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Three New Oleanane-Type Triterpenoidal Glycosides from Impatiens balsamina and Their Biological Activity

Three new oleanane-type triterpenoidal glycosides, imbalosides A–C (1–3), were isolated from the white flowers of Impatiens balsamina. The structures of these phytochemical constituents (1–3) were elucidated through 1D and 2D Nuclear Magnetic Resonance (NMR) and Mass Spectrometry (MS) data analyses followed by chemical methods. All the characterized compounds (1–3) were evaluated for their antiproliferative activity against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and BT549) and their anti-neuroinflammatory activity on the basis of inhibition levels of nitric oxide (NO) in the lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cell lines.


Introduction
Impatiens balsamina L., known as garden balsam or rose balsam, is an annual plant belonging to the family Balsaminaceae and is widely distributed in Korea, Japan, and mainland China. Diverse parts of I. balsamina, including flowers, stems, and leaves, have long been used as traditional medicines to treat various diseases. The flowers of I. balsamina have been used as remedies for lumbago, burns, and scalds [1], whereas its aerial parts have been used to treat articular rheumatism, abscesses, and tumors [2]. In the previous research on this plant, 1,4-naphthoquinone derivatives showed a variety of pharmacological effects such as antitumor, anti-inflammatory, and hepatoprotective activities [3][4][5].
As part of the continuing studies to identify the bioactive constituents from Korean medicinal plants [6][7][8][9][10][11], we previously conducted a phytochemical investigation on the MeOH extract of the white flowers of I. balsamina, which led to the isolation and characterization of phenolic compounds including mono-and biflavonoids with cytotoxic, anti-inflammatory, and neuroprotective activities [12,13]. In order to discover bioactive molecules in other structural classes from this plant, we further investigated its EtOAc-soluble layer and identified three new oleanane-type triterpenoidal glycosides (1-3) (Figure 1). The chemical structures of the new compounds (1)(2)(3) were established on the basis of spectroscopic (1D and 2D NMR) and spectrometric [High Resolution Fast Atom Bombardment MS (HRFABMS)] analyses as well as chemical methods. The isolates (1)(2)(3) were tested for their cytotoxicity against four human tumor cell lines and anti-neuroinflammatory activity using lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cell lines.

Structure Elucidation
Imbaloside A (1) was isolated as a colorless gum. The molecular formula of 1 was determined as C41H66O14 based on the [M -H] -ion peak at m/z 781.4368 (calcd for C41H65O14 -, 781.4380, error = 1.5 ppm) from the HRFABMS analysis. The 1 H NMR spectrum of 1 showed a broad singlet at δH 5.30 for an olefinic proton, overlapped signals from δH 3.20 to 4.38 for oxygenated methine/methylene protons, seven singlets at δH 1.40, 1.09, 1.05, 1.00, 0.97, 0.93, and 0.88 for methyl protons, and the others in the region from δH 2.28 to 0.82. Among a total of 41 carbons present in this molecule, 40 resonances were observed in the 13 C NMR spectrum of 1, including 30 peaks for typical oleanane-type triterpenoids [14][15][16][17][18] with two olefinic carbons at δC 144.6 and 124.2 and four oxygenated carbons at δC 91.1, 78.8, 70.4, and 65.8, and ten peaks for β-glucuronic acid (δC 106.9, 75.6, 78.1, 73.8, and 76.8) and β-xylopyranose (δC 102.8, 74.5, 77.0, 71.2 and 66.5). These 1 H and 13 C NMR data implied that 1 is an oleanane-type triterpenoidal glycoside with two sugar moieties, and its core structure including the location of a double bond and four oxygenated carbons was established through analysis of the Distortionless Enhancement by Polarization Transfer (DEPT), correlation spectroscopy (COSY), Heteronuclear Single Quantum Correlation (HSQC), and Heteronuclear Multiple Bond Correlation (HMBC) spectra ( Figure 2, Supplementary materials). The β-configurations of the two anomeric carbons on the glucuronic acid and xylopyranose were assigned by the relatively large 3 J coupling constants (7.1 Hz) between H-1 and H-2 of both sugars. These two sugar units were confirmed to be connected at C-3 (glucuronic acid) and C-22 (xylopyranose) by observing HMBC cross-peaks of H-3/C-1′ and H-22/C-1′′ ( Figure 2). The relative configuration at C-3 was confirmed by the strong nuclear Overhauser effect (NOE) correlation of H-3 with H-5 along with the mild correlation of H-3 with H-1ax ( Figure 3A). The α-orientation of the hydroxyl group at C-16 was verified by the strong NOE spectroscopy (NOESY) cross-peaks of H-16 with both H-15a and H-15b ( Figure 3) and the relatively small coupling constant, 3.4 Hz, between H-  cytotoxicity against four human tumor cell lines and anti-neuroinflammatory activity using lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cell lines.

