New Antiproliferative Dibenzo-α-Pyrone From Whole Plants of Centella asiatica

Abstract

A new compound, 1,3,7,9-tetrahydroxy-6H-dibenzo[b,d]pyran-6-one (1), together with 6 known compounds (2-7), was isolated from the extract of whole plants of Centella asiatica. The chemical structure was elucidated on the basis of spectroscopic and mass spectrometric analyses. All the isolates were evaluated for their cytotoxicities against 4 human cancer cell lines (MCF-7, HepG2, Hela, and A549). Among them, compound 1 was found to exhibit significant cytotoxicity on MCF-7 and HepG2 cancer cells with half-maximal inhibitory concentration values of 4.1 and 7.2 µM, respectively.

Centella asiatica (L.) Urban, a stoloniferous, perennial, medicinal plant belonging to the family Umbelliferae, is well known as “Ji-Xue-Cao” and “Gotu kola” in China and the United States, respectively. This plant is widely distributed in tropical and subtropical regions such as India, Malaysia, Australia, Madagascar, Japan, and most areas of China. ¹ Centella asiatica has been traditionally used to treat diverse diseases in China, India, and Madagascar. ¹ In India, the species is used to treat dermatitis, diabetes, cough, cataract, and other eye troubles. ² For Malays, C. asiatica is used in the treatments of bronchitis, asthma, gastric catarrh, dysentery, kidney trouble, urethritis, and dropsy. ^3,4 Centella asiatica is widely cultivated as an edible vegetable and used as a dietary supplement in some countries, which indicates that it possesses low toxicity and has potential for an in-depth investigation. ⁵ Previous phytochemical studies of C. asiatica have resulted in the isolation of alkaloids, triterpenoids, tannin, flavonoids, triterpenoid, and flavonoid glycosides. ^6,7 Some of these compounds exhibited significant antitumor, anti-inflammatory, antioxidant, antimicrobial, and other activities. ^8,9

As part of our continuous effort for discovering bioactive compounds from C. asiatica, a new compound, 1,3,7,9-tetrahydroxy-6H-dibenzo[b,d]pyran-6-one (1), together with 6 known compounds (2-7), were isolated from the ethyl acetate (EtOAc) soluble fraction. Herein, we report the isolation, structural elucidation, and cytotoxic evaluation of these compounds on 4 human cancer cells (MCF-7, HepG2, Hela, and A549).

Results and Discussion

Compound 1 was obtained as a yellow amorphous powder and assigned the molecular formula C₁₃H₈O₆, according to the ion at m/z 260.0306 in its high-resolution electron ionization mass spectrometry (HREIMS) and nuclear magnetic resonance (NMR) spectroscopic data (Supplemental Figure S4). The ultraviolet (UV) spectrum showed 3 absorptions at 210, 231, and 236 nm. The infrared (IR) spectrum displayed absorptions attributable to hydroxyl (3425 cm⁻¹) and chelated carbonyl (1670 cm⁻¹) groups. The ¹H NMR spectrum (Supplemental Figure S1; Table 1) exhibited a significant hydroxyl proton at δ _H 11.76, and 2 sets of tetra-substituted aromatic rings at δ _H 7.24 (d, J = 1.8 Hz, H-10) and 6.37 (d, J = 1.8 Hz, H-8) and 6.64 (d, J = 2.4 Hz, H-4) and 6.24 (d, J = 2.4 Hz, H-2). The ¹³C NMR spectrum (Supplemental Figure S2; Table 1) indicated 13 carbons, including 12 aromatic carbons and a chelated carbonyl carbon at δ _C 164.5 (C-6). The NMR spectroscopic data of 1 were very similar to those of urolithin B, ¹⁰ revealing the same isocoumarin feature. In addition, 2 sets of m-coupled aromatic protons and 4 carbons in the low field at δ _C 165.9 (C-9), 165.2 (C-7), 159.5 (C-1), and 158.9 (C-3) were observed in the ¹H and ¹³C NMR data of 1, suggesting that 4 hydroxy groups were linked with C-9, C-7, C-1, and C-3, respectively. Furthermore, the key heteronuclear multiple bond correlations of δ _H 11.76 (OH-7) to δ _C 97.8 (C-6a), 165.2 (C-7), δ _H 6.24 (H-2) to δ _C 101.3 (C-4), 158.9 (C-3), 159.5 (C-1), and 135.0 (C-10b), and δ _H 6.64 (H-4) to δ _C 158.9 (C-3), 95.8 (C-2), 135.0 (C-10b), and 153.1 (C-4a) further confirmed the structure of 1 (Supplemental Figure S3, Figure 1). Therefore, the structure of 1 was defined as 1,3,7,9-tetrahydroxy-6H-dibenzo[b,d]pyran-6-one.

