Background

Millettia pyrrhocarpa Mattapha, Forest & Hawkins, sp. Nov. belongs to the family Fabaceae: Faboideae and is found in Nakorn Nayok province, Thailand. M. pyrrhocarpa is a woody climber plant that is known in Thai as “Nang Rong” [1]. Previous phytochemical analyses of the Milletia genus revealed that various secondary metabolites were present, such as alkaloids [2], coumarins [3], flavonoids [4], isoflavonoids [5], phenols [6], phytosterols [2], rotenoids [7–10], and triglycerides [11]. Among these, rotenoids are the most common in this genus [12], as observed in the leaves of Millettia oblate ssp. teitensis [13], Millettia brandisiana KURZ [14] and in the root bark of Millettia usaramensis [15] and Millettia speciosa [16]. Pharmacological investigation of the Millettia genus revealed that the crude extract and isolated compounds showed antimicrobial [2, 17, 18], antioxidant [2, 18], antiplasmodial [7, 10], immunomodulatory [19], anti-cholinesterase [20, 21], anthelmintic [22], anti-inflammatory [5, 23], antidiabetic [24], cytotoxic [6, 16, 20], and anticancer [6, 20, 25] activities. Since the phytochemical study and biological activity of M. pyyrhocarpa has not been reported, it is of great interest to study the secondary metabolites in the leaves and twigs of M. pyyrhocarpa and their antibacterial, anti-HIV1-RT, and cytotoxic activities. The preliminary evaluation of the bioactivity of the leaves and twigs of M. pyyrhocarpa might lead to the discovery of important substances that exhibit promising biological activity and potential to be developed for medicinal applications in the future.

Material

Plant material

Leaves and twigs of Millettia pyrrhocarpa were collected from Nakhon Nayok Province, Mueang district, Hin Tang subdistrict, Khao Yai National Park, Nang Rong waterfall in Thailand and identified by Dr. Narong Nantasean. Voucher specimens of M. pyrrhocarpa (M. pyrrhocarpa Mattapan, & BKF staff 1139B holotype BKF) were deposited at the Department of Chemistry, Faculty of Science and Center of Innovation in Chemistry, Mahidol University, Rama VI Road, Bangkok 10400, Thailand.

Cells line materials KKU-M213 (human cholangiocarcinoma) cells were kindly provided by Dr. Banchob Sripa from Liver Fluke and Cholangiocarcinoma Research Center, Department of Pathology, Faculty of Medicine, Khon Kean University. MMNK1 (human cholangiocyte) cells were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB, Osaka, Japan). FaDu (human hypopharyngeal carcinoma), HT29 (human colorectal adenocarcinoma), MDA-MB-231 (human mammary gland/breast adenocarcinoma), SH-SY5Y (human neuroblastoma), A549 (human lung carcinoma), and HepG2 (human hepatocellular carcinoma) cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Methods

General experimental procedures

Column chromatography (CC) was carried out on silica gel 60 H from E. Merck. 70–230 mesh ASTM, cat. No. 7734 and No.7736. Thin-layer chromatography (TLC) separations were carried out on silica gel 60 PF254 on aluminium sheets, and the isolated compounds were identified under ultraviolet light. Infrared spectra (IR) were recorded as KBr pellets using a Shimadzu 8900 FT-IR spectrophotometer. Melting points were recorded on a Büchi 322 micro melting point apparatus and are uncorrected. Mass spectra were recorded on a Thermo Finnigan Polaris Q mass spectrometer at 70 eV (probe), and EIMS was measured by a Brüker Esquire apparatus. X-ray absorption spectroscopy was carried out on a Bruker D8 QUEST CMOS PHOTON II. 1H (500 MHz), 13C (125 MHz), and 2D NMR spectra were recorded on a Bruker AV-500 spectrometer in deuterated chloroform (CDCl3) or deuterated methanol (CD3OD) solutions, and TMS was used as the internal standard.

