Conversion of Tomato Saponins to Pregnane Derivatives

Abstract

Here reports new conversions methods of tomato saponins, esculeoside A (1) and a mixture of esculeosides B-1 (2) and B-2 (3), (the latter two were obtained from tomato cans) into pregnane derivative (5) by an alkal treatment followed by acid treatment. Compound 1 or a mixture of 2 and 3 were each refluxed with 1 N KOH to afford a characteristic pyridine steroidal glycoside (4), which was then treated with 2 N HCl–MeOH to afford a pregnane derivative, 3β-hydroxy-5α-pregn-16-en-20-one (5). The results of the above two reactions indicated that tomato saponins are chemically closely related to pregnane hormones. We assume that the assimilated tomato saponins via the small intestine are metabolized into pregnane derivatives, demonstrating various bioactivities such as anti-cancer, anti-osteoporosis, and anti-menopausal disorder activities.

In 2003, Nohara and colleagues isolated a tomato saponin, named as esculeoside A (1),^1,2) from the mature fruits of tomato, Solanum lycopersicum L., and determined its structure. Tomato saponin is a significant constituent of ripe tomatoes and is present in approximately four-fold higher levels than lycopene. Thus far, the bioactivity of tomato has been attributed solely to lycopene. Therefore, the pharmacological evaluation of 1 is important.

Recently, Fujiwara et al.³⁾ reported that the oral administration of 1 to apolipoprotein E-deficient mice significantly reduced the serum levels of cholesterol glycerides, low-density lipoprotein cholesterol, and the severity of atherosclerotic lesions without any detectable side effects. In addition, we found an interesting chemical conversion: esculeogenin A, the sapogenol of 1, was easily converted to a pregnane derivative, 3β-hydroxy-5α-pregn-16-ene-20-one (5), by refluxing in aqueous pyridine,^4,5) as shown in Chart 1, indicating that tomato saponin is closely related to the pregnane hormone from the viewpoint of chemical reaction.

Chart 1

Furthermore, we found a new method for the respective conversions of 1 or a mixture of esculeosides B-1 (2) and B-2 (3)⁶⁾ to the pregnane derivative as follows:

Esculeoside A (1) was refluxed in 1 N KOH for 1 h. The reaction mixture was separated by silica gel column chromatography using a mixture of CHCl₃–MeOH–H₂O (8 : 2 : 0.2) to afford the major product (4), amorphous powder, [α]_D −96.2°(pyridine), in 32% yield. The high-resolution negative-ion fast-atom-bombardment mass spectroscopy (HR-FAB-MS) of 4 exhibited the [M−H]⁻ peak at m/z 1044.5009 [Calcd for C₅₀H₇₈O₂₂N: 1044.5016]. The ¹H-NMR (pyridine-d₅) spectrum showed four methyl groups at δ 0.63 (3H, s), 1.16 (3H, s), 1.59 (3H, d, J=6.9 Hz), and 2.11 (3H, s); two aromatic protons at δ 7.18 (1H, br s) and 8.18 (1H, br s); and four anomeric protons at δ 4.87 (1H, d, J=7.5 Hz), 5.17 (1H, d, J=7.5 Hz), 5.21 (1H, d, J=7.4 Hz), and 5.54 (1H, d, J=6.9 Hz). The ¹³C-NMR (pyridine-d₅) spectrum showed five aromatic carbons at δ 123,2, 131.3, 140.8, 150.0, and 151.0; two oxygen-bearing carbons at δ 77.2, and 77.4; four methyl carbons at δ 12.1, 13.7, 17.6, and 19.4; eight methylene carbons at δ 21.0, 28.8, 29.7, 32.1, 34.6, 35.2, 37.0, and 40.3; six methine carbons at δ 35.6, 37.0, 44.5, 54.4, 54.6, and 62.8; two quaternary carbons at δ 35.6, and 42.8; and a β-lycotetraosyl moiety at δ 102.2, 71.9, 74.9, 79.8, 75.4, 60.3 (β-galactopyranosyl C-1–C-6), 104.8, 81.2, 86.5, 70.5, 78.6, 61.5 (inner β-glucopyranosyl C-1–C-6), 105.0, 74.9, 78.5, 70.8, 78.5, 62.2 (terminal β-glucopyranosyl C-1–C-6), 104.7, 75.2, 76.0, 70.3, 67.2 (β-xylopyranosyl C-1–C-5). The heteronuclear multiple-bond correlation (HMBC) showed that the methyl group at δ 2.11 correlated to the three aromatic carbons at δ 123.2, 131.3, and 140.8, indicating this methyl group as C-27; the aromatic protons at δ 7.18 and 8.18 were assigned to H-24 and H-26, respectively. The HR-FAB-MS and HMBC results showed that the E-ring is open, and the hydroxyl groups are present at the C-16 and C-23 positions. Therefore, the structure of 4 was deduced to be a pyridine derivative as shown in Chart 2.

