Anti-Influenza Triterpene Saponins from the Bark of Burkea africana

In an in vitro cytopathic effect inhibition assay with the H3N2 influenza virus A/Hong Kong/68 (HK/68), the bark extract of Burkea africana was found to be a promising antiviral lead with an IC50 value of 5.5 μg/mL without noteworthy cytotoxicity in Madin Darby canine kidney cells. After several chromatographic steps, triterpene saponins of the lupane and oleanane types were identified as the bioactive principles. In total, eight new triterpene saponins (1–8) with four so far undescribed aglycone structures were isolated and characterized via HRESIMS, GC-MS, and 1D and 2D NMR spectroscopy. Their anti-influenza virus activity on HK/68 and the 2009 pandemic H1N1 strain A/Jena/8178/09 revealed the most potent effects by compounds 7 and 8, with IC50 values between 0.05 and 0.27 μM. This is the first time triterpene saponins have been reported as constituents of the investigated plant material.

Burkea af ricana Hook. is known under various names such as "wild syringa", "seringa tree", and "Rhodesian ash" in English or "mpulu" (Tsonga), "monato" (Tswana), "mufhulu" (Venda), and "wildesering" in Afrikaans. It is a flat-topped tree up to 20 m high, belonging to the family Leguminosae and widely distributed in the tropical and subtropical African regions. This species is difficult to grow and not yet planted on a commercial scale. 1 Traditionally, the roots of B. af ricana are used to treat toothache and stomach pain. The bark, rich in plant polyphenols, is used for leather tanning or pounded as a fish stupefiant and an arrow poison antidote. Bark decoctions are used for the treatment of colds, coughs, and stomach obstruction, while infusions are applied in the treatment of gonorrhea and syphilis. 2 The stem bark is documented as possessing antidiarrheal, antioxidant, and antibacterial activity against Salmonella typhi 3−5 and to inhibit the hyaluronidase of the black-necked spitting cobra, Naja nigricollis. 6 In the literature, little can be found on the phytochemical composition of B. af ricana. 7 Described compound classes, identified solely by using simple color reactions or by TLC comparison, comprise alkaloids, cardiac glycosides, flavonoids, phenolic acids, terpenoids and tannins, and both hydrolyzable polyphenols and proanthocyanidins. 1,4,7 To date, no triterpene saponins have been reported for this species.
Triterpene saponins are distributed widely as plant secondary metabolites with great structural diversity, which is also reflected in the variety of reported biological and pharmacological properties, such as antidiabetic, antifungal, cytotoxic, hepatoprotective, anticancer, chemopreventive, antiallergic, immunomodulatory, immunostimulatory, molluscicidal, hemolytic, and anti-inflammatory activities. 8−11 Moreover, antiviral activities against, for example, human respiratory syncytial virus (HRSV), human immunodeficiency virus (HIV), herpes simplex type 1 and 2 viruses (HSV), human rhinovirus (RV), tobacco mosaic virus (TMV), or hepatitis B virus (HBV) have been described. 12−17 So far, there are only a few reports on the anti-influenza virus activity of triterpene saponins. 18 Recently, Song et al. described the anti-influenza virus (H1N1) activity of uralsaponins isolated from Glycyrrhiza uralensis, 17 and Chen et al. demonstrated the anti-influenza A activity of saikosaponin A in vitro and in vivo. 19 In this study, eight triterpene saponins (1−8) were isolated from the ethanolic stem bark extract of B. af ricana and identified by means of HRESIMS, GC-MS, and 1D and 2D NMR spectroscopic methods. Herein, the isolation, structural elucidation, and the anti-influenza properties of these compounds are described.