Extraction and isolation

Air-dried powders of the leaves and twigs of M. pyrrhocarpa (2.8 kg) were extracted successively with hexane, EtOAc, and MeOH. Twelve litres of solvent were used in the extraction at room temperature for three days in triplicate. The solvents were filtered and evaporated under reduced pressure to give crude hexane (41.0 g), EtOAc (118.0 g), and MeOH (216.0 g) extracts. The crude hexane extract was fractionated using silica gel column chromatography (CC) using a gradient system of hexane–EtOAc and EtOAc–MeOH to generate ten fractions (A1-A10). Fraction A5 (13.6 g) was fractionated on a silica gel column and eluted with a gradient system of hexane–EtOAc and EtOAc–MeOH to give three subfractions (B1-B3). Subfraction B2 (5.1 g) was fractionated on a silica gel column and eluted with a gradient system of hexane–ethyl acetate and EtOAc–MeOH to give four subfractions, C1-C4. Subfraction C1 (0.06 g) was recrystallized with ethanol:ethyl acetate (2:1) to give compound (1) (70 mg) as a white solid. Subfraction C3 (0.41 g) was recrystallized with ethanol:ethyl acetate (2:1) to afford compound (3) (40 mg) as a white solid. The crude EtOAc extract (118.0 g) was fractionated on a silica gel column and eluted with a gradient system of hexane–EtOAc and EtOAc–MeOH to afford nine fractions (D1-D9). Fraction D2 (23.3 g) was fractionated on a silica gel column and eluted with a gradient system of hexane–ethyl acetate and EtOAc–MeOH to produce seven subfractions (E1-E7). Subfraction E4 (3.0 g) was recrystallized with ethanol:ethyl acetate (2:1) to generate compound (1) (86 mg) as a white solid. The crude MeOH extract (216.1 g) was fractionated on a silica gel column and eluted with a gradient system of hexane–EtOAc and EtOAc–MeOH to generate nine fractions (G1-G9). Fraction G3 (1.3 g) was recrystallized with ethanol to afford compound (2) (36 mg) as a white solid.

X-ray diffraction

The colourless plate of compound (2) was suitable for single-crystal X-ray diffraction with a size of 0.32 × 0.28 × 0.04 mm. The unit cell parameters and intensity data were recorded on a Bruker D8 QUEST CMOS PHOTON II diffractometer equipped with a graphite-monochromator Mo-Kα (λ = 071,073 Å) radiation at 296(2) K. Data reduction was performed using SAINT, and the SADABS-2016/2 scaling algorithm [26] was used for absorption correction. The structure was solved with the ShelXT structure solution program using combined Patterson and dual-space recycling methods [27]. The structure was refined by least squares using ShelXL [28]. All non-H atoms were refined anisotropically. The hydrogen atoms of solvent molecules were positioned geometrically with C—H = 0.93–0.98 Å and refined using a rigid model with fixed displacement parameters Uiso (H) = 1.5Ueq (C) for methyl groups and 1.2Ueq (C) for the other groups. The O–H hydrogen atoms were located on difference Fourier maps but refined with O–H = 0.82 ± 0.02 Å with Uiso (H) = 1.5Ueq (O). C19H20O6, FW = 344.35, orthorhombic space Group P21P21P21, unit cell dimensions a = 6.40000(10) Å, b = 9.9684(2) Å, c = 25.9394(6) Å, V = 1654.88(6) Å3, Z = 4, dcalcd = 1.382 g/cm3, µ = 0.103 mm−1, F(000) = 728. The 46671 measurements yielded 6568 independent reflections after equivalent data were averaged. The final refinement gave R1 = 0.0393 and wR2 = 0.0896 [I > 2σ(I)]. The crystallographic data of the compound have been deposited in the Crystallographic Open Database (COD) number 3000415. The molecular graphic was illustrated by ORTEP [29].

Antibacterial activity

Bacterial strains

The study on in vitro antibacterial activity was carried out against nine strains (S. aureus ATCC 25923 DMST 8840, E. aerogenes ATCC13048 DMST 8841, E. coli O157: H7 DMST 12743, E. coli Enterotoxigenic, ETEC DMST 30543, E. coli Enteropathogenic, EPEC DMST 30546, S. typhimurium ATCC 13311 DMST 562, S. flexneri DMST 4423, P. mirabilis DMST 8212, and V. cholera nonO1/nonO139 DMST 2873). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the crude extracts and isolated compounds were evaluated according to standard methods described in previous literature [30].

