Saponins Esculeosides B-1 and B-2 in Italian Canned Tomatoes

Abstract

Italian canned tomatoes contain the tomato glycosides esculeosides B-1 (1, 0.0052%) and B-2 (2, 0.0068%) without esculeoside A. Herein, the structure of esculeoside B-1 (1) is characterized to be 3-O-β-lycotetraosyl (5S,22R,23S,25S)-22,26-epimino-16β,23-epoxy-3β,23,27-trihydroxycholestane 27-O-β-D-glucopyranoside. We hypothesized that these substances might be derived from esculeoside A when the cans are prepared with treatment in boiling water. To confirm that hypothesis, we refluxed esculeoside A with water for 6.5 h, providing esculeosides B-1 (1) and B-2 (2) in yields of 25.8% and 31.0%, respectively.

In 2003, Nohara and colleagues^1,2) isolated a tomato saponin, called esculeoside A, from the ripe fruits of tomato, Solanum lycopersicum L. and determined its structure (Fig. 1). Tomato saponin is a significant component of ripened tomatoes and occurs at levels approximately 4-fold higher than those of lycopene. Thus far, the bioactivity of tomato has been attributed solely to lycopene. Therefore, carrying out pharmacological examinations of esculeoside A in the near future is important.

Fig. 1. Structure of Esculeoside A

Recently, Fujiwara et al.³⁾ have revealed that oral administration of esculeoside A to apolipoprotein E-deficient mice significantly reduces levels of serum cholesterol glycerides, low-density lipoprotein-cholesterol and the areas of atherosclerotic lesions without any detectable side effects. Our recent studies on the constituents of Solanum plants have revealed that pregnane glycosides are accompanied by normal spirostanol and furostanol glycosides^4–12); esculeogenin A is easily converted into a pregnane derivative by refluxing with aqueous pyridine,¹³⁾ and pregnane glycoside has been obtained from the over-ripe tomato fruit.¹⁴⁾ These facts strongly suggest that orally administered steroidal glycosides can be metabolized into pregnane derivatives, which are a type of steroidal hormone.

Therefore, in further experiments, men consumed tomatoes and their urine was collected and separated with various column chromatographies to afford 3 androstane derivatives.¹⁵⁾ These androsterone analogs are normally excreted; however, because no excretions were detected in the control samples, the occurrence of androsterone analogs indicated excretion via the production of progesterone by subjects that had consumed tomatoes. The tomato steroidal glycoside might stimulate the hormone secretor or perhaps is itself metabolized into the pregnane. We hypothesize that orally administered steroidal glycosides such as spirostanol, furostanol and spirosolane glycosides are metabolized, introducing a hydroxyl group at C-23. These intermediates may then be metabolized into pregnane derivatives.

Thus, we conclude that the tomato saponin esculeoside A is metabolized into various steroidal hormones such as pregnane, which has anti-osteoporosis, anti-menopausal disorder, and anti-tumor activity in the body.^16,17) In previous work, we performed qualitative analyses of fresh tomato, tomato boiled in water, tomato heated in a microwave oven, freeze-dried tomato, and processed tomatoes from plastic bottles and cans before their development for health foods.¹⁸⁾ In all cases, esculeoside A was a major component. In this work, we investigated the tomato saponins in Italian canned tomatoes.

Commercially available Italian cans, containing 400 g of ingredients, were chosen for the experiments in order to compare the respective ingredients in each cans. Trademarks of Matured Whole Tomatoes (Del Monte, i), Whole Tomatoes (Aeon Co., Ltd., ii), Matured Whole Tomatoes (Kagome Co., Ltd., iii), and Whole Peeled Italian Tomatoes (Spigadoro, iv) were used. Each was individually blended with water and passed through filter paper to produce a filtrate, which was then passed through a highly porous polystyrene gel (Diaion HP-20) and rinsed with water. Methanol was then passed through the gel to produce an eluate, which was evaporated to produce a residue. This methanolic residue was subjected to reversed phase silica gel column chromatography, octadecylsilane (ODS), and then eluted with 40%, 60%, and 100% methanol. The eluate from the 40% methanol contained aromatics, that from the 60% methanol was composed of tomato saponins, and the final methanol eluate included less polar substances. The respective yields are shown in Experimental.

The 60% methanol eluates from the ODS column apparently lacked esculeoside A but contained esculeoside B on thin-layer chromatography with CHCl₃–MeOH–water (7 : 3 : 0.5, 6 : 4 : 1). Therefore, they were combined to give a residue (390.6 mg, 0.024%), which was then passed through the ODS column again with 55% methanol to provide 2 tomato saponins: 1, 85.5 mg (0.0052%) and 2, 112.0 mg (0.0068%). Compound 2 was obtained as an amorphous powder showing [α]_D −49.2° (pyridine). The results of positive high-resolution fast atom bombardment mass spectrometry showed (m/z): 1228.5964 (Calcd for C₅₆H₉₃NO₂₈+H: 1228.5962). This compound was identified with the previously isolated esculeoside B, 3-O-β-lycotetraosyl (5S,22S,23R,25S)-22,26-epimino-16β,23-epoxy-3β,23,27-trihydroxycholestane 27-O-β-D-glucopyranoside,²⁾ and renamed esculeoside B-2. This sapogenol moiety is called a solanocapsine type,²⁾ and its glycoside is very rare.