Table 1

Nuclear Magnetic Resonance Spectroscopic Data of Compound 1 in Dimethyl Sulfoxide-d₆ (¹H: 600 MHz, ¹³C: 150 MHz).

No	δ C	δ H
1	159.5
2	95.8	6.24 d (2.4)
3	158.9
4	101.3	6.64 d (2.4)
4a	153.1
6	164.5
6a	97.8
7	165.2
8	102.1	6.37 d (1.8)
9	165.9
10	104.8	7.24 d (1.8)
10a	138.6
10b	135.0
7-OH		11.76 s

Figure 1

Key heteronuclear multiple bond correlation of compound 1.

Six known compounds (2-7) were also isolated from the whole plants of C. asiatica and identified as dihydroartomunoxanthone (2), ¹¹ artonol A (3), ¹² cyclocomunomethonol (4), ¹¹ artochamin B (5), ¹³ schweinfurthin E (6), ¹⁴ and vedelianin (7), ¹⁵ by comparing their NMR spectroscopic data with those in the literature (Figure 2).

Figure 2

The structures of compounds 1-7.

The in vitro cytotoxic effects of compounds 1-7 toward the MCF-7, HepG2, Hela, and A549 cancer cells were investigated by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using doxorubicin as the positive control (Table 2). Compounds 1, 2, 4, and 6 showed different levels of cytotoxicity to the 4 cancer cells with half-maximal inhibitory concentration (IC₅₀) values ranging from 4.1 to 48.5 µM; compounds 3 and 5 showed weak cytotoxicity to the 4 cancer cells with IC₅₀ values of over 50 µM. Compound 1 displayed significant cytotoxicity toward the MCF-7 and HepG2 cancer cells with IC₅₀ values of 4.1 and 7.2 µM, respectively. Combined with the previous article reporting that this kind of dibenzo-α-pyrone compound possessed moderate to high cytotoxicity to cancer cells, ¹⁶ these compounds might be considered as lead compounds for the development of anticancer drugs. However, few fully pharmacological studies on the anticancer activity of dibenzo-α-pyrones have been reported. Thus, further pharmacological investigation of compound 1 should be carried out.

Table 2

Cytotoxicities of Compounds 1-7 and the Positive Control Doxorubicin.

Compound	IC50 (μM)
	MCF-7	HepG2	Hela	A549
1	4.1	7.2	15.6	12.5
2	27.1	22.3	35.8	>50
3	>50	>50	>50	>50
4	38.9	43.5	>50	>50
5	>50	>50	>50	>50
6	48.5	42.7	47.2	>50
7	>50	>50	>50	>50
Doxorubicin	1.5	2.0	1.7	2.5

Abbreviation: IC₅₀, half-maximal inhibitory concentration.

IC₅₀ values represent the means ± SEM of 3 parallel measurements.

Positive control.

Experimental

General

UV spectra were recorded in methanol (MeOH) using a Shimadzu UV-2401PC spectrophotometer (Shimadzu, Tokyo, Japan), IR spectra with a JASCO FT-IR 620 spectrophotometer (JASCO, Tokyo, Japan), and NMR spectra with a Bruker Avance III 600 MHz spectrometer with tetramethylsilane as an internal standard (Bruker, Karlsruhe, Germany). HREIMS were obtained using a Bruker micro-TOF-Q mass spectrometer. 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). Thin-layer chromatography was performed with precoated silica gel GF₂₅₄ glass plates (Qingdao Marine Chemical Co., Ltd). A Waters 2535 high-performance liquid chromatography (HPLC) fitted with a 2998 Photodiode Array Detector and 2707 Autosampler (Waters) was used for the semipreparative separations.

