0.92 (4H, m), 1.14 (3H, s, H-19), 1.13 (3H,

201

d, J = 6.7 Hz, H-21), 0.86 (3H, d, J = 6.4 Hz, H-27), 0.82 (3H, t, J = 7.3 Hz, H-29), 0.81 (3H, d,

202

J = 6.4 Hz, H-26), and 0.71 (3H, s, H-18); positive ESIMS, m/z 455 [M + H]+.

203

Cholesterol acetate (24). Procedure A was followed by using cholesterol (386 mg, 1.0

204


205

(428 mg, yield: 100%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.36 (1H, d, J = 5.0 Hz,

206

H-6), 4.59 (1H, m, H-3), 2.34-2.28 (2H, m), 2.04-1.93 (2H, m), 2.03 (3H, s, OAc-3), 1.88-1.78

207

(3H, m), 1.65-1.40 (8H, m), 1.38-1.20 (4H, m), 1.18-0.90 (9H, m), 1.02 (3H, s, H-19), 0.90 (3H,

208

d, J = 6.5 Hz, H-21), 0.86 (3H, d, J = 6.6 Hz, H-27), 0.85 (3H, d, J = 6.6 Hz, H-26), and 0.66

209

(3H, s, H-18); positive ESIMS, m/z 429 [M + H]+.

210

7-Ketositosterol acetate (25). Procedure B was followed by using β-sitosterol acetate (25)

211

(200 mg, 0.44 mmol), CrO3 (658 mg, 6.58 mmol) and dimethylpyrazole (632 mg, 6.58 mmol) in

212

dry DCM (20 mL). The resulting residue was purified by pre-TLC (H/E = 8:1) to give the title

213

compound (138 mg, yield: 67%) as a white solid: positive ESIMS, m/z 471 [M + H]+.

214

7-Ketostigmasterol acetate (26). Procedure B was followed by using stigmasterol acetate (26)

215

(454 mg, 1.0 mmol), CrO3 (1.5 g, 15.0 mmol) and dimethylpyrazole (1.44 g, 15.0 mmol) in dry

216

DCM (50 mL). The resulting residue was purified by CC (H/E = 15:1, 10:1 and 8:1) to give the


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217

title compound (269 mg, yield: 57%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.68 (1H,

218


219

H-3), 2.55-2.51 (1H, m), 2.47-2.42 (1H, m), 2.39-2.33 (1H, m), 2.24-2.20 (1H, m), 2.04-1.93

220

(4H, m), 2.03 (3H, s, OAc-3), 1.79-1.48 (7H, m), 1.41-1.10 (8H, m), 1.20 (3H, s, H-19), 1.01

221

(3H, d, J = 6.7 Hz, H-21), 0.83 (3H, d, J = 6.3 Hz, H-27), 0.79 (3H, t, J = 7.3 Hz, H-29), 0.78

222

(3H, d, J = 6.3 Hz, H-26), and 0.68 (3H, s, H-18); positive ESIMS, m/z 469 [M + H]+.

223

7-Ketocholesterol acetate (27). Procedure B was followed by using cholesterol acetate (27)

224

(428 mg, 1.0 mmol), CrO3 (1.5 g, 15.0 mmol) and dimethylpyrazole (1.44 g, 15.0 mmol) in dry

225

DCM (50 mL). The resulting residue was purified by CC (H/E = 15:1, 10:1, and 8:1) to give the

226

title compound (260 mg, yield: 60%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.69 (1H, s,

227

H-6), 4.70 (1H, m, H-3), 2.55-2.51 (1H, m), 2.48-2.35 (2H, m), 2.26-2.18 (1H, m), 2.07-1.82 (4H,

228

m), 2.06 (3H, s, OAc-3), 1.71-1.47 (5H, m), 1.40-0.99 (13H, m), 1.20 (3H, s, H-19), 0.91 (3H, d,

229

J = 6.5 Hz, H-21), 0.86 (3H, d, J = 6.6 Hz, H-27), 0.85 (3H, d, J = 6.6 Hz, H-26), and 0.67 (3H, s,

230

H-18); positive ESIMS, m/z 443 [M + H]+.

231

7-Ketositosterol (3). Procedure C was followed by using 7-keto-β-sitosterol acetate (100 mg,

232

0.21 mmol) and LiOH (20 mg, 0.84 mmol) in THF/MeOH/H2O (3:1:1, 5 mL). The resulting

233

residue was purified by pre-TLC (H/E = 2:1) to give the title compound (3) (90 mg, yield: 100%).

234

7-Ketostigmasterol (4). Procedure C was followed by using 7-keto-stigmasterol acetate (181

235

mg, 0.39 mmol) and LiOH (37 mg, 1.55 mmol) in THF/MeOH/H2O (3:1:1, 5 mL). The resulting

236

residue was purified by pre-TLC (C/M = 20:1) to give the title compound (4) (166 mg, yield:

237

100%).



Page 12 of 47

238

7β-Hydroxysitosterol (5). Procedure D was followed by using 7-keto-β-stigmasterol (3) (30

239

mg, 0.07 mmol), CeCl3·7H2O (104 mg, 0.28 mmol) and NaBH4 (7 mg, 0.18 mmol) in MeOH (3

240

mL). The mixture was stirred at rt for 18 h. The residue was purified by pre-TLC (C/M = 20:1)

241

to give the title compound (5) (17 mg, yield: 57%) as a white solid: 1H NMR (600 MHz,

242

CD3OD+CDCl3) δ 5.24 (1H, s, H-6), 3.72 (1H, d, J = 8.0 Hz, H-7), 3.41 (1H, m, H-3), 2.26-2.18

243

(2H, m), 2.04-2.02 (1H, m), 1.87-1.78 (4H, m), 1.70-1.66 (1H, m), 1.55-1.33 (6H, m), 1.32-0.99

244

(13H, m), 1.06 (3H, s, H-19), 0.94 (3H, d, J = 6.5 Hz, H-21), 0.86 (3H, t, J = 7.4 Hz, H-29), 0.84

245

(3H, d, J = 6.8 Hz, H-27), 0.83 (3H, d, J = 6.8 Hz, H-26), and 0.72 (3H, s, H-18); 13C NMR (150

246

MHz, CD3OD+CDCl3) δ 144.0 (s, C-5), 127.4 (d, C-6), 73.7 (d, C-7), 72.1 (d, C-3), 57.7 (d, C-

247

17), 56.8 (d, C-14), 50.0, 47.2, 44.0, 42.5, 41.2, 41.0, 38.2, 37.5, 37.4, 35.1, 32.2, 30.3, 29.6, 27.3,

248

27.1, 24.1, 22.2, 20.2 (q, C-27), 19.5 (q, C-26), 19.4 (q, C-21), 19.3 (q, C-19), 12.3 (q, C-29),

249


250

7β-Hydroxystigmasterol (6). Procedure D was followed by using 7-keto-stigmasterol (4) (100

251


252

mL). The mixture was stirred at rt for 6 h. The residue was purified by pre-TLC (C/M = 20:1) to

253

give the title compound (6) (50 mg, yield: 50%) as a white solid: 1H NMR (600 MHz,