Chart 2

On the other hand, a mixture of esculeoside B-1 (2) and esculeoside B-2 (3) was refluxed in 1 N KOH for 1 h. After neutralizing with 1 N HCl, the reaction mixture was concentrated under reduced pressure and passed through Diaion HP-20 to afford the crude material, which was purified by using silica gel chromatography using a mixture of CHCl₃–MeOH–H₂O (8 : 2 : 0.2) to afford the major compound, amorphous powder, [α]_D −98.5°(pyridine), in 34% yield, as shown in Chart 3. The negative FAB-MS showed the [M−H]⁻ peak at m/z 1044.5012 [Calcd for C₅₀H₇₈O₂₂N]; the ¹H-NMR (pyridine-d₅) and ¹³C-NMR (pyridine-d₅) spectra were identical to those of compound 4.

Chart 3

Next, compound 4 was refluxed in 2 N HCl–MeOH for 1 h, and the resulting reaction mixture was purified by silica gel column chromatography using a mixture of n-hexane–acetone (5 : 1) to afford compound 5 (in 27% yield from 4). The HR positive FAB-MS showed the [M+H]⁺ peak at m/z 317.2484 (Calcd for C₂₁H₃₃O₂: 317.2481). The ¹H-NMR spectrum (in CDCl₃) showed three methyl groups at δ 0.77 (3H, s), 0.81 (3H, s), and 2.18 (3H, s); an oxygen-bearing methane proton at δ 3.53 (1H, m); and an olefinic proton at δ 6.62 (1H, dd, J=1.8, 3.1 Hz). Moreover, the ¹³C-NMR spectrum showed a total of 21 carbons including three methyl groups at δ 12.2, 15.9, and 28.5; one oxygen-bearing methine carbon at δ 71.3; two olefinic carbons at δ 144.4 and 155.5; and a carbonyl carbon at δ 196.8. Thus, which The product was identified as 3β-hydroxy-5α-pregn-16-en-3β-ol-20-one,⁷⁾ as shown in Chart 4. The above these reactions are summarized in Chart 5.

Chart 4

Chart 5

Our recent studies on the constituents of Solanum plants have revealed that pregnane glycosides are accompanied with normal spirostanol and furostanol glycosides.^8–16) Esculeogenin A was easily converted into a pregnane derivative, 3β-hydroxy-5α-pregn-16-ene-20-one (5) by refluxing in aqueous pyridine.^4,5) Both 1, and a mixture of esculeosides B-1 (2) and B-2 (3) were converted to the same pregnane derivative by refluxing in a KOH solution, followed by the reaction with HCl/MeOH. Moreover, a pregnane glycoside has been isolated from over-ripe tomato.¹⁷⁾ The above facts strongly indicate that orally administered steroidal glycosides can be metabolized into pregnane derivatives, a type of steroidal hormone. In further experiments, urine was collected from human subjects who had consumed tomatoes, and separated by various column chromatographic techniques to afford three androstane derivatives.¹⁸⁾ These androsterone analogs are normally excreted; however, because no such analogs were detected in the control samples, their occurrence indicated the production of progesterone by the subjects that had consumed tomatoes. We hypothesize that the orally administered steroidal glycosides such as spirostanol, furostanol and spirosolane glycosides are metabolized, thus introducing a hydroxyl group at C-23. These intermediates may then be metabolized into pregnane derivatives. We conclude that the tomato saponin 1 is metabolized into various steroidal hormones such as pregnane, with anti-osteoporosis, anti-menopausal disorder, and anti-tumor bioactivities in the body.^19,20)

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 Industries), and silica gel 60 (230–400 mesh, Merck). 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.

