Urease inhibitory isoflavonoids from different parts of Calopogonium mucunoides (Fabaceae)

Abstract

The dichloromethane-methanol (1:1) soluble part of Calopogonium mucunoides (Fabaceae) resulted in the isolation of 10 isoflavones (4′-O-methylalpinumisoflavone, 4′-O-methylderrone, alpinumisoflavone, daidzeine, Calopogonium isoflavone A, atalantoflavone, 2′,4′,5′,7-tetramethoxyisoflavone, 7-O-methylcuneantin, cabreuvin and 7-O-methylpseudobaptigenin) and a rotenoid (6a,12a-dehydroxydegueline). Among these, daidzeine, 7-O-methylcuneantin, atalantoflavone and 6a, 12a-dehydroxydegueline have been isolated for the first time from C. mucunoides while remaining are already reported from this source. Structures of all the isolated constituents were elucidated with the aid of NMR spectroscopic and mass spectrometric techniques. Among all the isolated constituents, nine were evaluated for their urease inhibitory potential. However, six were found potent. These include 4′-O-methylderrone, daidzeine, atalantoflavone, 2′,4′,5′,7-tetramethoxyisoflavone, 7-O-methylcuneantin and 6a, 12a-dehydroxydegueline.

Keywords::

• Calopogonium mucunoides
• Fabaceae
• isoflavonoids
• spectral characterization
• urease inhibition

Introduction

Calopogonium mucunoides (Desv.) is a tropical herb which belongs to the family Fabaceae distributed in Africa, America and the Pacific regions¹. C. mucunoides has been used as a folk empirical medicine for the treatment of anaemia and to protect against witchcraft². Previous phytochemical studies on the Calopogonium plants by various research groups resulted in the isolation of O- and C-prenylated isoflavones along with pyranoflavanones and pterocarpanes^3–6. Isoflavone type metabolites are well-known to exhibit wide range of interesting biological activities such as oestrogenic, antifungal, antioxidant, antibacterial, cytotoxic, antimonoamine oxidase, etc^7–9. But urease inhibitory properties of this class of phenolic secondary metabolites have attracted very little attention. The most prominent work described in the literature was due to Zhu-Ping and co-workers¹⁰ who reported the urease inhibitory potential of isoflavone-based compounds from synthetic source.

Urease enzyme is produced by Helicobacter pylori, bacterium in stomach which converts urea into ammonia and carbondioxide¹¹. Due to the production of ammonia, this bacterium counteracts the strong acidic environment of stomach. In this way, the urease action not only provides an environment with a pH suitable for H. pylori colonization of the stomach mucosal lining but also provides the mechanism for eventual gastric wall damage that increases the possibility of gastric ulcers¹².

The H. pylori infection breaks up in early childhood, persists lifelong if not eradicated, and is associated with chronic gastritis and an increased risk of peptic ulcer and gastric cancer¹³. Urease also serves as a virulence factor in human and animal infections of urinary and gastrointestinal tracts¹⁴. Urease inhibition by potent and specific compounds could lead to treatment of infections caused by the urease producing bacteria. That is why screening of natural and synthetic compounds has gained more attention recently to explore the compounds as antiulcer agents and to be used further for drug development in current medicinal research. A number of synthetic compounds including imidazoles, hydroxamic acids, and phosphazenes are effective urease inhibitors, but limited studies have been conducted on natural products¹⁵. Therefore this study is designed to determine the urease inhibitory potentials of isolated compounds of Calopogonium mucunoides (Desv.) and antiurease activities are reported for the first time in this study.

In the search of bioactive secondary metabolites from C. mucunoides, the CH₂Cl₂/MeOH soluble part of leaves, roots and seeds were investigated. As a result, altogether eleven constitutes could be purified and characterized. However, nine constituents were tested against urease enzyme.

