Archives of Toxicology

, Volume 81, Issue 3, pp 211–218

Isolation of chebulic acid from Terminalia chebula Retz. and its antioxidant effect in isolated rat hepatocytes


  • Hyun-Sun Lee
    • Department of Food Science, College of Life sciences and BiotechnologyKorea University
  • Sung-Hoon Jung
    • Department of Food Science, College of Life sciences and BiotechnologyKorea University
  • Bong-Sik Yun
    • Korea Research Institute of Bioscience and Biotechnology
    • Department of Food Science, College of Life sciences and BiotechnologyKorea University
Molecular Toxicology

DOI: 10.1007/s00204-006-0139-4

Cite this article as:
Lee, H., Jung, S., Yun, B. et al. Arch Toxicol (2007) 81: 211. doi:10.1007/s00204-006-0139-4


A hepatoprotective compound was isolated from the ethanolic extract of the fruits of Terminalia chebula Retz. by consecutive solvent partitioning, followed by silica gel and Sephadex LH-20 column chromatographies. The purified compound was identified as a mixture of chebulic acid and its minor isomer, neochebulic acid, with a ratio of 2:1 by spectroscopic analysis including 1D and 2D NMR and MS spectroscopy. To our knowledge, this is the first report on the protection of rat hepatocytes against oxidative toxicity by chebulic acid obtained from T. chebula Retz. This compound exhibited in vitro a free radical-scavenging activity and ferric-reducing antioxidant activity. Also, the specific ESR spectrum for the OOH radical signals consisting of three-line ESR spectra was within the field of 0.27 mT, whereas 2.5 and 0.25 mg/ml of chebulic acid significantly reduced the signal intensity of the ESR spectra to 0.06 mT and 0.11 mT, respectively. Using isolated rat hepatocyte experiment, we demonstrated that the treatment of hepatocytes with chebulic acid significantly reduced the tert-butyl hydroperoxide (t-BHP)-induced cell cytotoxicity, intracellular reactive oxygen species level, and the ratio of GSSH, oxidized form of glutathione (GSH) to the over total GSH (GSH + GSSG) (4.42%) as compared to that with t-BHP alone (8.33%).


Chebulic acidCytotoxicityAntioxidantHepatocytesOxidative stressRat


Terminalia chebula Retz. (Combretaceae) is a plant native to India and Southeast Asia, where it is extensively cultivated. Its dried ripe fruit has been traditionally used to treat various ailments in Asia (Perry 1980). In Myanmar, the medicinal terminalia fruit is used as a laxative and tonic agent (Cheng et al. 2003) and in Tibet, it is regarded as the “king of medicines”. Terminalia chebula (T. chebula) has been reported to exhibit a variety of biological activities including anticancer (Saleem et al.2002), antidiabetic (Sabu and Kuttan 2002), antimutagenic (Kaur et al. 1998; Arora et al. 2003), antibacterial (Aqil et al. 2005), antifungal (Vonshak et al. 2003) and antiviral activities (Badmaev and Nowakowski 2000).

Free radicals and their related species, which are mainly derived from reactive oxygen species (ROS) and reactive nitrogen species, have attracted a great deal of attention in recent years. It has been reported that they are generated endogenously in various physicochemical conditions or pathophysiological states (Devasagayam et al. 2004). Reactive oxygen species are the by-products of normal aerobic metabolism (Beckman and Ames 1998) because approximately 2–5% of the oxygen consumed by cells is subsequently converted to free radicals (Wickens 2001). The production of ROS is normally counterbalanced by cellular defensive systems (Halliwell and Gutteridge 1985; Freeman and Crapo 1982). However, approximately 1% of the ROS escapes from daily elimination, and gives rise to oxidative cellular damage (Berger 2005). The process by which the production of ROS is not effectively neutralized and thus leads to cellular damage is known as oxidative stress (Halliwell and Gutteridge 1985). Oxidative stress has been related to cardiovascular disease, cancer, and other chronic diseases (Willcox et al. 2004).

While the active principles such as chebulic acid from T. chebula have been reported (Juang et al. 2004), the biological activities of these compounds were not fully investigated. In our knowledge, this is the first report on the antioxidant properties and liver protecting effect of chebulic acid obtained from T. chebula against the oxidative stress induced by tert-butyl hydroperoxide (t-BHP). In our earlier paper, we reported on the protective effect of T. chebula extract on the liver, both in vivo and in vitro (Lee et al. 2005). We have now identified phenolcarboxylic acid compound, chebulic acid and its minor isomer as an active compound from T. chebula, and further investigated its liver protecting capacity and antioxidant activity using various biochemical measurements, such as cytotoxicity, lipid peroxidation, intracellular ROS formation and glutathione (GSH) redox status.

