Pterocarpans and isoflavones from the root bark of Millettia micans and of Millettia dura

doi:10.1016/j.phytol.2017.07.012

Phytochemistry Letters

Volume 21, September 2017, Pages 216-220

Dedicated to the late Professor Joseph Magadula.

https://doi.org/10.1016/j.phytol.2017.07.012 Get rights and content

Highlights

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From the root bark of two Millettia species, two new pterocarpans and seven known flavonoids were isolated.
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The structures were elucidated by spectroscopy including theoretical quantum chemical CD calculation.
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Some of the compounds were evaluated for cytotoxicity, antiplasmodial and translation inhibitory activities.

Abstract

From the CH₂Cl₂/CH₃OH (1:1) extract of the root bark of Millettia micans, a new pterocarpan, (6aR,11aR)-3-hydroxy-7,8,9-trimethoxypterocarpan (1), named micanspterocarpan, was isolated. Similar investigation of the CH₂Cl₂/CH₃OH (1:1) extract of the root bark of Millettia dura gave a further new pterocarpan, (6aR,11aR)-8,9-methylenedioxy-3-prenyloxypterocarpan (2), named 3-O-prenylmaackiain, along with six known isoflavones (3-8) and a chalcone (9). All purified compounds were identified by NMR and MS, whereas the absolute configurations of the new pterocarpans were established by chriptical data analyses including quantum chemical ECD calculation. Among the isolated constituents, calopogonium isoflavone B (3) and isoerythrin A-4′-(3-methylbut-2-enyl) ether (4) showed marginal activities against the 3D7 and the Dd2 strains of Plasmodium falciparum (70–90% inhibition at 40 μM). Maximaisoflavone B (5) and 7,2′-dimethoxy-4′,5′-methylenedioxyisoflavone (7) were weakly cytotoxic (IC₅₀ 153.5 and 174.1 μM, respectively) against the MDA-MB-231 human breast cancer cell line. None of the tested compounds showed in-vitro translation inhibitory activity or toxicity against the HEK-293 human embryonic kidney cell line at 40 μM.

Graphical abstract

Introduction

The genus Millettia (family Leguminosae, subfamily Papilionoideae) with approximately 260 species is widespread in Africa (139 species) and Asia (121 species) (Banzouzi et al., 2008). Out of the East African countries, the highest number of Millettia species, 25, is indigenous to Tanzania, followed by 6 species native to Kenya (Banzouzi et al., 2008). The genus is a rich source of secondary metabolites such as chalcones, isoflavones, rotenoids (Dagne et al., 1989, Yenesew et al., 2003), isoflavans (Khalid and Waterman, 1983), flavanones, coumarins (Baruah et al., 1984) and pterocarpans (Sritularak et al., 2002). Some of these metabolites inhibit nitric oxide formation or possess larvicidal, pesticidal, cytotoxic, anti-inflammatory, antimicrobial and cancer chemopreventive activities (Banzouzi et al., 2008).

Millettia dura (Dunn), growing in both Tanzania and Kenya, is used as food for livestock, as a source of firewood and charcoal, and as timber for construction since it is tough and resistant to termites (Orwa et al., 2009). Traditionally, in various parts of Africa, it is used to treat hernia, diarrhea, menstrual irregularities and for healing wounds (Banzouzi et al., 2008). Its seeds, seed pods, stem and root bark have been reported to contain rotenoids and isoflavones (Dagne et al., 1991, Derese et al., 2003, Ollis et al., 1967, Yenesew et al., 1996, Yenesew et al., 1997). In contrast, Millettia micans (Taub), a vulnerable small tree endemic to Tanzania, has no documented traditional uses and has not yet been phytochemically explored.

Here, we report the isolation and characterization of two new pterocarpans, named micanspterocarpan (1) and 3-O-prenylmaackiain (2), from the root bark of Millettia micans and that of Millettia dura, respectively. From the roots of Millettia dura, the known compounds (3-9) were also identified. The antiplasmodial and cytotoxicty profiles of some of the isolated compounds are also presented.

