Cytotoxic effect and antioxidant activity of pterocarpans from Millettia aboensis root

Abstract Chemical analysis of the methanol extract of the root bark of Millettia aboensis led to the isolation of homopterocarpin (1), secundiflorol I (2), and maackain (3). The structures of these compounds were elucidated based on their MS and NMR spectra. The crude methanol root extract was screened for its cytotoxic activity on mouse lymphoma cell line (L5178Y), and the isolated compounds were tested for their antioxidant activity using a 2, 2-diphenylhydrazyl (DPPH) radical scavenging model. The crude methanol root extract gave a percentage growth inhibition of 87.5% on the mouse lymphoma cell line (L5178Y). Compound 3 gave the highest antioxidant activity with an IC50 of 83 µg/ml. These compounds can serve as leads for anticancer agents. Graphical Abstract


Introduction
Natural products of African origin have been shown to contain novel therapeutic compounds (Okoye et al. 2015). These novel compounds have great use as anticancer candidates e.g. echitamine, eugenol and 1,2-benzene dicarboxylic acid from Alstomia boonei; methyl gallate, anacardicin, zoapatanolide A and agathisflavone from Anacardium occindentale; ellagic acid, castalagin and flavogallonic acid from Anogeissus leiocarpus and more than 50% of these candidates are currently utilized for treatment and curation of diseases (Newman and Cragg 2016;Santos et al. 2016;Ohiagu et al. 2021). Compounds isolated from natural sources have been screened daily for anticancer leads and these compounds have always proven to be cytotoxic (Fregene and Newman 2005;Baliga and Dsouza 2011;Zingue et al. 2016). Millettia aboensis, a member of the Leguminosae family with approximately 260 species worldwide and 139 species in Africa, is one of these medicinal plants with a history of disease curing (Banzouzi et al. 2008;Marco et al. 2017;Ikuesan et al. 2020). Several species in this genus has been utilized in alternative medicine for curing illnesses such as rheumatic arthritis, skin diseases, cardiovascular diseases, and gynecological diseases (Ngamga et al. 2007;Huan et al. 2016). Also, they have been shown to be potent sources of bioactive natural products like coumarins, pterocarpans, flavanones, rotenoids, chalcones, flavones, and isoflavones (Marco et al. 2016). The genus Millettia has been confirmed as a means for the treatment of cancer or to be cytotoxic (Ito et al. 2004;Rayanil et al. 2011;Zingue et al. 2016). The chemistry of M. aboensis has been studied previously. Previous reports indicate the isolation of orobol 8-C-glucoside, genistein 6-C-glucoside, isopranetin 8-C-glucoside, genistein 8-C-glucoside, 3,3-phenylchromen-4-one, peniciaculin B, epicathechin-(2b!O!7, 4b!8)-cathechin, epicathechin-(2b!O!7, 4b!8)epicathechin, etc. Ajaghaku et al. 2020;Ezeh et al. 2020;Ikuesan et al. 2020). It has been observed that all pterocarpanoids are mostly isolated from plants whose genus is Leguminosae (Fabaceae) (Al-Hamini and Alkhathlan 2000). Hence, this led to the isolation, characterization, cytotoxic effect and antioxidant screening of these pterocarpans resident in M. aboensis since it is of the genus Leguminosae (Fabaceae).

