Antioxidant and Cytoprotective Effects of New Diarylheptanoids from Rhynchanthus beesianus

from Rhynchanthus beesianus

Article

Antioxidant and Cytoprotective Eﬀects of New Diarylheptanoids

Pei-Feng Zhu, Gui-Guang Cheng, Lan-Qin Zhao, Afsar Khan, Xing-Wei Yang, Bu-Yun Zhang,

Meng-Cheng Li, Ya-Ping Liu,* and Xiao-Dong Luo*

Cite This: J. Agric. Food Chem. 2021, 69, 6229 −6239

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ABSTRACT: Rhynchanthus beesianus (Zingiberaceae) has been an important food spice and vegetable in southern China. Fifteen

phenolic compounds (1−15) including three new diarylheptanoids, rhynchanines A−C (1−3) and one new phenylpropanoid, 4-O-

methylstroside B (9), were isolated from R. beesianus rhizomes. The structures of new compounds were elucidated by comprehensive

analyses through NMR, HRMS technique, acid hydrolysis, and Mosher’s reaction. Among them, compound 5 is the ﬁrst isolated

natural product and its NMR data are reported. Most of the isolated compounds, especially 3−6 and 8, showed signiﬁcant

antioxidant activities on DPPH, ABTS radical scavenging, and FRAP assays. Furthermore, the antioxidant phenolic compounds

were evaluated for their cytoprotective capacity against H O -induced oxidative stress in HepG-2 cells. Compounds 3 and 5 could

signiﬁcantly inhibit reactive oxygen species production, and compounds 3, 5, and 6 could remarkably prevent the cell apoptosis.

Then, the R. beesianus rhizome, which contained phenolic compounds, might serve as a functional food for potential application on

preventing oxidative stress-connected diseases.

KEYWORDS: Rhynchanthus beesianus, diarylheptanoid, antioxidant activity, intracellular reactive oxygen species, cell apoptosis

INTRODUCTION

The genus Rhynchanthus Hook.f. (Zingiberaceae) is a small

■

genus with about six species. Rhynchanthus beesianus is the

Reactive oxygen species (ROS), the major etiological factor of

oxidative stress, could cause cell damage and apoptosis when

these are excessively produced. The excessive production of

ROS accelerates aging and increases the risk of diseases, for

example, Alzheimer’s disease, diabetes, atherosclerosis, Parkin-

son’s disease, and cancer. Fortunately, antioxidants could

eﬃciently retard the process of these disorders. A number of

antioxidants, including phenols, ﬂavonoids, polysaccharides,

and tannins, have been proved to reduce oxidative damage

through scavenging radicals, metal chelation, and promotion of

only species of this genus that is distributed in southern

China. It is an edible, ornamental, and medicinal plant with

tuberous rhizomes, having unique aroma and taste compared

with other edible gingers. Besides being an important blending

agent in kitchen, the tender leaves and tuberous rhizomes of R.

beesianus are widely and traditionally consumed as vegetables

20,21

and edible spices.

Additionally, R. beesianus rhizomes have

been traditionally used as folk medicine for regulating various

gastrointestinal disorders including abdominal discomfort,

9,20

dyspepsia, belching, and bloating.

Previous studies

the expression of antioxidant and phase II metabolic enzymes.

Studies have shown that the intake of natural antioxidants has

reported that the essential oil from R. beesianus rhizomes

19,20

exhibited anti-inﬂammatory and antibacterial activities.

good protective eﬀects on oxidative stress-induced degener-

However, until now, there is no report on the nonvolatile

components of R. beesianus rhizome and their antioxidant

activity. Therefore, the aim of this research was to illustrate the

chemical components from R. beesianus rhizomes and evaluate

their antioxidant and cytoprotective activities.

ative diseases.

As a vital class of natural antioxidants,

phenolics have attracted particular attention for their high

−9

contents in daily diets.

Diarylheptanoids, composed of two aromatic rings linked by

Phytochemical investigation on R. beesianus rhizomes

resulted in 15 polyphenols, including three new diary-

lheptanoids rhynchanines A−C (1−3) and one new phenyl-

propanoid, 4-O-methylstroside B (9). Compound 5 had been

a heptane skeleton, are an important group of natural

polyphenols with various bioactives. The diarylheptanoids

are commonly present in linear and cyclic forms, and the latter

usually contain a six-membered tetrahydropyran moiety.

reported only by LC−MS identiﬁcation from Curcuma longa;

Previous pharmacological investigations demonstrated that the

diarylheptanoids have remarkable antioxidant, antitumor,

antidepressant, antitubercular, neuroprotective, and estrogenic

Received: February 13, 2021

Revised: April 30, 2021

Accepted: May 3, 2021

Published: May 24, 2021

0−15

eﬀects.

been isolated from edible plants of Curcuma, Alpinia, Zingiber

Zingiberaceae), Myrica (Myricaceae), Corylus (Betulaceae),

Garuga (Burseraceae), and Juglans (Juglandaceae) gen-

In recent decade, various diarylheptanoids have

(

1,16,17

era.

2021 American Chemical Society

Journal of Agricultural and Food Chemistry

J. Agric. Food Chem. 2021, 69, 6229−6239

229

Article

Figure 1. Compounds 1−15 from R. beesianus.

herein, 5 is isolated as a pure monomer and its NMR data are

assigned for the ﬁrst time. The antioxidant capacity of

solvent signals. MS data were measured on Waters Xevo TQS or

Waters AutoSpec Premier P776 mass spectrometers. Semipreparative

HPLC was performed on a Waters 600 with a COSMOSIL C18 (10 ×

250 mm, NacalaiTesque Corporation, Japan) column and analytical

HPLC was performed on a Shimadzu SIL-20A Series HPLC system

^e^q^uⁱ^p^p^e^d^wⁱ^t^h^a^r^e^v^e^r^s^e^-^p^h^a^s^e^C^O^S^M^O^S^I^L^C18 column (4.6 mm ×

compounds 1−15 was assessed by DPPH, ABTS , and

FRAP assays. Additionally, the antioxidant phytochemicals

were tested for their protective eﬀect on oxidative stress in

H O -induced human liver HepG-2 cells. The cytoprotective

50 mm, 5 μm, NacalaiTesque Corporation, Japan). Silica gel (200−

capacity was assessed for regulating the intracellular ROS

production and cell apoptosis by ﬂow cytometry. Compounds

300 mesh, Qingdao Marine Chemical Co., Ltd., China), MCI-gel

(

75−150 μm, Mitsubishi Chemical Corporation, Japan), YMC-gel

⁵⁰^μ^m^,^Y^M^C^C^o^.^,^L^t^d^.^,^J^a^p^aⁿ⁾^,^aⁿ^d^R^P^-^C18 column (40−63 μm,

and 5 signiﬁcantly inhibited ROS production, while

compounds 3, 5, and 6 remarkably prevented the cell

apoptosis.

Merck Drugs & Biotechnology, Germany) were used for column

chromatography. The PMP (Chengdu Aikeda Chemical Reagent Co.,

Ltd., China), 2,2-diphenyl-1-picryhydrazyl (DPPH) radical, methyl-

thiazol-2-yl-2,5-diphenyltetrazoliumbromide (MTT), 2,2′-azino-bis

MATERIALS AND METHODS

■

General Experimental Procedures and Reagents. Optical

rotations were measured on a JASCO P-1020 polarimeter. The UV

spectra were recorded on a Shimadzu UV-2401PC spectrometer. The

IR spectra were measured on a Bruker FT-IR Tensor-27 infrared

spectrophotometer with KBr disk. The NMR spectra were obtained

on Bruker DRX-400, DRX-500, and DRX-600 spectrometers.