Section snippets

Results and discussion

Column chromatographic separation of the CH₂Cl₂/CH₃OH (1:1) extract of the root bark of Millettia micans resulted in the isolation of a new pterocarpan (1, Fig. 1). The HRMS (EI) spectrum indicated the molecular formula C₁₈H₁₈O₆ (m/z obs, 330.1115 [M]⁺, calcd 330.1103), whereas the UV absorption maxima at 268, 269 and 281 nm, and the ¹H NMR (H-6ax, H-6eq, H-6a, H-11a) and ¹³C NMR (C-6, C-6a, C-11a) spectral data (Table 1) are consistent with a pterocarpan skeleton (Tanaka et al., 1997, Yenesew

General

Melting points were obtained on a Büchi Melting point B-545 instrument. NMR spectra were acquired on Varian MR-400, Bruker Avance III 600 and Bruker Avance III HD 800 MHz spectrometers. All spectra were processed using MestReNova 10.0 using the residual solvent peak as indirect chemical shift reference. HRMS (EI) spectra were obtained on a Micromass GC-TOFmicro mass spectrometer (Micromass, Wythenshawe, Waters Inc., UK), using direct inlet and 70 eV ionization voltage. LC–MS (ESI) chromatograms

Conclusions

Two new pterocarpans were characterised from the CH₂Cl₂/CH₃OH (1:1) root bark extracts of Millettia micans and Millettia dura, along with seven known compounds from the latter extract. Marginal antiplasmodial activity was observed for calopogonium isoflavone B (3), isoerythrin A-4′-(3-methylbut-2-enyl) ether (4), maximaisoflavone B (5) and 7,2′-dimethoxy-4′,5′-methylenedioxyisoflavone (7) against the chloroquine-sensitive (3D7) and -resistant (Dd2) strains of Plasmodium falciparum. Whereas 5

Acknowledgments

We are grateful to Mr. P. C. Mutiso of the Herbarium, Botany Department, University of Nairobi and Mr. F. Mbago, the botanist from the Department of Botany, University of Dar es Salaam, for the identification of the plant species used in this study. M.M. and T.D. are grateful to the Natural Products Research Network for Eastern and Central Africa (NAPRECA) and the German Academic Exchange Service (DAAD) for a scholarship. The Swedish Research Council (Swedish Research Links, 2012-6124), the