Materials and methods


Leibovitz’s L-15 (L-15) medium was obtained from Gibco Life Technologies (Paisley, UK, USA). Percoll was purchased from GE Healthcare Amersham Biosciences (Uppsala, Sweden). Bovine serum albumin (BSA), 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT), streptomycin, penicillin, insulin, dexamethasone, galactose, sodium selenite, tert-butyl hydro peroxide (t-BHP), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma (St. Louis, MO). All other chemicals used were of the highest grade. 1H and 13C NMR spectra were recorded on a Bruker AMX 500 NMR spectrometer (Bruker, Rheinstetten, Germany) using DMSO-d6 with tetramethylsilane (TMS) as the internal standard. Two-dimensional NMR spectra such as 1H-1H COSY, ROSEY, HMQC and HMBC were also measured using the Bruker AMX 500 NMR spectrometer (Bruker Analytik GmbH, Rheinstetten, Germany). The EI mass spectrum was measured on a JEOL JMS-700 M Station mass spectrometer (JEOL, Tokyo, Japan).

Plant material

The medicinal dried ripe fruit of T. chebula, purchased from a local market (Kyungdong Herb-Market, Seoul, Korea), was identified by Prof. B.W. Kang (College of Life Sciences and Biotechnology, Korea University).

Extraction and isolation

The dried ripe fruits of T. chebula (1.5 kg) were ground with a mortar, and extracted twice with 97% ethanol. The extract was combined, lyophilized, resuspended in H2O and then, extracted successively with n-hexane, chloroform, ethyl acetate (EtOAc) and n-butanol. The EtOAc-soluble portion (20 g) was applied to a column of silica gel and eluted by a gradient with increasing amount of methanol in ethyl acetate. The active fraction of EtOAc and methanol (70:30, v/v) was further purified by Sephadex™ LH-20 (Amersham Biosciences, Uppsala, Sweden) column chromatography using methanol as the eluent, followed by PR-μ-BondaPak C18 (300 × 3.9 mm, 10 μm) (Waters, Milford, MA, USA) column chromatography to give the active compound.

Antioxidant power assay

The antioxidant activity was measured by determining the ferric-reducing antioxidant power (FRAP) and DPPH activity. The FRAP assay was performed according to the standard method (Lee et al. 2005; Jiménez-Escrig et al. 2001). The radical scavenging properties of the extract were evaluated by assessing the DPPH scavenging activity (Kitagaki and Tsugawa 1999). The SC50 values calculated denote the concentration of the sample required to scavenge 50% of the DPPH radicals.

Superoxide anion scavenging activity assay in electron spin resonance (ESR)

The superoxide radical scavenging activity of the active compound from T. chebula extract was also evaluated using ESR spin trapping using N-tert-butyl-α-phenylnitron (BPN) as a spin trap. The ESR assay was performed using Fenton’s reagents and hydroxyl radicals (Wei et al. 2002). Briefly, reaction mixtures containing 300 μM FeSO4, 50 μM BPN and the indicated concentration of the active compound were prepared. The reaction was initiated by the addition of t-BHP (final concentration 250 μM). Then, the reaction mixture was transferred into a quartz capillary, and its ESR spectra were recorded at exactly 90 s with ESR spectrometer (JEOL, JES-FA200 spectrometer, Tokyo, Japan). The ESR spectrometry conditions used to estimate the O2•− and ascorbyl radical with spin-trapping reagent were as follows: microwave frequency = 9,385.501–9,392.20 MHz, microwave power = 4.00 mW, field center 334.297–335.32 mT, sweep width ±5.00 mT, modulation frequency width = 0.0800 mT/100.00 kHz, sweep time = 4.0–8.0 min, amplitude 500–1,500, and time constant = 0.3–1.0 s, at room temperature.


The laboratory animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals (Committee on Care and Use of Laboratory Animals, 1985). Male Sprague-Dawley rats (200 ± 10 g) were purchased from Samtako Bio Korea Co. (Gyeonggi, Korea), and allowed free access to a standard diet (Samyang-Feed Co. Ltd., Inchon, Korea) and tap water.