254

CD3OD+CDCl3) δ 5.24 (1H, s, H-6), 5.16 (1H, dd, J = 15.1, 8.8 Hz, H-23), 5.03 (1H, dd, J =

255

15.1, 8.8 Hz, H-22), 3.71 (1H, d, J = 8.2 Hz, H-7), 3.42 (1H, m, H-3), 2.29-2.20 (2H, m), 2.06-

256

1.99 (2H, m), 1.87-1.69 (4H, m), 1.58-1.36 (7H, m), 1.31-1.11 (9H, m), 1.06 (3H, s, H-19), 1.03

257

(3H, d, J = 6.7 Hz, H-21), 0.85 (3H, d, J = 6.3 Hz, H-27), 0.81 (3H, t, J = 7.1 Hz, H-29), 0.80

258

(3H, d, J = 6.3 Hz, H-26), and 0.73 (3H, s, H-18); 13C NMR (150 MHz, CD3OD+CDCl3) δ 144.0

259

(s, C-5), 139.7 (d, C-22), 130.5 (d, C-23), 127.3 (d, C-6), 73.7 (d, C-7), 72.0 (d, C-3), 57.7 (d, C-

260

17), 56.7, 52.7, 50.0, 43.8, 42.4, 41.8, 41.1, 40.8, 38.2, 37.5, 33.1, 32.2, 30.3, 27.3, 26.5, 22.2,


Page 13 of 47


261

21.9 (q, C-27), 21.5 (q, C-26), 19.5 (q, C-21), 19.4 (q, C-19), 12.7 (q, C-29), and 12.6 (q, C-18);

262

positive ESIMS, m/z 429 [M + H]+.

263

7-Ketocholesterol (7). Procedure C was followed by using 7-keto-cholesterol acetate (260

264

mg, 0.61 mmol) and LiOH (59 mg, 2.44 mmol) in THF/MeOH/H2O (3:1:1, 5 mL). The resulting

265

residue was purified by pre-TLC (CHCl3/MeOH = 15:1) to give the title compound (7) (223 mg,

266

yield: 91%) as a white solid: 1H NMR (600 MHz, CD3OD+CDCl3) δ 5.63 (1H, s, H-6), 3.55 (1H,

267

m, H-3), 2.48-2.45 (1H, m), 2.41-2.35 (2H, m), 2.30-2.26 (1H, m), 2.06-2.04 (1H, m), 1.98-1.96

268

(1H, m), 1.90-1.87 (2H, m), 1.63-1.46 (5H, m), 1.39-0.99 (13H, m), 1.22 (3H, s, H-19), 0.94 (3H,

269

d, J = 6.4 Hz, H-21), 0.87 (3H, d, J = 6.6 Hz, H-27), 0.86 (3H, d, J = 6.6 Hz, H-26), and 0.71

270

(3H, s, H-18); 13C NMR (150 MHz, CD3OD+CDCl3) δ 204.5 (s, C-7), 168.8 (s, C-5), 126.3 (d,

271

C-6), 71.0 (d, C-3), 56.1 (d, C-17), 51.3 (d, C-14), 51.2 (d, C-9), 46.5, 44.2, 42.6, 40.6, 39.9, 39.5,

272

37.5, 37.3, 36.9, 31.7, 29.5, 29.0, 27.3, 24.9, 23.2 (q, C-27), 23.0 (q, C-26), 22.2, 19.4 (q, C-21),

273

17.1 (q, C-19), and 12.4 (q, C-18); positive ESIMS, m/z 401 [M + H]+.

274

7β-Hydroxycholesterol (8). Procedure D was followed by using 7-keto-cholesterol (7) (100

275


276

mL). The mixture was stirred at rt for 4 h. The residue was purified by pre-TLC (C/M = 15:1) to

277

give the title compound (8) (58 mg, yield: 58%) as a white solid: 1H NMR (600 MHz,

278

CD3OD+CDCl3) δ 5.24 (1H, s, H-6), 3.72 (1H, d, J = 8.2 Hz, H-7), 3.42 (1H, m, H-3), 2.27-2.18

279

(2H, m), 2.05-2.02 (1H, m), 1.89-1.78 (4H, m), 1.56-1.25 (10H, m), 1.18-0.99 (9H, m), 1.06 (3H,

280

s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.87 (3H, d, J = 6.6 Hz, H-27), 0.86 (3H, d, J = 6.6 Hz,

281

H-26), and 0.71 (3H, s, H-18); 13C NMR (150 MHz, CD3OD+CDCl3) δ 143.9 (s, C-5), 127.3 (d,

282

C-6), 73.6 (d, C-7), 72.0 (d, C-3), 57.6 (d, C-17), 56.9 (d, C-14), 49.9, 43.9, 42.4, 41.1, 40.9, 40.6,



Page 14 of 47

283

38.2, 37.4, 37.3, 37.0, 32.2, 29.6, 29.0, 27.2, 24.9, 23.2 (q, C-27), 23.0 (q, C-26), 22.2, 19.5 (q,

284

C-21), 19.4 (q, C-19), and 12.4 (q, C-18); positive ESIMS, m/z 403 [M + H]+.

285

Synthesis of Sterol Ferulates 13 and 13a.

286

3-O-(4-Acetoxyferuloyl)-7β-hydroxycholesterol (28). Trans-4-acetoxy ferulic acid was

287

prepared followed procedure A by using trans-ferulic acid (582 mg, 3.0 mmol), Ac2O (2.8 mL,

288

30.0 mmol) and Py (12 mL, 150 mmol). The mixture was incubated at 37 oC overnight. The

289

organic solvent was removed in vacuo to give trans-4-acetoxy ferulic acid (700 mg, yield: 100%)

290

as a white solid: 1H NMR (600 MHz, CDCl3) δ 7.73 (1H, d, J = 15.9 Hz, H-1), 7.14 (1H, d, J =

291

8.1 Hz, H-6'), 7.12 (1H, s), 7.06 (1H, d, J = 8.1 Hz, H-6'), 6.39 (1H, d, J = 15.9 Hz, H-2), 3.87

292

(3H, s, OCH3-3'), and 2.32 (3H, s, OAc-4'); positive ESIMS, m/z 237 [M + H]+.

293

Procedure E was followed by using trans-4-acetoxy ferulic acid (20 mg, 0.08 mmol), 7β-

294

hydroxycholesterol (31 mg, 0.08 mmol), DCC (50 mg, 0.24 mmol) and DMAP (24 mg, 0.2

295

mmol) in dry DCM (1 mL). The resulting residue was purified by pre-TLC (H/E = 4:1) to give

296

the title compound (21 mg, yield: 42%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.34 (1H,

297

s, H-6), 4.75 (1H, m, H-3), 3.86 (1H, d, J = 8.3 Hz, H-7), 2.48-2.32 (2H, m), 2.15-0.99 (24H, m),

298

1.06 (3H, s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.87 (3H, d, J = 6.5 Hz, H-27), 0.86 (3H, d, J

299

= 6.5 Hz, H-26), 0.70 (3H, s, H-18), and 4-acetyl ferulate [7.62 (1H, d, J = 15.9 Hz, H-7'), 7.10

300

(1H, d, J = 8.1 Hz, H-6'), 7.09 (1H, s, H-2'), 7.04 (1H, d, J = 8.1 Hz, H-5'), 6.35 (1H, d, J = 15.9

301

Hz, H-8'), 3.85 (3H, s, OMe-3'), and 2.31 (3H, s, OAc-4')]; positive ESIMS, m/z 621 [M + H]+.