Chemical shifts (δ) were expressed in ppm with reference to the

(

3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), 2′,7′-dichloro-

ﬂuorescindiacetate (DCFH-DA), and 1,3,5-tri(2-pyridyl)- 2,4, 6-

triazine (TPTZ) were purchased from Sigma-Aldrich (Shanghai,

China). A ﬂow cytometer (Guava easyCyte 6-2L, Millipore, Billerica,

USA) and Annexin V-FITC/PI apoptosis kit were purchased from

Beijing 4A Biotech Co., Ltd. (Beijing, China).

230

J. Agric. Food Chem. 2021, 69, 6229−6239

Journal of Agricultural and Food Chemistry

Article

Plant Materials. The R. beesianus rhizomes, identiﬁed by Mr.

Buyun Zhang, were purchased from Dali City of Yunnan province,

China, on January 25, 2018, kept dry, and ventilated. A voucher

specimen (no. 20180125) was stored in a cool and dried environment

of Kunming Institute of Botany, CAS.

the calculated ECD curves were obtained by SpecDis version 1.63

software.

Preparation of the (R)- and (S)-MTPA Ester Derivatives of

Compound 9a. Mosher’s reaction was performed in accordance with

the previous method with slight modiﬁcations. Compound 9a (the

aglycone of compound 9) (1 mg) was dissolved in 500 μL of

anhydrous tetrahydrofuran (THF), and then 2 equiv of 4-

(dimethylamino) pyridine (4-DMAP), 2 equiv of DCC, and 2

equiv of (R)-MTPA chloride were added under vacuum. The mixture

was dried and stirred at 25 °C for 24 h. Then, the solvent was

removed, the mixture was puriﬁed using a Sephadex LH-20 column

(MeOH), and the (R)-MTPA ester of 9aR was obtained. The (S)-

MTPA ester of compound 9aS was prepared by the same procedure.

Extraction and Isolation. The air-dried R. beesianus rhizomes

(

8.0 kg) were kibbled and extracted with 90% aqueous EtOH (50 L ×

, each time for 3 h), and then the solvent was evaporated in vacuum.

The extract (622 g) was chromatographed on a silica gel column

⃗

using sequential gradient elution of CHCl −Me CO (1:0 0:1) to

obtain six fractions. Fraction II (23.9 g) was separated on a YMC

column, eluted successively with MeOH/H O (75:25 100:0), and

then puriﬁed on a silica gel column (petroleum ether−Me CO, 30:1

⃗

20:1) to give compounds 12 (14.4 mg), 14 (212.3 mg), and 8 (11.2

The H NMR spectra of the (R)- and (S)-MTPA esters were obtained

mg). Fraction IV (15.8 g) was applied on an MCI column, eluted

after each reaction.

⃗

successively with MeOH/H O (40:60 100:0), and then chromato-

Rhynchanine A (1). White amorphous powder; [α]²_D² −28.6° (c

.17, MeOH); UV (MeOH) λ (log ε) 196 (3.9), 244 (4.0), 268

graphed over a silica gel column elution with CHCl /Me CO (10:1)

max

to obtain 13 (4.5 mg). Fraction V (114 g) was subjected to an RP-C

(

4.2) nm; IR (KBr) ν 3439, 2922, 1614, 1514, 1266, 1224, 1033,

16 cm ; H NMR (CD OD, 600 MHz) and C NMR (CD OD,

3 3

⃗

max

column with a gradient of MeOH/H O (30:70 85:15) to aﬀord ﬁve

−1

subfractions V-1 to V-5. Subfraction V-1 (12.4 g) was chromato-

50 MHz) data, see Table 1; positive HRESIMS m/z 723.3142 [M +

graphed on a silica gel column (CHCl /MeOH, 8:1) to get

Na] (calcd. for C H O Na , 723.3140).

48 10

compound 10 (279.1 mg) and a mixture. The latter was further

separated over HPLC to give 11 (15.8 mg; t = 12 min; CH CN/

Rhynchanine B (2). White amorphous powder; [α]²²−21.2° (c

0.25, MeOH); UV (MeOH) λ_m_a_x(log ε) 196 (4.8), 244 (4.0), 267

H O, 7:93; 3.0 mL/min). Subfraction V-3 (11 g) was separated on a

silica gel column (CHCl /MeOH, 20:1) and then puriﬁed over

(4.2) nm; IR (KBr) ν 3431, 2923, 1614, 1514, 1265, 1225, 1033,

max

HPLC to yield compounds 1 (8.5 mg; t = 40.5 min; CH CN/H O,

−1

820 cm ; H NMR (CD OD, 600 MHz) and C NMR (CD OD,

5:65; 1.0 mL/min), 2 (7.3 mg; t = 43.5 min; CH CN/H O, 35:65;

R 3 2

150 MHz) data, see Table 1; positive HRESIMS m/z 723.3145 [M +

.0 mL/min), and 15 (8.1 mg; t = 27.5 min; CH CN/H O, 40:60;

Na] (calcd. for C H O Na , 723.3140).

41 48 10

.0 mL/min). Subfraction V-4 (20.5 g) was separated on a silica gel

Rhynchanine C (3). White amorphous powder; [α]²⁶−14.9° (c

0.10, MeOH); UV (MeOH) λmax (log ε) 196 (3.7), 264 (3.1), 308

(3.1), 337 (3.2) nm; IR (KBr) νmax 3417, 2920, 1637, 1614, 1518,

⃗

column (CHCl /MeOH, 20:1 6:1) to give compound 7 (15.3 mg)

and a mixture, which was puriﬁed by HPLC to aﬀord compound 9

(

9.6 mg; t = 52 min; MeOH/H O, 25:75; 2.5 mL/min). Subfraction

−

V-5 (13.8 g) was subjected on a silica gel column (CHCl −MeOH,

1276, 1076, 1034 cm ; H NMR (DMSO-d , 600 MHz) and

⃗

:1 2:1) to aﬀord compounds 6 (113.0 mg) and 4 (24.5 mg) and

NMR (DMSO-d , 150 MHz) data, see Table 2; positive HRESIMS

subfractions V-5-1 to V-5-3. Subfraction V-5-1 was separated on a

m/z 591.2048 [M + Na] (calcd. for C H O Na , 591.2048).

27 36 13

silica gel column (CHCl /MeOH, 6:1) and then isolated on a

Tsaokopyranol G (3a). White amorphous powder; [α] −19.5°

Sephadex LH-20 column with MeOH to obtain 3 (6.8 mg).

Subfraction V-5-2 was applied on a silica gel column (CHCl₃/

MeOH, 6:1) and then isolated on a Sephadex LH-20 column

(

c 0.032, MeOH); H NMR (CD OD, 400 MHz) δ : 7.09 (1H, s, H-

′), 6.90 (1H, s, H-2″), 6.68−6.77 (4H, overlap, H-5′, 6′, 5″, 6″),

.80 (1H, d, J = 6.8 Hz, H-7), 4.55 (1H, d, J = 11.3 Hz, H-1), 4.26

1H, m, H-3), 3.93 (3H, s, 3′-OCH ), 3.72 (3H, s, 5′-OCH ), 3.64

1H, m, H-5), 3.43 (1H, dd, J = 8.0, 1.9 Hz, H-6), 2.07 (1H, t, J =

13.2 Hz, H-4a), 1.81 (1H, d, J = 14.0 Hz, H-2a), 1.73 (1H, q, J = 12.2

Hz, H-2b), 1.47 (1H, d, J = 14.1 Hz, H-4b).

