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Abstract

A new flavonol glycoside derivate named kaempferol 3-O-trans-p-coumaroyl-7-O-α-ʟ-rhamnoside (1), together with five known compounds (2–6), was isolated from the EtOAc-soluble extract of the leaves of Tripterygium wilfordii. The structure of new compound was elucidated with spectroscopic and physico-chemical analyses. All the isolates were evaluated for in vitro cytotoxic activity against four human cancer cells including HepG2, Hep3B, A549 and MCF-7. Among them, compound 1 exhibited the significant cytotoxic activities against HepG2 and Hep3B cells with IC₅₀ values of 6.8 ± 1.6 and 4.2 ± 1.3 μM, respectively. The above data demonstrated the potential of 1 for the treatment of liver cancer. In addition, 4 also showed moderate inhibitory activity on HepG2 and Hep3B cells with IC₅₀ values of 16.1 ± 2.3 and 10.7 ± 1.8 μM, respectively.

Keywords

Celastraceae cytotoxic activity flavonol glycoside Hep3B

Introduction

Tripterygium wilfordii Hook. f. (Celastraceae) is a vine shrub, which is widely distributed in China, Korea and Japan.¹ The roots of T. wilfordii, also known as ‘Lei-Gong-Teng’ in China, were used to treat inflammatory and autoimmune diseases as a traditional Chinese medicine.² Pharmacological studies confirmed that T. wilfordii possessed anti-inflammatory,³ immunosuppressive, anti-tumour⁴ and neuroprotective activities.⁵ And a large number of phytochemical investigations on the roots of T. wilfordii revealed the presence of dihydroagarofuran sesquiterpenoids,⁶ sesquiterpene alkaloids,⁷ abietane diterpenoids,⁸ triterpenoids,⁹ megastigmane glycosides¹⁰ and a small amount of flavonoids and lignans.^11,12 However, there are less chemical studies on the leaves of T. wilfordii compared with its roots. Thus, a series of studies focused on the leaves of T. wilfordii were carried out. As a result, a new flavonol glycoside derivate named kaempferol 3-O-trans-p-coumaroyl-7-O-α-ʟ-rhamnoside (1), together with five known compounds (2–6), was isolated. Herein, we report the isolation and structural elucidation of these compounds. In addition, their cytotoxic activities on HepG2, Hep3B, A549 and MCF-7 cells were also evaluated.

Results and discussion

Structural elucidation

Compound 1 was obtained as a yellow amorphous powder, and the molecular formula was established as C₃₀H₂₆O₁₂ based on the HRESIMS at m/z 577.1207 for the [M − H]⁻(Supplemental Figure S5). The IR spectrum showed typical absorption bands of hydroxy and carbonyl groups at 3425 and 1690 cm⁻¹, respectively. Its ¹H NMR spectrum (Table 1, Supplemental Figure S1) revealed the presence of two sets of AA′XX′-type aromatic protons at δ_H 7.76 (2H, d, J = 8.4 Hz, H-2′/6′), 6.92 (2H, d, J = 8.4 Hz, H-3′/5′), 7.49 (2H, d, J = 8.4 Hz, H-2″′/6″′) and 6.81 (2H, d, J = 8.4 Hz, H-3″′/5″′), a pair of m-coupled aromatic protons at δ_H 6.42 (1H, d, J = 1.8 Hz, H-6) and 6.22 (1H, d, J = 1.8 Hz, H-8), two trans-olefinic protons at δ_H 7.51 (1H, d, J = 15.6 Hz, H-7″′) and 6.81 (1H, d, J = 15.6 Hz, H-8″′). In addition, the presence of α-ʟ-rhamnopyranoside unit could be indirectly deduced by the ¹H NMR data at δ_H 5.30 (1H, d, J = 1.2 Hz, H-1″), 3.98 (1H, m, H-2″), 3.48 (1H, dd, J = 9.0, 3.0 Hz, H-3″), 3.16 (1H, m, H-4″), 3.30 (1H, dd, J = 9.0, 6.0 Hz, H-5″) and 0.80 (3H, d, J = 6.0 Hz, H-6″) combined with ¹³C NMR data at δ_C 101.8 (C-1″), 70.3 (C-2″), 70.6 (C-3″), 71.1 (C-4″), 70.0 (C-5″) and 17.5 (C-6″) (Supplemental Figure S2). And this sugar moiety could be further confirmed by acid hydrolysis assay. The low-field region of ¹³C NMR spectrum of 1 revealed 24 carbons including 2 carbonyl carbons at δ_C 177.7 (C-4) and 167.7 (C-9″′), 18 aromatic carbons and 4 olefinic carbons at δ_C 164.2–93.7. The comparison of the 1D NMR data of 1 and kaempferol 7-O-α-ʟ-rhamnoside revealed that the skeleton of 1 was flavonol glycoside.¹³ Furthermore, the structure of trans-coumaroyl moiety could be determined by the key HMBC correlations of δ_H 7.49 (H-2″′/6″′) with δ_C 125.1 (C-1″′), 115.0 (C-3″′), 159.2 (C-4″′) and 144.6 (C-7″′), δ_H 6.81 (H-3″′/5″′) with δ_C 125.1 (C-1″′), 115.0 (C-2″′) and 159.2 (C-4″′), δ_H 7.51 (H-7″′) with δ_C 125.1 (C-1″′), 115.0 (C-2″′/6″′), 114.5 (C-8″′), and 167.7 (C-9″′), and δ_H 6.32 (H-8″′) with δ_C 125.1 (C-1″′), 144.6 (C-7″′) and 167.7 (C-9″′) (Figure 1, Supplemental Figure S3 and S4). Although the position of trans-p-coumaroyl moiety could not be determined by the HMBC correlations, two hydroxy signals with larger chemical shifts at δ_H 11.1 and 10.9 could indirectly reveal that the trans-p-coumaroyl moiety was linked with C-3. In addition, the aglycone isolated from the acid hydrolysis assay of 1 was identified as p-coumaroylkaempferol (2) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods, and it further demonstrated the structure of 1. Thus, the structure of compound 1 was assigned as kaempferol 3-O-trans-p-coumaroyl-7-O-α-ʟ-rhamnoside.