■ RESULTS AND DISCUSSION
Screening for Anti-Influenza Natural Materials. To identify anti-influenza natural leads, some 160 lead-like enhanced extracts of various plant and fungal species were prepared as described recently. 20,21 Briefly, defatted materials were extracted successively with dichloromethane and methanol. The two resulting extracts were combined in order to cover a wide and drug-like range of polarity within one extract. Finally, tannin depletion via polyamide gel was carried out in order to remove common assay interfering compounds.
In a cytopathic effect (CPE) inhibitory assay with the H3N2 influenza virus A/Hong Kong/68 (HK/68) in Madin Darby canine kidney (MDCK) cells, the bark extract of B. af ricana (BA-E) was identified as a strong inhibitor of the CPE with an IC 50 of 5.5 μg/mL. Moreover, this extract was not cytotoxic to MDCK cells (Table S1, Supporting Information).
Assessment of Anti-Influenza Virus Activity of B. africana Bark. For the phytochemical workup, an ethanolic extract of the bark material of B. af ricana was generated in a larger scale and chromatographically separated via polyamide to obtain 10 fractions, whereby fractions A3 to A10 contained increasing amounts of tannins as monitored via TLC. The fractions were well tolerated by MDCK cells (Table S1 and Figure S1, Supporting Information). In the CPE assay, the tannin-rich fractions A3 to A10 showed activity rising parallel to their polarity. This might have been related to the increasing presence of polyphenols, which have been previously described as anti-influenza agents. 22−24 Since the focus of this study was to identify novel anti-influenza scaffolds, polyphenol-enriched fractions were not considered in the subsequent phytochemical workup. Hence, the tannin-depleted fractions (A1 and A2), which showed distinct antiviral effects against IAV HK/68 with IC 50 values of 4.0 and 3.0 μg/mL, respectively, were pooled for further phytochemical workup.
Microfractionation of the Tannin-Depleted Fraction: Activity Profiling and Dereplication. With a small aliquot of a tannin-depleted fraction, a time-based microfractionation via flash chromatography was performed to give 13 fractions (B1 to B13). These were then tested in the CPE inhibition assay as well as in the cytotoxicity assay (Table S2 and Figure  S1, Supporting Information). Fractions B7 to B9 displayed potent anti-influenza virus activity with IC 50 values of 0.17, 0.13, and 0.17 μg/mL, respectively. Moreover, the calculated selectivity indices (SI, CC 50 /IC 50 ) for B7 to B9 were between 6 and 14, suggesting a virus-specific activity of the contained compounds.
Dereplication of the bioactive microfractions using HR-ESIMS suggested triterpenoid saponins as the major constituents based on characteristic neutral losses of carbohydrate residues and fragment ions typical for C 30 aglycones.
Isolation and Identification of Triterpene Saponins from B. africana. For the targeted isolation of triterpene saponins, 28 g of the tannin-depleted extract were dissolved in water and partitioned sequentially between petroleum ether, ethyl acetate (EtOAc), and n-butanol. By using HRESIMS analysis, the EtOAc fraction was found to be enriched with the triterpene saponins of interest. Hence, this fraction was subjected to several chromatographic steps including size exclusion, supercritical fluid, and flash CC, resulting in the isolation of eight pure triterpene saponins (1−8). Their structures were identified by means of HRESIMS as well as 1D and 2D NMR methods.
The full assignments of the 13 C and 1 H NMR spectroscopic data of isolated compounds 1−8 are listed in Tables 1 to 4. The respective NMR and HRESIMS spectra are provided in the Supporting Information.

Journal of Natural Products
Article five methines, nine methylenes, and five quaternary carbons. By comparison with the literature, the aglycone was identified as alphitolic acid. 25 An increased δ value of C-3 at 96.5 ppm and the HMBC correlation of the anomeric hexose H-1′ with C-3 of the aglycone suggested the presence of a disaccharide unit at C-3. The linkage of the pentose unit to the hexose was established from the HMBC correlation between the resonances of H-1″ (δ H 4.60) and C-2′ (δ C 82.1). Both sugars were determined to be in the pyranose form, and according to the large coupling constants of the anomeric protons (J = 5.7 Hz, H-1′; J = 5.5 Hz, H-1″), their orientation was deduced to be β. By NMR data comparison of the sugar residues of 1 and 2, they were identified as Glc and Xyl units. Hydrolysis of 1 and conversion of the sugars to chiral diastereomers followed by GC-MS analysis confirmed their absolute configuration as Dglucose and D-xylose. Based on these data, compound 1 was identified as , 911.5010, Δ = 0.6 ppm), was also obtained as a white, amorphous powder. The MS/MS and NMR spectra were closely comparable to those of 1, indicating the same sapogenin and sugar residues with the difference of an additional deoxyhexose unit (loss of 146 Da). Detection of a C 2 fragment ion at m/z 481.1520 (intact carbohydrate chain), a Y 1α (loss of a deoxyhexose), a Y 1β (loss of a pentose), and a Y 1α/1β (combined loss of a deoxyhexose and a pentose) fragment ion upon CID of the [M + Na] + ion demonstrated the presence of a branched trisaccharide unit with the pentose and deoxyhexose moieties both being terminal. The findings from the HRESIMS analysis were confirmed by the NMR data, which displayed six additional carbons to the otherwise analogous resonances for the sapogenin alphitolic acid with the Xyl and Glc unit analogous to 1. The δ value of the additional anomeric carbon , 781.4380, Δ = −0.2 ppm). The fragmentation pattern clearly located the additional oxygen on the aglycone. The NMR data of 3 were generally in accordance with those of 1, but in contrast, the sapogenin of 3 showed only five methyl groups in the NMR spectra. Instead, C-27 was characterized by an oxygenated methylene group (δ C 61.0, δ H 3.76/4.15). This was confirmed by a significant increase of the δ values of positions C-14 (δ C 47.4) and C-15 (δ C 36.5, δ H 1.46/1.72) and a slight increase in positions C-13 (δ C 40.2, δ H 2.40) and C-8 (δ C 42.7). So far, this sapogenin has not been described in the literature and was assigned as 27-hydroxyalphitolic acid. GC-MS analysis and the NMR spectra indicated that 3 contains the same sugar moiety as compound 1, and consequently, it was assigned as 3-O-β-Dxylopyranosyl-(1→2)-β-D-glucopyranosyl-27-hydroxyalphitolic acid.
Compound 4 was isolated as a whitish, amorphous powder with the molecular formula C 42 H 68 O 15