(

MeOH) to yield 5 (8.9 mg) (Figure 1).

Acid Hydrolysis of Compounds 3 and 9 and HPLC Analysis.

The acid hydrolysis of compounds 3 and 9 was carried out by a

previously reported procedure, with minor modiﬁcations. Com-

pound 3 (2.0 mg) was reﬂuxed with 2 mL of solvents (2 M TFA) on

an oil bath for 2 h at 120 °C. Then, the aglycone was extracted with

4-O-methylstroside B (9). White amorphous powder;[α] +16.2°

CHCl (1 mL, three times). Next, 60 μL of NaOH (0.3 mol/L) and

(c 0.07, MeOH); UV (MeOH) λ_m_a_x(log ε) 197 (4.4), 252 (2.7), 278

60 μL of PMP (0.5 mol/L in methanol) were added to derivatize the

(

3.2) nm; IR (KBr) ν 3424, 2924, 1631, 1516, 1262, 1075, 1027

cm ; H NMR (DMSO-d , 500 MHz) and C NMR (DMSO-d₆,

25 MHz) data, see Table 2; positive HRESIMS m/z 397.1468 [M +

max

sugar in the aqueous portion and reacted at 75 °C for 60 min. Then,

the reaction was quenched with 60 μL of HCl (0.3 mol/L) and the

−1

reaction mixture was extracted with CHCl (1 mL, three times). The

Na] (calcd. for C H O Na , 397.1469).

17 26 9

aqueous layer was further analyzed over HPLC (t = 18.5 min; 18%

(R)-3-3(3,4-Dimethoxyphenyl)propane-1,2-diol (9a). White

acetonitrile:82% sodium phosphate (pH 6.8); 1.5 mL/min). The acid

hydrolysis and HPLC analysis of 9 were performed by the same way

as that of 3. Likewise, the standard monosaccharide D-Glc (1 mg) was

derivatized with PMP the same way as 3 and 9, and HPLC analysis

was performed under the same conditions as 3 and 9. The sugar

moieties of compounds 3 and 9 were identiﬁed as D-Glc, showing

retention times (t_R= 18.5 min) consistent with the standard

monosaccharide (D-glucose) derivative.

amorphous powder; [α]²³+35.1° (c 0.043, MeOH); ¹H NMR

(CD

OD, 400 MHz) δ

: 6.77 (1H, d, J = 8.2 Hz, H-5), 6.75 (1H, d, J

= 2.0 Hz, H-2), 6.68 (1H, dd, J = 8.2, 2.0 Hz, H-6), 3.73 (3H, s, 3-

OCH ), 3.70 (3H, s, 4-OCH ), 3.67 (1H, overlap, H-8), 3.37 (2H,

dd, J = 11.1, 5.4 Hz, H-9), 2.67 (1H, dd, J = 13.8, 5.6 Hz, H-7a), 2.52

(1H, dd, J = 13.8, 7.5 Hz, H-7b).

Antioxidant Activity Assays. DPPH Radical Scavenging

ECD Calculations. The ECD calculations of 3a (the aglycone of

compound 3) were achieved using Gaussian 16. More speciﬁcally, the

Activity. The DPPH scavenging capacity of the compounds was

26,27

carried out by a previous method with slight modiﬁcations.

D structure of 3a was ﬁrst conﬁrmed by its ROESY spectrum and

Brieﬂy, 50 μL of the sample (12.5, 25, 50, 75, and 100 μM) and 200

μL of DPPH (0.1 mM in 95% ethanol) were put in a 96-well plate and

then passed to conformational analysis, which was applied for

CONFLEX software (Conﬂex Corp., Tokyo, Japan) including a

molecular mechanics force ﬁeld (MMFF94s). At the B3LYP/6-

measured as (A ). The blank contained 50 μL of the sample (12.5, 25,

50, 75, and 100 μM) and 200 μL of 95% ethanol and was measured as

31+G(d) level in the gas phase, the available conformers were further

(A ), while the control included 50 μL of ethanol and 200 μL of

optimized by the density functional theory (DFT). Using time-

dependent DFT (TDDFT) at the B3LYP/6-311+G(d) level, ECD

calculations were used for the optimized conformations, and ﬁnally,

DPPH (0.1 mM in 95% ethanol) and was observed as (A ). All the

mixtures were shaken thoroughly at 30 °C for half an hour in the dark,

and then the absorbance values were recorded later (at 517 nm). The

231

J. Agric. Food Chem. 2021, 69, 6229−6239

Journal of Agricultural and Food Chemistry

Article

Table 1. H (600 MHz) and ¹³C NMR (150 MHz)

_o_f_t_h_e_s_a_m_p_l_e₍₁₂_.₅_,₂₅_,₅₀_,₇₅_,_a_n_d₁₀₀_μ_M₎_a_n_d₂₀₀_μ_L_o_f_A_B_T_S+

solution was taken in a 96-well plate and measured as (A ). The blank

Spectroscopic Data for 1 and 2 in CD OD

contained 25 μL of the sample (12.5, 25, 50, 75, and 100 μM) and

200 μL of 95% ethanol and was measured as (A ), while the control

included 25 μL of ethanol and 200 μL of ABTS solution and was

position

δ_H(mult., J in Hz)

δ_C

δ_H(mult., J in Hz)

δ_C

observed as (A ). All the mixtures were shaken thoroughly at 30 °C in

4.72 d (9.7)

4.15 dd (9.7, 2.8)

4.30 m

77.8 4.71 d (9.7)

84.0 4.14 dd (9.7, 2.9)

66.7 4.28 m

39.1 1.94 ddd (14.2, 3.7,

2.0)

77.9

83.9

66.7

39.0

the dark for 6 min, and then the absorbance values were tested later

(at 734 nm). All the solutions were prepared on the same day for the

assay. The antioxidant activity (RSA) of all samples was calculated as

follows:

1.95 ddd (14.1, 3.7,

.1)

.75 overlap

1.74 overlap

72.4 3.96 m

38.9 1.78 m

A − (A − A )

RSA (%) =

× 100%

3.95 m

1.79 m

72.4

38.9

A₀

.70 overlap

1.69 overlap

32.1 2.56 m

The positive control was ascorbic acid (V ), and all data were the

averages of triplicate measurements.

2.62 m

32.2

133.2

112.8

148.8

148.0

115.9

122.2

56.5

134.2

130.4

116.3

156.7

116.3

130.4

133.2

129.9

80.8

′

132.7

Ferric Reducing Antioxidant Power (FRAP) Assay. The FRAP

assay was assessed by a previously reported procedure with minor

7.00 d (2.0)

112.8 6.98 d (1.9)

148.8

modiﬁcations. The acetate buﬀer (300 mM, pH 3.6) and

147.8

tripyridyltriazine solution (10 mM) in 40 mM HCl were mixed

with FeCl ·6H O (20 mM) in proportion of 10:1:1, and then the

6.76 d (8.1)

6.93 dd (8.1, 2.0)

3.78 s

115.9 6.74 d (8.1)

122.2 6.91 dd (8.1, 1.9)

56.5 3.77 s

resultant FRAP reagent was kept in a water bath for 0.5 h at 37 °C.