Plant Material

Centella asiatica was collected in Dali, Yunnan Province, People’s Republic of China, and authenticated by Professor Jing Chen (College of Pharmacy, Dalian Medical University). A voucher specimen of the plant (No. 20181108) was deposited at the College of Pharmacy, Dalian Medical University, Liaoning, People’s Republic of China.

Extraction and Isolation

Dried whole plants of C. asiatica (10.0 kg) were extracted with MeOH at room temperature for 24 hours, and the solution was concentrated to obtain a crude extract (1.7 kg). This was suspended in water (H₂O) and partitioned successively with light petroleum, dichloromethane (CH₂Cl₂), EtOAc, and n-butanol. The EtOAc fraction displayed moderate in vitro cytotoxicity to 4 of the cancer cell lines and was, therefore, investigated.The EtOAc fraction (152.3 g) was chromatographed over a silica gel column using a gradient of CH₂Cl₂-MeOH (from 80:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 2:1 to 0:1) and was separated into 10 fractions (Fr.1-Fr.10). Fr.5 (14.9 g) was separated into 12 fractions (Fr.5.1-Fr.5.12) by column chromatography on silica gel using a gradient of CH₂Cl₂-MeOH (from 100:1 to 1:1). Fr.5.4 (1.7 g) was subjected to RP-18 column chromatography using MeOH-H₂O as the eluting solvent to afford 9 fractions (Fr.5.4.1-Fr.5.4.9). Fr.5.4.6 (95.3 mg) was applied to a C₁₈ reversed-phase HPLC column and eluted with a gradient of MeOH-H₂O (45:55‐55:45) at a flow rate of 3.0 mL/min over 70 minutes. This resulted in the isolation of compound 1 (8.1 mg, t _R = 31.2 minutes) and compound 2 (6.3 mg, t _R = 47.6 minutes). Fr.5.4.7 (134.7 mg) was eventually purified by HPLC, using a gradient solvent system of 50-70% MeOH in H₂O over 120 minutes to yield compounds 4 (8.2 mg, t _R = 35.0 minutes), 5 (7.5 mg, t _R = 44.1 minutes), 3 (5.7 mg, t _R = 50.1 minutes), 7 (7.2 mg, t _R = 65.2 minutes), and 6 (8.8 mg, t _R = 85.6 minutes).

1,3,7,9-Tetrahydroxy-6H-dibenzo[b,d]pyran-6-one (1)

Yellow amorphous powder; UV (MeOH) λ_max (log ε): 210 (4.02), 231 (4.35), 236 (3.78) nm; IR (potassium bromide disc) ν _max 3425, 1670, 1631, 1598, 1571 cm^{−1; 1}H (600 MHz) and ¹³C NMR (150 MHz) spectral data in DMSO-d ₆, see Table 1; HREIMS: m/z 260.0306 [M]⁺ (calcd for C₁₃H₈O₆, 260.0321).

Cytotoxic Activity

The MTT assay was used to determine the cytotoxicities of compounds 1-7 against 4 cancer cells according to the method previously described. ¹⁷ The cells were cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum, 100 unit/mL penicillin, and 100 µg/mL streptomycin in a humidified 37 °C incubator supplied with 5% carbon dioxide. A series of concentrations of compounds 1-7 and positive control were added to the cultures and incubated for another 24 hours. The absorbance of each well was measured at 570 nm with a liquid scintillation counter (MultiskanFC, Thermo, Shanghai). DMSO was used as the negative control and doxorubicin as the positive control.

Supplemental Material

Supplementary Material 1 - Supplemental material for New Antiproliferative Dibenzo-α-Pyrone From Whole Plants of Centella asiatica

Supplemental material, Supplementary Material 1, for New Antiproliferative Dibenzo-α-Pyrone From Whole Plants of Centella asiatica by Yi Liu, Wei Wang and Jian Miao in Natural Product Communications