Isolation and cultivation of rat hepatocytes

Rat hepatocytes were prepared by collagenase perfusion, as described previously (Lee et al. 2005; Bissell et al. 1973) with several modifications. The cell viability was determined by tryptophan blue exclusion, and was found to be greater than 90%. The cells were incubated in a humidified incubator at 37°C (Vision, Korea) in an airy atmosphere. The medium was replaced with fresh identical medium 4 h after plating. At 20 h after plating, the media were replaced with L-15 media containing the test chemicals. The cells were either seeded onto 24-well plates at 1.8 × 105 cells/well for cytotoxicity studies [lactate dehydrogenase (LDH) leakage and MTT assay], or 6-well plates at 9 × 105 cells/well for malondialdehyde (MDA) measurement.

Assessment of cell viability

All treatments were performed 20 h after cell attachment to allow for monolayer formation. After cell attachment, the hepatocytes were washed, and then incubated in L-15 medium for 30 min containing the active compound and 1.5 mM t-BHP. The cell viability was measured by the MTT assay (Lee et al. 2005; Skehan et al. 1990). Membrane damage that results in LDH leakage is generally considered irreversible and, therefore, LDH leakage was used as a biomarker of cellular viability (Lee et al. 2005; Tseng et al. 1996). The lipid peroxidation product, MDA was assayed according to an improved thiobarbituric acid (TBA) fluorometric method involving emission at 552 nm and excitation at 515 nm using 1,1,3,3-tetramethoxypropane as a standard (Yagi 1987).

DCF assay for oxidative stress

ROS were determined by the 2′,7′-dichlorofluorescien diacetate (DCFH-DA) fluorescence assay (Wang and Joseph 1999) 100 μM DCFH-DA-loaded hepatocytes were incubated for 30 min containing the active compound and 1.5 mM t-BHP. The fluorescence intensity was determined with excitation at 485 nm and emission at 530 nm using a plate reader (VICTOR, Wallac, Finland).

GSH assay

The intracellular reduced GSH and its oxidized form, GSSG in the hepatocytes, and liver tissues were determined by HPLC and UV detector (Varian Prostar model 320 and Varian model 210, Palo Alto, CA, USA) (Asensi et al. 1994).

Statistical analysis

The results obtained were expressed as mean ± standard deviation (SD). The student’s t-test was used to perform the statistical comparison between the groups by one-way analysis of variance. Significant differences were taken as P < 0.05.


Structure determination of hepatoprotective compound

The hepatoprotective compound was isolated from the ethanolic extract of the fruits of T. chebula by organic solvent partitioning, followed by silica gel, Sephadex LH-20, as shown in Fig. 1. The active compound was obtained as yellow powder with a yield of 0.08% (Table 1). The IR spectrum showed the presence of hydroxyl groups (3,404 cm−1), ester carbonyl groups (1,712 cm−1) and aromatic rings (1,608 and 1,520 cm−1 data not shown). The molecular weight was deduced by the observation of quasi-molecular ions at m/z 379 [M + Na]+ and m/z 357 [M + H]+ by FAB mass measurement in positive mode. The 1H NMR data in DMSO-d6 revealed two sets of peaks with different intensities with a ratio of 2:1, implying that the active principle was a mixture of two compounds not capable of being separated by the above isolation protocol. Therefore, the chemical structure of the active principle was elucidated as a mixture by two-dimensional NMR experiments. In the 1H NMR spectrum of the major component 1, an aromatic methine proton at δ 6.91, three methine protons at δ 5.10 (1H, s), 3.61 (1H, d, J = 8.8 Hz) and 2.87 (1H, d, J = 10.8, 8.8, 3.6 Hz), an asymmetrical methylene proton at δ 2.70 (1H, dd, J = 16.8, 10.8 Hz) and 2.10 (1H, dd, J = 16.8, 3.6 Hz) and four broad exchangeable protons quenched by the addition of D2O were observed. The 13C NMR spectrum revealed two sets of 14 carbons, which were comprised of four carbonyl carbons, six sp2 carbons, three methines and a methylene. The above NMR data implied that the two substances were in a stereoisomeric relationship with each other. A partial structure, CH2–CH–CH–, was established by the DQF-COSY experiment, and all of the proton-bearing carbons were assigned from the HMQC spectrum, as shown in Table 2. The structures of 1 and 2 were established by the HMBC experiment, which displayed common long-range couplings for these compounds from H-8 to C-1, C-4a, C-6 and C-7, from H-3 to C-1, C-4a and C-12, from H-4 to C-8a, C-5 and C-13 and from H-10 to C-11 and C-13 (Fig. 2). Therefore, compounds 1 and 2 were determined to have the same planar structure, as shown in Fig. 2. In an extensive literature search based on their purported chemical structures, 1 and 2 were identified as chebulic acid and its minor isomer, neochebulic acid, respectively. Since compound 1 was the predominant constituent among the two isomeric forms in the T. chebula extract, chebulic acid as the representative active component was used for the subsequent biochemical measurements in our experiment.
Fig. 1

Scheme used for the extraction of chebulic acid from Terminalia chebula Retz.