The test mixtures including 20 μL of the sample (12.5, 25, 50, 75, and

′-OMe

″

‴

00 μM) and 180 μL of FRAP reagent were kept in a 96-well plate

134.3

and measured as (A ). The blank contained 200 μL of 95% ethanol

6.96 d (8.5)

6.64 d (8.5)

130.4 6.98 d (8.5)

116.2 6.66 d (8.5)

156.6

and 20 μL of distilled water and was measured as (A ). All the

mixtures were kept at 37 °C in the dark for 10 min, and then the

absorbance values were taken later (at 593 nm). Ferrous sulfate

6.64 d (8.5)

6.96 d (8.5)

6.47 d (15.9)

5.86 dd (15.9, 8.3)

3.90 td (8.3, 7.0)

1.83 dd (7.0, 2.1)

116.2 6.66 d (8.5)

130.4 6.98 d (8.5)

134.3 6.42 d (15.9)

129.3 5.91 dd (15.9, 8.0)

82.4 3.93 td (8.5, 3.7)

44.1 1.73 overlap

1.56 ddd (14.2, 9.0,

(

FeSO ·7H O) was used to protract the standard curve (y = 1.3334x

4 2

+ 0.08). All the solutions were prepared freshly before use.

Intracellular Antioxidant Capacity against H O -Induced

HepG-2 Cells. Cell Culture and MTT Assay. HepG-2 cells were

obtained from Kunming Cell Bank, CAS, and maintained in DMEM

medium using 10% (v/v) fetal bovine serum (FBS), supplemented

with 1% antibiotics, which were a mixture of penicillin (100 U/mL)

and streptomycin (100 mg/mL), and then cultured in a humidiﬁed

45.0

.64 m

.7)

environment containing 5% CO /95% air at 37 °C. The viability of

‴

3.62 td (8.5, 4.4)

1.68 m

2.53 m

69.3 3.75 overlap

41.0 1.66 m

32.2 2.64 m

56.4 3.27 s

68.2

41.3

32.1

the cells was tested by MTT assay in accordance with a reported

method. The HepG-2 cells were seeded in a 96-well plate (5000

cells/well) and then cultured overnight. After treating with diﬀerent

concentrations of compounds (2.5, 5, 10, 20, and 40 μM) and

enduring for 6 h, 20 μL of MTT (5 mg/mL) was added to the wells,

and then the cells were cultured at 37 °C for another 4 h. At last, the

culture medium was discarded, and then DMSO (100 μL) was added

to thoroughly dissolve the blue crystals in each well. The absorbance

was subsequently measured at 570 nm.

‴-OMe

3.26 s

56.7

⁗

133.3

133.4

111.4

152.4

148.3

120.2

120.8

56.6

134.3

130.5

116.3

156.6

116.3

130.5

6.93 d (2.0)

111.4 6.95 d (2.0)

152.4

148.3

6.51 d (8.3)

6.77 dd (8.3, 2.0)

⁗-OMe 3.79 s

120.2 6.50 d (8.3)

120.9 6.76 dd (8.3, 2.0)

56.6 3.78 s

Assessment of Intracellular Reactive Oxygen Species (ROS). A

ﬂuorescence DCFH-DA probe was used to quantify the intracellular

,27

ROS content according to the reported instructions.

HepG-2 cells

⁗′

134.3

were seeded in six-well plates (2 × 10 cells/well) and were cultured

⁗′

6.99 d (8.5)

6.68 d (8.5)

130.5 6.99 d (8.5)

116.3 6.67 d (8.5)

156.6

116.3 6.67 d (8.5)

130.5 6.99 d (8.5)

for 24 h. Then, compounds 1−8 (10 μM) or V (10 μM, positive

control) was used to treat the cells for another 24 h. The cells were

collected and washed with cold PBS, then0.7 mM H O was added,

6.68 d (8.5)

6.99 d (8.5)

and the cells were incubated for another 6 h. After that, the culture

medium was discarded, serum-free medium and 1 mL of 10 μM

DCFH-DA were added, and then the cells were cultured in the dark at

7 °C. After incubating for 20 min, the cells were washed and

DPPH radical scavenging activity (RSA) of the samples was calculated

by the following formula:

detected with ﬂow cytometry.

Assessment of Cytoprotective Activity against H O -Induced

HepG-2 Cell Apoptosis. Apoptotic and necrotic cells were monitored

using the Annexin VFITC/PI detection kit in line with a previous

study with slight modiﬁcations. HepG-2 cells were incubated and

A − (A − A )

RSA (%) =

× 100%

A₀

The positive control was ascorbic acid (V ), and all data were the

averages of triplicate measurements.

treated with the compounds. Subsequently, the cells were collected,

washed with cold PBS, and then resuspended in 100 μL of buﬀer.

After staining the cells with Annexin V-FITC (5 μL) at room

temperature for 5 min, the cells were stained with 10 μL of PI (20 μg/

mL) and reacted for 5 min in an ice bath. Afterward, the percentage of

apoptosis and necrosis of HepG-2 cells was analyzed by ﬂow

cytometery (BD Biosciences, USA).

ABTS Radical Scavenging Activity. To ﬁnd out the antioxidant

capacity of the samples, the ABTS assay was carried out with an

improved method as reported previously. In short, the working

solution (ABTS solution) was diluted to an absorbance of 0.70 ±

.02 when measured at 734 nm. The test mixture consisting of 25 μL

232

J. Agric. Food Chem. 2021, 69, 6229−6239

Journal of Agricultural and Food Chemistry

Article

Table 2. H and ¹³C NMR Spectroscopic Data for 3, 5, and 9 in DMSO-d₆

position

δ_H(mult., J in Hz)

δ_C

δ_H(mult., J in Hz)

δ_C

position

δ_H(mult., J in Hz)

δ_C

4.58 dd (11.5, 3.3)

1.74 ddd (13.8, 3.3, 2.8)

73.1

39.2

7.56 d (15.8)

7.09 d (15.8)

142.6

122.6

132.0

113.7

148.8

147.5

112.1

121.6

39.4

6.85 d (1.9)

.54 ddd (13.8, 11.5, 2.8)

4.09 m

1.67 ddd (14.4, 11.4, 2.8)

62.7

33.6

188.4

124.5

6.63 d (15.2)

6.84 d (8.1)

6.74 dd (8.1, 1.9)

2.71 dd (13.6, 5.7)

2.54 dd (13.6, 7.3)

3.80 m

3.68 dd (10.0, 4.2)

3.27 dd (10.0, 6.7)

3.73 s

3.71 s

4.13 d (7.7)

2.99 t (7.8)

3.08 m

3.06 m

.44 ddd (14.4, 3.1, 2.8)

4.10 overlap

3.69 dd (5.6, 3.6)

4.74 d (5.6)

71.5

82.3

71.9

7.50 d (15.2)

7.00 d (15.2)

6.97 d (15.2)

143.9

128.4

142.3

126.2

114.3

146.0

147.7

116.3

120.4

70.9

73.5

′

134.0

110.5

147.2

145.5

115.0

118.3

55.6

133.8

111.7

146.9

145.2

114.7

119.5

55.3

6.91 d (1.9)

6.99 d (1.9)

3-OMe

4-OMe

55.9

55.8

103.9

74.0

77.3

70.4

76.8

61.4

6.72 d (8.1)

6.75 dd (8.1, 1.9)

3.76 s

6.76 d (8.1)

6.89 dd (8.1, 1.9)

1′

2′

3′

4′

5′

6′

′-OMe

″

128.2

130.9

116.3

160.3

116.3

130.9

6.97 d (1.9)

7.62 d (8.4)

6.83 d (8.4)

3.14 t (8.6)

3.64 dd (11.7, 1.8)

3.43 dd (11.7, 5.3)

6.68 d (8.1)

6.78 dd (8.1, 1.9)

3.70 s

4.33 d (7.7)

2.99 m

3.04 dd (17.1, 8.6)

3.14 dd (17.1, 8.6)

3.42 overlap

6.83 d (8.4)

7.62 d (8.4)

″-OMe

‴

101.9

73.5

77.0

70.1

76.4

3.67 dd (11.7, 1.8)

61.4

.43 overlap

Compounds 3 and 5 were measured at H (600 MHz) and ¹³C NMR (150 MHz); compound 9 was measured at H (500 MHz) and ¹³C NMR

(125 MHz).