Results and discussion
The secondary metabolites from the root of M. aboensis with voucher specimens deposited in the herbarium section of the Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, with specimen number PCG/474/A/021 were extracted exhaustively using methanol. The crude methanol extract inhibited the growth (87.5%) at 10 mg/ml of mouse lymphoma cell line (L5178Y) (Sigma), which was grown in Eagle's minimal essential medium supplement with 10% horse serum in roller tube culture. Three bioactive compounds were isolated from the ethyl acetate fraction of the methanol extract of M. aboensis. These compounds were obtained from the semi-preparative HPLC gradient elution using methanol and water, after rigorous chromatographic separations on silica gel and Sephadex LH-20. This gave rise to three known pterocarpans: homopterocarpin (1), secundiflorol I (2), and maackain (3) (Figure 1).
Compound 1 was isolated as a yellow oil. The molecular formula was deduced as C 17 H 16 O 4 based on the observed ESI-MS molecular ion peak at m/z 269.4 [M-CH 3 ] þ . It showed UV maxima at 216.0, 229.6, and 283.3 nm, which is suggestive of a pterocarpan nucleus and [a] 20 D À47.17 (c, 0.6, MeOH). The proton NMR spectrum of 1 showed features very similar to the previously reported homopterocarpin (Maekawa and Kitao 1970;Higgins and Ingham 1981;Hemaa et al. 2011). These signals include the aromatic signals of the characteristic ABX coupling pattern at d 7.25 (d, J ¼ 8.3 Hz), 6.53 (dd, J ¼ 8.3, 2.2 Hz), and 6.37 (d, J ¼ 2.2 Hz) assigned to H-5, H-6, and H-8, and another aromatic ABX coupling pattern at d 7.37 (dd, J ¼ 8.5, 0.6 Hz), 6.63 (dd, 8.5, 2.5 Hz), and 6.43 d, J ¼ 2.6 Hz) assigned to H-6 0 , H-5 0 , and H-3 0 . The 1 H NMR spectrum of compound 1 also revealed two methine proton signals at d 5.24 (br s, H-4), and 3.83 (m, H-3), two diastereotopic methylene proton signals at, d 3.95 (d, Ha-2) and 4.14 (dd, Hb-2); two methoxy singlets at 3.75 (s, 7-OCH 3 ) and 3.76 (s, 4 0 -OCH 3 ). The attachments of the methoxy groups at positions C-7 and C-4 0 were supported by the observed cross peaks of 3.75 (7-OCH 3 ) with C-7 (161.4) and 3.76 (4 0 -OCH 3 ) with C-4 0 (161.7) in the HMBC spectrum. The observed cross peaks of the three protons at d H 5.24, 4.14, and 3.95 with d C 156.18 indicate d C 156.18 belongs to C-9. Based on the above information, and the 13 C NMR spectral data as shown in Table S1, compound 1 was characterized as homopterocarpin. This is the first report ever of the isolation of homopterocarpin from the root of M. aboensis.
Compound 2 was isolated as brown needles. Its observed ESI-MS molecular ion peak at m/z 301.4 [M þ H] þ , 16 amu larger than that of compound 1, indicates a gain of an oxygen atom and hence a molecular formula of C 17 H 17 O 5 þ . It showed UV maxima at 211.1, 287.6, and 295.3 nm, which is suggestive of a pterocarpan nucleus and [a] 20 D À47.17 (c, 0.6, MeOH). The proton NMR spectrum of 2 showed features very similar to that of compound 1. These signals include the aromatic signals of characteristic ABX coupling pattern assigned to d 7.28 (d,J ¼ 8.4 Hz,6.49 (dd,J ¼ 8.4,2.4 Hz,and 6.30 (d,J ¼ 2.4 Hz,. However, the presence of two meta coupled aromatic singlets at d 6.98 (s, H-3 0 ) and 6.52 (s, H-3), in place of the usual ABX COSY system of the second aromatic ring, suggested a further substitution on this aromatic nucleus as found in a previously reported secundiflorol I (Tanaka et al. 1998;Tian and McLaughlin 2000). The 1 H NMR spectrum of compound 2 (Table S2) also revealed two methine proton at d 5.46 (d, J ¼ 6.5 Hz, H-4), and 4.25 (m, Ha-3), two diastereotopic methylene proton signals at d 3.58 (d, J ¼ 10.8 Hz, Ha-2) and 3.74 (d, J ¼ 2.4 Hz, Hb-2) and two methoxy groups at 3.79 (s, H-5 0 ) and 3.79 (s, H-4 0 ). Compound 2 was thus elucidated as the previously reported secundiflorol I (Tanaka et al. 1998;Tian and McLaughlin 2000).
Compound 3 was isolated as a yellow oil. The molecular formula was deduced as  (Table S2) showed features very similar to that of compound 2. These signals include the aromatic signals of ABX coupling pattern assigned to d 7. 26 (m,6.48 (dd,J ¼ 8.4,2.4 Hz,and 6.30 (d,J ¼ 2.4 Hz,, and two meta-coupled aromatic protons at d 6.81 (s, H-3 0 ) and 6.38 (d, J ¼ 1.5 Hz, H-6 0 ). The 1 H NMR spectrum of compound 3 also revealed the presence of two methine proton at d 5. 45 (d,J ¼ 6.6 Hz,and 4.22 (m,, and two diastereotopic methylene protons at d 3.53 (m, Ha-2) and 3.91 (m, Hb-2). However, the major differences are the absence of two methoxy singlets and the presence of a highly deshielded methylene proton signals at d 5.88 (d, J ¼ 1.1 Hz, Ha-1 0 ) and 5.85 (d, J ¼ 1.1 Hz, Hb-1 0 ), which suggest the presence of a methylenedioxy (OCH 2 O) moiety. Compound 3 was thus elucidated as the previously reported maackain (Kinoshita et al. 1990).
To the best of our knowledge, this is the first report ever of the isolation of compounds 1, 2, and 3 from the root of M. aboensis.
All the pterocarpans isolated in this study had similar negative [a] D values, thus suggesting the same biogenetic origin. Most other pterocarpans isolated from natural sources always have negative [a] D values (Al-Hamini and Alkhathlan 2000).
The three compounds exhibited good antioxidant activity (Table 1), with compound 3 being the most active. The activity of compound 3 was comparable to that of the standard drug, ascorbic acid. This is in agreement with a study carried out by Bhandarkar et al. 2015.
Natural product compounds have been shown to offset cancer basically through antioxidant influence. No wonder these compounds, apart from being cytotoxic, they also have antioxidant activity (Al-Dabbagh et al. 2018)

Conclusion
The current analysis showed that the methanol root extract of M. aboensis possesses strong cytotoxic effects against mouse lymphoma cell line (L5178Y). Also, the isolation of three pterocarpans confirmed that the root has cytotoxic effects of therapeutic importance. The antioxidant properties were in agreement with the cytotoxicity of the extract. The research is suggestive of a new lead toward the use of M. aboensis as an anticancer agent.

Funding
The author(s) reported there is no funding associated with the work featured in this article.