Dihydrochalcone Glycosides From Balanophora harlandii

Abstract

Dihydrochalcone class of compounds displays good performance in taste modulation on several natural non-caloric/low-caloric sweeteners. Compounds such as hesperetin dihydrochalcone (HDC) and hesperetin dihydrochalcone-4′-O-β-d-glucoside (HDCG) were reported to be found in nature. However, the evidence was not complete to support their natural presence. The Coca-Cola Company has been working on the identification of natural sources of reported dihydrochalcones, as well as the exploration of new natural dihydrochalcones. During the investigation on plant Balanophora harlandii, the natural presence of HDCG was confirmed, and 3 new analogs have been identified. In this paper, detailed structural elucidation of known HDCG and 3 new analogs have been described.

Nowadays, many natural, non-caloric/low-caloric sweeteners have been applied to satisfy an increasing demand by consumers in the food and beverages. But none of them can really replace sucrose. Terpene glycosides, such as rebaudiosides from Stevia rebaudiana and mogrosides from Siraitia grosvenorii (monkfruit, Luo Han Guo), are natural sweeteners available on the market. The major components (e.g., rebaudioside A and mogroside V) from these sweeteners, however, are reported to exhibit sweet linger, bitter aftertaste, or licorice-like taste. ^1,2 Addition to the discovery and development of minor components with improved taste profiles (e.g., rebaudioside M, siamenoside I), ^2,3 another strategy is to design sweetener systems containing taste modulators to modify the taste perceptions.

Since the discovery and application of the sweet tasting agent, neohesperidin dihydrochalcone (NHDC), ⁴ in the food industries, a couple of decades ago, the exploration of dihydrochalcones (DCs) has never been stopped. NHDC is a semisynthetic derivative of neohesperidin which can be isolated from the Citrus species. It was reported to be 250-1800 times sweeter than different concentration levels of sucrose. ⁴ Although NHDC has a slower sweet onset than sucrose and lingering aftertastes, it performs great in bitterness blocking, sweetness enhancing, and mouthfeel improving. ^4,5 Its analogs, hesperetin dihydrochalcone (HDC) and hesperetin dihydrochalcone-4′-β-d-glucoside (HDCG), share similar characteristics in sweetness and taste improvement. ⁵

Few publications reported the natural presence of NHDC in Oxytropis myriophylla ⁶ and HDCG in Balanophora harlandii. ⁷ However, neither of the paper provided supportive data to differentiate the substituent groups on C-3 and C-4 (Figure 1). Balanophora harlandii is a parasitic plant widely distributed in the southwest of China. ⁸ Several other DCs, such as phloretin and its analogs have been identified from the species. ^7,8 During our investigation on B. harlandii, the natural source of HDCG (1) has been confirmed. Additionally, 3 new (2-4) and 4 known dihydrochalcone glycosides, phlorizin (5), phloretin-4′-β-d-glucopyranoside (6), 3-hydroxyphloretin-2′-O-β-d-glucopyranoside (7), and 3-hydroxyphloretin-4′-O-β-d-glucopyranoside (8) have been purified.

Figure 1

Structures of dihydrochalcone glycosides from B. harlandii.