RESULTS AND DISCUSSION

The long-range H− H COSY connectivity of δ 2.62 (H-

■

)/1.79 (H-6)/3.95 (H-5)/1.95 (H-4)/4.30 (H-3)/4.15 (H-

)/4.72 (H-1) and the HMBC correlations from δ 4.72 (1H,

Structure Elucidation of Puriﬁed Compounds 1−3

and 9. Compound 1 was obtained as a white amorphous

powder. Its molecular formula was assigned on C H O on

d, J = 9.7 Hz, H-1) to δ 132.7 (s, C-1′), 112.8 (d, C-2′), 122.2

48 10

(

d, C-6′), and 72.4 (d, C-5) and from δ 3.95 (1H, m, H-5) to

the basis of the positive HRESIMS ion at m/z 723.3142 [M +

δ 77.8 (d, C-1), 66.7 (d, C-3), and 32.1 (t, C-7) presented a

Na] (calcd. for C H O Na , 723.3140), indicating 18

48 10

heptane chain having a tetrahydropyran ring linking C-1 to C-5

in unit I of 1 (Figure 2). Meanwhile, the other long-range

degrees of unsaturation. The H NMR spectrum (Table 1)

showed the presence of two AA′BB′ systems (δ 6.64 and

.96, each d, J = 8.5 Hz; δ 6.68 and 6.99, each d, J = 8.5 Hz)

H− H COSY connectivity of H-7‴ (δ 2.53) and H-1‴ (δ

(

.47) to the adjacent protons and the chemical shifts of C-1‴

and two ABX systems (δ 6.76, d, J = 8.1 Hz; 6.93, dd, J = 8.1,

δ 134.3, d), C-2‴ (δ 129.3, d), C-3‴ (δ 82.4, d), and C-5‴

.0 Hz; 7.00, d, J = 2.0 Hz; and δ 6.51, d, J = 8.3 Hz; 6.77, dd,

δ 69.3, d) conﬁrmed the other heptane chain with a double

J = 8.3, 2.0 Hz; 6.93, d, J = 2.0 Hz), which suggested the

bond and two oxygenated methines in unit II of 1. The unit I

of 1 was similar to hedycoropyran B except the absence of a

hydroxyl group at C-6, which was suggested by HMBC

correlations of δ 1.79 (1H, m, H-6) with δ 39.1 (t, C-4) and

presence of two 1,4-disubstituted and two 1,3,4-trisubstituted

phenyl groups. Moreover, the H NMR spectrum of 1

displayed resonance signals for a pair of oleﬁnic protons, three

methoxy groups, and six oxygenated methine protons (Table

34.3 (s, C-1″) and the chemical shift of C-6 (δ 38.9, t).

). The C NMR spectrum displayed 41 carbon signals,

Meanwhile, the unit II of 1 resembled neohexahydrocurcumin

with the exception of an additional methoxy group at C-3‴ and

the absence of a methoxy group C-3⁗′, as conﬁrmed by

HMBC correlations of δ 3.26 (3H, s) with δ 82.4 (d, C-3‴)

including three methoxy groups, six aliphatic methylenes, six

oxygenated methine groups, and 26 aromatic carbon

resonances (16 methines and 10 quaternary carbons, including

four benzene rings and a double bond) (Table 1). These data

suggested that 1 was a diarylheptanoid dimer with two C6−

and of δ 6.99 (2H, d, J = 8.5 Hz, H-2⁗′ and 6⁗′) to δ 156.6

(s, C-4⁗′). The other two methoxy groups were located at C-

3′ and C-3⁗, which were supported by HMBC correlations

from δ 3.78 (3H, s) to δ 148.8 (s, C-3′) and from δ 3.79

C7−C6 units, corresponding to the structures similar to

hedycoropyran B (unit I) and neohexahydrocurcumin (unit

II) (Figure 1).

(3H, s) to δ 152.4 (s, C-3⁗), respectively. Finally, the HMBC

233

Journal of Agricultural and Food Chemistry

J. Agric. Food Chem. 2021, 69, 6229−6239

of C-3 ‴ and C-5 ‴ in 1 . Therefore, compound 2 was elucidated

Article

as the diastereomer of 1 at C-3‴ and/or C-5‴ and named as

rhynchanine B.

Structurally, compounds 1 and 2 had two chiral carbons

with uncertain conﬁguration (C-3‴ and C-5‴), and the

possible relative conﬁgurations of C-3‴ and C-5‴ were 3‴R

‴R, 3‴S 5‴S, 3‴R 5‴S, and 3‴S 5‴R. Thus, compounds 1

and 2 were two of four diastereomers and were isolated as

highly pure monomers, which were supported by 1D and 2D

NMR data. To determine the absolute conﬁguration, the para-

toluene sulfonyl ester derivatives of 1 and 2 were synthesized

to grow their crystals. Unfortunately, the crystals were not

formed. Moreover, the experimental electron circular dichro-

ism (ECD) and calculated curves did not coincide because of

the ﬂexible heptane chain in unit II of 1 and 2.

The molecular formula of 3 was determined as C H O

36 13

Figure 2. Key H− H COSY and HMBC correlations of compounds

−3 and 9.

by the molecular ion peak at m/z 591.2048 [M + Na] in the

HRESIMS, suggesting 10 degrees of unsaturation. The H

NMR spectrum (Table 2) showed that 3 had resonances for

six aromatic protons at δ 6.68 (1H, d, J = 8.1 Hz), 6.78 (1H,

dd, J = 8.1, 1.9 Hz), 6.97 (1H, d, J = 1.9 Hz), 6.72 (1H, d, J =

correlation from δ 4.15 (1H, dd, J = 9.7, 2.8 Hz, H-2) to δ_C

48.3 (s, C-4⁗) implied that the two units were connected via

30,31

8.1 Hz), 6.75 (1H, dd, J = 8.1, 1.9 Hz), and 6.91 (1H, d, J = 1.9

an ether linkage between C-2 and C-4⁗.

Thus, the planar

Hz), presenting two 1,3,4-trisubstituted benzene rings. An

anomeric proton at δ 4.33 (1H, d, J = 7.7 Hz), an oxygenated

structure of compound 1 was elucidated and named as

rhynchanine A.

methylene group at δ 3.67 (1H, dd, J = 11.7, 1.8 Hz) and 3.43

The ROESY correlation of H-1 with H-5, of H-2 with H-3

and H -4, and of H-3 with H -4 (Figure 3) indicated that the

(

1H, overlapped), along with ﬁve oxygenated methine carbons

at δ 101.9, 77.0, 76.4, 73.5, and 70.1, and an oxygenated

tetrahydropyran ring of unit I of 1 was in a chair conformation

31,33

methylene carbon at δ 61.4 indicated that a β-glucose moiety

and H-1 and H-5 were in an axial orientation.