Structure Elucidation
Imbaloside A (1) was isolated as a colorless gum. The molecular formula of 1 was determined as  The β-configurations of the two anomeric carbons on the glucuronic acid and xylopyranose were assigned by the relatively large 3 J coupling constants (7.1 Hz) between H-1 and H-2 of both sugars. These two sugar units were confirmed to be connected at C-3 (glucuronic acid) and C-22 (xylopyranose) by observing HMBC cross-peaks of H-3/C-1′ and H-22/C-1′′ ( Figure 2). The relative configuration at C-3 was confirmed by the strong nuclear Overhauser effect (NOE) correlation of H-3 with H-5 along with the mild correlation of H-3 with H-1ax ( Figure 3A). The α-orientation of the hydroxyl group at C-16 was verified by the strong NOE spectroscopy (NOESY) cross-peaks of H-16 with both H-15a and H-15b ( Figure 3) and the relatively small coupling constant, 3.4 Hz, between H- The β-configurations of the two anomeric carbons on the glucuronic acid and xylopyranose were assigned by the relatively large 3 J coupling constants (7.1 Hz) between H-1 and H-2 of both sugars. These two sugar units were confirmed to be connected at C-3 (glucuronic acid) and C- 2.01 and 1.43 for 1) and the coupling constant (broad singlet at C-16 for camelliagenone and broad triplet with the small coupling constant 3.4 Hz for 1), which possess the same α-orientation of the hydroxyl group at C-16 [19]. The β-configuration of the alkoxy group at C-22 was assigned by NOESY cross-peak of H-22 with H-19ax ( Figure 3A) and coupling constant analysis ( Figure Figure 3C). Generally, the coupling constant of two protons in anti (or axial-axial) orientation in cyclohexane is over 9-10 Hz, which excluded the possibility of an α-OXyl epimer for 1 since the observed coupling constants between H-22 and H-21ax,eq were both smaller than 8 Hz (7.6 and 3.9 Hz, respectively). In fact, camelliagenone has an α-OH at C-22 and the coupling constant between H-22 and H-21α was 12.0 Hz [19]. The hydroxymethyl group at C-28 was confirmed as β-form by the NOE correlations of H-28a with H-18 and H-28b with H-15 and H-26 ( Figure 3A). The absolute configurations of the glucuronic acid and xylopyranose were assigned as D-form by comparing retention times of their chiral derivatives with those of authentic samples [20]. Thus, the structure of 1 was defined as 3-O-β-D-glucuronyl-3β,16α,22β,28-tetrahydroxyolean-12-ene-22-O-β-D-xylopyranoside.
Plants 2020, 9, x FOR PEER REVIEW 3 of 9 16 and both H-15a and H-15b, indicating that H-16 should be in equatorial position rather than axial position. This initial assignment of α-OH at C-16 was supported by observing a similar NMR chemical shift pattern around C-16 of camelliagenone (e.g., δH-15 2.02 and 1.42 for camelliagenone and δH-15 2.01 and 1.43 for 1) and the coupling constant (broad singlet at C-16 for camelliagenone and broad triplet with the small coupling constant 3.4 Hz for 1), which possess the same α-orientation of the hydroxyl group at C-16 [19]. The β-configuration of the alkoxy group at C-22 was assigned by NOESY cross-peak of H-22 with H-19ax ( Figure 3A) and coupling constant analysis ( Figure Figure 3C). Generally, the coupling constant of two protons in anti (or axial-axial) orientation in cyclohexane is over 9-10 Hz, which excluded the possibility of an α-OXyl epimer for 1 since the observed coupling constants between H-22 and H-21ax,eq were both smaller than 8 Hz (7.6 and 3.9 Hz, respectively). In fact, camelliagenone has an α-OH at C-22 and the coupling constant between H-22 and H-21α was 12.0 Hz [19]. The hydroxymethyl group at C-28 was confirmed as βform by the NOE correlations of H-28a with H-18 and H-28b with H-15 and H-26 ( Figure 3A). The absolute configurations of the glucuronic acid and xylopyranose were assigned as D-form by comparing retention times of their chiral derivatives with those of authentic samples [20]. Thus, the structure of 1 was defined as 3-O-β-D-glucuronyl-3β,16α,22β,28-tetrahydroxyolean-12-ene-22-O-β-Dxylopyranoside. Imbaloside B (2) was obtained as a colorless gum, and its molecular formula was determined as C45H70O16 from the deprotonated HRFABMS ion peak at m/z 865.4580 (calcd for C45H69O16 -, 865.4591, error = 1.3 ppm). The 1 H and 13 C NMR data were quite similar to those of compound 1 ( Table 1), suggesting that compound 2 possesses the same core structure as compound 1, but apparent differences existed at the β-D-xylopyranose moiety in terms of the presence of two acetoxy groups (δH 2.08 and 2.02; δC 172.2, 171.8, 21.0 and 20.7) ( Table 1). The connectivity of the two acetoxy groups were verified on the basis of the HMBC cross-peaks from H-3′′ (δH 5.09) to OAc-3′′ (δC 172.2) and H-4'' (δH 4.87) to OAc-4'' (δC 171.8), respectively ( Figure 2). According to the similar 1 H and 13 C NMR data and NOESY correlations to those of compound 1, the relative configuration of compound 2 was confirmed to be identical with that of 1. Thus, the chemical structure of 2 was assigned as 3-O-β-Dglucuronyl-3β,16α,22β,28-tetrahydroxyolean-12-ene-22-O-(3,4-O-diacetyl)-β-D-xylopyranoside. Imbaloside B (2) was obtained as a colorless gum, and its molecular formula was determined as C 45 Figure 2). According to the similar 1 H and 13 C NMR data and NOESY correlations to those of compound 1, the relative configuration of compound 2 was confirmed to be identical with that of 1. Thus, the chemical structure of 2 was assigned as 3-O-β-D-glucuronyl-3β,16α,22β,28 -tetrahydroxyolean-12-ene-22-O-(3,4-O-diacetyl)-β-D-xylopyranoside.