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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.
Rotenoids and isoflavones from the leaf and pod extracts of Millettia brandisiana Kurz
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M. extensa (Benth.) Baker and M. dura Dunn have been used to treat wound healing infections (Sharma et al., 2014; Marco et al., 2017), while M. dielsiana Harms ex Diels has been used for rheumatoid arthritis (Dat et al., 2019). Several Millettia species, including M. brandisiana Kurz, M. dorwardii Collett & Hemsl., and M. pinnata (L.) Panigrahi have been used for the treatment of anti-inflammation (Bora et al., 2014; Pailee et al., 2019; Chen et al., 2018).
Phytochemical investigations of the leaf and pod extracts of Millettia brandisiana Kurz led to the isolation and identification of four previously undescribed rotenoids, (–)-(6aS,12aS)-millettiabrandisins A–C and (–)-(6aS,12aS)-6-deoxyclitoriacetal, two previously undescribed isoflavones, millettiabrandisins D and E, and 20 known compounds. The structures of previously undescribed compounds were determined on the basis of NMR and MS data. The absolute configurations of (–)-(6aS,12aS)-millettiabrandisins A–C were determined from the comparison of their experimental and calculated ECD spectra. (–)-(6aR,12aR)-12a-Hydroxy-α-toxicarol was also confirmed by X-ray crystallographic data. Some isolated compounds were evaluated for their cytotoxicity against three cancer cell lines, including lung cancer (A549), colorectal cancer (SW480), and leukemic cells (K562). Of these, α-toxicarol displayed the best cytotoxicity against lung cancer (A549) and leukemic cells (K562) with the IC₅₀ values of 104.4 and 67.5 μM, respectively. 6″,6″-Dimethylchromene-[2″,3″:7,8]-flavone showed the highest cytotoxicity against colorectal cancer (SW480) with an IC₅₀ value of 97.2 μM.
Phytochemical analysis and preclinical toxicological, antioxidant, and anti-inflammatory evaluation of hydroethanol extract from the roots of Harpalyce brasiliana Benth (Leguminosae)
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Another result observed was that two compounds isolated from the extract of the root bark of Millettia dura (Leguminosae), maximaisoflavone B and 7,2-dimethoxy-4,5′-methylenedioxyisoflavone, demonstrated cytotoxicity effect against cells originated from human breast cancer (MDA-MB-231) presenting IC50 of 153.5 and 174.1 μM, respectively. On the other hand, those bioactive compounds did not cause significant cytotoxicity in normal cells originated from human embryonic kidney (HEK-293), demonstrating to be safe in normal cells and cytotoxic for cancer cells (Marco et al., 2017), corroborating with our results. The extract no evidence of cytotoxic or hemolytic activities at the concentrations evaluated, the next step was to evaluate its acute toxicity in mice.
Harpalyce brasiliana Benth (Leguminosae) is a shrub endemic to Brazil, popularly known as “snake's root.” This species is used in folk medicine for the treatment of inflammation and snakebites. However, up to now there is no scientific research to justify its popular use. The study aimed to characterize the phytochemical profile of the hydroethanol extract from the roots of H. brasiliana (Hb), to evaluate its antioxidant and anti-inflammatory potential, as well as to investigate its cytotoxicity and acute toxicity.
The extract was obtained by maceration method using a solution of ethanol:water (70: 30, v/v). The phytochemical profile was obtained by liquid chromatography coupled to mass spectrometry. The cytotoxicity of extract (31–2000 μg/mL) was evaluated in vitro, by the 3-methyl-[4-5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method using murine macrophage and fibroblast cell lines (RAW 247.6 and 3T3, respectively) and by the hemolytic assay. For the in vivo acute toxicity, the extract (2000 mg/kg) was administered and after 14 days the weight (body and organs) and hematological and biochemical parameters were analyzed. Chemical free radical scavenging effect of the extract (125–2000 μg/mL) was investigated through diphenylpicryl hydrazine reduction, total antioxidant capacity, reducing power, hydroxyl radical scavenging, and iron and copper chelating assays. In vitro anti-inflammatory effect of the extract (125, 500, and 2000 μg/mL) was demonstrated through of nitric oxide (NO) analyzed in lipopolysaccharides stimulated RAW 264.7 cells. In vivo anti-inflammatory activities were evaluated in carrageenan-induced paw edema and zymosan-air-pouch models, with gavage administration (post-treatment) of extract at 100, 200, and 400 mg/kg. For the first animal model, the anti-edematogenic activity and myeloperoxidase (MPO) levels were investigated, while in the zymosan-air-pouch model the leukocyte number, MPO, total protein and pro-inflammatory cytokine (IL-1β, IL-6, and TNF-α) levels were quantified. In addition, the oxidative parameters such as malondialdehyde (MDA) and reduced glutathione (GSH) were determined.
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A new isoflavone glucoside and other compounds from Poiretia bahiana C. Mueller: Chemophenetics, fragmentation pattern and biogenetic implications
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This compound, obtained as a colorless crystal (mp 154-155 °C), was optically active [α]D –66.7 (c 0.07, MeOH). The known compounds (2–15) were identified by comparison of their NMR spectral data (Supplementary Figs. 12–55) with the literature [15–28]. However, the fragmentation pattern of isoflavones have drawn great attention, especially for large-scale metabolite profiling studies [13,29,30].