The J₁_,₂

was present in 3. The sugar unit was identiﬁed as D-glucose (t

.7 Hz and J₂_,₃= 2.8 Hz values indicated that H-1 and H-2

20.5 min) by acid hydrolysis and HPLC analysis. Besides

were in an axial orientation and H-3 was in an equatorial

orientation, as conﬁrmed by ROESY correlations of H-1 with

H-2⁗. The large coupling constants (J = 15.9 Hz) between H-

the β-D-glucose moiety and two 1,3,4-trisubstituted benzene

rings, there were signals for ﬁve oxygenated methine groups in

33,34

the C NMR spectrum.

Comparison of NMR spectro-

‴ and H-2‴ suggested an E conﬁguration of the double bond.

scopic data of 3 indicated that its aglycone resembled

tsaokopyranol G, except that the major diﬀerence was one

Although the ROESY correlation of H-3‴ with H-5‴ could be

obviously observed, the conﬁgurations of H-3‴ and H-5‴

could not be determined because of the ﬂexible heptane chain

in unit II of 1.

more β-D-glucose moiety in 3. The β-D-glucose moiety was

attached at C-6 of the aglycone via an ether linkage, as

The molecular formula of 2 was established as C H O by

supported by the HMBC correlations from H-1‴ to δ 82.3 (d,

48 10

the positive HRESIMS spectrum ([M + Na] at m/z

23.3145), which was similar to that of 1. They had the

C-6).

The ROESY correlation of H-1 with H-5, of H-2 with H-3

and Hax-4, and of H-3 with Hax-4 (Figure 3) indicated that the

tetrahydropyran ring of unit I of 1 was in a chair conformation

same physical data in the UV and IR spectra, suggesting the

presence of the same functional groups. The 1D and 2D NMR

spectra of 2 showed almost the same behavior to those of 1

31,33

and H-1 and H-5 were in an axial orientation.

Two protons

(

−

Table 1), except that the chemical shifts of C-3‴ and C-5‴ in

[δ 1.74 (ddd, J = 13.8, 3.3, 2.8 Hz) and 1.54 (ddd, J = 13.8,

slightly shifted (Δδ +0.03 ppm and +0.13 ppm and Δδ

11.5, 2.8 Hz)] were linked to C-2; the former should be

equatorial and the latter should be axial. Meanwhile, the

1.6 ppm and −1.1 ppm, respectively), compared with those

proton at δ 1.67 (ddd, J = 14.4, 11.4, 2.8 Hz, H-4) should be

Figure 3. Key ROESY correlations of compounds 1−3.

234

J. Agric. Food Chem. 2021, 69, 6229−6239

Journal of Agricultural and Food Chemistry

the structure of 3 was determined and named rhynchanine C.

Article

axial and the other proton at δ 1.44 (ddd, J = 14.4, 3.1, 2.8

HMBC correlations from δ 3.73 (3H, s) to δ 147.5 (s, C-4).

H C

The β-D-glucose moiety was attached at C-9 as indicated by

Hz, H-4) should be equatorial. The J₁_,₂= 11.5 Hz, J₂_,₃= 2.8

Hz, and J₃_,₄= 3.1 Hz values indicated that H-1, H -2, and H -

HMBC correlations from H-1‴ to δ_C73.5 (d, C-9). The

were axially oriented and H-3 was in an equatorial

orientation, as supported by the ROESY correlations of H_a_x-

with H -4 and of H -2/H -4 with H-3. To assign the

optical rotation value ([α] _D+16.2°) was in good agreement

with that of stroside B ([α] +8.0°), which suggested that the

absolute conﬁgurations, the hydrolysis of 3 aﬀorded the

aglycone (compound 3a) and a sugar unit, and the absolute

conﬁguration of 3a was determined as 1R,3S,5S,6S,7R by the

ECD calculations, which showed a high agreement between

the experimental and calculated ECD spectra (Figure 4). Thus,

absolute conﬁguration of C-8 was R. To conﬁrm the surmise,

the hydrolysis of 9 aﬀorded the aglycone (compound 9a) and a

sugar unit, and the optical rotation value of compound 9a (

[

α] +35.1°) was in good agreement with that of (+)-3-(3,4-

dimethoxyphenyl)propane-1,2-diol ([α]

+18°), while the

optical rotation value of the (−)-isomer was the opposite (

[

α]

−23°), which further veriﬁed that the absolute

conﬁguration of C-8 was R. In addition, Mosher’s ester

was also synthetized from the aglycone (compound 9a) to

determine the absolute conﬁguration of C-8. Treatment with

(

R)- and (S)-MTPA chlorides led to esteriﬁcation of the OH-8

group to aﬀord the (S)- and (R)-MTPA derivatives,

respectively. Unfortunately, in the H NMR spectra, there

was no diﬀerence in the chemical shifts between (R)-MTPA

ester and (S)-MTPA ester; it might be due the inﬂuence of 9-

OH. Consequently, the structure of 9 was determined and

named as 4-O-methylstroside B.

Figure 4. Experimental and calculated ECD of compound 3a (the

aglycone of compound 3).

Eleven known compounds were identiﬁed as plantagineoside

C (4), 7-(3,4-di-hydroxyphenyl)-1-(4-hydroxyphenyl)-1,4,6-

heptatrien-3-one (5), 3,5-dihydroxy-1-(3,4-dihydroxyphen-

Compound 9 was isolated as a white amorphous powder. It

yl)-7-(4-hydroxyphenyl)-heptane (6), pinoresinol (7),

possessed a molecular formula of C H O as established by

kaempferol (8), citrusin C (10), tachioside (11), 3,4-

dimethoxycinnamaldehyde (12), 3-methoxy-4-hydroxycinna-

its HRESIMS ion at m/z 397.1468 [M + Na] . The H NMR

spectrum (Table 2) showed the presence of a 1,3,4-

39 44

trisubstituted benzene ring. An anomeric proton signal at

δ_H4.13 (1H, d, J = 7.7 Hz), ﬁve oxygenated methine carbons

at δ_C103.9, 77.3, 76.8, 74.5, and 70.4 and an oxygenated

maldehyde (13), eugenol (14), and 4-hydroxy-benzalace-

tone (15) by comparison with the previously reported data

in the literature (Figures S50 −S71, Supporting Information).

Antioxidant Activities In Vitro. The antioxidant capacity

methylene carbon at δ 61.4 indicated the presence of a β-

glucose moiety in 9. The sugar unit was identiﬁed as D-glucose

of compounds (1−15) was assessed by DPPH, ABTS , and

FRAP assays (Table 3). In the DPPH assay, compounds 4

(SC = 18.41 ± 1.27 μM), 3 (SC = 19.25 ± 1.28 μM), and 6

using the same method as for 3. Detailed analysis of the H

and C NMR spectra of 9 showed that these were similar to

that of stroside B, except for a methoxy group at C-4 in 9

(SC₅₀= 25.86 ± 1.41 μM) showed a stronger eﬃciency as

compared with ascorbic acid (SC₅₀= 29.81 ± 1.474 μM). In

instead of a hydrogen in stroside B, which was indicated by

Table 3. Antioxidant Activity of Compounds 1−15 from R. beesianus

ABTS⁺^a^,^b

sample

DPPH

FRAP

rhynchanine A (1)

rhynchanine B (2)

rhynchanine C (3)

plantagineoside C (4)