Results and discussion

The leaves, roots and seeds of Calopogonium mucunoides extracted with a mixture of CH₂Cl₂/MeOH (1:1) were subjected to the flash chromatography. The obtained crude fractions were purified using silica gel column chromatography resulting in the isolation of eleven compounds. These compounds were identified with the aid of their obtained spectral data (one and two dimension NMR and MS) and further confirmed by comparison of published data. The identified compounds include 4′-O-methylalpinumisoflavone (1), 4′-O-methylderrone (2)³, alpinumisoflavone (3)⁴, atalantoflavone (4)¹⁶, Calopogonium isoflavone A (5)^3,4, daidzeine (6)¹⁷, 2′,4′,5′,7-tetramethoxyisoflavone (7)⁴, 7-O-methylcuneantin (8)¹⁸, cabreuvin (9) and 7-O-methylpseudobaptigenin (10)⁴ and 6a,12a-dehydroxydegueline (11)¹⁹. Among all characterized constituents, daidzeine, 7-O-methylcuneantin, atalantoflavone and 6a, 12a-dehydroxydegueline have been isolated for the first time from C. mucunoides.

Urease inhibitory activity

When the isolated compounds from Calopogonium mucunoides were evaluated against urease, most of them showed excellent urease inhibition as shown in Table 1. Compounds 2, 4, 6, 7, 8 and 11 showed potent urease inhibitory activity even higher than the standard inhibitor (thiourea). Thiourea was used as positive control in this study. It binds at biometallic active site of the enzyme (urease) and inhibits its activity.

Table 1.  Urease Inhibition Studies of compounds 1–8 and 11.

Compound n°	1	2	3	4	5	6	7	8	11	Thiourea*
IC50 (µM)	25.4 ± 0.15	12.0 ± 0.22	232.0 ± 0.08	17.3 ± 0.10	94.4 ± 0.13	7.9 ± 0.19	12.6 ± 0.12	8.4 ± 0.14	11.1 ± 0.24	21.6 ± 0.12

Each IC₅₀ value is mean of three experiments performed.

*Compound used as positive control.

From the results presented in Table 1, it may be suggested, except for compound 11 which is a rotenoid type isoflavanoid, that the urease inhibitory potential of isoflavones tested (1, 2, 3, 4, 5, 6, 7, 8) depends on the presence and the nature of the fusion of chromen moiety on ring A on one hand, and on the other hand on the number of the methoxyl groups in the molecule. In fact it appeared that, all the isoflavones tested (6, 7 and 8) without the chromen moiety on their ring A, were more active than those processing this moiety (1–5). Moreover, while among the isoflavones tested not having the chromen moiety (6–8), the urease inhibitory activity increase with decreasing number of methoxyl groups, in the isoflavones tested processing chromen unit, the activity increase when the nature of the fusion of this chromen unit with ring A is angular. The most active compound tested is daidzeine (6), which did not have in its structure neither chromen moiety nor methoxyl group but hydroxyl groups.

These results demonstrated that, the structure-activity relationship of isolated compounds may be attributed to coordinating capabilities of the R substituents with the metallocentre (nickel) of enzyme²⁰. So, in compounds having methoxyl and chromen groups, the binding of the compound at the active site of the enzyme is strongly reduces while the presence of hydroxyl groups increases this binding. In another words, the stronger will be the binding of the R substituent with the metallocentre of the enzyme, the more active will be the compound. Therefore, hydroxyl groups may be essential for urease inhibitory activity of isoflavone type compounds.

Conclusion

The above results let to the conclusion that compounds 1, 2, 4, 6, 7, 8 and 11 have excellent urease inhibitory potential. Therefore, further in vivo studies are recommended for these compounds so that they may be used in drug development for ulcer.

Materials and methods

General experimental procedures

The melting points were determined on a Barnstead Electro thermal apparatus (Digital Melting Point IA-90). Column chromatography was carried out using silica gel (Merck 70–230 and 230–400 mesh). Thin layer chromatography was performed on percolated 0.5 mm thick Merck Si gel 60 F₂₅₄ aluminium sheets. TLC plates were sprayed with 5% (v/v) aqueous solution of H₂SO₄, containing 4% (w/v) phosphomolybdic acid and 0.5% (w/v), ceric ammonium sulphate and heated at 120°C on a hot plate to visualize the spots. The mass spectrums where recorded on a JEOL MS Route instrument and nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DPX-400 instrument, ¹H and ¹³C-NMR probes operating at 400 and 100 MHz, respectively with tetramethylsilane as an internal standard.

