Anticancer Activity of Compounds Isolated from Engelhardtia serrata Stem Bark

Abstract

Two triterpenoid acids, betulinic acid and ursolic acid, were purified from the ethanol extract of Engelhardtia serrata Bl. by bioassay-guided isolation. Their cytotoxic and apoptosis-inducing activities on the K562 cell line were evaluated by MTT and flow cytometric analysis. Betulinic acid and ursolic acid inhibited the growth of K562 tumor cell line with IC₅₀ values of 6.25 and 12.5 μg/ml and also induced 35% and 13% apoptosis at 25 μg/ml, respectively.

Keywords:

• Apoptosis-inducing effect
• betulinic acid
• cytotoxicity
• Engelhardtia serrata
• ursolic acid

Introduction

The stem bark of Engelhardtia serrata Bl. (Juglandaceae), whose leaves are used as a popular sweet-tea in the Guangdong province of China, is usually used as a folk medicine for the treatment of rheumatism and diarrhea. Little work has been done on the chemical components of this plant. The 60% ethanol extract of E. serrata was successively partitioned by chloroform and ethyl acetate, respectively. The ethyl acetate–soluble portion was identified to contain a large amount of polyphenolic components. In a previous paper, we reported the isolation and identification of gallic acid, phlorizine, ( − )-gallocatechin, ellagic acid, and 3,3′-di-O-methyl ellagic acid-4-O-β-D-xylopyranoside (Liu et al., 2003). The chloroform-soluble portion presented good activity in inducing apoptosis on K562 cells in our screening for anticancer chemical entities from traditional Chinese herbal medicine. Bioassay-guided fractionation led to isolation of two bioactive compounds (1 and 2, Fig. 1). They were determined to be betulinic acid (1) and ursolic acid (2) by detailed analysis of Mass Spectral (MS) and NMR data including ¹H NMR and ¹³C NMR spectra and comparison with published data (Mu et al., 1996; Huang et al., 1998). Their cytotoxic and apoptosis-inducing activities on the K562 cell line were investigated by MTT and flow cytometric analysis.

Figure 1 Structures of compounds 1 and 2.

Materials and Methods

General experimental procedure

Melting points: Yanaco MP-S3 micro-melting point apparatus, uncorrected. IR: Bruker IFS-55 (KBr). Optical rotations: Perkin-Elmer 241 polarimeter. ¹H NMR and ¹³C NMR: JEOL JNM-GX400 (¹H 300 MHz, ¹³C 75 MHz) spectrometer. CC: silica gel H (10–40 μm, Qingdao Haiyang Chemical Factory). TLC: silica gel G (10–40 μm, Qingdao Haiyang Chemical Factory). All other chemicals for bioassay were purchased from Sigma.

Plant material

Stem bark of E. serrata was collected in Yunnan province of China by Qishi Sun in October 1998. A voucher specimen (99036) is deposited at the Division of Pharmacognosy, Shenyang Pharmaceutical University (Shenyang, China).

Extraction and isolation

The air-dried stem bark (3 kg) of E. serrata was extracted with C₂H₅OH/H₂O (6:4, 2 × 10l) under reflux. The extracts were concentrated, and the aqueous suspension was partitioned with chloroform and ethyl acetate, respectively. The ethyl acetate extract containing the polyphenolic components was successively fractionated by gel-filtration and octadecytsilane, C₁₈ (ODS) chromatography to afford gallic acid, phlorizine, (−)-gallocatechin, ellagic acid, 3,3′-di-O-methyl ellagic acid-4-O-β-d-xylopyranoside. The chloroform fraction (2 g) was subjected to silica gel chromatograph eluted with a gradient of cyclohexane and ethyl acetate (100:1, 98:2, 93:7, 95:5, 90:10, 80:20, 70:30). Compounds 1 (20.2 mg) and 2 (8.4 mg) were obtained from fractions eluted with 90:10 and 80:20 of cyclohexane/ethyl acetate by recrystallization from methanol, respectively.