29.96 ± 1.26

29.88 ± 1.26

19.25 ± 1.28

18.41 ± 1.27

30.65 ± 1.49

25.86 ± 1.41

235.66 ± 2.37

56.92 ± 1.76

32.26 ± 1.24

33.18 ± 1.24

31.26 ± 1.50

35.17 ± 1.55

118.25 ± 2.01

35.85 ± 1.55

118.67 ± 2.07

18.59 ± 1.27

2446.54 ± 41.63

2146.59 ± 61.09

1699.25 ± 55.58

2399.88 ± 119.99

2573.21 ± 162.88

2833.20 ± 152.74

1613.25 ± 61.10

3526.49 ± 64.29

<100

-(3,4-di-hydroxyphenyl)-1-(4-hydroxyphenyl)-1,4,6-heptatrien-3-one (5)

[

(3R,5R)-3,5-dihydroxy-7-(4-hydroxyphenyl) heptyl]-1,2-benzenediol (6)

pinoresinol (7)

kaempferol (8)

-O-methylstroside B (9)

citrusin C (10)

tachioside (11)

<100

548.31 ± 33.29

<100

539.98 ± 30.00

1084.95 ± 56.79

426.63 ± 11.52

3351.32 ± 49.35

118.25 ± 2.07

178.17 ± 2.25

135.26 ± 1.55

126.85 ± 1.43

31.00 ± 1.49

,4-dimethoxycinnamaldehyde (12)

-methoxy-4-hydroxycinnamaldehyde (13)

eugenol (14)

-hydroxybenzalacetone (15)

244.25 ± 2.39

29.81 ± 1.47

ascorbic acid

Values represent means ± SD (n = 3). DPPH and ABTS : concentration in μM required to scavenge 50% of the radical. FRAP: concentration in

μM Trolox equivalents/g. Positive control. NI: SC ≥ 500 μM.

235

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Journal of Agricultural and Food Chemistry

Article

addition, compared with ascorbic acid, compounds 1 (SC₅₀

9.96 ± 1.26 μM), 2 (SC = 29.88 ± 1.26 μM), and 5 (SC =

50 50

0.65 ± 1.49 μM) displayed potent antioxidant capacity, while

compound 8 (SC₅₀= 56.92 ± 1.76 μM) presented slightly

weaker antioxidant capacity than ascorbic acid (Table 3). In

the ABTS assay, compound 8 (SC₅₀= 18.59 ± 1.27 μM)

exhibited signiﬁcant antioxidant activity compared with

ascorbic acid (SC₅₀= 31.00 ± 1.49 μM), and compounds

−4 and 6 displayed potent antioxidant capacity, with SC

values close to that of ascorbic acid. Meanwhile, in FRAP

assay, compound 8 (3526.49 ± 64.29 μmol Trolox/g DW)

showed the highest ferric reducing power ability at 25 μM

concentration, compared with ascorbic acid (3351.32 ± 49.35

μmol Trolox/g DW), and compounds 1, 2, and 4−6 exhibited

a good ferric reducing power ability at the same concentration.

The results suggested that the diarylheptanoids had stronger

DPPH radical scavenging activity than the other phenolic

compounds (with an activity order of 1−6 > 7−15), while

mono-diarylheptanoids displayed higher scavenging activity

Figure 5. Inhibitory eﬀect of intracellular ROS in H O -induced

HepG-2 cells of the compounds (1−8) from R. beesianus. All the

values are presented as mean ± SD (n = 3). Mean (bar values) with

diﬀerent letters are signiﬁcantly diﬀerent (p < 0.05). BC: blank

control; M: model group (H O ); V : positive control (V ).

2 2 C C

than bis-diarylheptanoids. In ABTS assay, compound 8

1.94%, compared to that of blank control (5.51 ± 0.69%) (p <

presented the best antioxidant capacity and compound 6

showed higher antioxidant capacity than compound 5, which

may be due to more hydroxy groups in 6. Furthermore, in

FRAP assay, the phenolic compounds with more phenolic

hydroxy groups exhibited a higher ferric reducing power ability.

Inhibitory Eﬀect on Intracellular ROS Generation in

H O -Induced HepG-2 Cells. ROS, which can activate

0.05). Furthermore, comparing the H O -treated group

(model group, p < 0.05), the apoptosis ratios were signiﬁcantly

reduced with diﬀerent compound-treated groups. All the test

compounds (1−8, with a concentration at 10 μM) had a

signiﬁcant cytoprotective eﬀect on the H O -induced HepG-2

cell apoptosis, compared with the model group. Compound 3

showed the strongest eﬃciency on protecting HepG-2 cells

from H O toxicity, and the cell apoptosis ratio of 3 (5.96 ±

cellular damage when they are excessively produced, are the

major etiological factor of oxidative stress and can activate

0.36%) was close to that of the V group (positive control,

apoptosis. H O initiates abnormal accumulation of intra-

9.76 ± 0.51%) and slightly higher than that of the blank

control (Figure 6). In addition, compounds 5 and 6 also had

considerably better cytoprotective eﬀect, and their cell

apoptosis ratios were slightly lower than that of the V_C

group. Meanwhile, compounds 4, 7, and 8 displayed a

moderate cytoprotective eﬀect, and their cell apoptosis ratios

are shown in Figure 6. Moreover, diﬀerent compounds have

diﬀerent protective eﬀects against the H O -induced apoptosis

cellular ROS and induces apoptosis, which is generally used to

generate intracellular ROS in in vitro models. Based on the

above results, compounds 1−8 exhibited considerable radical

scavenging capacity and were chosen to evaluate intracellular

ROS generation in H O -induced HepG-2 cells. The

cytotoxicity of compounds 1−8 was performed on HepG-2

cells using MTT assay (at 2.5, 5, 10, 20, and 40 μM

concentrations), which implied that compound 7 had toxicity

at 20 μM and all tested samples had no inhibitory eﬀect on

HepG-2 cells at 10 μM. Thus, 10 μM of each compound was

selected as the test concentration for evaluating the inhibitory

eﬀect of intracellular ROS production. After incubating the

HepG-2 cells for 24 h, the levels of intracellular ROS of each

compound were measured, as shown in Figure 5. The ROS

generation ratio was signiﬁcantly increased to 60.8 ± 1.95% in

the H O -treated group (model group), compared with the

of HepG-2 cells. The diﬀerences of antioxidant capacity may

be attributed to the number of phenolic hydroxyl groups and

their attachment positions.

CONCLUSIONS

The DPPH, ABTS radical scavenging, and FRAP assay study

■

results indicated that compounds 1−6 and 8 from R. beesianus

rhizomes displayed signiﬁcant antioxidant activities, while

compounds 9−13 and 15 were inactive under the same

concentration. Hence, structure−antioxidant activity relation-

ships of the diarylheptanoids (1−6) were discussed with

varying degrees of radical scavenging eﬀects. Compounds 3, 4,

and 6 presented signiﬁcant antioxidant activities on DPPH

radical scavenging, which suggested that the phenolic hydroxyl

groups and the glycoside moiety may play a vital role in

scavenging the radicals. Furthermore, compounds 3, 4, and 6

exhibited a slightly lower SC₅₀value than compounds 1−2,

which suggested that mono-diarylheptanoids had better

blank control (24.3 ± 1.19%). Compared with the model

group, compounds 1−8 displayed an inhibitory eﬀect on the

generation of intracellular ROS with varying degrees (p <

.05). Additionally, compound 3 displayed the strongest

inhibitory eﬀect on intracellular ROS production (p < 0.05),

with the ROS generation ratio close to that of ascorbic acid,

followed by compound 5. Furthermore, compound 3 showed

higher inhibitory eﬀect on intracellular ROS production than

compound 4, which may be attributed to hydroxyl groups at C-

and C-7 or the replacement of glycoside group.

antioxidant activities than bis-diarylheptanoids. In the ABTS

Cytoprotective Activity against H O -Induced HepG-

Cell Apoptosis. Apoptosis is a deﬁning process of cell death

that is adjusted to proliferation, growth, and mutation of cells.

and FRAP assays, the diarylheptanoids (1−6) except 5 and 8

dramatically scavenged the radicals, while simple phenolic

compounds (7 and 9−15) did not, which suggested that

diarylheptanoids and ﬂavonols probably exhibited better

antioxidant activities than simple phenolic compounds.