Plant collection

The leaves and roots of C. mucunoides were collected in July 2007 and the seeds in January 2010 from the central region of Cameroon (Eloumden and Melen). These plant materials were identified at the National Herbarium, Yaoundé, Cameroon, where vouchers specimen is deposited under accession number 17456/SFR/CAM (C. mucunoides Desv.).

Extraction and isolation

The collected plant materials were air-dried, ground and extracted at room temperature in a mixture of CH₂Cl₂/MeOH (1:1) for 48 h. The extracts were concentrated on a rotary evaporator to afford 102, 325.7 and 364 g of roots, seeds and leaves crude extracts of C. mucunoides, respectively.

The crude extract of the roots of C. mucunoides was subjected to silica gel column chromatography eluting with a mixture of n-Hex/AcOEt and pure acetate.

Elution with a mixture of n-Hex/AcOEt (97:3) afforded a yellow powder of 4′-O-methylalpinumisoflavone (1) (10 mg); with n-Hex/AcOEt (19:1) another yellow powder of 4′-O-methylderrone (2) (1.2 g) was obtained and elution with n-Hex/AcOEt (37:3) afforded yellow crystals of alpinumisoflavone (3) (1.7 g). Subsequent elution with n-Hex/AcOEt (7:3) led to the isolation of daidzeine (6) (40 mg).

The crude extract of the leaves of C. mucunoides (362 g) was subjected to flash chromatography, using silica gel (70–230 mesh) and eluted with a gradient of increasing polarity of n-Hex/AcOEt and finally, pure AcOEt, resulting four fractions labelled: A (117 g); B (15 g); C (14.4 g) and D (3.9 g).

Repeated column chromatography of fraction A using n-Hex/AcOEt (19:1) gave 1 (8.2 g) and 2 (9 g). The elution with n-Hex/AcOEt (17:1) led to 3 (22 g) and two more compounds: Calopogonium isoflavone A (5) (5 g, white crystals) and atalantoflavone (4) (40 mg, yellow crystals).

The workup of the other fractions (B, C and D) yielded to the same compounds mentioned above.

The crude extract of the seeds of C. mucunoides was subjected to flash silica gel chromatography eluted with a gradient of increasing polarity of n-Hex/AcOEt and AcOEt/MeOH to yield four fractions: A (18.6 g), B (14.4 g), C (3.9 g) and D (36.8 g).

Fraction A, eluted with n-Hex/AcOEt (7:3) led to the isolation of 7,2,4′,5′-tetramethoxyisoflavone (7) (400 mg) as a white powder. Fractions 79–104 were rechromatographed using n-Hex/AcOEt (9:1) to afford a yellow powder of 2′-O-methylcuneantin (8) (10 mg). Whereas, subsequent elution with n-Hex/AcOEt (4:1) gave a white powder of 6a,12a-dehydroxydegueline (11) (5 mg).

Treatment of the remaining three fractions (B, C and D) yielded to the isolation of cabreuvin (9) (200 mg) and 7-O-methylpseudobaptigenin (10) (250 mg).

4′-O-methylalpinumisoflavone (1)

Pale yellow powder; m.p. 131–133°C (Litt.³: m.p. 135–136 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 13.10 (1H, s, chelated hydroxyl, OH-5), 7.80 (1H, s, H-2), 7.47 (2H, d, J = 9.7 Hz, H-2′,6′), 6.90 (2H, d, J = 9.7 Hz, H-3′,5′), 6.70 (1H, d, J = 10.0 Hz, H-4′′), 6.30 (1H, s, H-8), 5.56 (1H, d, J = 10.0 Hz, H-3′′), 3.82 (3H, s, OCH₃-4′), 1.45(6H, s, CH₃-2′′). ¹³C-NMR (CDCl₃, 100 MHz), δ 180.9 (C-4), 159.7(C-7), 159.5(C-4′), 156.5 (C-5), 152.5 (C-8a), 152.4 (C-2), 130.1 (C-2′,6′), 127.1 (C-3′′), 123.5 (C-1′), 122.9 (C-3), 116.0 (C-4′′), 114.0 (C-3′,5′), 106.1 (C-4a), 105.5 (C-6), 94.8 (C-8), 78.0 (C-2′′), 55.3 (OCH₃-4′), 28.3 (2CH₃-2′′). All those ¹H and ¹³C-NMR data matched well with literature values³. HRESIMS (positive mode) m/z 351, 1225 [M+H]⁺ (calcd for C₂₁H₁₉O₅, 351.1233).