Anti-HIV1-RT and cell-based assay for anti-HIV-1

Anti-HIV1-RT assays of the crude extracts and isolated compounds were evaluated according to the standard methods described in previous literature [31–33]. Cell-based assays for anti-HIV-1 RT of the crude extracts and isolated compounds were evaluated according to the standard methods described in previous literature [34, 35]

Cytotoxicity assay

Cytotoxicity assays of the crude extracts and isolated compounds were evaluated according to the standard methods described in previous literature [36, 37].

Results

Isolation and purification

The crude hexane, ethyl acetate, and methanol extracts of the leaves and twigs of M. pyrrhocarpa were subjected to repeated chromatography over silica gel 60 and silica gel 60 PF254 to yield three pure compounds, (1–3). Compounds (1–3) were identified as elliptinol (1) [38, 39], munduserol (2) [40], and dehydromunduserone (3) [40] by comparison of their spectral data with those in the literature. The 1H and 13C-NMR data of. The 1H and 13C-NMR data of (1), (2), and (3) are shown in Table 1.

Table 1 1H and 13C data for compounds (1–3) (500 MHz, 125 MHz, CDCl3 or CD3OD, δ in ppm)
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6aS, 12aS, 12S-Elliptinol (1): white solid (EtOH:EtOAc); mp = 230 − 231 °C;  \({\left[\alpha \right]}_{589}^{26.9}\) : + 173.36 (c 0.38, CHCl3); UV (MeOH) λmax (log ε) 235 (1.37), 281 (1.24), 341 (1.09), 447 (1.35), 494 (0.93), 671 (0.91) nm; IR (KBr) υmax 3470, 3102, 2969, 2938, 1620, 1586, 1502, 1273, 1253, 1190, 1142, 1119, 1092, 1041, 989 cm−1; for 1H and 13C NMR spectroscopic data, see Table 1. EIMS m/z 340.31 (base peak), 226.22, 81.05.

6aS, 12aS, 12S-Munduserol (2): white solid (EtOH:EtOAc); mp = 240 − 241 °C; \({\left[\alpha \right]}_{589}^{\text{26.}{2}}\) : + 160.03 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 231 (1.73), 450 (1.33), 507 (1.23), 680 (1.19) nm; IR (KBr) υmax 3460, 2918, 2851, 1655, 1637, 1618, 1508, 1292, 1157, 1045 cm−1; for 1H and 13C NMR spectroscopic data, see Table 1. EIMS m/z 345.31 (M + H)+, 192.17 (base peak).

Dehydromunduserone (3): white solid (MeOH); mp = 202–204 °C [lit. 41, mp = 210 − 211 °C]; UV (MeOH) λmax (log ε) 231 (1.59), 275 (1.46), 306 (1.36), 356 (1.12), 457 (1.01), 507 (0.92), 677 (0.90) nm; IR (KBr) υmax 3441, 3098, 2999, 2955, 2837, 1761, 1726, 1634, 1601, 1566, 1508, 1452, 1441, 1408, 1290, 1246, 1199, 1163, 1105, 1049, 795 cm−1; for 1H and 13C NMR spectroscopic data, see Table 1. EIMS m/z 354.37, 192.29 (base peak), 179.31.

Antibacterial activity

The three crude extracts and isolated compounds (1–3) were found to inhibit the growth of V. cholerae with MIC/MBC values of 12.5/25 mg/mL, while the EtOAc extract inhibited the growth of E. coli (ETEC), E. coli (EPEC), and S. flexneri with MIC/MBC values of 6.25/6.25 mg/mL, and the MeOH extract was found to inhibit the growth of E. coli (ETEC) with MIC/MBC values of 6.25/6.25 mg/mL. The isolated compounds (1–3) were found to inhibit the growth of nine bacterial strains with MIC/MBC values of 3 < p3 mg/mL with chloramphenicol as the positive control. These results are shown in Figs. 1 and 2 and were consistent with previous reports [2, 17, 18].