Compound 1 was obtained as an amorphous powder showing [α]_D −42.6° (pyridine). Positive high-resolution fast atom bombardment mass spectrometry exhibited a quasi-molecular ion at m/z 1228.5966 (Calcd for C₅₆H₉₃NO₂₈+H: 1228.5962). The signals on the ¹H- and ¹³C-NMR of 1 were very similar to those of 2. In particular, the signals originating from the sugar moieties of 1 were almost superimposable on those of 2. The sapogenol moiety of 1 showed 3 methyl groups at δ 0.72 (3H, s, H₃-19), 0.85 (3H, s, H₃-18), and 1.29 (3H, d, J=6.1 Hz, H₃-21) in the ¹H-NMR spectrum (pyridine-d₅), and it exhibited signals assigned to C-22–C-27 on the sapogenol moiety at δ 61.9, 96.6, 40.2, 33.2, 42.3, and 70.4, respectively, in the¹³C-NMR (pyridine-d₅) spectrum. Therefore, the structure of 1 was also deduced to be a solanocapsine type glycoside—that is, a stereo-isomer of 2. The signal assigned to H₃-21 appeared at δ 1.29 higher field than that in 2, in this signal appeared at δ 1.62 owing to 1,3-diaxial configuration. Therefore, in 1, the 21-Me group does not lie in a 1,3-diaxial configuration. Moreover, nuclear Overhauser effect was observed between H-20 and H-22 in 2 but not in 1. Consequently, the structure of 1 was deduced to be 3-O-β-lycotetraosyl (5S,22R,23S,25S)-22,26-epimino-16β,23-epoxy-3β,23,27-trihydroxycholestane 27-O-β-D-glucopyranoside, which was designated as esculeoside B-1.

Fig. 2. Structures of Esculeoside B-1 (1)

Thus, because the saponins esculeosides B-1 (1) and B-2 (2) were isolated from Italian canned tomatoes, we hypothesized that the esculeoside A in Italian tomatoes transforms into esculeosides B-1 and B-2 when these tomatoes are canned via sterilization with boiling water. Therefore, we considered whether esculeoside A would change to esculeoside B when refluxed with water. To investigate this question, we refluxed esculeoside A (104.0 mg) with water in an oil bath for 6.5 h. Using thin-layer chromatography, we found that esculeoside A was almost quantitatively converted to a more polar substance. The reaction mixture was evaporated in vacuo to give a residue (98.1 mg, 97.4%), which was passed through an ODS column, yielding in 2 compounds identified as esculeoside B-1 (1, 26.0 mg, 25.8%) and B-2 (2, 31.2 mg, 31.0%). Thus, esculeoside A is transferred into esculeosides B-1 and B-2 when Italian tomatoes are produced in cans. Although we have reported that esculeoside A is partly converted to esculeogenins B-1 and B-2 via refluxing with HCl in 50% aqueous dioxane,¹⁹⁾ conversion of 1 directly into esculeoside B-1 (1) and B-2 (2) as the glycoside only via refluxing with water has been attained for the first time. The proposed reaction mechanism is shown in Chart 1.²⁰⁾

Chart 1. Transformation of Esculeoside A into Esculeosides B-1 (1) and B-2 (2)

Thus, we showed that Italian canned tomatoes contain tomato glycosides esculeosides B-1 (1) and B-2 (2), and we concluded that these substances might be derived from esculeoside A when the cans are prepared through treatment with boiling water.

Experimental

General Procedure

Optical rotations were measured with a JASCO P-1020 (l=0.5) automatic digital polarimeter. FAB-MS were obtained with a glycerol matrix in the positive ion mode using a JEOL JMS-DX300 and a JMS-DX 303 HF spectrometer. The ¹H- and ¹³C-NMR spectra were measured in pyridine-d₅ with JEOL α-500 spectrometer, and chemical shifts are given on a δ (ppm) scale with tetramethylsilane (TMS) as the internal standard. Column chromatographies were carried out on a Diaion HP-20 (Mitsubishi Chemical Ind.), silica gel 60 (230–400 mesh, Merck), and ODS (preparative C₁₈, 55–105 µm, waters). TLC was performed on silica gel plates (Kieselgel 60 F₂₅₄, Merck) and RP C₁₈ silica gel plates (Merck). The spots on TLC were visualized by UV light (254/366 nm) and sprayed with 10% H₂SO₄, followed by heating.