4′-O-methylderrone (2)

Pale yellow powder; m.p. 164–166°C (Litt.³: m.p. 166 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 13.10 (1H, s, chelated hydroxyl, OH-5), 7.80 (1H, s, H-2), 7.43 (2H, d, J = 9.7 Hz, H-2′,6′), 6.90 (2H, d, J = 9.7 Hz, H-3′,5′), 6.70 (1H, d, J = 10.0 Hz, H-4′′), 6.28 (1H, s, H-6), 5.56 (1H, d, J = 10.0 Hz, H-3′′), 3.82 (3H, s, OCH₃-4′), 1.45(6H, s, 2CH₃-2′′). ¹³C-NMR (CDCl₃, 100 MHz), δ 180.9 (C-4), 159.7 (C-7),159.5 (C-4′), 156.5 (C-5), 152.5 (C-8a), 152.4 (C-2), 130.1 (C-2′,6′), 127.1 (C-3′′), 123.5 (C-1′), 122.9 (C-3), 116.0 (C-4′′), 114.0 (C-3′,5′), 106.1 (C-4a), 105.5 (C-6), 94.8 (C-8), 78.1 (C-2′′), 55.4 (OCH₃-4′), 28.3 (2CH₃-2′′). All those ¹H and ¹³C-NMR data matched well with literature values³. HRESIMS (positive mode) m/z 351, 1225 [M+H]⁺ (calcd for C₂₁H₁₉O₅, 351.1233).

Alpinumisoflavone (3)

Pale yellow crystals; m.p. 213–214°C (Litt.⁴: m.p. 210–213 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 12.98 (1H, s, chelated hydroxyl, OH-5), 7.90 (1H, s, H-2), 7.43 (2H, d, J = 8.3 Hz, H-2′,6′), 6.92 (2H, d, J = 8.3 Hz, H-3′,5′), 6.71 (1H, d, J = 10.4 Hz, H-4′′), 6.31 (1H, s, H-8), 5.61 (1H, d, J = 10.4 Hz, H-3′′), 4.90 (1H,s, free OH), 1.52(3H, s, CH₃-2′′), 1.51 (3H, s, CH₃-2′′). ¹³C-NMR (CDCl₃, 100 MHz), δ 181.0 (C-4), 162.2 (C-7), 159.6 (C-5), 156. 1 (C-8a), 153.3(C-4′), 152.5 (C-2), 130.3 (C-2′,6′), 127.5 (C-3′′), 124.3 (C-1′), 123.6 (C-3), 115.6 (C-3′, 5′), 114.6 (C-4′′), 106.0 (C-4a), 101.6 (C-6), 95.6 (C-8), 78.1 (C-2′′), 28.2 (2CH₃-2′′). All those ¹H and ¹³C-NMR data matched well with literature values⁴. HREIMS m/z 336.2 [M⁺] (calcd for C₂₀H₁₆O₅, 336.0999).

Atalantoflavone (4)