Table 1

Protection of rat hepatocytes against oxidative toxicity by fractions from Terminalia chebula Retz.

Purification step

Cell viability (% control)

Yield (%)

EtOH extracts


90.2 ± 4.7


Ethyl acetate layer


98.6 ± 1.8


Silica gel fraction


61.6 ± 4.5


Sephadex LH-20


59.3 ± 1.3


Cells were treated with 10 μg/ml of each extract and 1.5 mM t-BHP for 30 min. Cell viability was determined by MTT assay. Results are expressed as mean ± SD (n = 3)

Table 2

1H NMR and 13C NMR data of the antioxidant from T. chebula Retz.















5.10 (1H, s)


4.91 (1H, s)



3.61 (1H, d, J = 8.8 Hz)


3.94 (1H, d, J = 4.4 Hz)























6.91 (1H, s)


6.95 (1H, s)








2.87 (1H, ddd, J = 10.8, 8.8, 3.6)


3.20 (1H, ddd, J = 12.0, 4.4, 2.8)



2.70 (1H, dd, J = 16.8, 10.8)


2.57 (1H, dd, J = 16.8, 12.0)


2.10 (1H, dd, J = 16.8, 3.6)


1.87 (1H, dd, J = 16.8, 2.8)

















12.39 (br s)


9.80 (br s)


9.57 (br s)


9.18 (br s)


Chemical shifts in ppm from tetramethylsilane (TMS) as internal standard

1H and 13C NMR spectra were measured at 400 MHz and 100 MHz, respectively
Fig. 2

Structure of chebulic acid isolated from T. chebul Retz.

Antioxidant activities of chebulic acid

The antioxidant activities of the pure compound, chebulic acid were evaluated by two different methods (DPPH, and FRAP), and compared to those of epigallocatechin gallate (EGCG). The results of the DPPH assays demonstrated that the antioxidant activity of chebulic acid was 2.5 times lower than that of EGCG, but their activities were found to be similar in the FRAP assay (Table 3). Also the antioxidant activity of chebulic acid was further evaluated by the ESR spin-trapping technique, in order to examine its in situ superoxide radical scavenging activity. Superoxide radicals were generated via a Fe2+-mediated Fenton reaction according to the protocol described in the Materials and methods section. The specific ESR spectrum for the radical signals consisted of a three-line ESR spectra within the field of 0.27 mT, whereas the addition of 2.5 or 0.25 mg/ml of chebulic acid significantly reduced the signal intensity of the ESR spectra (0.06 mT or 0.11 mT) in a dose dependent manner (Fig. 3).
Table 3

Antioxidant activities of chebulic acid in vitro


DPPH SC50a (μg DMb ml−1)

FRAPc(mmol FeSO4·7H2O g−1 DM

Chebulic acid

48.5 ± 1.3

360.6 ± 1.4


19.5 ± 1.7

417.5 ± 10.4

Each value is mean ± SD of three replicate experiments (n = 3)

aAmount of sample necessary to decrease the initial DPPH concentration by 50%

bDM, dry matter

cFerric-reducing antioxidant power
Fig. 3

ESR spectra produced from reaction of chebulic acid with t-butyl hydroperoxide (t-BHP). ESR spectrum observed after incubation for 90 s in the presence of 50 μM N-tert-butyl-α-phenylnitron (BPN). a Only vehicle, b 0.25 M t-BHP, c 2.5 mg/ml chebulic acid and 0.25 M t-BHP, d 0.25 mg/ml chebulic acid and 0.25 M t-BHP

Hepatoprotective effect of chebulic acid against oxidative stress

The treatment of the hepatocytes with different concentrations of chebulic acid in the presence of 1.5 mM t-BHP protected the hepatocytes against the cytotoxicity of t-BHP in a dose-dependent manner (Fig. 4). Compared with the control, the t-BHP treated cells showed similar cell viability (Fig. 4a), LDH leakage (Fig. 4b), and MDA formation (Fig. 4d) at a dosage of 100 μg/ml. We also measured the intracellular ROS level using DCF formation (Fig. 4c). The intracellular ROS levels decreased in a dose dependent manner. The ROS levels of the cells treated with t-BHP were found to be reduced to about 17% of those of the cells treated with t-BHP alone.
Fig. 4