Extraction of Tomato Cans

Commercially available Italian cans (containing 400 g of ingredients) were used for the experiments. Trademarks of Matured Whole Tomatoes (Delmonte, i), Whole Tomatoes (Yeon Co., Ltd., ii), Matured Whole Tomatoes (Kagome Co., Ltd., iii), and Whole Peeled Italian Tomatoes (Spigadoro, iv)] were used. Each was individually blended with water and passed through by filter paper to produce a filtrate, which was then passed through a highly porous polystyrene gel (Diaion HP-20) and rinsed with water. Methanol was then passed through the gel to produce an eluate, which gave a residue after evaporation. This methanolic residue was subjected to reversed phase silica gel column chromatography, ODS, and then eluted with 40%, 60%, and 100% methanol. The eluate from the 40% methanol contained aromatics, that from the 60% methanol was composed of tomato saponins, and the final methanol eluate included less polar substances. The respective yields: the MeOH eluate from Diaion, i 559.2 mg, ii 635.7 mg, iii 557.1 mg, iv 851.0 (total 2603.0 mg, 0.163%); the 40% MeOH eluate from ODS column, i 352.0 mg, ii 405.8 mg, iii 280.0 mg, iv 488.7 mg (total 1526.5 mg, 0.095%); the 60% MeOH eluate from ODS column, i 42.0 mg, ii 101.6 mg, iii 60.8 mg, iv 186.2 mg (total 390.6 mg, 0.024%); the MeOH eluate from ODS column, i 26.2 mg, ii 64.0 mg, iii 63.6 mg, iv 104.6 mg (total 258.4 mg, 0.016%). The 60% methanol eluates from the ODS column were combined to give a residue (390.6 mg, 0.024%), which was then passed through a further ODS column with 55% methanol to provide two tomato saponins, 1, 85.5 mg, 0.0052%, and 2, 112.0 mg, 0.0068%.

Esculeoside B-1 (1)

An amorphous powder, [α]_D¹⁵ −42.6° (c=1.0, pyridine), positive high resolution (HR)-FAB-MS (m/z): 1228.5966 (Calcd for C₅₆H₉₃NO₂₈+H: 1228.5962). ¹H-NMR (pyridine-d₅) δ: 0.72 (3H, s, H₃-19), 0.85 (3H, s, H₃-18), 1.29 (3H, d, J=6.1 Hz, H₃-21), 2.77 (1H, m, H-20), 3.42 (1H, d, J=10.2 Hz, H-22), 4.80 (1H, d, J=7.9 Hz, gal H-1), 4.92 (1H, d, J=7.9 Hz, 27-O-glc H-1), 5.11 (1H, d, J=7.9 Hz, inn. glc H-1), 5.18 (1H, d, J=7.9 Hz, xyl H-1), and 5.52 (1H, d, J=8.5 Hz, ter. glc H-1). ¹³C-NMR (pyridine-d₅) δ: sapogenol C-1–27: 37.2, 29.6, 77.7, 34.5, 44.7, 28.7, 32.2, 35.0, 54.4, 35.6, 21.1, 37.2, 41.9, 53.2, 33.1, 79.8, 61.1, 15.1, 12.3, 27.2, 16.6, 61.9, 96.6, 40.2, 33.2, 42.3, and 70.4; gal C-1–C-6: 102.2, 72.6, 75.0, 79.7, 74,6, and 60.9; inn-glc C-1–6: 104.1, 80.3, 87.0, 70.3, 77.3, and 62.3, ter-glc C-1–6: 104.3, 75.3, 78.0, 71.0, 78.4, and 62.0, xyl C-1–5; 103.8, 74.4, 77.5, 69.7, and 66.6; 27-O-glc C-1–6: 103.7, 75.0, 77.7, 71.2, 77.6, and 62.3. This was named as esculeoside B-1.

Esculeoside B-2 (2)

An amorphous powder, [α]_D¹⁵ −49.2° (c=1.0, pyridine), positive HR-FAB-MS (m/z): 1228.5964 (Calcd for C₅₆H₉₃NO₂₈+H: 1228.5962). This was identified with esculeoside B and renamed as esculeoside B-2.

Conversion of Esculeoside A into Esculeosides B-1 (1) and B-2 (2)

A solution of esculeoside A (104.0 mg) in dist. water (5 mL) was refluxed on the oil bath for 6.5 h. The reaction mixture was evaporated in vacuo to dryness (98.1 mg, 97.4%), which was then chromatographed on ODS by eluting with 55% MeOH to obtain two substances. They were identified with esculeoside B-1 (1, 26.0 mg, 25.8%) and esculeoside B-2 (2, 31.2 mg, 31.0%) by measurring ¹H- and ¹³C-NMR spectra.

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