Conversion of Esculeoside A (1) into Pyridine Derivative (4)

A solution of esculeoside A (1, 432 mg) in 1 N KOH (22 mL) was refluxed for 1 h on the oil bath. After neutralization with 1 N HCl, the reaction mixture was concentrated under reduced pressure to give the reaction mixture, to which was water added and passed through Diaion HP-20. First, it was washed with water and next eluted with methanol to afford the crude material, which was separated and purified by using silica gel chromatography with CHCl₃–MeOH–H₂O (8 : 2 : 0.2) to provide major compound (4, 114 mg), an amorphous powder, [α]_D −96.2° (c=0.5, pyridine), in a yield of 32%. HR negative FAB-MS of 4: [M−H]⁻ at m/z 1044.5009 [Calcd for C₅₀H₇₈O₂₂N: 1044.5016]. ¹H-NMR (pyridine-d₅) δ: 0.63 (3H, s, 19-H₃), 1.16 (3H, s, 18-H₃), 1.59 (3H, d, J=6.9 Hz, 21-H₃), 2.11 (3H, s, 27-H₃), 4.87 (1H, d, J=7.5 Hz, galactosyl H-1), 5.17 (1H, d, J=7.5 Hz, inner glucosyl H-1), 5.21 1H, d, J=7.4 Hz, xylosyl H-1), 5.54 (1H, d, J=6.9 Hz, terminal glucosyl H-1), 7.18 (1H, br s, H-24), 8.18 (1H, br s, H-26). ¹³C-NMR (pyridine-d₅): aglycone moiety C-1–C-27, δ 37.0, 29.7, 77.2, 34.6, 44.5, 28.8, 32.1, 35.6, 54.4, 35.6, 21.0, 40.3, 42.8, 54.6, 35.2, 77.4, 62.8, 13.7, 12.1, 37.0, 19.4, 150.0, 151.0, 123.2, 131.3, 140.8, 17.6; galactopyranosyl C-1–C-6 at δ 102.2, 71.9, 74.9, 79.8, 75.4, 60.3; inner glucopyranosyl C-1–C-6 at δ 104.8, 81.2, 86.5, 70.5, 78.6, 61.5; terminal glucopyranosyl C-1–C-6 at δ 105.0, 74.9, 78.5, 70.8, 78.5, 62.2; xylopyranosyl C-1–C-5 at δ 104.7, 75.2, 76.0, 70.3, 67.2.

Conversion of a Mixture of Esculeosides B-1 (2) and B-2 (3) into Pyridine Derivative (4)

A mixture of eesculeoside B-1 (2, 255 mg) and esculeoside B-2 (3) was refluxed with 1 N KOH for 1 h. After neutralization with 1 N HCl, the reaction mixture was concentrated under reduced pressure and passed through Diaion HP-20. It was first washed with water and next eluted with methanol to give the crude material, which was purified by using silica gel chromatography with CHCl₃–MeOH–H₂O (8 : 2 : 0.2) to provide major compound, an amorphous powder (74 mg), [α]_D −98.5°(pyridine), in 34% yield. The negative FAB-MS exhibited m/z 1044.5009 [Calcd for C₅₀H₇₉O₂₂N–H]⁻], of which ¹H-NMR (pyridine-d₅) and ¹³C-NMR (pyridine-d₅) spectra were identical with tose of compound 4.

Conversion of Pyridine Derivative (4) into Pregnane Derivative (5)

Compound 4 (165 mg) was refluxed with 2 N HCl–MeOH for 1 h and neutralized with 2 N KOH–MeOH, concentrated under reduced pressure to give a syrup, which was added with water, and passed through Diaion HP-20. First water eluate was discarded and second methanolic eluate was evaporated to dryness, which was then separated and purified with silica gel column chromatography with n-hexane–acetone (5 : 1) to give compound 5 (14 mg, in 27% yield from 4). Colorless needles, mp 203–205°C, [α]_D²² +48.2° (c=1.0, CHCl₃). HR-FAB-MS (m/z): m/z 317.2484 [M+H]⁺ (Calcd for C₂₁H₃₃O₂: 317.2481). ¹H-NMR spectrum (CDCl₃) δ: 0.81 (3H, s, H₃-18), 0.77 (3H, s, H₃-19), 2.18 (3H, s, H₃-21), 3.53 (1H, m, H-3), 6.62 (1H, dd, J=1.8, 3.1 Hz, H-16). ¹³C-NMR spectrum (CDCl₃) δ: 33.7 (C-1), 32.2 (C-2), 71.3 (C-3), 36.7 (C-4), 45.0 (C-5), 27.1 (C-6), 31.5 (C-7), 31.9 (C-8), 56.3 (C-9), 35.6 (C-10), 21.0 (C-11), 38.1 (C-12), 46.3 (C-13), 54.8 (C-14), 34.7 (C-15), 144.4 (C-16), 155.5 (C-17), 15.9 (C-18), 12.2 (C-19), 196.8 (C-20), 28.5 (C-21), which was identified with 3β-hydroxy-5α- pregn-16-ene-3β-ol-20-one.