Pale yellow crystals; m.p. 292–294°C (Litt.²¹: m.p. 289–290 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 12.98 (1H, s, chelated hydroxyl, OH-5), 7.90 (1H, s, H-2), 7.43 (2H, d, J = 8.3 Hz, H-2′,6′), 6.92 (2H, d, J = 8.3 Hz, H-3′,5′), 6.71 (1H, d, J = 10.4 Hz, H-4′′), 6.61 (1H, s, H-6), 5.61 (1H, d, J = 10.4 Hz, H-3′′), 4.90 (1H,s, free OH), 1.52 (3H, s, CH₃-2′′), 1.51 (3H, s, CH₃-2′′). ¹³C-NMR (CDCl₃, 100 MHz), δ 181.0 (C-4), 162.2 (C-7), 159.6 (C-5), 156.1 (C-8a), 153.3 (C-4′), 152.5 (C-2), 130.3 (C-2′, 6′), 127.5 (C-3′′), 124.3 (C-1′), 123.6 (C-3), 115.6 (C-3′, 5′), 114.6 (C-4′′), 106.0 (C-4a), 101.6 (C-6), 95.6 (C-8), 78.1 (C-2′′), 28.2 (2CH₃-2′). All those ¹H and ¹³C-NMR data matched well with literature values¹⁶. HREIMS m/z 336.2 [M⁺] (calcd for C₂₀H₁₆O₅, 336.0999).

Calopogonium isoflavone A (5)

White crystals; m.p. 135–137°C (Litt.^3,4: m.p. 131.91 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 8.04 (1H, d, J = 8.9 Hz, H-5), 7.91 (1H, s, H-2), 7.47 (2H, dd, J = 9.4 and 1.7 Hz, H-2′,6′), 6.95 (2H, dd, J = 9.4 and 1.7 Hz, H-3′,5′), 6.84 (1H, d, J = 8.9 Hz, H-4), 6.75 (1H, d, J = 10.0 Hz, H-5), 5.70 (1H, d, 10.0 Hz, H-4′′), 3.82 (3H, s, OCH₃-4′), 1.48(6H, s, 2CH₃-2′′). ¹³C-NMR (CDCl₃, 100 MHz), δ 176.0 (C-4), 159.6 (C-4′), 157.3 (C-7), 151.9 (C-8a), 151.7 (C-2), 130.3 (C-2′,6′), 130.2 (C-3′′), 126.7 (C-5), 124.3 (C-1′), 122.4 (C-3), 115.0 (C-3′,5′), 114.9 (C-4′′), 114.0 (C-6), 109.8 (C-8), 109.2 (C-4a), 77.7 (C-2′′), 55.4 (OCH₃-4′), 28.2 (2CH₃-2′′). All those ¹H and ¹³C-NMR data matched well with literature values^3,4. HREIMS m/z 334 [M⁺] (calcd for C₂₁H₁₈O₄, 334.1206).

Daidzeine (6)

Yellow powder; m.p. 330°C (Litt.¹⁷: m.p. 328–332 °C). ¹H-NMR (CDCl₃, 400 MHz): 8.44 (2H, d, J = 9.0 Hz, H-5), 8.14 (1H, s, H-2), 7.79 (2H, dd, J = 8.4 and 1.8 Hz, H-2′,6′), 7.27 (2H, dd, J = 8.4 and 1.8 Hz, H-3′,5′), 7.21 (1H, dd, J = 9.0 and 2.4 Hz, H-6), 7.10 (1H, d, J = 2.4 Hz, H-8). ¹³C-NMR (CDCl₃, 100 MHz), δ 176.4 (C-4), 164.1(C-7), 159.0 (C-4′), 158.6 (C-8a), 152.5 (C-2), 131.0 (C-2’,6′), 128.3 (C-5), 124.4 (C-3), 117.0 (C-1′), 118.0 (C-4a), 116.2 (C-3′,5′), 115.8 (C-6), 103.1 (C-8). All those ¹H and ¹³C-NMR data matched well with literature values¹⁷. HRESIMS m/z 254.0 [M+H]⁺ (calcd for C₁₅H₁₁O_4, 25.0580).

7,2′,4′,5′-tetramethoxyisoflavone (7)