302

3-O-(4-Acetoxyferuloyl)-7-ketocholesterol (29). Procedure E was followed by using trans-4-

303

acetoxy ferulic acid (18 mg, 0.075 mmol), 7-keto-cholesterol (20 mg, 0.05 mmol), DCC (31 mg,

304

0.15 mmol) and DMAP (16 mg, 0.13 mmol) in dry DCM (1 mL). The resulting residue was 14 ACS Paragon Plus Environment

Page 15 of 47


305

purified by pre-TLC (H/E = 4:1) to give the title compound (18 mg, yield: 58%) as a white solid:

306

1

307

2.51 (1H, m), 2.42-2.38 (1H, m), 2.26-2.21 (1H, m), 2.08-1.98 (2H, m), 1.93-1.87 (1H, m), 1.77-

308

1.74 (1H, m), 1.58-1.48 (5H, m), 1.37-0.99 (13H, m), 1.20 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz,

309

H-21), 0.85 (3H, d, J = 6.5 Hz, H-27), 0.84 (3H, d, J = 6.5 Hz, H-26), 0.68 (3H, s, H-18), and 4-

310

acetyl ferulate [7.63 (1H, d, J = 15.9 Hz, H-7'), 7.10 (1H, d, J = 8.2 Hz, H-6'), 7.09 (1H, s, H-2'),

311

7.04 (1H, d, J = 8.2 Hz, H-5'), 7.35 (1H, d, J = 15.9 Hz, H-8'), 3.84 (3H, s, OMe-3'), and 2.31

312

(3H, s, OAc-4')]; positive ESIMS, m/z 619 [M + H]+.

H NMR (600 MHz, CDCl3) δ 5.72 (1H, s, H-6), 4.85 (1H, m, H-3), 2.64-2.61 (1H, m), 2.56-

7-Ketocholesterol

313

ferulate

(13).

Procedure

F

was

followed

by

using

3-O-(4-

314

acetoxyferuloyl)-7-ketocholesterol (15 mg, 0.024 mmol) and K2CO3 (0.52 mg, 0.0049 mmol) in

315

DCM/MeOH (2:1, 3 mL). The residue was purified by pre-TLC (H/E = 3:1) to give the title

316

compound 13 (10 mg, yield: 71%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.72 (1H, s,

317

H-6), 4.84 (1H, m, H-3), 2.65-2.51 (2H, m), 2.44-2.37 (1H, m), 2.28-2.20 (1H, m), 2.10-1.85 (4H,

318

m), 1.80-1.48 (3H, m), 1.40-1.20 (8H, m), 1.18-0.99 (6H, m), 1.23 (3H, s, H-19), 0.91 (3H, d, J

319

= 6.5 Hz, H-21), 0.86 (3H, d, J = 6.5 Hz, H-27), 0.85 (3H, d, J = 6.5 Hz, H-26), 0.68 (3H, s, H-

320

18), and ferulate moiety [7.60 (1H, d, J = 15.8 Hz, H-7'), 7.06 (1H, d, J = 8.2 Hz, H-6'), 7.02 (1H,

321

s, H-2'), 6.90 (1H, d, J = 8.2 Hz, H-5'), 6.26 (1H, d, J = 15.8 Hz, H-8'), and 3.92 (3H, s, OMe-3')];

322

13

323

50.0, 49.8, 45.4, 43.1, 39.5, 38.7, 38.4, 37.9, 36.2, 36.1, 35.7, 28.5, 28.0, 27.5, 26.3, 23.8, 22.8 (q,

324

C-27), 22.6 (q, C-26), 21.2, 18.9 (q, C-21), 17.3 (q, C-19), 12.0 (q, C-18), and ferulate moiety

325

[166.4 (s, C=O, C-9'), 148.0 (s, C-4'), 146.8 (s, C-3'), 145.1 (d, C-7'), 126.9 (s, C-1'), 123.1 (d, C-

326

6'), 115.4 (d, C-8'), 114.7 (d, C-5'), 109.3 (d, C-2'), and 55.9 (q, OMe-3')]; positive ESIMS, m/z

327

577 [M + H]+.

C NMR (150 MHz, CDCl3) δ 202.0 (s, C-7), 164.0 (s, C-5), 126.7 (d, C-6), 72.1 (d, C-3), 54.8,



328

Page 16 of 47

7β-Hydroxycholesterol ferulate (13a). Procedure F was followed by using 3-O-(4-

329

acetoxyferuloyl)-7β-hydroxycholesterol (21 mg, 0.034 mmol) and K2CO3 (1.0 mg, 0.0068 mmol)

330

in DCM/MeOH (2:1, 3 mL). The residue was purified by pre-TLC (H/E = 3:1) to give the title

331

compound 13a (16 mg, yield: 80%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.34 (1H, s,

332

H-6), 4.76 (1H, m, H-3), 3.87 (1H, d, J = 7.8 Hz, H-7), 2.48-2.37 (2H, m), 2.05-1.79 (5H, m),

333

1.70-1.22 (10H, m), 1.18-0.99 (9H, m), 1.09 (3H, s, H-19), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.87

334

(3H, d, J = 6.5 Hz, H-27), 0.86 (3H, d, J = 6.5 Hz, H-26), 0.70 (3H, s, H-18), and ferulate moiety

335

[7.60 (1H, d, J = 15.9 Hz, H-7'), 7.06 (1H, dd, J = 8.2, 1.6 Hz, H-6'), 7.03 (1H, d, J = 1.6 Hz, H-

336

2'), 6.91 (1H, d, J = 8.2 Hz, H-5'), 6.27 (1H, d, J = 15.9 Hz, H-8'), and 3.92 (3H, s, OMe-3')]; 13C

337

NMR (150 MHz, CDCl3) δ 142.5 (s, C-5), 126.3 (d, C-6), 73.4 (d, C-7), 73.3 (d, C-3), 55.8 (d),

338

55.4 (d), 48.2, 42.9, 40.8, 39.6, 39.5, 37.8, 36.7, 36.6, 36.2, 35.7, 28.5, 28.0, 27.9, 26.4, 23.8,

339

22.8 (q, C-27), 22.6 (q, C-26), 21.0, 19.2 (q, C-19), 18.8 (q, C-19), 11.8 (q, C-18), and ferulate

340

moiety [166.6 (s, C=O, C-9'), 147.9 (s, C-4'), 146.7 (s, C-3'), 144.7 (d, C-7'), 127.0 (s, C-1'),

341

123.1 (d, C-6'), 115.9 (d, C-8'), 114.7 (d, C-5'), 109.3 (d, C-2'), and 55.9 (q, OMe-3')]; positive

342

ESIMS, m/z 579 [M + H]+.

343

Synthesis of Aza-oxysterols 7a and 8a.

