A new β -hydroxydihydrochalcone from Tephrosia uniflora , and the revision of three β -hydroxydihydrochalcones to flavanones

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Introduction
Tephrosia species (Leguminosae) are widely distributed and utilized in herbal medicine for the treatment of a variety of disorders including stomach ache [1], diarrhoea [2], asthma [3,4], inflammation and respiratory problems [1,5].They have also been used to treat snake bites [6].The genus is known to generate prenylated flavonoids [5] and isoflavonoids [7] that possess antimicrobial [8], anticancer [9], antiinflammatory [10], antiplasmodial and cytotoxic [11] effects.Some flavonoids, especially isoflavonoids, exert their antimicrobial effect [12] by inhibiting DNA synthesis, metabolism, or membrane formation of bacteria [13].Motivated by our interest in the identification of bioactive metabolites from Kenyan plants, we have investigated the stems of T. uniflora for its constituents.It is a perennial herb with axillary flowers and small seeds, and is found, for example, in the Amboseli ecosystem, Kenya [14,15].Previous phytochemical investigation of this plant provided an isoflavone, a rotenoid and phytosterols [6].Herein, we report the isolation of a new β-hydroxydihydrochalcone (1) and of three known compounds (2)(3)(4) from its stem, and the evaluation of the antibacterial activity and the cytotoxicity of elongatin (2), one of the isolated constituents.
We also note that the oxygenation pattern of ring A in compound 7 [24] and (ring B in compound 7a) do not correspond to the reported NMR data (Table 2 and Table 3).Ring A in 7 (ring B in 7a) is symmetrical and hence C-2 and C-6 (C-2 ′ and 6 ′ in 7a) are chemically equivalent and should resonate at the same frequency.In contrast, different chemical shifts have been reported [24] for these positions (Tables 2 and 3) suggesting that the originally assigned substitution pattern of this ring in this compound is erroneous.The reported NMR data is not in agreement Fig. 1.The structures of the compounds isolated from the stem of Tephrosia uniflora, and of the compounds revised herein.

C. Chepkirui et al.
with oxygenation at C-2 ′ and C-6 ′ but that of C-2 ′ and C-5 ′ .As we only have access to pdf copies of the NMR data of this compound, we recommend the re-analysis of 7a to ensure the accuracy of our suggestion for correction.
Having a β-hydroxydihydrochalcone skeleton as a working structure, the interpretation of the mass spectrometric data may easily lead to the misassignment of flavanones to β-hydroxydihydrochalcones, despite the molecular weight of flavanones being 18 amu lower.For compound 5, the HRESIMS data showing the m/z 469.26742 signal corresponding to the protonated molecular ion has allegedly been used to establish its molecular formula; however, the spectrum has not been attached as supplementary material [23] and hence this information cannot be confirmed.For compound 6 [22], the HRESIMS signal m/z 297.07603 [M + Na] + was used to establish the molecular formula, corresponding to the β-hydroxydihydrochalcone structure 7.However, the spectrum shown in the supplementary material of this report does not show this signal [24], but instead m/z 289.0696 that was (erroneously) interpreted as "a quasi-molecular ion peak [M+H− H 2 O] + " of β-hydroxydihydrochalcone 7.This mass signal corresponds to the protonated molecular ion of flavanone 7a, supporting the NMR-based revision of 7 to 7a.This MS-based misassignment is analogous to that of 4,2 ′ ,4 ′ ,βtetrahydroxy-6 ′ -methoxy-α,β-dihydrochalone [31], which showed m/z 286.0823 (HREIMS) that was reported as [M − H 2 O] + instead of recognizing it as the molecular ion peak of the corresponding flavanone, 4 ′ ,7-dihydroxy-5-methoxyflavanone [17].
Elongatin (2) isolated from T. uniflora was evaluated for activity against the model bacteria B. subtilis and E. coli as well as for cytotoxicity against the MCF-7 human breast cancer cell line.It showed moderate antibacterial activity (EC 50 of 25.3 μM, EC 90 of 32.8 μM) against B. subtilis and cytotoxicity (EC 50 41.3μM), which is in agreement with a previous report on related flavonoids [12].

Plant material
The stem of Tephrosia uniflora was collected along the Emali-Loitokitok road, Makueni County, in July 2016.The plant was authenticated by Mr. Pactrick C. Mutiso of the University Herbarium, Department of Biology, University of Nairobi, where a voucher specimen (PCM-2016/010) was deposited.