Table 1.

¹H and ¹³C NMR spectral data of compound 1 in DMSO-d₆ (¹H: 600 MHz, ¹³C: 125 MHz).

No	1
No	δ_H (mult, J in Hz)	δ_C (mult)
2		156.5
3		134.2
4		177.7
5		161.3
6	6.42, d (1.8)	98.7
7		164.2
8	6.22, d (1.8)	93.7
9		157.2
10		104.1
1′		120.5
2′	7.76, d (8.4)	130.6
3′	6.92, d (8.4)	115.4
4′		160.0
5′	6.92, d (8.4)	115.4
6′	7.76, d (8.4)	130.6
1″	5.30, d (1.2)	101.8
2″	3.98, m	70.3
3″	3.48, dd (9.0, 3.0)	70.6
4″	3.16, m	71.1
5″	3.30, dd (9.0, 6.0)	70.0
6″	0.80, d (6.0)	17.5
1″′		125.1
2″′	7.49, d (8.4)	129.5
3″′	6.81, d (8.4)	115.0
4″′		159.2
5″′	6.81, d (8.4)	115.0
6″′	7.49, d (8.4)	129.5
7″′	7.51, d (15.6)	144.6
8″′	6.32, d (15.6)	114.5
9″′		167.7
12-OH	12.63, s

Figure 1.

Key HMBC correlations of compound 1.

Moreover, five known compounds (2–6) were obtained from the leaves of T. wilfordii and identified as p-coumaroylkaempferol (2),¹⁴ tripteryol B (3),¹⁵ 3β-O-trans-coumaroylbetulinic acid (4),¹⁶ camaldulenic acid (5)¹⁷ and crategolic acid (6)¹⁸ based on the NMR data and comparison with literature data (Figure 2).

Figure 2.

Structures of compounds 1–6.

Analysis of biological activity results

The in vitro cytotoxic activity of compounds 1–6 against HepG2, Hep3B, A549 and MCF-7 cells was evaluated by the MTT method, and doxorubicin was used as the positive control. As shown in Table 2, compounds 1, 3–6 showed different levels of cytotoxic activity with IC₅₀ values ranging from 4.2 ± 1.3 to 47.8 ± 2.0 μM. Among these compounds, 1 showed the significant cytotoxic activity on HepG2 and Hep3B cells with IC₅₀ values of 6.8 ± 1.6 and 4.2 ± 1.3 μM, respectively. In addition, 4 also exhibited moderate cytotoxic activity towards Hep3B cells with IC₅₀ value of 10.7 ± 1.8 μM. In general, the potential structure–activity relationships could be deduced. The comparison of cytotoxic activity on four cancer cells between 1 and 2 indicated that the increasing cytotoxic activity might be associated with the α-ʟ-rhamnoside moiety which linked with C-7 of 1. Based on the significant cytotoxic activity of 1 on Hep3B cells, the deeply pharmacological investigations on 1 should be further carried out.