344

Pregnenolone acetate (30). Procedure A was followed by using pregnenolone (1.58 g, 10.0

345

mmol) and Ac2O (4.7 mL, 50.0 mmol) in Py (20 mL), giving the title compound (3.58 g, yield:

346

100%) as a white solid: 1H NMR (600 MHz, CDCl3) δ 5.35 (1H, m, H-6), 4.32 (1H, m, H-3),

347

2.58-2.52 (2H, m), 2.39-2.31 (1H, m), 2.23-2.12 (1H, m), 2.12 (3H, s, H-21), 2.04 (3H, s, OAc-

348

3), 2.0-1.2 (16H, m), 1.37 (3H, s, H-19), and 0.64 (3H, s, H-18); positive ESIMS, m/z 359 [M +

349

H]+.


Page 17 of 47


350

20-Aza-cholesterol acetate (31). Pregnenolone acetate (358 mg, 1.0 mmol) was dissolved in

351

10 mL methanol, and isopentylamine (522 mg, 6.0 mmol) was added. pH value of the solution

352

was adjusted to around 6 by adding HOAc (1.0 mL). Then 10 mL of THF and NaBH3CN (82 mg,

353

1.1 mmol) in methanol (2 mL) were added, the reaction mixture was stirred under reflux

354

overnight. After evaporation in vacuo, the resulted residue was resuspended in 10 mL water, and

355

the pH was adjusted to 8 by 10% Na2CO3 solution. The solution was extracted with DCM (10

356

mL × 3), washed with water (10 mL) and brine (10 mL), dried over Na2SO4, and concentrated

357

under reduced pressure. The residue was purified by CC (H/E = 5:1; H/E/TEA = 5:1:0.1 and

358

3:1:0.1) to give the title compound (446 mg, yield: 85%) as a colorless gum: 1H NMR (600 MHz,

359

CDCl3) δ 5.37 (1H, m, H-6), 4.60 (1H, m, H-3), 2.90-2.75 (1H, m, H-20), 2.38-2.28 (2H, m, H-

360

23), 2.05 (3H, s, OAc-3), 2.02-1.92 (1H, m), 1.90-1.80 (2H, m), 1.75-1.41 (12H, m), 1.40-1.10

361

(8H, m), 1.01 (3H, s, H-19), 0.98-0.84 (9H, m, H-21, H-26 and H-27), and 0.73 (3H, s, H-18);

362

positive ESIMS, m/z 430 [M + H]+.

363

7-Keto-20-aza-cholesterol acetate (32). Procedure B was followed by using 20-aza-

364

cholesterol acetate (446 mg, 1.04 mmol), CrO3 (1.56 g, 15.6 mmol) and dimethylpyrazole (1.50

365

g, 15.6 mmol) in dry DCM (50 mL). The mixture was stirred at rt for 6 h. After addition of EA,

366

the mixture was filtered through a short pad of Celite eluted by a mixture of EA and TEA (30:1),

367

and the filtration was concentrated in vacuo. The resulted residue was purified by CC (H/E = 5:1;

368

H/E/TEA = 5:1:0.1 and 3:1:0.1) to give the title compound (314 mg, yield: 68%) as a yellow

369

gum: 1H NMR (600 MHz, CDCl3) δ 5.70 (1H, m, H-6), 4.71 (1H, m, H-3), 2.98-2.80 (1H, m, H-

370

20), 2.58-2.41 (2H, m, H-23), 2.30-2.10 (2H, m), 2.05 (3H, s, OAc-3), 2.02-1.20 (19H, m), 1.18

371

(3H, s, H-19), 0.98-0.84 (9H, m, H-21, H-26 and H-27), and 0.75 (3H, s, H-18); positive ESIMS,

372

m/z 444 [M + H]+.



Page 18 of 47

373

7-Keto-20-aza-cholesterol (7a). Procedure C was followed by using 7-keto-20-aza-

374

cholesterol acetate (314 mg, 0.71 mmol) and LiOH (68 mg, 2.84 mmol) in THF/MeOH/H2O

375

(3:1:1, 5 mL). The mixture was stirred at rt for 1 h. After removal of the organic solvent, the

376

residue was partitioned between water (5 mL) and DCM (5 mL). DCM extracts were washed

377

with water and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by

378

CC (H/E/TEA = 5:1:0.5, 3:1:0.5, 2:1:0.5 and 1.5:1:0.5) to give the title compound (7a) (240 mg,

379

yield: 85%) as a white solid: 1H NMR (700 MHz, CDCl3) δ 5.69 (1H, s, H-6), 3.67 (1H, m, H-3),

380

2.97-2.86 (1H, m, H-20), 2.53-2.49 (2H, m, H-23), 2.41-2.38 (1H, m), 2.25-2.21 (1H, m), 1.96-

381

1.87 (2H, m), 1.85-1.77 (2H, m), 1.70-1.66 (2H, m), 1.64-1.58 (2H, m), 1.57-1.50 (1H, m), 1.45-

382

1.21 (10H, m), 1.18 (3H, s, H-19), 0.94 (3H, d, J = 6.5 Hz, H-21), 0.93 (6H, d, J = 6.5 Hz, H-

383

26/27), and 0.78 (3H, s, H-18); positive ESIMS, m/z 402 [M + H]+.

384

7β-Hydroxy-20-aza-cholesterol (8a). Procedure D was followed by using 7-keto-20-aza-

385

cholesterol (7a) (284 mg, 0.71 mmol), CeCl3·7H2O (1.59 g, 4.26 mmol) and NaBH4 (108 mg,

386

2.84 mmol) in MeOH (10 mL) at -10 oC. The mixture was stirred at -10 oC for 5 min and

387

quenched with water (1 mL). After removal of most of the organic solvent, the residue was

388

partitioned between water (10 mL) and DCM (10 mL). DCM extracts were washed, dried and

389

concentrated. The resulted residue was purified by CC (H/E/TEA = 5:1:0.1, 2:1:0.1, 2:1:0.5, and

390

1:1:0.5) to give the title compound (8a) (100 mg, yield: 35%) as a white solid: 1H NMR (700

391

MHz, CDCl3) δ 5.27 (1H, s, H-6), 3.83 (1H, d, J = 8.1 Hz, H-7), 3.54 (1H, m, H-3), 2.99 (1H, m,

392

H-20), 2.88 (1H, m, H-23a), 2.68 (1H, m, H-23b), 2.35-2.31 (1H, m), 2.28-2.22 (1H, m), 1.98-

393

1.83 (5H, m), 1.66-1.57 (4H, m), 1.53-1.46 (4H, m), 1.42-1.30 (4H, m), 1.25-1.16 (4H, m), 1.16

394

(3H, s, H-19), 0.94 (3H, d, J = 6.7 Hz, H-21), 0.93 (3H, d, J = 6.5 Hz, H-27), 0.92 (3H, d, J = 6.5

395

Hz, H-26), and 0.73 (3H, s, H-18); 13C NMR (175 MHz, CDCl3) δ 143.5 (s, C-5), 125.6 (d, C-6),


Page 19 of 47


396

73.3 (d, C-7), 71.3 (d, C-3), 63.2 (d, C-20), 55.2 (d, C-17), 48.1, 45.9, 43.5, 42.9, 41.7, 40.6, 37.7,