Betulinic acid (1): white needles, m.p. 318–320°C; [α_D²⁴] + 80° (concentration 0.18, ethanol); IR ν_max (KBr) cm⁻¹:3440, 2930, 2860, 2600, 1640, 1375.

Ursolic acid (2): white needles; m.p. 260–262°C;[α_D²⁴] + 63° (concentration 0.1, ethanol); IR ν_max (KBr) cm⁻¹:3400, 2980, 2840, 1690, 1450, 1380.

Cytotoxicity assay

K562 cell line was cultured as previously reported (Hu et al., 1996). Aliquots of 2 × 10⁵ exponentially growing cells were seeded on 96-well flat microtiter plates. The cell viability was determined in the presence or absence of compound or cisplatin, as a positive control, using the standard MTT assay, a colorimetric assay based on the ability of viable cells to reduce yellow 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) to blue formazan. Briefly, 24 h after seeding, 100 μl of new media or a test compound were added, and the plates were incubated for 24 h again. Then, 10 μl of MTT solution (5 mg/ml in PBS, filter sterilized) were added to each well and incubated for a further 4 h at 37°C. The formazan product was solubilized by the addition of 100 μl DMSO. The optical density of each well was read on a SPECTRA MAX plus plate reader (Molecular Devices Corporation, USA) at 570 nm.

Apoptosis-inducing assay by flow cytometry

Approximately 1 × 10⁶ cells of K562 were collected, washed twice with ice-cold PBS, and then fixed overnight in 70% ethanol. After RNase digestion for 30 min at 37°C, cells were collected by centrifugation and stained with propidium iodide (PI) water solution (50 μg/ml PI, 0.1% sodium citrate, and 0.2% Nonidet P-40 in water) for 30 min at 4°C in the dark and were analyzed using a Coulter EPICS XL-MCL flow cytometer with an argon ion laser and 625-nm filter to determine the cell cycle phase based on cellular DNA content. Flow cytometry data were integrated using MultiCycle AV software (Phoenix Flow Systems, San Diego, CA, USA).

Results and Discussion

Betulinic acid and ursolic acid are pentacyclic triterpenoid compounds that occur naturally in a large variety of vegetarian foods, medicinal herbs, and plants. Betulinic acid (BetA) has selective cytotoxicity against many tumor cell lines (Zuco et al., 2002s). It has been reported that betulinic acid can induce apoptosis on neuroblastoma cells (Fulda & Debatin, 2000s). Ursolic acid (UA) is of interest to scientists in the area of oncology because of its cytotoxicity, induction of differentiation, antimutagenic, antiviral, and anti-invasive activities (Hollosy et al., 2000, 2001; Novotny et al., 2001). In our study, the apoptosis-inducing effect and cytotoxic activity of the betulinic and ursolic acids on the K562 cell line was studied. The cells treated with 1.56–25 μg/ml of betulinic acid resulted in a dose- and time-dependent decrease in cell number, as evidenced by MTT assay together with morphological changes. The highest dose (25 μg/ml) of betulinic acid resulted in approximately 80% inhibition in tumor cell growth after 24 h of treatment and 35% apoptosis. When treated with ursolic acid, only 58% inhibition of cell growth and 13% apoptosis were found. During the experiment, cisplatin was used as positive control, which presented 80% inhibition in tumor cell growth and 29.6% apoptosis at the concentration of 50 μg/ml. Comparing the IC₅₀ value of cytotoxicity and apoptosis percentage at 25 μg/ml, it can be found that betulinic acid (IC₅₀, 6.25 μg/ml) showed stronger activity in both cytotoxicity and apoptosis-inducing activity than ursolic acid did (IC₅₀, 12.5 μg/ml). Consequently, the difference in their structures should contribute to the divergence in their anticancer activity.

Acknowledgment

We appreciate the kind help of Prof. Qishi Sun at Shenyang Pharmaceutical University for his collection and identification of the plant material.