Compound 1 was obtained as a white amorphous powder and had a molecular formula C₂₂H₂₆O₁₁ by high-resolution electrospray ionization mass spectrometry (HRESIMS) analysis ([M − H]⁻ at m/z 465.1412, calcd. 465.1402, Δ = 2.1 ppm, supplemental figure S1). A loss of 1 unit of 162 Da at m/z 303.0873 observed from MS/MS as well as an anomeric proton (δ 4.85, d, J = 7.7 Hz, H-1″) suggested 1 β-glucose moiety in compound 1. The 1-dimensional nuclear magnetic resonance (1D NMR) spectra of its aglycone (Table 1, supplemental figures S2-S3) was similar to that of 3-hydroxyphloretin-4′-O-β-d-glucoside ⁹ except for a methoxy group instead of a hydroxyl group, indicating the presence of a phloroglucinol ring (δ 6.00 [s, H-3′ and H-5′]), a catechol ring (δ 6.65 [d, J = 1.8 Hz, H-2], 6.79 [d, J = 8.2 Hz, H-5], and 6.59 [dd, J = 1.8, 8.2 Hz, H-6]). Two methylene groups were mutually coupled in ¹H-¹H COSY experiment (δ 3.26 [t, J = 7.6 Hz, H₂-8] and 2.74 [t, J = 7.6 Hz, H₂-7], supplemental figure S6). The key rotating frame overhauser effect spectroscopy (ROESY) correlations of OCH₃/H-5 fixed the methoxy group at C-4 ( supplemental figure S7), which was further determined by the correlation networks of OCH₃/C-4, H-2/C-4, and H-6/C-4 in the heteronuclear multiple bond correlation (HMBC) experiment (Figure 2, supplemental figure S5). The glucosyl unit was connected to C-4′ by the correlations of H-1″/C-4′ (Figure 2). Taken together, 1 was identified as hesperetin dihydrochalcone-4′-O-β-d-glucoside.

Table 1

NMR Spectra of Compounds 1-4 (in DMSO-d ₆; ¹H: 600 MHz; ¹³C: 150 MHz, Please See supplemental figures S1-S30).

Position	1		2		3		4
	δH (multiplicity, J in hz)	δC	δH (multiplicity, J in hz)	δC	δH (multiplicity, J in hz)	δC	δH (multiplicity, J in hz)	δC
1	-	134.3	-	132.2	-	132.2	-	132.0
2	6.65 (d, 1.8)	115.9	6.80 (d, 1.8)	112.6	6.80 (d, 1.2)	112.3	6.79 (d, 1.6)	112.3
3	-	146.4	-	147.4	-	147.4	-	147.4
4	-	146.0	-	144.6	-	144.5	-	144.5
5	6.79 (d, 8.2)	112.5	6.66 (d, 8.0)	115.3	6.65 (d, 8.0)	115.2	6.65 (d, 8.0)	115.2
6	6.59 (dd, 8.2, 1.8)	118.9	6.61 (dd, 8.0, 1.8)	120.3	6.62 (dd, 8.0, 1.3)	120.4	6.62 (dd, 8.0, 1.7)	120.3
7	2.74 (2H, t, 7.6)	29.6	2.79 (2H, t, 7.6)	29.8	2.80 (t, 8.0)	29.6	2.81 (t, 7.5)	29.3
8	3.26 (2H, t, 7.6)	45.6	3.28 (2H, t, 7.6)	45.7	3.43,3.34 (m)	44.8	3.45,3.32 (m)	45.2
9	-	205.1	-	205.1	-	204.5	-	205.2
-OCH3	3.70 (s)	55.9	3.73 (s)	55.5	3.74 (s)	55.6	3.74 (s)	55.5
1’	-	105.6	-	105.4	-	104.8	-	107.1
2’	-	164.4	-	163.7	-	160.8	-	159.8
3’	6.00 (s)	95.0	6.04 (s)	95.0	6.15 (d, 1.7)	94.5	6.35 (d, 2.3)	94.3
4’	-	163.5	-	163.4	-	165.5	-	162.7
5’	6.00 (s)	95.0	6.04 (s)	95.0	5.92 (d, 1.7)	96.9	6.16 (d, 2.3)	97.5
6’	-	164.4	-	163.7	-	165.5	-	163.8
1’’	4.85 (d, 7.7)	99.6	4.87 (d, 7.7)	99.5	4.93 (d, 7.1)	100.8	5.01 (d, 7.4)	100.5
2’’	3.19 (m)	73.2	3.20 (m)	73.0	3.29 (m)	73.3	3.29 (m)	73.3
3’’	3.26 (m)	76.6	3.26 (m)	76.4	3.33 (m)	76.7	3.30 (m)	76.6
4’’	3.16 (m)	69.6	3.16 (m)	69.4	3.19 (m)	69.4	3.27(m)	69.8
5’’	3.29 (m)	77.2	3.30 (m)	77.1	3.33 (m)	77.2	3.44 (m)	77.2
6’’	3.46 (m), 3.65 (m)	60.6	3.48 (m), 3.67 (m)	60.5	3.69,3.50 (m)	60.5	3.70,3.44 (m)	60.8
1’’’	-	-	-	-	-	-	4.96 (d, 7.4)	99.3
2’’’	-	-	-	-	-	-	3.24 (m)	73.0
3’’’	-	-	-	-	-	-	3.26 (m)	76.5
4’’’	-	-	-	-	-	-	3.45 (m)	69.7
5’’’	-	-	-	-	-	-	3.11 (m)	77.0
6’’’	-	-	-	-	-	-	3.70, 3.44 (m)	60.7

