Stigmastane-type steroid saponins from the leaves of Vernonia amygdalina and their α-glucosidase and xanthine oxidase inhibitory activities

Abstract Two new vernonioside K (1) and vernonioside L (2) and four known Δ7,9(11) stigmastane-type steroidal saponins—vernonioside B2 (3), vernoniacum B (4), vernonioside B1 (5), and vernoamyoside A (6)—were isolated from the leaves of Vernonia amygdalina. Their structures were determined by comprehensive spectroscopic analysis with one-dimensional nuclear magnetic resonance, two-dimensional nuclear magnetic resonance, and high-resolution mass spectrometry. All isolated compounds (1–6) were evaluated to determine their inhibitory effects on α-glucosidase and xanthine oxidase. Among them, two new compounds 1 and 2 showed significant inhibition of α-glucosidase with IC50 values of 78.56 ± 7.28 and 14.74 ± 1.57 (μM), respectively, comparable with acarbose as a positive control (127.53 ± 1.73 μM); none of these compounds inhibited xanthine oxidase activity. Compounds 1 and 2 are promising candidates for the development of antidiabetic agents from natural sources. Graphical Abstract


Introduction
Vernonia amygdalina Del. (Asteraceae), also known as 'bitter leaf,' is a small shrub with dark green leaves and rough bark that is native to Africa.The leaves of this plant have been regarded as leafy vegetables and used in traditional medicine for the treatment of tonsillitis, fever, malaria, diabetes, pneumonia, jaundice, anemia, stomach problems, and ascariasis (Oyeyemi et al. 2018).V. amygdalina is a rich source of secondary metabolites, such as terpenoids, steroids, coumarins, flavonoids, phenolic acids, lignans, saponins, sesquiterpene lactones, tannins, and anthraquinones (Izevbigie 2003;Tona et al. 2004;Muraina et al. 2010;Oyeyemi et al. 2018;Okoduwa et al. 2021;Zhao et al. 2021).Among these, the stigmastane-type steroids are considered characteristic active components of V. amygdalina; they have a wide range of biological activities, including anti-inflammatory (Quasie et al. 2016;Nguyen et al. 2021), antidiabetic (Okon andUmoren 2017, Anh et al. 2021), antimalarial (Masaba 2000), and anti-tumor effects (Wong et al. 2013).Here, we describe the identification of six compounds, including two new compounds, from the leaves of V. amygdalina, along with their inhibitory activities against α-glucosidase and xanthine oxidase.
Compound 2 was isolated as a white amorphous solid.S1).Furthermore, an anomeric proton of the glucopyranosyl unit [δ H 5.02 (1H, d, J = 7.7 Hz, H-1′)] was observed in the 1 H NMR spectrum.The β configuration of this proton was determined by the large J 1 ′ ,2 ′ value coupling constant (J = 7.7 Hz) (Table S1); the absolute configuration was deduced as β-d-glucopyranoside by acid hydrolysis and comparison of the R f with authentic d-glucose (TLC, MC: MeOH:H 2 O = 8:5:1, R f = 0.3) (Vu et al. 2021).The HMBC correlation from δ H 5.02 to δ C 77.3 showed that the sugar moiety was connected to C-3 of aglycone.The 13 C NMR spectrum of 2 exhibited 35 carbon signals, of which 29 and 6 signals were ascribable to the steroidal aglycone and to β-d-glucose, respectively.The 1 H and 13 C NMR data of 2 revealed the typical Δ 7,9(11) stigmastane-type steroidal skeleton (Zhao et al. 2021) and were similar to 3 except for the absence of a hydroxy group at C-16 and a methoxy group at C-28, based on the obvious upfield shifts of C-16 (δ C 27.7) and C-28 (δ C 84.7) in 2 compared with C-16 (δ C 76.2) and C-28 (δ C 113.4) in 3 [5,6].The NOESY spectrum of 2 showed correlations from H-3 to H-5, from H-14 to H-17, and from H-18 to H-19 and H-20 suggesting the trans fusion of the rings A/B and C/D; α-orientation of H-3, H-5, H-14, and H-17; and β-orientation of H-18, H-19, and H-20.The NOESY cross-peaks from H-20 to H-18/H-21, from H-21 to H-18/H-26, and from H-26 to H-22/H-23/H-28 indicated that these protons were in the β configuration.The NOESY correlation between H-17 and H-14/H-29 showed that H-29 was in the α configuration.Therefore, the structure of compound 2 was elucidated as shown in Figure S1 and named vernonioside L.
α-Glucosidase is used in the treatment of type 2 diabetes mellitus (Kumar et al. 2011, Ha et al. 2018).α-Glucosidase inhibitors act by competitive inhibition of α-glucosidase at the brush borders of the intestinal epithelium.As a result, the digestion of complex carbohydrates is delayed and the absorption of glucose is shifted, thereby allowing the sluggish insulin secretion to 'catch up' with carbohydrate absorption (Hossain and Pervin 2018).In this study, the anti-α-glucosidase activities of compounds 1-6 were evaluated, using acarbose as a positive control.Among the compounds tested, the two new compounds 1 and 2 showed significant inhibitory effects on α-glucosidase with IC 50 values of 78.56 ± 7.28 and 14.74 ± 1.57 μM, respectively, which were stronger than the positive control (acarbose 127.53 ± 1.73 μM).These observations showed that compounds 1 and 2 have the potential for use in treatments for type 2 diabetes mellitus via the inhibition of α-glucosidase activity.However, further in vivo studies and clinical trials are required.
Xanthine oxidase is a key enzyme that catalyzes the last step in the conversion of purines to uric acid; it plays a vital role in the development of hyperuricemia and gout (Lund 2010).Allopurinol is a xanthine oxidase inhibitor prescribed for the treatment of gout.Therefore, the inhibitory effects of compounds 1-6 against xanthine oxidase activity were evaluated.As shown in Table S2, none of these compounds significantly inhibited xanthine oxidase activity.

Conclusion
Two new Δ 7,9(11) stigmastane-type steroidal saponins, vernonioside K and L, together with four known compounds: vernonioside B2 (3), vernoniacum B (4), vernonioside B1 (5), and vernoamyoside A (6) were isolated from the leaves of V. amygdalina.The structures of the new compounds were elucidated from one-dimensional NMR, two-dimensional NMR, and MS data.Compounds 1-6 were examined for inhibitory effects on α-glucosidase and xanthine oxidase.Novel compounds 1 and 2 exhibited significant anti-α-glucosidase activity with IC 50 values of 78.56 ± 7.28 and 14.74 ± 1.57 μM, respectively, which were comparable with the positive control (acarbose, 127.53 ± 1.73 μM).However, none of the examined compounds showed inhibition of xanthine oxidase.This study provided phytochemical evidence for further development of new anti-α-glucosidase lead compounds, along with investigations of their chemical modifications and mechanisms of action.