Nevertheless, abnormal apoptosis may lead to untoward eﬀects

on the organism. In this study, abnormal apoptosis was

induced by 1.0 mM H O in HepG-2 cells, and the cell

apoptosis percentage was dramatically increased to 30.61 ±

In summary, 15 phenolic compounds (1−15) including

three new diarylheptanoids rhynchanines A−C (1−3) and one

236

Journal of Agricultural and Food Chemistry

J. Agric. Food Chem. 2021, 69, 6229−6239

https://pubs.acs.org/doi/10.1021/acs.jafc.1c00869.

Article

functional food (antioxidant nutraceuticals) containing anti-

oxidants besides being a kitchen spice.

ASSOCIATED CONTENT

sı Supporting Information

■

The Supporting Information is available free of charge at

D and 2D NMR spectra; HRESIMS, UV, and IR

spectra of compounds 1−3 and 9; 1D NMR spectra of

a, 9a, 9aR, 9aS, 4−8, and 10−15; and HPLC proﬁles

of compounds 1−15 (PDF)

AUTHOR INFORMATION

■

Corresponding Authors

Ya-Ping Liu − State Key Laboratory of Phytochemistry and

Plant Resources in West China, Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming 650201, China;

Faculty of Agriculture and Food, Kunming University of

Science and Technology, Kunming 650500, China;

orcid.org/0000-0002-2164-2489 ; Phone: +86-0871

65223188; Email: liuyaping@kust.edu.cn

Xiao-Dong Luo − State Key Laboratory of Phytochemistry

and Plant Resources in West China, Kunming Institute of

Botany, Chinese Academy of Sciences, Kunming 650201,

China; Key Laboratory of Medicinal Chemistry for Natural

Resource, Ministry of Education and Yunnan Province, School

of Chemical Science and Technology, Yunnan University,

Kunming 650091, China; orcid.org/0000-0002-6768-

679 ; Phone: +86-0871 65223177; Email: xdluo@

Figure 6. Cytoprotective eﬀect of the compounds (1−8) of R.

beesianus on apoptosis in H O -induced HepG-2 cells. (A) Flow

mail.kib.ac.cn

cytometry analysis; (B) apoptotic cell percentage of diﬀerent groups.

All the values are presented as mean ± SD (n = 3). Mean (bar values)

with diﬀerent letters are signiﬁcantly diﬀerent (p < 0.05). BC: blank

control; M: model group (H O ); V : positive control (V ).

Authors

Pei-Feng Zhu − State Key Laboratory of Phytochemistry and

Plant Resources in West China, Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming 650201, China;

University of Chinese Academy of Sciences, Beijing 100049,

China

Gui-Guang Cheng − Faculty of Agriculture and Food,

Kunming University of Science and Technology, Kunming

new phenylpropanoid, 4-O-methylstroside B (9), were isolated

from R. beesianus rhizomes. Most of the isolated compounds

_d_i_s_p_l_a_y_e_d_s_i_g_n_i_ﬁ_c_a_n_t_a_n_t_i_o_x_i_d_a_n_t_a_c_t_i_v_i_t_y_o_n_D_P_P_H_,_A_B_T_S+

650500, China

radical scavenging, and FRAP assays. Furthermore, compounds

Lan-Qin Zhao − State Key Laboratory of Phytochemistry and

Plant Resources in West China, Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming 650201, China;

University of Chinese Academy of Sciences, Beijing 100049,

China

Afsar Khan − Department of Chemistry, COMSATS

University Islamabad, Abbottabad 22060, Pakistan

Xing-Wei Yang − State Key Laboratory of Phytochemistry and

Plant Resources in West China, Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming 650201, China;

orcid.org/0000-0002-9578-2986

Bu-Yun Zhang − State Key Laboratory of Phytochemistry and

Plant Resources in West China, Kunming Institute of Botany,

Chinese Academy of Sciences, Kunming 650201, China;

University of Chinese Academy of Sciences, Beijing 100049,

China

Meng-Cheng Li − Faculty of Agriculture and Food, Kunming

University of Science and Technology, Kunming 650500,

China

3, 5, and 6 could prevent oxidative stress damage through a

decrease in ROS content and cell apoptosis in H O -induced

HepG-2 cells. The antioxidant capacity of diﬀerent compounds

had diﬀerent eﬀects on DPPH, ABTS radical scavenging, and

FRAP assays. Compound 8 showed the best antioxidant

activity in ABTS and FRAP assays, while it presented

moderate antioxidant capacity in DPPH assay. Furthermore,

compound 8 moderately inhibited the intracellular ROS

production and prevented the cell apoptosis. The results may

be attributed to diﬀerent assays for antioxidant activity. But in

most cases, the results of diﬀerent assays were consistent.

Compounds 3, 5, and 6 not only displayed signiﬁcant

antioxidant activity but also prevented oxidative stress damage

by inhibiting the intracellular ROS production and cell

apoptosis in H O -induced HepG-2 cells. Meanwhile, com-

pound 3 possessed more hydroxyl groups and a glycoside

moiety, which might be the main promotional factors for

antioxidant ability. The diarylheptanoids, especially mono-

diarylheptanoids, had stronger antioxidant capacity than the

other phenolic compounds (compounds 1−6 > 7−15).

Therefore, R. beesianus rhizomes might be regarded as a

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jafc.1c00869

237

J. Agric. Food Chem. 2021, 69, 6229−6239

Journal of Agricultural and Food Chemistry

Author Contributions

comosa , in breast cancer cells, pre-osteoblast cells, and rat uterine

Article

tissues. J. Agric. Food Chem. 2017, 65, 3490−3496.

16) Haniadka, R.; Saldanha, E.; Sunita, V.; Palatty, P. L.; Fayad, R.;

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(17) Wohlmuth, H.; Deseo, M. A.; Brushett, D. J.; Thompson, D. R.;

P.-F.Z. and G.-G.C. contributed equally to this work.

(

Funding

The authors are grateful to Yunnan Major Science and

Technology Project in China (2019ZF010, 2019ZF003, and

MacFarlane, G.; Stevenson, L. M.; Leach, D. N. Diarylheptanoid from

019FY003004) for the ﬁnancial support.

Pleuranthodium racemigerum with in Vitro prostaglandin E2 inhibitory

and cytotoxic activity. J. Nat. Prod. 2010, 73, 743−746.

Notes

The authors declare no competing ﬁnancial interest.

18) Gao, J.-Y.; Yang, Z.-H.; Ren, P.-Y.; Li, Q.-J. Reproductive

ecology of Rhynchanthus beesianus W. W Smith (Zingiberaceae) in

south yunnan, China: a ginger with bird pollination syndrome. J.

Integr. Plant Biol. 2006, 48, 1294−1299.

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