Effect of chebulic acid obtained from T. chebula against oxidative stress induced by 1.5 mM t-BHP for 30 min in primary cultured rat hepatocytes. Values are expressed as mean ± SD *P < 0.05, **P < 0.01, compared with cell treated with t-BHP alone

Levels of GSH and GSSG

We measured the levels of GSH and GSSH in the cells treated with 100 μg/ml of chebulic acid and/or t-BHP. The group of cells treated with t-BHP alone showed a significantly increased ratio of GSSH to the total GSH (GSH + GSSG) than the control, with the ratios being 8.33 ± 0.23% and 0.05 ± 0.01% for the treated and control cells, respectively. However, the group treated with both chebulic acid and t-BHP showed a significantly reduced GSH redox status (4.42 ± 0.24%) (Fig. 5).
Fig. 5

Protection effect of chebulic acid on reduced glutathione level in primary rat hepatocytes. Cells were treated with 100 μg/ml chebulic acid and/or only 1.5 mM t-BHP for 30 min. Values are expressed as mean ± SD *P < 0.05, **P < 0.01, compared with cell treated with t-BHP alone


T. chebula fruit extract has a high phenolic content and strong biological activities (Bajpai et al. 2005), including anticancer (Saleem et al. 2002), anti-lipid peroxidation and anti-diabetic (Sabu and Kuttan 2002) activities. Juang et al. (2004), reported the structures of the 14 hydrolysable tannins and their related compounds, which had been isolated from T.chebula in an omnibus report. They were isolated from the n-butanol layer, and their structures and molecular weights were more complex and larger than those of phenolcarboxylic acid. The EtOAc layer separated from the ethanolic extracts of T. chebula has a similar activity to that of the n-butanol layer. Juang et al. (2004) reported that chebulic acid belongs to phenolcarboxylic acid group. Some previously reported biological activities of T. chebula were possible partially related to its antioxidant activity (Cheng et al. 2003). Also, these activities were in line with the known ability of moiety with gallic acid or galloyl glycosides to scavenge DPPH radicals and an increasing number of hydroxyl groups capable of donating hydrogen to the radical should enhance the capability of a substrate to scavenge the radical (Naik et al. 2004; Chevalley et al. 1999).

It has been proposed that oxidative stress causes damage to cellular and extracellular macromolecules, such as proteins, lipids and nucleic acids, which results from the balance tipping towards prooxidant status. Therefore, antioxidants are closely related to the prevention of degenerative illnesses, including cardiovascular, and neurological diseases, such as Alzheimer’s disease (Youdim and Joseph 2001). The liver is the largest organ in the body and it effectively filters the blood coming from the gastrointestinal tract. Thus, the liver is the major organ susceptible to ROS or chemical-induced tissue injury.

Mitochondria and cytochrome P450 enzymes are the main sources of ROS in hepatocytes acutely and/or chronically exposed to a toxic injury due to drugs, alcohol, viruses, etc. (Gonzalez 2005). ROS plays a key role in both normal biological function and in the pathogenesis of certain human diseases. Thus, it has been suggested that the antioxidant activity of chebulic acid would play a role in the relief of liver damage.

GSH is known to play a protective role against t-BHP-induced toxicity, and the oxidative stress of the tissues generally involves the GSH system (Ochi and Miyaura 1989). GSH has multiple functions in disease prevention and the detoxification of chemicals and drugs while its depletion is associated with increased risks of toxicity and disease. A deficiency of hepatic GSH and its oxidized form, GSSH, and/or an increase in the amount of toxic free radical species, may contribute to the progression of liver disease (Jaeschke 1990). Thus, GSH could play a useful role in the management of the liver or hepatocytes suffering from oxidative stress.

To summarize, we identified chebulic acid isolated from T. chebula Retz., and exhibited its in vitro antioxidant activities and protection of isolated rat hepatocytes against oxidative toxicity. We have demonstrated that: (1) chebulic acid showed in vitro DPPH radical scavenging activity, ferric reducing antioxidant activity and the ESR spectrum for theOOH radical signals, (2) the treatment of rat hepatocytes with chebulic acid significantly reduced t-BHP-induced cell cytotoxicity, intracellular ROS levels and the ratio of GSSH to the over total GSH as compared to that with t-BHP alone.


This work was supported by grant number 20050401-034-749-180-02-00 from the BioGreen 21 program, Rural Development Administration, Republic of Korea Research Fund (K0401771) in 2004–2005 and Ministry of Education and Human Resources Development in Korea (2005).

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