References

• 1) Fujiwara Y., Yahara S., Ikeda T., Ono M., Nohara T., Chem. Pharm. Bull., 51, 234–235 (2003).
• 2) Fujiwara Y., Takaki A., Uehara Y., Ikeda T., Okawa M., Yamauchi K., Ono M., Yoshimitsu H., Nohara T., Tetrahedron, 60, 4915–4920 (2004).
• 3) Fujiwara Y., Kiyota N., Hori M., Matsushita S., Iijima Y., Aoki K., Shibata D., Takeya M., Ikeda T., Nohara T., Nagai R., Arterioscler. Thromb. Vasc. Biol., 27, 2400–2406 (2007).
• 4) Matsushita S., Yoshizaki M., Fujiwara Y., Ikeda T., Ono M., Okawara T., Nohara T., Tetrahedron Lett., 46, 3549–3551 (2005).
• 5) Nohara T., Okamoto K., Matsushita S., Fujiwara Y., Ikeda T., Miyashita H., Ono M., Yoshimitsu H., Kansui H., Kunieda T., Chem. Pharm. Bull., 56, 1015–1017 (2008).
• 6) Manabe H., Fujiwara Y., Ikeda T., Ono M., Murakami K., Zhou J.-R., Yokomizo K., Nohara T., Chem. Pharm. Bull., 61, 764–767 (2013).
• 7) Yoshihara T., Nagaoka T., Ohra J., Sakamura S., Phytochemistry, 27, 3982–3984 (1988).
• 8) Nohara T., Yabuta H., Suenobu M., Hida R., Miyahara K., Kawasaki T., Chem. Pharm. Bull., 21, 1240–1247 (1973).
• 9) Zhu X., Tsumagari H., Honbu T., Ikeda T., Ono H., Nohara T., Tetrahedron Lett., 42, 8043–8046 (2001).
• 10) Dong M., Feng X., Wang B., Wu L., Ikejima T., Tetrahedron, 57, 501–506 (2001).
• 11) Tran Q. L., Tezuka Y., Banskota A. H., Tran Q. K., Saiki I., Kadota S., J. Nat. Prod., 64, 1127–1132 (2001).
• 12) Yokosuka A., Mimaki Y., Sashida Y., J. Nat. Prod., 65, 1293–1298 (2002).
• 13) Mimaki Y., Watanabe K., Sakagami H., Sashida Y., J. Nat. Prod., 65, 1863–1868 (2002).
• 14) Yin J., Kouda K., Tezuka Y., Tran Q. L., Miyahara T., Chen Y., Kadota S., J. Nat. Prod., 66, 646–650 (2003).
• 15) Liu H., Xiong Z., Li F., Qu G., Kobayashi H., Yao X., Chem. Pharm. Bull., 51, 1089–1091 (2003).
• 16) Ikeda T., Tsumagari H., Okawa M., Nohara T., Chem. Pharm. Bull., 52, 142–145 (2004).
• 17) Fujiwara Y., Yoshizaki M., Matsushita S., Yahara S., Yae E., Ikeda T., Ono M., Nohara T., Chem. Pharm. Bull., 53, 584–585 (2005).
• 18) Noguchi E., Fujiwara Y., Matsushita S., Ikeda T., Ono M., Nohara T., Chem. Pharm. Bull., 54, 1312–1314 (2006).
• 19) Nohara T., Ikeda T., Fujiwara Y., Matsushita S., Noguchi E., Yoshimitsu H., Ono M., J. Nat. Med., 61, 1–13 (2006).
• 20) Nohara T., Ono M., Ikeda T., Fujiwara Y., El-Aasr M., J. Nat. Prod., 73, 1734–1741 (2010).