Yellow powder; m.p. 193–194°C (Litt.⁴: m.p. 191.33 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 8.19 (1H, d, J = 8.0 Hz, H-5), 7.95 (1H, s, H-2), 6.98 (1H, dd, J = 8.0 et 3.2 Hz, H-6), 6.94 (1H, s, H-5′), 6.86 (1H, d, J = 3.2 Hz, H-8), 6.62 (1H, s, H-3′), 3.93 (3H, s, OCH₃-2′), 3.91 (3H, s, OCH₃-7), 3.85 (3H, s, OCH₃-5′), 3.77 (3H, s,OCH₃-4′). ¹³C-NMR (CDCl₃, 100 MHz), δ 176.6 (C-4), 164.2 (C-7), 158.3 (C-8a), 154.6 (C-2), 152.2 (C-2′), 150.1 (C-4′), 143.3 (C-5′), 128.1 (C-5), 122.1 (C-3), 118.8 (C-4a), 115.6 (C-6′), 114.7 (C-6), 112.5 (C-1′), 100.5 (C-8), 98.6 (C-3′), 57.2 (OCH₃-2′), 56.9 (OCH₃-5′), 56.5 (OCH₃-7), 56.1 (OCH₃-2′). All those ¹H and ¹³C-NMR data matched well with literature values⁴. HREIMS m/z 342.1104 [M⁺] (calcd for C₁₉H₁₈O₆, 342.1105).

2′-O-methylcuneantin (8)

Yellow powder; m.p. 210–212°C (Litt.^18,22: m.p. 210–212 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 8.17 (1H, d, J = 8.8 Hz, H-5), 7.88 (1H, s, H-2), 6.97 (1H, dd, J = 8.8 and 2.4 Hz, H-6), 6.85 (1H, d, J = 2.4 Hz, H-8), 6.81 (1H, s, H-6′), 6.61 (1H, s, H-3′), 5.94 (2H, s, OCH₂O-4′,5′), 3.90 (3H, s, OCH₃-7), 3.72 (3H, s, OCH₃-2′). ¹³C-NMR (CDCl₃, 100 MHz), δ 176.0 (C-4), 164.2 (C-7), 158.3 (C-8a), 154.4 (C-2), 154.4 (C-2′), 148.7 (C-4′), 141.5 (C-5′), 128.1 (C-5), 122.4 (C-1′), 118.7 (C-4a), 114.7 (C-6), 113.1 (C-3), 111.5 (C-6′), 101.5 (OCH₂O-4′,5′), 100.4 (C-8), 95.7 (C-3′), 57.2 (OCH₃-7), 56.1 (OCH₃-2′). All those ¹H and ¹³C-NMR data matched well with literature values^18,22. HRESIMS m/z 327.0861 [M+H]⁺ (calcd for C₁₈H₁₇O₆, 327.0863).

Cabreuvin (9)

Yellow powder; m.p. 166–167°C (Litt.⁴: m.p. 165 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 8.20 (1H, d, J = 11.1 Hz, H-5), 7.94 (1H, s, H-2), 7.21 (1H, d, J = 2.0 Hz, H-2′), 7.05 (1H, dd, J = 8.9 et 2.0 Hz, H-6′), 7.0 (1H, dd, J = 11.1 and 2.2 Hz, H-6), 6.92 (1H, d, 8.9 Hz, H-5′), 6.85 (1H, d, J = 2.2 Hz, H-8), 3.92 (3H, s, OCH₃-3′), 3.91 (3H, s, OCH₃-4′), 3.90 (3H, s, OCH₃-7), ¹³C-NMR (CDCl₃, 100 MHz), δ 175.2 (C-4), 163.4 (C-7), 157.3 (C-8a), 151.6 (C-2), 148.2 (C-3′), 148.0 (C-4′), 127.1 (C-5), 124.3 (C-3), 124.1 (C-1′), 120.4 (C-6′), 118.0 (C-4a), 114.0 (C-6), 112.0 (C-2′), 110.5 (C-5′), 99.5 (C-8), 55.4 (OCH₃-4′), 55.3 (OCH₃-3′), 55.2 (OCH₃-7). All those ¹H and ¹³C-NMR data matched well with literature values⁴. HRESIMS m/z 312.0999 [M⁺] (calcd for C₁₈H₁₇O₅, 312.1105).