Journal of Natural Products
Article compounds 3 and 4 was found in the second sugar moiety of the glycoside, where an additional carbon signal in the 13 C NMR spectrum and slightly different chemical shifts of the neighboring carbons indicated the replacement of the pentose unit by a hexose moiety. This was confirmed by the MS/MS data, which showed a disaccharide consisting of two hexoses. HMBC correlations again supported a 1→2 linkage between the sugar units. After hydrolysis, conversion of the sugars to chiral diastereomers followed by GC-MS analysis indicated the presence of D-glucose. Therefore, both sugar units were assigned as D-glucose, and accordingly, 4 was identified as 3-

Journal of Natural Products
Article Supporting Information) were in accordance with those of 5, When compared to the spectroscopic data of 6, the sapogenin of 7 differed only by the lack of a hydroxy group at position 2. This was corroborated by the upfield shift of C-2 (δ C 27.2), its additional proton signal (δ H 1.69/1.80), and the upfield shifts of the signals of the neighboring atoms [δ C 39.8 (C-1); δ C 90.7 (C-3); δ C 40.3 (C-4); δ C 37.9 (C-10)]. For the saccharide unit, the HRESIMS analysis was consistent with the presence of a linear trisaccharide, with a pentose bound directly to the aglycone, followed by an intermediate pentose and a terminal deoxyhexose moiety. Sixteen carbon resonances of the 13 C NMR spectrum could be assigned to two pentose units and one deoxyhexose, with δ values of 106.0, 106.2, and 102.5 for the anomeric carbons of pentose′, pentose″, and of the deoxyhexose, respectively. HMBC correlations between C-1′ of the first pentose unit with C-3 of the sapogenin supported the attachment of the trisaccharide at this position by analogy with compounds 1−6. The interglycosidic linkages were determined as 1→2 between the two pentoses as well as between the deoxyhexose and the pentose, according to the correlations of the HMBC spectrum. Hydrolysis of 7 and    Anti-Influenza Virus Activity of Triterpene Saponins from B. africana and their Aglycones. Besides the extract and fractions, all isolated compounds (1−8) including their aglycones were tested for their anti-influenza virus potential in the CPE inhibition assay using HK/68. Additionally, an isolate of the H1N1 influenza pandemic 2009 (A(H1N1)pdm09), the influenza virus A/Jena/8178/09 (Jena/8178), was included in these studies to further prove the antiviral activity of the isolated compounds against a currently circulating H1N1 strain (Table 5).
The oleanane-type triterpene saponins 5 and 6, both comprising a branched trisaccharide moiety, were identified as potent anti-influenza virus agents with IC 50 values against HK/68 and/or Jena/8178 in the low micromolar range. The most pronounced anti-influenza virus effect was observed for the oleanane-type triterpene saponins 7 (linear trisaccharide residue) and 8 (branched tetrasaccharide moiety), which reduced the CPE of HK/68 or Jena/8178 by 50% at nanomolar concentrations of 0.05 and 0.27 μM or 0.17 and 0.16 μM, respectively.
Since after oral intake the sugar moieties of triterpene saponins are most likely removed in the gastrointestinal tract, 27 we were further interested in the anti-influenza virus activity of the aglycones of 1 to 8 obtained by hydrolysis. As a general trend, they showed better compatibility and lower or no antiviral activity compared to their glycosidic counterparts. Intriguingly, the sapogenins of the most potent compounds 6, 7 , and 8 , namely, the so f ar unknown 21pm e t h o x y c i n n a m o y l o x y m a s l i n i c a c i d a n d 2 1pmethoxycinnamoyloxyoleanolic acid, lost their cytotoxicity in MDCK cells, while still demonstrating an anti-influenza virus activity against both tested strains in the range of 7−11 μM.
An anti-influenza mechanism via the inhibition of the viral surface protein neuraminidase can be excluded (data not shown). Recent studies performed with a series of similar synthetic 3-O-β-chacotriosyl oleanane-and ursane-type triterpenoids identified this compound class as entry inhibitors targeting viral hemagglutinin. 28−31 The chacotriose residue, a branched trisaccharide moiety comprising two rhamnose units and one glucose unit, was reported to be essential for the observed bioactivity, which is in agreement with our results of at least three sugars as a mandatory feature. Whether linear or branched sugar chains are more preferable for anti-influenza virus activity of the investigated triterpene saponins could not be concluded in this study. Thus, an interaction with the viral envelope protein hemagglutinin being a key factor for viral entry into the host cell could be involved in the anti-influenza activity of the so far undiscovered triterpene saponins (1 to 8) from B. af ricana bark. However, this is only a hypothesis, which warrants further investigation through another study.

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