397

37.5, 37.0, 36.6, 31.5, 28.2, 26.7, 26.3, 22.6, 22.4 (q, C-26/27), 20.8 (q, C-21), 19.1 (q, C-19),

398


399

LC/MS Analysis. LC/MS analysis was carried out with a Thermo-Finnigan Spectra System

400

consisting of an Accela high speed MS pump, an Accela refrigerated autosampler, and an LCQ

401

Fleet ion trap mass detector (Thermo Electron, San Jose, CA, USA) incorporated with an

402

electrospray ionization (ESI) interface. A Gemini-NX C18 column (150 mm × 3.0 mm i.d., 5 µm,

403

Phenomenex) was used to analyze authentic sterol oxides and the sterol-enriched fraction with a

404

flow rate of 0.3 mL/min. The binary mobile phase system consisted of water with 0.2% acetic

405

acid (AA) as phase A and methanol with 0.2% AA as phase B. The column was eluted by a

406

gradient progress (80 to 90% B from 0 to 10 min; 90 to 95% B from 10 to 20 min; 95-99% B

407

from 20 to 30 min; 99-100% B from 30 to 45 min; 100% B from 45 to 65 min, and then 80% B

408

from 65 to 70 min). The injection volume was 10 µL for each sample. The column temperature

409

was maintained at 20 °C. To optimize the mass spectrometric parameters, each standard

410

dissolved in methanol (10 µM) was infused in the ESI source by a syringe pump (flow rate 10

411

µL/min) and analyzed in positive ion mode. The optimized parameters for analysis of

412

oxyphytosterols are: capillary voltage 16 V, spray voltage 4.6 kV, tube lens offset 70 V, capillary

413

temperature 260 oC, and sheath gas (nitrogen) flow rate 25 (arbitrary units). The structural

414

information of compounds 1-21 were obtained by tandem mass spectrometry (MS/MS) through

415

collision-induced dissociation (CID) with a relative collision energy setting of 35%. Data

416

acquisition was performed with Xcalibur version 2.0 (Thermo Electron, San Jose, CA, USA).

417 418

Nuclear Magnetic Resonance (NMR). 1H,

13

C NMR, and two-dimensional (2-D) NMR

spectra were recorded on a Bruker AVANCE 600 MHz or 700 MHz spectrometer (Bruker Inc., 19 ACS Paragon Plus Environment


Page 20 of 47

419

Silberstreifen, Rheinstetten, Germany). Compounds were analyzed in CDCl3, CD3OD or

420

Pyridine-d5. Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), and br

421

(broad). The 13C NMR spectra are proton decoupled.

422

MTT Assay. Cell growth inhibition was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-

423

diphenyltetrazolium bromide (MTT) colorimetric assay.21 Human colon cancer cells HCT-116

424

and HT-29 were plated in 96-well microtiter plates with 5000 cells/well and allowed to attach for

425

24 h at 37 ºC. The test compounds in dimethyl sulfoxide (DMSO) were added to cell culture

426

medium to the desired final concentrations (final DMSO concentrations for control and

427

treatments were 0.1 %). After the cells were cultured for 24 h, the medium was aspirated and

428

cells were treated with 200 µL fresh medium containing 2.41 mmol/L MTT. After incubation for

429

3 h at 37 ºC, the medium containing MTT was aspirated, 100 µL of DMSO was added to

430

solubilize the formazan precipitate, and plates were shaken gently for an hour at room

431

temperature. Absorbance values were derived from the plate reading at 550 nm on a Biotek

432

microtiter plate reader (Winooski, VT). The reading reflected the number of viable cells and was

433

expressed as a percentage of viable cells in the control. Both HCT-116 and HT-29 cells were

434

cultured in McCoy’s 5A medium. All of the above media were supplemented with 10 % fetal

435

bovine serum, 1 % penicillin/streptomycin, and 1 % glutamine, and the cells were kept in a 37 ºC

436

incubator with 95% humidity and 5% CO2.

437

Apoptotic Assay. The Cell Death Detection ELISA (Enzyme-linked immunoabsorbant assay)

438

Plus kit from Roche (Mannheim, Germany) was used. HCT-116 and HT-29 cells were seeded in

439

96-well plates at 3000 cells/well and incubated at 37 oC in 5% CO2 incubator. After 24 hours,

440

fresh media supplemented with DMSO (control), 5, 6, 7, or 8 (5, 10, 20 or 30 µM; final DMSO

441

concentration for control and treatments was 0.1%) were added to the wells. After 48 h, the 20 ACS Paragon Plus Environment

Page 21 of 47


442

microplate was centrifuged for 10 min at 1200 rpm, and the supernatant was removed. Then, 200

443

µL of the lysis buffer was added in each well and the microplate was incubated for 30 min at

444

room temperature. The plate was then centrifuged for 10 min at 1200 rpm and 20 µL of the

445

supernatant was transferred to streptavidin-coated microwells. ELISA assay was performed

446

according to manufacturer’s instruction. Absorbance in each well was measured at 405 nm in

447

absorbance units (AU), and the enrichment factor (EF) in small nucleosomes was calculated with

448

the formula EF = AU(treated)/AU(DMSO). The experiment was repeated independently to confirm the

449

results.

450 451

Statistical Analysis. For simple comparisons between two groups, two-tailed Student’s t-test was used. A p-value of less than 0.05 was considered statistically significant in all the tests.

452 453

RESULTS

454

Structural Elucidation of Oxyphytosterols 1-4. The chemical investigation on the sterol-

455

enriched fraction yielded two unusual 3-ketophytosterols 1 and 2, and two 7-ketophytosterols 3

456

and 4 (Figure 1). Compound 1 had a molecular formula of C29H48O2 based on positive ESI-MS

457

at m/z 411 [M – H2O + H]+ and NMR data. One tertiary proton adjacent to oxygen atom, one

458

olefinic proton and six methyl groups in the 1H NMR spectrum of 1, and a ketone group, four

459

double bond signals and an oxygenated tertiary carbon in the 13C NMR spectrum of 1, suggested

460

1 is oxyphytosterol.22 Heteronuclear multiple bond correlation (HMBC) established a partial

461

structure of 4-en-6-ol-3-one in 1 (Figure 2). The fragment ion at m/z 253/393/429 [M – 2H2O –

462

C10H20 + H]+ in the MS3 spectra of 1 (Table 1), corresponding to the loss of two water molecules

463

followed by the cleavage of the side chain, further confirmed 1 as being stigmast-4-en-6β-ol-3-

464

one, as previously described in extracts of wheat straw.23



465

Page 22 of 47

Compound 2 possessed a molecular formula of C29H46O2 based on positive ESI-MS at m/z

466

409 [M – H2O + H]+ and NMR data. Its 1H and

13

467

except for additional olefinic signals [δH 5.09 (1H, dd, J = 15.2, 8.8 Hz) and δH 5.24 (1H, dd, J =

468

15.2, 8.8 Hz); and δC 130.0 (d) and δC 139.2 (s)] in 2, indicating 2 is stigmast-4,22-dien-6β-ol-3-

469

one, as previously described in extracts of wheat straw.23

C NMR data were very similar to those of 1

470

Compound 3 had the same molecular formula of C29H48O2 to that of 1 based on positive ESI-

471

MS at m/z 411 [M – H2O + H]+ and NMR data. The 1H and 13C NMR spectra of 3 were similar

472

to those of 1, suggesting 3 is also an oxyphytosterol. HMBC correlations established a partial

473

structure of 5-en-3-ol-7-one in 3 (Figure 2). The fragment ion at m/z 253/393/429 [M – 2H2O –

474

C10H20 + H]+ in the MS3 spectra of 3, corresponding to the loss of two water molecules followed

475

by the side chain, further confirmed 3 as being stigmast-5-en-3β-ol-7-one, namely 7-

476

ketositosterol, as previously reported from marine sponges.24 This is the first report of 7-

477

ketositosterol 3 in WB.