Fig. 1
figure 1

Determination of MIC for crude extracts and pure compounds from leaves and twigs of M. Pyrrhocarpa

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Fig. 2
figure 2

Determination of MBC for crude extracts and pure compounds from leaves and twigs of M. Pyrrhocarpa

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Anti-HIV-1 RT activity

The hexane and EtOAc extracts showed the highest %inhibition of HIV-1 RT, at values of 81.27 and 66.97 at 200 µg/mL, while the MeOH extract was inactive. Compounds (1) and (2) reduced syncytium formation in 1A2 cells at EC50 values of 4.48 and 4.99 μM, respectively. AZT was used as a positive control, as shown in Figs. 3 and 4.

Fig. 3
figure 3

Anti-HIV-1 RT of crude extracts and pure compounds from leaves and twigs of M. Pyrrhocarpa. aAnti-HIV-1 RT activity express as %inhibition at 200 µg/mL: very active (VA) =  > 70% inhibition, moderately active (MA) = 50% to 69% inhibition, weakly active (WA) = 30% to 50% inhibition and inactive (I) =  < 30% inhibition; For determination of IC50 in the HIV-1 RT assay, the coefficients of determination, R2, were 0.98–0.99 in all assays for 50% end point

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Fig. 4
figure 4

Anti-syncytium MC99 + 1A2 of crude extracts and pure compounds from leaves and twigs of M. Pyrrhocarpa. bAnti-syncytium (MC99 + 1A2) EC50 = dose of compound that reduced 50% syncytium formation by ΔTat/RevMC99 virus in 1A2 cells. AZT, averaged from three experiments, EC50 3.95 × 10− 3 μM;.cTI, Therapeutic Index: IC50/EC50

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Cytotoxicity activity

The hexane extract showed cytotoxicity against KKU-M213, FaDu, HT-29, A549, SH-SY5Y, MNN-K1, and Hep G2 cells at ED50 values of 3.42, 6.20, 3.37, 4.45, 7.21, 12.63, and 1.23 µg/mL, respectively, while the ethyl acetate extract showed cytotoxicity against KKU-M213, HT-29, A549, SH-SY5Y, MNN-K1, and Hep G2 cells at ED50 values of 6.20, 14.99, 8.84, 11.79, 11.00 and 3.31 µg/mL, respectively. 6aS, 12aS, 12S-elliptinol (1) showed cytotoxicity against A549 and HepG2 at ED50 values of 2.27 and 3.97 µg/mL. Ellipticine [41, 42] was used as a positive control, as shown in Fig. 5.

Fig. 5
figure 5

Cytotoxicity study of crude extracts and isolated compounds from leaves and twigs of M. Pyrrhocarpa. dCytotoxic assay: ED50 less than 20 µg/mL were considered active for extracts and ED50 less than 4 µg/mL were considered active for pure compounds. Cancer cell lines: KKU-M213 (Human intrahepatic cholangiocarcinoma) FaDu (Human squamous cell carcinoma) HT-29 (Human colon adenocarcinoma) MDA-MB-231 (Human mammary gland/breast adenocarcinoma) A 549 (Human lung adenocarcinoma) SH-SY5Y (Human neuroblastoma) MNN-K1 (highly differentiated immortalized human cholangiocyte cell line) Hep G2 (Human hepatocellular carcinoma)

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Discussion

Structure elucidation

Although the spectral data of compounds (1) and (2) were consistent with previous reports [38–40], it was noted that the coupling constants and proton orientation of compounds (1) and (2) at positions 6a, 12a, and 12 of MOM-protected munduserol [43], elliptinol [38, 39], 6a, 12a-cis-12, 12a-cis-12-acetoxy-6,6a,12,12a-tetrahydrorotoxen [44], compounds (1), and (2) are quite different, and the structures and 1H-NMR data of the four compounds are shown in Fig. 6 and Table 2.