Table 2.

Cytotoxic activities of compounds 1–6 on four cell lines.

Compounds	IC₅₀ (μM)^a
Compounds	HepG2	Hep3B	A549	MCF-7
1	6.8 ± 1.6	4.2 ± 1.3	35.1 ± 2.7	47.0 ± 2.1
2	>50	>50	>50	>50
3	45.0 ± 1.7	>50	>50	42.8 ± 1.4
4	16.1 ± 2.3	10.7 ± 1.8	23.2 ± 2.5	18.0 ± 1.6
5	40.9 ± 1.4	>50	>50	47.8 ± 2.0
6	>50	44.2 ± 2.5	>50	38.6 ± 1.4
Doxorubicin^b	1.2 ± 0.8	0.8 ± 1.0	1.4 ± 0.6	1.7 ± 1.3

IC₅₀ values were determined by regression analyses and expressed as means ± SD of three replicates.

Positive control.

Conclusion

In this study, a new flavonol glycoside derivate named kaempferol 3-O-trans-p-coumaroyl-7-O-α-ʟ-rhamnoside (1), together with five known compounds (2–6), was isolated from the EtOAc-soluble extract of the leaves of T. wilfordii. All compounds were tested for their in vitro cytotoxic activity against HepG2, Hep3B, A549 and MCF-7 cells. It was noted that compound 1 showed the significant cytotoxic activity on HepG2 and Hep3B cells with IC₅₀ values of 6.8 ± 1.6 and 4.2 ± 1.3 μM, respectively. This investigation confirmed that p-coumaroyl and caffeoyl moieties could highly increase the cytotoxic effect of alternariol on cancer cells. The previous studies mainly reported the presence of various dihydroagarofuran sesquiterpenoids with potent cytotoxic activity from the roots of T. wilfordii. This study provided a prediction that this kind of phenylpropanoid-conjugated flavonol glycoside might be considered as lead compounds to treat liver cancer. Thus, the in-depth pharmacological investigations on 1 should be carried out.

Experimental

General

Optical rotations were determined on a Perkin-Elmer 241MC automatic digital polarimeter (PerkinElmer, Waltham, USA). UV spectra were conducted on a JASCO V-650 spectrometer (JASCO, Tokyo, Japan). IR spectra were recorded with a JASCO FT-IR 620 spectrophotometer (JASCO, Tokyo, Japan). Nuclear magnetic resonance (NMR) spectra were acquired on a Bruker AV-600 MHz spectrometer with tetramethylsilane (TMS) as an internal standard (Bruker, Karlsruhe, Germany). Mass spectra were obtained on a QTOF2 high-resolution mass spectrometer (Micromass, Wythenshawe, UK). Column chromatography was conducted using silica gel 60 (200 μm particle size, Yantai Xinde Chemical Co., Ltd, Yantai, China) and RP-18 (150-63 μm particle size, Merck, Darmstadt, Germany). TLC was performed with precoated silica gel GF₂₅₄ glass plates (Qingdao Marine Chemical Co., Ltd). A Shimadzu LC-6AD HPLC with a SPD-20A detector (Shimadzu, Tokyo, Japan) and an YMC Pack C₁₈ column (250 mm × 10 mm, i.d., 5 μM, YMC Co. Ltd., Japan) were used for the semi-preparative separations.

Plant material

The leaves of T. wilfordii were collected in Maanshan, Anhui province, China, and authenticated by Professor Hongxiang Lou (College of Pharmacy, Shandong University). A voucher specimen of the plant (No. 20200910) was deposited at the Cheeloo College of Medicine, Shandong University, Shandong, China.