478

Compound 4 had a molecular formula of C29H46O2 based on positive ESI-MS at m/z 409 [M

479

– H2O + H]+ and NMR data. Its 1H and 13C NMR data were very similar to those of 3 except for

480

additional olefinic signals [δH 5.01 (1H, dd, J = 15.1, 8.7 Hz) and δH 5.15 (1H, dd, J = 15.1, 8.7

481

Hz); and δC 130.3 (d) and δC 139.0 (d)] in 4, indicating 4 is an oxidation product of stigmasterol.

482

Fragment ion at m/z 251/391/427 [M – 2H2O – C10H20 + H]+ in the MS3 spectra of 4 (Table 1),

483

following the same fragmentation patterns as 3, established 4 as being stigmast-5,22-dien-3β-ol-

484

7-one, namely 7-ketostigmasterol, as previously described elsewhere.25 This is the first report of

485

7-ketostigmasterol 4 in WB as well.


Page 23 of 47


486

Synthesis of Sterol Oxides 3-8, Sterol Ferulates 13 and 13a, and Aza-oxysterols 7a and

487

8a. Sterol oxides 3-8 were synthesized in house in quantities, following the method of McCarthy

488

with some modifications (Figure 3A).26 Briefly, acetylation of sterols yielded sterol acetates

489

22− −24. The acetates were treated by the combination of chromium trioxide and dimethylpyrazole

490

to give 7-ketosterol acetates 25− −27. After hydrolysis using lithium hydroxide, 7-ketosterols 3, 4

491

and 7 were obtained. Luche reduction of 7-ketosterols afforded 7β-hydroxysterols 5, 6 and 8.

492

Coupling reaction of 7-ketocholesterol 7 with 4-acetoxy ferulic acid utilizing N,N′-

493

dicyclohexylcarbodiimide

494

deacetylation yielded 7-ketocholesterol ferulate 13. Likewise, 7β-hydroxycholesterol ferulate

495

13a was obtained from 7β-hydroxycholesterol 8. The synthesis of aza-oxysterols 7a and 8a was

496

illustrated in Figure 3B. Reductive amination of pregnenolone acetate 30 with isopentylamine

497

catalyzed by NaBH3(CN) in MeOH/THF at pH 6 produced 20-aza-cholesterol acetate 31.27

498

Oxidation of 31 by chromium trioxide followed by treatment with lithium hydroxide afforded 7-

499

keto-20-aza-cholesterol 7a with a yield of 60%. Luche reduction of 7a gave 7β-hydroxyl-20-aza-

500

cholesterol 8a with a yield of 35%. This is the first report of synthesis for 7-keto-20-aza-

501

cholesterol 7a and 7β-hydroxy-20-aza-cholesterol 8a.

(DCC)

and

4-dimethylaminopyridine

(DMAP)

followed

by

502

Characterization of Synthetic Sterol Oxides 5-8, Sterol Ferulates 13 and 13a, and Aza-

503

oxysterols 7a and 8a. Compound 5 was reduced from 7-ketositosterol 3. Its molecular ion at m/z

504

431 [M + H]+ is two mass units higher than that of 3, indicating 5 is the reduction product of 3.

505

The appearance of one additional signal adjacent to oxygen atom [δH 3.72 (1H, d, J = 8.0 Hz)

506

and δC 73.7 (d)] in 5 confirmed the conversion of the carbonyl group in 3 into the hydroxyl group

507

in 5. The fragment ion at m/z 241/395/431 [M – 2H2O – C10H20 – CH2 + H]+ in the MS3 spectra

508

of 5 (Table 1), corresponding to the loss of two water molecules followed by the side chain and 23 ACS Paragon Plus Environment


Page 24 of 47

509

one methyl group, further supported that 5 was stigmast-5-en-3β,7β-diol, namely 7β-

510

hydroxysitosterol, as previously reported in wheat flour.18 Likewise, compound 6 was the

511

product of the reduction of 4 and subsequently established as being stigmast-5,22-dien-3β,7β-

512

diol, namely 7β-hydroxystigmasterol, as previously reported elsewhere.25

513

Compound 7, owning a molecular formula of C27H44O2 based on positive ESI-MS at m/z 383

514

[M – H2O + H]+ and NMR data, was synthesized from cholesterol. Its 1H and

13

515

were very similar to those of 3, suggesting 7 is an oxidation product of cholesterol. The fragment

516

ion at m/z 253/365/401 [M – 2H2O – C8H16 + H]+ in the MS3 spectra of 7 (Table 1), in

517

agreement with the loss of two water molecules followed by the side chain, provided 7 as

518

cholest-5-en-3β-ol-7-one, namely 7-ketocholesterol, as previously described in marine sponges.24

519

Likewise, compound 8, as the reduction product of 7, was elucidated as being cholest-5-en-

520

3β,7β-diol, namely 7β-hydroxycholesterol, as previously reported in marine sponges.24

C NMR data

521

Compound 13, possessing a molecular formula of C37H52O5 based on its positive ESI-MS at

522

m/z 383 [M – C10H10O4 + H]+ and NMR data, was a ferulic acid conjugate of 7. HMBC

523

correlation between H-3 (δH 4.84) and the carbonyl group (C-9') of ferulate (δC 166.4)

524

established the linkage at C-3 between ferulate moiety and 7-ketocholesterol (Figure 2). The

525

fragment ion at m/z 383/577 [M – C10H10O4 + H]+ in the MS2 spectra of 13, corresponding to the

526

loss of a ferulic acid moiety, further confirmed 13 as being 7-ketocholesterol ferulate. Compound

527

13a was synthesized by the coupling reaction of ferulic acid with 8. Its NMR spectra were

528

similar to those of 13 except for an additional oxygenated signal [δH 3.87 (1H, d, J = 7.8 Hz) and

529

δC 73.4 (d)], coinciding 13a being 7β-hydroxycholesterol ferulate.


Page 25 of 47


530

Compound 7a, having a molecular formula of C26H43NO2 based on its positive ESI-MS at

531

m/z 402 [M + H]+ and NMR data, was synthesized from pregnenolone through four steps. The 1H

532

NMR spectra of 7a was similar to that of 7-ketocholesterol 7. The major difference is the

533

appearance of two multiplets at δH 2.97-2.86 (1H, m, H-20) and δH 2.53-2.49 (2H, m, H-23) in

534

7a compared to those of 7, indicating 7a is 7-keto-20-aza-cholesterol. Compound 8a was the

535

reductive product of 7a. The 1H and

536

signal [δH 3.83 (1H, d, J = 8.1 Hz) and δC 73.3 (d)] compared to 7a, suggesting 8a is 7β-

537

hydroxy-20-aza-cholesterol.