The genus Poiretia belongs to the Fabaceae (Leguminosae) family and it encompasses twelve species of flowering plants. The chemistry of this genus is scarcely investigated, although some studies have demonstrated the potential of Poiretia species to produce important bioactive compounds. Herein, we describe the phytochemical investigation of P. bahiana C. Mueller leaves. A new isoflavone glucoside named as 2′,4′,5′-trimethoxyisoflavone-7-O-β-D-glucopyranoside (1), along with six known isoflavones (2–7), two rotenones (8–9), cyclitol 3-O-methyl-chiro-inositol (10), the amino acid proline (11), a mixture of sitosterol (12) and stigmasterol (13), and a mixture of the triterpenes lupeol (14) and β-amirine (15) were obtained from P. bahiana leaves. The structures were established by extensive analysis of their spectroscopic data, which included ¹H NMR, ¹³C NMR, DEPT, and 2D-NMR (¹³C¹H HETCOR and ¹³C¹H COLOC). Two isoflavones (3 and 5) and two rotenones (8–9) exhibited antifungal activity against the plant pathogenic fungus Cladosporium sphaerospermum. Furthermore, the biogenetic implications of the oxygenation pattern of the B-ring of the isoflavones, and the chemophenetics and fragmentation pattern of the isoflavones and rotenones are discussed.
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The genus Millettia (Fabaceae) is represented by six species in Kenya namely: M. dura, M. leucantha, M. lasiantha, M. usaramensis ssp. usaramensis, M. oblata, and M. tanaensis (Maundu and Tengnäs, 2005). In our previous phytochemical investigation of Kenyan Millettia species, we reported chalcones (Buyinza et al., 2019; Deyou et al., 2015; Yenesew et al., 1998), flavanones (Deyou et al., 2015), flavanonols (Deyou et al., 2015), flavonols (Buyinza et al., 2019), isoflavones (Buyinza et al., 2019; Derese et al., 2003, 2014; Deyou et al., 2017; Marco et al., 2017; Yenesew et al., 1996, 1997, 1998, 2003a), pterocarpans (Marco et al., 2017) and rotenoids (Deyou et al., 2015; Yenesew et al., 1998, 2003b). In continuation of our study on Kenyan Millettia species, herein we report the first phytochemicals (Table 1, Fig. 1) from M. lasiantha (Fig. 2) a plant traditionally used in Kenya as an aphrodisiac (Kokwaro, 2009).
A review on genus Millettia: Traditional uses, phytochemicals and pharmacological activities
2020, Saudi Pharmaceutical Journal
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The CH2Cl2/MeOH (1:1) extract of the stem bark of M. oblate ssp. teitensis was evaluated, which confirmed about the in vitro antiplasmodial activity using non-radioactive assay against the chloroquine-sensitive Sierra Leone 1 (D6) and chloroquine-resistant Indochina 1 (W2) strains of P. falciparum with an IC50 value of 12.0 ± 1.2 and 10.0 ± 2.3 μM each (Derese et al., 2014). The antiplasmodial activity of CH2Cl2/CH3OH (1:1) extract of the root bark of M. dura Dunn was investigated and the minimal activity was found for compounds viz. calopogonium isoflavone B, maximaisoflavone B and 7, 2′-dimethoxy-4′, 5′-methylenedioxyisoflavone against the chloroquine-sensitive 3D7 and the chloroquine-resistant Dd2 P. falciparum strain where the activity found in between 70 and 90% inhibition at 40 μM (Marco et al., 2017). A novel polysaccharide fraction (MSCP2) was extracted and isolated from the roots of M. Speciosa Champ.
The genus Millettia belongs to Fabaceae includes 200 species which are distributed in tropical and subtropical regions of the world. Plants belong to this genus are used as folkloric medicine, for the treatment of different ailments like in wound healing, boil, sores, skin diseases, snake bite, muscle aches, pains, rheumatic arthritis, and gynaecological diseases. The aim of the review is to provide updated, comprehensive and categorized information on the aspects of ethnobotanical, phytochemical, pharmacological uses and toxicity of genus Millettia in order to identify their therapeutic potential and generate space for future research opportunities. The present study comprises of isolated flavonoids, phenolic compounds, phytosterols, saponins, alkaloids, polysaccharides, terpenoids and resins and pharmacological activities of various Millettia species. The relevant data were searched by using the keyword “Millettia” in different scientific databases like, “Google Scholar”; “NISCAIR repository”; “Pub Med”; “Science Direct”; “Scopus” and the taxonomy is validated by “The Plant List”. This review discusses the existing information of the traditional evaluation as well as phytochemical and pharmacological evaluation of the extract and active constituents of the genus “Millettia”. This review confirms that several Millettia species have emerged as a high-quality medicine in a traditional system for arthritis, wound healing, inflammation, skin diseases. Numerous conventional uses of Millettia species have been validated by modern pharmacology research. Intensive investigations of the genus Millettia relating to phytochemistry and pharmacology, especially their mechanism of action, safety, and efficacy could be the future research interests by the researcher in the area of phytomedicine.

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Short communicationPterocarpans and isoflavones from the root bark of Millettia micans and of Millettia dura

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Results and discussion

General

Conclusions

Acknowledgments

Phytochemistry

Chem. Phys. Lett.

Phytochemistry

Phytochemistry

Tetrahedron

Phytochemistry

Phytochemistry

Phytochemistry

Phytochemistry

Phytochemistry

Phytochemistry

Chiroptical properties from time-dependent density functional theory. I. Circular dichroism spectra of organic molecules

J. Chem. Phys.

Traditional uses of the African Millettia species (Fabaceae)

Int. J. Bot.

Density-functional thermochemistry III. The role of exact exchange

J. Chem. Phys.

Short communication
Pterocarpans and isoflavones from the root bark of Millettia micans and of Millettia dura