DMSO-d ₆, dimethyl sulfoxide-d ₆.

Figure 2

Key HMBC and ROESY correlations of 1.

Compound 2 was obtained as a white amorphous powder and had the same molecular formula as 1 by HRESIMS analysis ([M − H]⁻ at m/z 465.1408, calcd. 465.1402, Δ = 1.3 ppm, supplemental figure S8). Comprehensive comparative analysis of its ¹H-NMR and ¹³C-NMR spectra with 1 (Table 1, supplemental figures S9-S10) indicated that the only difference was the location of methoxy and hydroxy group in the catechol ring. The methoxy group was connected to C-3 instead of C-4 by the cross-peaks of OCH₃/H-2 in ROESY (supplemental figure S14), which was further determined by the correlation networks of OCH₃/C-3 and H-5/C-3 in the HMBC experiments (Figure 3, supplemental figure S12). Based on the above evidences, 2 was deduced as a new compound and named 3-methoxyphloretin-4′-O-β-d-glucopyranoside.

Figure 3

Key HMBC and ROESY correlations of 2.

Compound 3 was obtained as a white amorphous powder and had the same molecular formula as 2 by HRESIMS analysis ([M − H]^- at m/z 465.1414, calcd. 465.1402, Δ = 2.6 ppm, supplemental figure S15). The 1D NMR spectra of 3 were similar to those of compound 2 (Table 1, supplemental figures S16-17). Slight shifts of δ_C and δ_H on methylene groups, phloroglucinol ring, as well as glucose moiety, indicate its glucose moiety is on a different position comparing to 2. The key ROESY correlations of OCH₃/H-2 fixed the methoxy group at C-3 (supplemental figure S21), which was further determined by the correlation networks of OCH₃/C-3, H-2/C-4, and H-5/C-3 in the HMBC experiment (supplemental figure S19). The glucosyl unit was connected to C-2′ by the correlations of H-1″/H-3′ in the ROESY and H-1″/C-2′ in the HMBC experiment (supplemental figure S22). The absolute configuration of glucose moiety was determined to be d-configured using the methods listed below. Based on the above evidences, 3 was deduced as a new compound and named 3-methoxyphloretin-2′-O-β-d-glucopyranoside.

Compound 4 was obtained as a white amorphous powder. Its HRESIMS exhibited a quasimolecular ion peak at m/z 627.1931 [M − H]⁻, calcd. 627.1931, Δ = 0.1 ppm, which was compatible with the molecular formula C₂₈H₃₆O₁₆ (supplemental figure S23). The ¹H NMR spectrum (Table 1, supplemental figure S24) showed two group signals of sugar moiety and the coupling constants of the sugar moiety protons ([δ 5.01, d, J = 7.4 Hz, H-1″] and [δ 4.96, d, J = 7.4 Hz, H-1″′]) suggested the presence of two β-linked glucose. The 1D NMR spectra of its aglycone moiety (Table 1, supplemental figures S24-S25) was similar to those of compound 3, except for some slight shifts of δ_C and δ_H on the phloroglucinol ring. The key ROESY correlations of OCH₃/H-2 (supplemental figure S29) fixed the methoxy group at C-3, which was further determined by the correlation networks of OCH₃/C-3, H-2/C-4, and H-5/C-3 in the HMBC experiment (supplemental figure S27). Similarly, 1 glucosyl unit was linked to C-4′ by the correlations of H-1″′/C-4′, the other one was linked to C-2′ by the correlations of H-1″/C-2′ in the HMBC experiment (supplemental figure S30). The absolute configuration of glucose moieties was determined to be d-configured using the methods listed below. Based on the above evidences, 4 was deduced as a new compound and named 3-methoxyphloretin-2′,4′-O-β-d-diglucopyranoside.