13

C NMR spectra of 8a showed an additional oxygenated

538

Fragmentation Patterns of Authentic Sterol Oxides. There are two major fragmentation

539

patterns of the authentic 7-ketosterols 3, 4 and 7 (Table 1) under ESI positive ionization mode,

540

as exemplified by compound 3 in Figure 4, pattern A): loss of two water molecules from parent

541

ion forms a stable resonance structure with the positive charge at C-7 position in MS2 spectra

542

(Figure 4A); and pattern B): cleavage of the side chain at the C17-C20 bond in the fragments

543

obtained from pattern A via hydrogen shifts and inductive cleavages produces a typical daughter

544

ion in MS3 spectra (Figure 4B). These fragmentation patterns are consistent with the conclusions

545

that Jiang et al. reported previously.28 The observation from 7β-hydroxysterols 5, 6 and 8 led to a

546

characteristic fragmentation pattern C): the cleavage of the side chain followed by an additional

547

methyl group in the fragments obtained from pattern A gives characteristic tertiary daughter ions

548

in MS3 spectra (Table 1).

549

Identification of Steroids 1-21 in WB by LC-ESI/MSn Spectra. Oxyphytosterols 1-4 were

550

isolated from WB extract and identified by NMR experiments. Sterol oxides 5-8 and 13 in WB

551

were recognized by comparison of their retention times and tandem mass spectra with those of

552

the synthetic standards (Table 1). Compounds 9-12 and 14-20 were tentatively elucidated in WB 25 ACS Paragon Plus Environment


Page 26 of 47

553

based on the analysis of their tandem mass spectra, referring to fragmentation patterns observed

554

from known standards (Table 1 and Figure 4). Stigmasterol 21 was confirmed in WB by

555

comparison with its commercial authentic standard.

556

As per the knowledge obtained above, compound 9 had two peak ions with loss of H2O at

557

m/z 397 [M – H2O + H]+ and 379 [M – 2H2O + H]+ in its MS2 spectra (pattern A), and one

558

daughter ion with loss of the alkyl side chain at m/z 253 [M – 2H2O – C9H18 + H]+ in its MS3

559

spectra (pattern B) (Table 1), indicating 9 being campest-5-en-3β-ol-7-one, namely 7-

560

ketocampesterol.29 Compound 10, with two peak ions at m/z 399 [M – H2O + H]+ and 381 [M –

561

2H2O + H]+ in its MS2 spectra (pattern A), and one major daughter ion at m/z 241 [M – 2H2O –

562

C9H18 – CH2 + H]+ in its MS3 spectra (pattern C) (Table 1), was identified as campest-5-en-

563

3β,7β-diol, namely 7β-hydroxycampesterol.29 Similarly, compound 11, having two peak ions at

564

m/z 395 [M – H2O + H]+ and 377 [M – 2H2O + H]+ in its MS2 spectra (pattern A), and one

565

tertiary ion at m/z 251 [M – 2H2O – C9H18 + H]+ in its MS3 spectra (pattern B), was proposed as

566

ergost-4-en-3,6-dione, as reported previously in wheat straw.30 Compound 12 owned two peak

567

ions at m/z 401 [M – H2O + H]+ and 383 [M – 2H2O + H]+ in its MS2 spectra (pattern A), and

568

one daughter ion at m/z 243 [M – 2H2O – C9H18 – CH2 + H]+ in its MS3 spectra (pattern C), was

569

tentatively suggested as ergostane-3β,7β-diol, as described previously elsewhere.31

570

Compound 14, with a base peak at m/z 397 [M – C10H10O4 + H]+ in its MS2 spectra and a

571

major ion at m/z 379 [M – C10H10O4 – H2O + H]+ in its MS3 spectra, had similar fragmentation

572

patterns (loss of ferulic acid moiety) as that of analogue 13 (Table 1), indicating 14 is a sterol

573

ferulate. The molecular weight of 14 at m/z 591 [M + H]+ was 14 mass units higher than that of

574

13, suggesting that 14 is 7-ketocampesterol ferulate, which has never been reported before. In a

575

similar manner, compounds 15-17, with major peak ions at m/z 383 [M – C10H10O4 + H]+, 395 26 ACS Paragon Plus Environment

Page 27 of 47


576

[M – C10H10O4 + H]+ and 397 [M – C10H10O4 + H]+ in their respective MS2 spectra,

577

corresponding to loss of ferulic acid moiety, were identified as campesterol ferulate 15,

578

stigmasterol ferulate 16 and sitosterol ferulate 17, respectively. Of which, campesterol ferulate

579

15 and sitosterol ferulate 17 have been reported in WB previously,19 whereas stigmasterol

580

ferulate 16 was here described in WB for the first time. Peak ions at m/z 415 [M – 162 + H]+ and

581

397 [M – 180 + H]+ in MS2 spectra of 18 corresponded to the loss of a hexanose. Moreover, peak

582

ion at m/z 379 [M – 180 – H2O + H]+ in its MS3 spectra, similar to the fragmentation of 7-

583

ketocampesterol 9, tentatively suggested that 18 is 3-O-glucopyranosyl-7-ketocampesterol,

584

which has never been reported before. Compound 19, bearing two peak ions at m/z 427 [M – 162

585

+ H]+ and 409 [M – 180 + H]+ in its MS2 spectra and one peak ion at m/z 391 [M – 180 – H2O +

586

H]+ in its MS3 spectra, was therefore proposed as 3-O-glucopyranosyl-7-ketostigmasterol, as

587

reported previously in Rosa laevigata.32 Similarly, parent ion at m/z 591 [M + H]+ of 20 was 14

588

mass units higher than that of 18, indicating that 20 is 3-O-glucopyranosyl-7-ketositosterol, as

589

described previously in Rosa laevigata.32

590

Consequently, a total of twenty-one steroids 1-21 were identified and characterized from WB

591

extract, using NMR experiments, chemical synthesis approaches, and LC-ESI/MSn techniques,

592

eventually leading to a comprehensive profile of steroids in WB, as shown in Figure 5. Among

593

them, 7-ketositosterol 3 and 7-ketostigmasterol 4 were found to be the major oxyphytosterols in

594

WB.

595

In Vitro Cytotoxic Activity. Two human colon cancer cell lines HCT-116 and HT-29 were

596

treated with eight sterol oxides 1-8 and the non-oxidized stigmasterol 21, with concentrations

597

ranging from 0 to 120 µM. Seven sterol oxides 2-8 were found to be active against the growth of

598

both HCT-116 and HT-29 human colon cancer cells (Figures 6A and 6B). Non-oxidized 27 ACS Paragon Plus Environment


Page 28 of 47

599

stigmasterol was inactive in both cells (IC50 >120 µM, Figures 6A and 6B). Unusual 3-

600

ketophytosterols 1 and 2 were less active than 7-ketophytosterols 3, 4 and 7 in both cancer cell

601

lines, indicating an entity of 3-ol-5-en-7-one in the structure is more favorable for anti-cancer

602

activity than a 6-ol-4-en-3-one. In HCT-116 cell lines, 7β-hydroxycholesterol 8 was found to be

603

the most active compound with an IC50 value of 13.20 µM, followed by 7β-hydroxystigmasterol

604

6 (IC50: 15.73 µM), 7β-Hydroxylsitosterol 5 (IC50: 16.64 µM), 7-ketocholesterol 7 (IC50: 24.78

605

µM), 7-ketositosterol 3 (IC50: 32.66 µM), and 7-ketostigmasterol 4 (IC50: 43.47 µM) (Figure 6A).