Extraction and isolation

The dried leaves of T. wilfordii (10.0 kg) were extracted three times with 75% MeOH (80.0 L × 2 h × 3) under reflux, and the solution was concentrated in vacuo to yield a black extract (1.2 kg). This extract was suspended in H₂O (10.0 L), partitioned successively with petroleum ether (PE), CH₂Cl₂, EtOAc and n-BuOH (each 8.0 L × 3). The EtOAc extract of T. wilfordii displayed the moderate in vitro cytotoxicity on four human cancer cell lines. Therefore, the EtOAc fraction (123.4 g) was chromatographed over a silica gel column using a gradient of CH₂Cl2-MeOH (from 100:0 to 0:1) and was separated into 17 fractions (Fr.1-Fr.17). Fr.11 (8.5 g) was fractionated by column chromatography on silica gel using a gradient of CH₂Cl₂-MeOH (from 100:1 to 1:1), and was separated into 12 fractions (Fr.11.1-Fr.11.12). Furthermore, Fr.11.4 (1.1 g) was subjected to the RP-18 column and using the elution of MeOH-H₂O (3:7 to 1:0) to afford 13 fractions (Fr.11.4.1-Fr.11.4.13). Fr.11.4.4 (143.1 mg) was eventually purified by semi-preparative HPLC, using a gradient solvent system 50%–55% MeOH in H₂O over 60 min yielded compounds 1 (8.1 mg, t_R = 30.3 min), 3 (10.7 mg, t_R = 42.7 min) and 2 (6.4 mg, t_R = 53.9 min). Fr.11.4.9 (101.2 mg) was separated via preparative HPLC using an isocratic solvent system of 80% MeCN in H₂O to yield compounds 6 (6.2 mg, t_R = 34.1 min), 5 (7.2 mg, t_R = 47.2 min) and 4 (4.2 mg, t_R = 58.2 min).

Kaempferol 3-O-trans-p-coumaroyl-7-O-α-ʟ-rhamnoside (1). A yellow amorphous powder; UV (MeOH) λ_max (log ε): 261 (3.10), 275 (3.57), 298 (2.12), 323 (2.30), 357 (2.52) nm; IR (KBr disc) ν_max 3425, 2945, 2841, 1690, 1590, 1504, 1401 cm⁻¹; ¹H (600 MHz) and ¹³C NMR (150 MHz) spectral data in DMSO-d₆, see Table 1; HRESIMS: m/z 577.1207 [M − H]⁻ (calcd for C₃₀H₂₆O₁₂, 578.1424).

Acid hydrolysis of compound 1

The sample of compound 1 (2.0 mg) was added to MeOH solution (3.0 mL) and refluxed with 2 M HCl (3.0 mL) at 100 °C for 4 h. After removal of MeOH under reduced pressure, the reaction mixture was diluted with H₂O (5.0 mL) and neutralized with NaHCO₃ solution, followed by extraction with EtOAc (3 × 10 mL). Next, the collected organic phase was evaporated and subjected to PTLC using CHCl2-MeOH (5:1) as eluent to yield the aglycone. The water layer was concentrated and submitted to PTLC (CHCl₂/MeOH/H₂O = 7:3:0.5) to yield the sugar. The identification of ʟ-rhamnose was carried out by the comparison of Rf values in TLC (CHCl₂/MeOH/H₂O = 7:3:0.5) between the obtained sugar and authentic sample. Spot was detected by spraying with vanillin-sulfuric acid followed by heating. In addition, their similar optical rotation could also confirm the type of sugar (obtained sugar sample: ${[α]}_{D}^{20}$ +8.4; authentic standard: ${[α]}_{D}^{20}$ +8.2).^13,19

Cytotoxicity assay

The MTT assay was used to determine the cytotoxicity of the compounds against human cancer cell lines according to the method previously described.²⁰ Briefly, the cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% foetal bovine serum, 100 U mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin in a humidified 37 °C incubator supplied with 5% CO₂. Then, cells were seeded at 5 × 10³ cells mL⁻¹ per well onto 96-well flat bottom plate and incubated for 12 h at 37 °C with 5% CO₂. Compounds 1–6 were dissolved in DMSO to obtain a initial concentration (10 mM) and diluted to obtain a series of concentrations. Next, cells were treated and continuously exposed to different concentrations of compounds for 48 h. MTT (5.0 mg mL⁻¹) was added to each well. After 4 h, the crystalline formazan formed was dissolved in DMSO (150 μL), and the absorbance at 570 nm of each well was measured with liquid scintillation counting. All experiments were performed at least three times and the values of IC₅₀ were measured by GraphPad Prism 8.0 software. DMSO was used as the negative control and doxorubicin was used as a positive control.

Supplemental Material

sj-doc-1-chl-10.1177_17475198211062836 – Supplemental material for Chemical constituents from the leaves of Tripterygium wilfordii and their cytotoxic activity

Supplemental material, sj-doc-1-chl-10.1177_17475198211062836 for Chemical constituents from the leaves of Tripterygium wilfordii and their cytotoxic activity by Zhi-Chao Jiang, Zi-Heng Li and Peng-Yu Li in Journal of Chemical Research

Footnotes

Acknowledgements

We are grateful to the Department of Instrumental Analysis of Shandong University for the measurement of the UV, IR, HRESIMS and NMR.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship and/or publication of this article.

ORCID iD

Peng-Yu Li

Supplemental material

Supplemental material for this article is available online.

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