7-O-methylpseudobaptigenin (10)

Yellow powder; m.p. 177–178°C (Litt.⁴: m.p. 179–180 °C). ¹H-NMR (CDCl₃, 400 MHz): δ 8.17 (1H, d, J = 8.8 Hz, H-5), 7.88 (1H, s, H-2), 6.98 (1H, d, J = 1.7 Hz, H-2′), 6.97 (1H, dd, J = 8.8 and 2.4 Hz, H-6), 6.85 (1H, d, J = 2.4 Hz, H-8), 6.75 (1H, dd, J = 8.9 and 1.7 Hz, H-6′), 6.61 (1H, J = 8.9 Hz, H-5′), 5.94 (2H, s, OCH₂O-3′,4′), 3.90 (3H, s, OCH₃-7). ¹³C-NMR (CDCl₃, 100 MHz), δ 176.0 (C-4), 164.2 (C-7), 158.3 (C-8a), 154.4 (C-2), 153.5 (C-2′), 148.7 (C-4′), 141.5 (C-5′), 128.1 (C-5), 122.4 (C-1′), 118.7 (C-4a), 114.7 (C-6), 113.1 (C-3), 111.5 (C-6′), 101.5 (OCH₂O-3′,4′), 100.4 (C-3′), 95.7 (C-8), 57.2 (OCH₃-7), 56.1 (OCH₃-2′). All those ¹H and ¹³C-NMR data matched well with literature values⁴. HRESIMS m/z 297.0759 [M+H] ⁺ (calcd for C₁₇H₁₃O₅, 297.0757).

6a,12a-dehydrodegueline (11)

Yellow powder; m.p. 232–233°C (Litt.^19,23: m.p. 232–233°C). ¹H-NMR (CDCl₃, 400 MHz): δ 8.47 (1H, s, H-1), 8.00 (1H, d, J = 7.2 Hz, H-11), 6.84 (1H, d, J = 7.2 Hz, H-10), 6.75 (1H, d, J = 9.0 Hz, H-4′), 6.58 (1H, s, H-4), 5.71 (1H, d, J = 9.0 Hz, H-5′), 5.00 (2H, s, H-6), 3.92 (3H, s, OCH₃-2), 3.85 (3H, s, OCH₃-3), 1.47 (6H, s, 2CH₃-5′). ¹³C-NMR (CDCl₃, 100 MHz), δ 174.6 (C-12), 157.5 (C-9), 156.5 (C-6a), 151.3 (C-7a), 146.5 (C-4a), 144.2 (C-3), 130.9 (C-3′), 126.6 (C-11), 118.7 (C-11a), 115.6 (C-10), 114.9 (C-4′), 112.6 (C-12a),110.8 (C-12b), 109.4 (C-1), 100.6 (C-4), 78.0 (C-6′), 65.1 (C-6), 56.5 (OCH₃-2), 56.2 (OCH₃-3), 28.3 (2CH₃-2′′). All those ¹H and ¹³C-NMR data matched well with literature values^19,23. HRESIMS m/z 392.126 [M⁺] (calcd for C₂₁H₁₈O₅, 392.1261).

Urease inhibition assay

Urease Inhibition activity was determined by modified indophenol method based on measurement of product (ammonia) formed during the reaction²⁴. Phosphate buffer of pH 8.2 (0.01 M K₂HPO₄. 3H₂O, 1 mM EDTA and 0.01 M LiCl₂) was taken in 96 well plate wells marked as Blank, Control and Inhibitor. Urease (Jack bean) solution (10 µL) was added and mixed with the 5 µL of test compound of concentration ranging from 5 µM to 500 µM and incubated at 30°C for 15 min. 50 µL each of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to wells. Increase in absorbance was measured after 50 min at 630 nm on microtitre plate reader (Spectramax Plus 384 Molecular Device, USA). All reactions were performed in triplicates. The standard used in this assay was Thiourea and percentage inhibitions were calculated by formula:

The IC₅₀ (inhibitor concentration that inhibits 50% activity of enzyme) values were then calculated using the EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, MA, USA).

Acknowledgments

Authors are grateful to the Third Word Academy of Sciences (TWAS) and the International Center for Chemical and Biological Sciences (ICCBS) for the award of ICCBS-TWAS fellowship to VTS. Further acknowledgement is due to Michèle Meyer from “Museum National d’Histoire Naturelle de Paris, France” for some spectral scanning.

Declaration of interest

The authors report no conflicts of interest.