Cytotoxicity Assessment
The cytotoxicity was assessed based on the inhibitory effects of the compounds (1-3) on the growth of the four human tumor cell lines A549, SK-OV-3, SK-MEL-2, and BT549 using a sulforhodamine B (SRB) assay. As shown in Table 2 (Table 2).

Anti-Neuroinflammatory Activity
The potential anti-neuroinflammatory activity of the new compounds (1-3) was also evaluated by measuring the nitric oxide (NO) production levels in the LPS-stimulated murine microglia BV-2 cell line. The tested compounds (1-3) exerted moderate inhibition levels of NO production with IC 50 values ranging from 33.8 to 41.0 µM without significant cell toxicity. L-NMMA (IC 50 21.3 µM) was used as a positive control (Table 3). Many oleanane-type triterpenoids and their glycosides have shown potent cytotoxicity and anti-neuroinflammatory activity that are consistent with our current study [16,[21][22][23][24]. This implied that the well-known oleanane-type triterpenoids are still good sources for future drug candidates to treat cancer or inflammation-related diseases.

General Experimental Procedures
Optical rotation data were recorded using a JASCO P-1020 polarimeter (JASCO, Easton, MD, USA). The NMR studies were accomplished employing a Bruker AVANCE III 700 NMR spectrometer (Bruker, Karlsruhe, Germany) and resultant spectra were processed using MestReNova (Mnova) (version 14.1.2-25024) with default weighting functions. HRFABMS data were acquired on a Waters SYNAPT G2 (Milford, MA, USA). The HPLC-DAD-MS data were measured using an Agilent 1260 Infinity HPLC system (Agilent, Santa Clara, CA, USA) with a Kinetex C 18

Plant Material
The air-dried white flowers of I. balsamina were collected in Asan, Korea, in August 2014, and the plant was identified by one of the authors (K.R.L.). A voucher specimen (SKKU-NPL 1406) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.