General experimental procedure
ECD experiments were run on a JASCO J-810 spectropolarimeter.UV spectroscopy was performed on a Shimadzu UV-1650 spectrophotometer.NMR spectra were acquired on a Bruker Avance NEO 500 MHz spectrometer equipped with a TXO cryogenic probe.MestreNova (v14.0.0) software was used to process the spectra.TLC analyses was performed on Merck pre-coated silica gel 60 F 254 aluminum plates.Preparative reversed-phase HPLC chromatography was carried out using a Waters 600E HPLC system using the Chromulan software (v.0.88,Pikron Ltd) and an RP-C8 Kromasil® column (250 mm × 25 mm, 5 μm).

Extraction and isolation from the stem of Tephrosia uniflora
The air-dried stems of Tephrosia uniflora (500 g) were extracted (4 × 1 L) with CH 2 Cl 2 /MeOH (1:1) at room temperature.The crude extract (60 g) was partitioned between EtOAc and H 2 O.The EtOAc layer was concentrated on a rotary evaporator to yield 30 g of crude extract.The crude EtOAc extract was adsorbed on silica gel, loaded onto a 300 g silica gel column, and eluted with isohexane containing increasing amounts of EtOAc (1% to 99% v/v).Based on their TLC profiles, the eluates were then pooled into 15 fractions.The fraction that was eluted with 3% EtOAc in isohexane was purified by column chromatography over Sephadex LH-20 (CH 2 Cl 2 /MeOH, 1:1), followed by further purification on Preparative TLC (isohexane/EtOAc, 7:3) to yield compound 2 (20 mg) as a white amorphous solid.The fraction that was eluted with

Antibacterial assays
Antibacterial activity of the isolated compound 2 was determined against the Gram-positive B. subtilis and Gram-negative E. coli bacteria following standard procedures [32].In summary, compound 2 was dissolved in 100% DMSO to 10 mg/mL concentration and stored at − 20 • C. Bacterial cultures of B. subtilis and E. coli were grown in Mueller-Hinton (HiMedia Laboratories Pvt.Limited, Mumbai, India) broth for 24 h until reaching the optical density (O⋅D = 0.5) [33], then diluted 10 times in pre-warmed medium and the compound added to a final concentration of 35 μg/mL into a 384-well microtiter plate and incubated for 24 h at 37 • C without agitation.The resazurin assay for assessing viability was performed as described [34], by adding 10 μL of Ala-marBlue solution to each well, followed by a 1 h incubation at 37 • C without agitation.Viability was determined using POLARstar Omega (BMG Labtech, Cape Town, S.A.) set at excitation λ = 540 nm and emission filter λ = 590 nm, where the fluorescence bleed-through between the wells was controlled through a chest-like plate design.In all assays, the known antibiotic ampicillin was used as a positive control and DMSO as negative control, following the same 2-fold dilution concentration range as the compounds under testing.Effective concentrations (EC 50 and EC 90 ) values were calculated from three independent replicate experiments using 2-fold dilution intervals.Non-linear regression dose-response inhibition following a log(agonist) vs. response was performed using GraphPad Prism version 9.2.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.
and kept in exponential growth in Dulbecco's Eagle's medium (Modified Medium) supplemented with 10% foetal calf serum and reseeded into 96-well microtiter plates to settle for 24 h as pre-assay preparation.The stock solution of the compound was added to a final concentration of 0.35% v/v of DMSO in the culture medium.The cell viability was determined using PrestoBlue (ThermoFisher) cell viability reagent following the manufacturer's instructions and for a 24 h incubation period.The fluorescence from resorufin was measured using POLARstar Omega (BMG Labtech, Cape Town, S.A) set at excitation λ = 540 nm and emission filter λ = 590 nm.Each assay contained a DMSO control at the equivalent starting concentration, positive control (uninhibited cell growth) and negative control (cell medium only).Cell viability was expressed as a percentage of solvent-only control with half maximal effective concentration (EC 50 ) values, associated standard deviation (SD) and standard error (SE) range, calculated from three independent replicate experiments using 2-fold dilution intervals.Non-linear regression dose-response inhibition following a log(agonist) vs. response -Find ECanything was performed using GraphPad Prism version 9.2.0 for Windows, GraphPad Software, San Diego, California USA, www.graphpad.com.

Table 2 1
H NMR spectral data of the skeletons of β-hydroxydihydrochalcones and flavanones a .