The absolution configuration of sugar moieties on compounds 3 and 4 was analyzed using acid hydrolysis, followed by derivatization of sugar moieties and comparison to derivatives of d- and l-glucose standards using GC–MS, respectively. As a result, glucose moieties of both 3 and 4 are d-configured.

Compound 5 was obtained as a white amorphous powder. Its HRESIMS exhibited a quasimolecular ion peak at m/z 435.1318 [M − H]⁻, calcd. 435.1297, Δ = 4.9 ppm, which was compatible with the molecular formula C₂₁H₂₄O₁₀. A close comparison of the ¹H-NMR and ¹³C-NMR (supplemental figure S31-S32) with the literature ¹⁰ allowed 5 to be identified as phlorizin.

Compound 6 was obtained as a white amorphous powder and had the same molecular formula of C₂₁H₂₄O₁₀ with compound 5 by HRESIMS analysis (m/z 435.1310 [M − H]⁻, calcd. 435.1297, Δ = 3.1 ppm). A close comparison of the ¹H-NMR and ¹³C-NMR (supplemental figures S33-S34) with the literature ¹¹ allowed 6 to be identified as phloretin-4′-O-β-d-glucopyranoside.

Compound 7 was obtained as a white amorphous powder. Its HRESIMS exhibited a quasi-molecular ion peak at m/z 451.1248 [M − H]⁻, calcd. 451.1246, Δ = 0.5 ppm, which was compatible with the molecular formula C₂₁H₂₄O₁₁. A close comparison of the ¹H-NMR and ¹³C-NMR (supplemental figures S35-S36) with the literature ¹² allowed 7 to be identified as 3-hydroxyphloretin-2′-O-β-d-glucoside.

Compound 8 was obtained as a white amorphous powder and had the molecular formula C₂₁H₂₄O₁₁ based on (-)-HRESIMS ion peak at m/z 451.1267 [M − H]⁻ (calcd. 451.1246, Δ = 4.7 ppm). A close comparison of the ¹H-NMR and ¹³C-NMR (supplemental figures S37-S38) with the literature ¹³ allowed 8 to be identified as 3-hydroxyphloretin-4′-O-β-d-glucoside.

The sensory profile of compound 1 (HDCG) was evaluated at 20 ppm with 250 ppm of either rebaudioside (Reb) M or siamenoside (Sia) I in acidified (citric acid) water. The two panelists assessed the samples containing rebaudioside M or siamenoside I and found them to be less sweet and less mouthfeel than the samples with the addition of 20 ppm HDCG, respectively. The panelists assessed that the sweetness increased by approximately 1% to 3% sucrose sweetness equivalence with the addition of 20 ppm HDCG.

Experimental

Chemicals and Reagents

Ninety-five percent ethanol (EtOH), methanol (MeOH), n-butanol (n-BuOH), and ethyl acetate (EtOAc) (all in AR grade) were purchased from Cinc High Purity Solvents (Shanghai) Co., Ltd. Hydrochloric acid, sodium hydroxide (NaOH), pyridine, and n-hexane (all in AR grade) were purchased from Sinopharm Chemical Reagent Co. Ltd. Acetonitrile for semipreparative high-performance liquid chromatography (HPLC grade) was provided by Merck KGaA. l-cysteine methyl ester hydrochloride, 1-trimethylsilylimidazole, d-glucose Reference Standard and l-glucose Reference Standard (all in LR grade) were purchased from Shanghai Macklin Biochemical Co., Ltd. NHDC was purchased from Sigma Aldrich. HDC and HDCG reference compounds were prepared from NHDC by The Coca-Cola Company. ⁵