Fig. 6
figure 6

Structures of MOM-protected munduserol [43], elliptinol [38, 39], and 6a, 12a-cis-12, 12a-cis-12-acetoxy-6,6a, 12, 12a-tetrahydrorotoxen [44]

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Table 2 Comparison of chemical shift and coupling constant values of MOM-protected munduserol [43], elliptinol [38, 39], 6a, 12a-cis-12, 12a-cis-12-acetoxy-6,6a,12,12a-tetrahydrorotoxen [44], compound (1) and (2)
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From Table 2, the coupling constant between H12a and H12 was found to be 10.2 Hz in compound (1) and 10.0 Hz in compound (2), which suggested that H12a and H12 in compound (1) and compound (2) were trans to each other. This conclusion was contradictory to the findings in previous reports [38, 39, 43, 44]. To settle this discrepancy, compound (2) was subjected to X-ray analysis, which confirmed that H6a, H12a and H12 were in fact H6aβ, H12aα, and H12β, respectively. The absolute configuration of compound (2) was therefore 6aS, 12aS, 12S. Comparison of the 1H-NMR data of compound (1) with those of 6aS, 12aS, 12S-munduserol (2) led to the conclusion that compound (1) was 6aS, 12aS, 12S-elliptinol (1). In this work, 6aS, 12aS, 12S-elliptinol (1) and 6aS, 12aS, 12S-munduserol (2) from the leaves and twigs of M. pyrrhocarpa afforded useful spectroscopic and XRD data, making it possible to assign the protons at the 6a, 12a, and 12 positions. The key COSY ( ) and HMBC ( ) correlations of compounds (1–3) are shown in Fig. 7, and the X-ray crystal structure of compound (2) is shown in Fig. 8.

Fig. 7
figure 7

Key COSY ( ) and HMBC ( ) correlations of 6aS, 12aS, 12S-elliptinol (1), 6aS, 12aS, 12S-munduserol (2) and dehydromunduserone (3)

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Fig. 8
figure 8

X-ray crystal structure of 6aS, 12aS, 12S-munduserol (2)

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Bioactivities

Regarding antibacterial activity, the crude EtOAc extract from M. pyrrhocarpa showed good antibacterial activity against E. Coli and S. flexneri at MIC and MBC values ​​of 6.25 mg/mL. Compounds (1–3) inhibited 9 strains of bacterial cells at MIC and MBC values of 3 and > 3 mg/mL, which was more prominent than other Milletia genera tested in previous reports [45, 46]. In anti-HIV activity, compounds (1) and (2) from M. pyrrhocarpa showed anti-syncytium activity with better IC50 and EC50 values than that of the pure compound found in Ventilago harmandiana [47]. In in vitro cytotoxic activity, crude hexane, ethyl acetate, and compound (1) from M. pyrrhocarpa showed cytotoxicity against A549 (human lung adenocarcinoma) cells with better ED50 values at 4.45, 8.84, and 2.27 µg/mL than those of the crude extract and pure compounds from Garcinia speciosa Wall [48]. In addition, compared to phenylacetylshikonin analogues, compound (1) showed comparable and better cytotoxicity to A549 cells [49]. Extracts and purified compounds (1–3) from M. pyrrhocarpa showed bioactivity, especially compound (1), which showed potent anticancer activity. This work does possess inherent limitations. The scarcity of these compounds makes it impossible to test other biological activities, such as anti-inflammatory and antidiabetic activities, and perform in vivo studies. Further molecular modelling and computational studies, such as molecular docking and molecular dynamics techniques, are desirable. Through these studies, the interaction between the tested compounds and cells could be predicted to obtain suitable structures so that appropriate syntheses can be carried out.

Conclusion

This is the first phytochemical investigation of the leaves and twigs of M. pyrrhocarpa and the first study to examine their biological activity. Interestingly, previous reports did not establish the absolute configuration of rotenol (1) and (2) [38,39,40]. This report established the complete structure, spectral data of compounds (1) and (2), and X-ray data of (2). Furthermore, bioassays such as antibacterial, anti-HIV virus, and cytotoxicity on rotenol (1), (2), and rotenone (3) provided significant data that could be used for further study.