606

The reduction products 5, 6 and 8 showed 2-fold higher inhibitory effects than their respective 7-

607

keto precursors 3, 4 and 7 (Figure 6A), suggesting an entity of 5-en-3,7-diol in the structure

608

greatly increases the activity compared to an partial structure of 3-ol-5-en-7-one. 7β-

609

Hydroxycholesterol 8, with eight carbons in the alkyl side chain, showed a better inhibitory

610

effect than 7β-hydroxysterols 5 and 6, both bearing ten carbons in alkyl side chains, indicating

611

that increasing the numbers of carbon in the alkyl side chain decreases the activity. In HT-29 cell

612

lines, 7β-hydroxycholesterol 8 also showed the most potency with IC50 value of 10.09 µM

613

(Figure 6B). Whereas, the rest of the sterol oxides 2-7 showed moderate inhibitory effects, with

614

IC50 values ranged from 29.57-60.46 µM (Figure 6B). Notably, the two major oxyphytosterols 3

615

and 4 found in WB exerted great antiproliferative property in both cancer cell lines, indicating

616

oxyphytosterols may contribute to the observed chemopreventive effects of WB.

617

We also measured the growth inhibitory effects of the ferulate conjugates of sterols, 7-

618

ketocholesterol ferulate 13 and 7β-hydroxycholesterol ferulate 13a. Our results indicated that

619

they were inactive in both cell lines (IC50s > 60 µM), indicating a free hydroxyl group at C-3β in

620

both entities of 3-ol-5-en-7-one and 5-en-3,7-diol plays a crucial role in the observed activity.


Page 29 of 47


621

In order to increase the polarity of oxyphytosterols, we synthesized two aza-oxysterols 7a

622

and 8a by introducing an –NH group into C-20 in the side chain of oxysterols 7 and 8,

623

respectively. In HT-29 cells, MTT assays showed that 7a exerted a similar activity (IC50: 35.33

624

µM) to that of 7 (IC50: 36.23 µM), and the activity of 8a was attenuated while still remaining

625

active (IC50: 54.26 µM), compared to 8 (IC50: 10.09 µM). In HCT-116 cells, the activity of both

626

aza-oxysterols 7a and 8a were decreased (IC50 > 60 µM for both) compared to oxysterols 7 and 8.

627

Induction of Apoptosis in Colon Cancer Cells. Apoptosis is a mechanism often responsible

628

for the induction of cell death in response to internal or external stress. In order to clarify

629

whether oxyphytosterols exert antiproliferative properties via a pathway of apoptosis, we tested

630

the four most active sterol oxides 5-8 for apoptotic effects in human colon cancer cells HCT-116

631

and HT-29. The results are summarized in Figures 6C and 6D. In HCT-116 cells, compounds 5-

632

7 showed significant apoptotic effects at the concentration of 30 µM (Figure 6C), while

633

compound 8 exerted significant apoptotic effect at an even low concentration (20 µM) compared

634

to DMSO control. In particular, the induction of apoptosis by 8 was significantly superior to

635

those of the rest 5-7 at the same concentration (20 µM). In HT-29 cells, compound 8 was also

636

found to be the most potent agent to induce apoptosis (Figure 6D). Compound 6 displayed a

637

significant apoptotic effect at the concentration of 30 µM (Figure 6D). Compounds 5 and 7

638

showed more or less apoptotic effects at the concentration of 30 µM.

639

640

DISCUSSION

641

In the present study, we outlined a comprehensive chemical profile of steroids in WB.

642

Notably, we observed that seventeen (1-14 and 18-20) out of twenty-one steroids (1-21) in WB 29 ACS Paragon Plus Environment


Page 30 of 47

643

are sterol oxides. This suggests that WB or whole grain wheat may be a dietary source of

644

oxyphytosterols.33 Daily consumption of whole grain wheat or diet supplemented with WB

645

allows oxyphytosterols to contribute to health effects of the whole grain. Interestingly, three

646

oxidation products of cholesterol, 7, 8 and 13, were also found in WB as minor components. This

647

observation could be demonstrated by the fact that many plants contain cholesterol both in free

648

and esterified forms.34, 35 In addition, it is worth emphasizing that most phytosterols are present

649

in WB in oxidized form or ferulate conjugates in this study, and stigmasterol is the only free

650

plant sterol detected in WB so far.

651

Phytosterols are liable to oxidation giving rise to a family of oxyphytosterols. Thus,

652

oxyphytosterols are most often present in the proximity of their parent compounds phytosterols.

653

The two main pathways elucidated for oxidation of sterols are enzymatic oxidations and non-

654

enzymatic oxidations. Exposure to reactive oxygen and free radical species, or radiation, ring

655

oxidation products are formed.36,

656

enzymatic reactions, the principal enzymes including cytochrome P450 monooxygenases,

657

dehydrogenases, epoxidases and hydroxylases.36, 37 Physiological effects of cholesterol oxidation

658

products have been well documented.38 However, the biological data on oxyphytosterols is

659

relatively scarce. Most reports available so far suggest that oxyphytosterols possess cytotoxic

660

effects in vitro and in vivo, cholesterol- and lipid-lowering effects, anti-diabetic properties and

661

anti-inflammatory activities.39 The main barrier to progress in this area was the lack of

662

availability of individual oxyphytosterols. In the present study, nine sterol oxides 1-8 and 13

663

obtained in quantities from direct purification or chemical synthesis enabled us to evaluate for

664

their antiproliferative properties. MTT assays showed that seven sterol oxides 2-8 exerted

665

significant inhibitory effects against human colon cancer cells HCT-116 and HT-29, compared to

37

Side chain oxidation is believed to be due mainly to


Page 31 of 47


666

the parent stigmasterol 21. Our results demonstrated that sterol oxides in WB may greatly

667

participate in the observed chemopreventative properties of the grain as an active ingredient.

668

It is known that phytosterols are hardly absorbed ( 120 2 41.91 3 32.66 4 43.47 5 16.64

60 40 20 0

DMSO

1

40 µM

60 µM

4

5

6

60 40

120 µM

7

8

21

IC50's (µM)

80

5

37.39

6 7 8 21

33.61 36.23 10.09 > 120

IC50's (µM) 1 > 120 2 60.46 3 57.76 4 29.57

DMSO

10 µM

20 µm

40 µM

60 µM

120 µM

Compounds (24 h Incubation)

Compounds (24 h Incubation)

C)

3

100

0 20 µM

2

120

20

10 µM

HT-29

140

% Viability

A)

Page 46 of 47

D)

Figure 6.

838


Page 47 of 47


GRAPHIC TOC


Oxyphytosterols as Active Ingredients in Wheat Bran Suppress Human

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