Instruments

1D and 2D NMR data were recorded on a Bruker 600 MHz Avance Ⅲ HD spectrometer, and the ¹H and ¹³C NMR chemical shifts were referenced to the residual solvent peaks for dimethyl sulfoxide-d ₆ (δ_H 2.50 and δ_C 39.52). HPLC was performed on an Agilent 1290 system using Agilent Eclipse plus C₁₈ column (1.8 µm, 2.1 × 50 mm). HRMS was performed in the negative ion mode with a Sciex Triple TOF 4600 spectrometer. MS/MS data were acquired by using data-dependent acquisition mode. GC–MS was performed on an Agilent 7890A/5977A GC/MSD System using an Agilent HP-5MS column (30 m × 0.25 mm × 0.25 μm). Semipreparative HPLC was carried out on an Agilent 1260 infinity with a UV detector using a Boston Green ODS-AQ column (10 × 250 mm, 5 µm). Preparative HPLC was carried out on a Shimadzu LC-20AP system using a YMC Actus Hydrosphere C18 column (30 × 250 mm, 5 µm). Column chromatography (CC) was performed using AB-8 resin (Sunresin New Materials Co. Ltd., China), MCI gel CHP-20 (Mitsubishi Chemical Systems, Inc. Japan), and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden). Lyophilization was carried out on a Scientz-18N freeze dryer (Ningbo Scientz Biotechnology Co., Ltd.).

Plant Material

The whole plant of B. harlandii was collected from AnShun, GuiZhou during November 2017. All the materials were dried under the sun. The identification and description of B. harlandii are according to the “Flora of China” ¹⁴ and “Flora of GuiZhou”. ¹⁵ Plants are dioecious. The rhizome is yellowish to brownish. Scapes are red (especially in females) to yellow. Leaves are about 6-12, yellow to reddish, usually clustered on the base of scape, decussate, subopposite, or spiraled, scaly. Male inflorescences are subspheroid to ovoid-ellipsoid, 1.8-2.5 cm × 1.5-2 cm. Bracts truncate with expanded liplike margin, fused side by side into a hexagonal alveolus. Male flowers: pedicellate, inserted basally in the alveolus, trimerous, 1.5-3 mm in diameter. Perianth lobes broadly deltoid. Synandria subdiscoid; anthers 3, transversely dehiscent. Female inflorescence is ovoid to ellipsoid. Spadicles obovoid, shortly stiped; cuticular ridges of apical cells labyrinthlike. Female flowers: only on the main axis of inflorescences (supplemental figure S39).

Plant Extraction

The dried powder of B. harlandii (10 kg) was extracted with 80% EtOH (100 L) at 70 °C for 4 hours. After the evaporation of EtOH, the solution was concentrated to a small volume (5 L).

LC-MS-Guided Isolation

The concentrated solution was subjected to an AB-8 resin column and eluted with 10% EtOH (100 L) and 95% EtOH (60 L). The 95% EtOH eluates were applied to an MCI column and gradually eluted with MeOH/water (H₂O) (1:4→4:1) to afford 5 fractions.

Approximately 200 µg/mL of NHDC, HDC, HDCG reference compounds, and 10 mg/mL of each fraction were prepared in MeOH, respectively. After being centrifuged at 12,000 rpm for 5 minutes, each supernatant was subjected to the LC–MS system using conditions listed in supplemental table S1. HRMS of NHDC, HDC, and HDCG were extracted and MS/MS data of each extracted ion chromatogram (XIC) matched signal was used to identify potential target compounds in the fractions. Candidates with m/z (either fragment or quasimolecular ion) match to the HRMS of HDCG were observed from Fr.1 to Fr.4, among which the signal with Rt = 4.99 in Fr.4 was identified as HDCG by comparing to reference compound (supplemental figures S40-S43). One candidate with m/z match to the HRMS of HDC was detected in Fr.5 (supplemental figure S44). However, further isolation of this candidate was not pursued due to limited yield.

Fr.1 (70 g) was dissolved with H₂O (3 L) and pH was adjusted to 1 with hydrochloric acid, partitioned with EtOAc (3 L). The water fraction (30 g) pH was adjusted to 7 with 1% NaOH solution and concentrated to 20 mL. The 20 mL sample was subjected to a Sephadex LH-20 column, followed by preparative HPLC (YMC Actus Hydrosphere C₁₈, 30 × 250 mm, 5 µm) with H₂O–acetonitrile (74:26) at 30 mL/min to yield compound 4 (180 mg, from all biomass). The EtOAc fraction (40 g) was evaporated to dryness, then dissolved with 50 mL MeOH–H₂O (1:1), chromatographed over an MCI column, further separated using preparative HPLC with H₂O–acetonitrile (70:30) at 30 mL/min to produce compound 7 (24 mg).

Fr.2 (60 g) was dissolved in water (3 L) and pH was adjusted to 1 with hydrochloric acid, then extracted with EtOAc (3 L) two times. The EtOAc fractions were combined and concentrated to get semi-solid (20 g), which was dissolved in 50 mL MeOH–H₂O (1:1) and chromatographed on an MCI column, a Sephadex LH-20 column, finally further purified on preparative HPLC with H₂O–acetonitrile (79:21) at 30 mL/min to obtain compounds 3 (12 mg) and 5 (40 mg).

Fr.3 (100 g) was isolated on preparative HPLC with H₂O–acetonitrile (78:22) at 30 mL/min, yielding compounds 6 (180 mg) and 8 (17 mg).

Fr.4 (60 g) was dissolved with 4% NaOH and pH was adjusted to 10 with hydrochloric acid, subsequently extracted with n-BuOH (4 L). The water phase pH was adjusted to 5 and partitioned with n-BuOH (4 L) one more time. The second n-BuOH fraction (25 g) was chromatographed over Sephadex LH-20, eluting with 50% EtOH to yield mixture (430 mg) containing compounds 1 and 2. The mixture was further purified on semipreparative HPLC using Boston Green ODS-AQ (10 × 250 mm, 5 µm) with H₂O–acetonitrile (74:26) at 3.5 mL/min to yield compounds 1 (2.3 mg) and 2 (220 mg).

Sugar Analysis

A solution of each compound (3, 2.4 mg and 4, 2.6 mg) in 1 M hydrochloric acid (H₂O, 5 mL) was heated at 100°C to reflux for 1.5 hours, respectively. The mixture was cooled to room temperature and extracted twice with EtOAc (5 mL/each time). The water layer was evaporated to dryness under reduced pressure to furnish a glucose residue. The residue/d-glucose/l-glucose was dissolved in pyridine (0.5 mL), respectively, to which 0.5 mL l-cysteine methyl ester hydrochloride was added. The mixture was kept at 60°C for 2 hours, dried under reduced pressure, and trimethylsilylated with 1-trimethylsilylimidazole (0.5 mL) for 2 hours. The mixture was partitioned between n-hexane and water (1.0 mL each), and the hexane extract was analyzed by GC–MS under the following conditions: column temperature, 230°C; injection temperature, 250°C; carrier, He gas; split ratio, 10:1; flow rate, 0.8 mL/min; MS scan range, 50-500.

Sensory Evaluation of HDCG

Samples of rebaudioside M (Reb M), HDCG with Reb M, siamenoside I (Sia I), and HDCG with Sia I in acidified (citric acid) water were prepared, respectively, as described in the patent WO2018200663 A1. ⁵ Taste tests were carried out with two panelists. Panelists were asked to evaluate the sweetness of samples and describe the difference of taste profile using 3%, 5%, and 8% sucrose in acidified (citric acid) water solution as references. Panelists were instructed to sip, evaluate the sweetness, and then spit the samples. Mineral water was given to panelists to rinse their mouth before tasting and between tasting different samples. Unsalted crackers were also given to panelists to eat followed by rinsing their mouth with mineral water before tasting the next sample.

Supplemental Material

Supplementary material - Supplemental material for Dihydrochalcone Glycosides From Balanophora harlandii

Supplemental material, Supplementary material, for Dihydrochalcone Glycosides From Balanophora harlandii by Indra Prakash, Yong Qian, Bin Wang, Zhenqiang Xin, Gil Ma, Zhou Yang, Juvenal Higiro, Goran B. Petrovic, Wenshuai Tian and Tianpei Xie in Natural Product Communications