Correlation Between Antioxidative Power and Anticancer Activity in Herbs from Traditional Chinese Medicine Formulae with Anticancer Therapeutic Effect

Abstract

Lobeliae chinensis. Herba (“ban bian lian”), Rheum officinale. Baill. (“da huang”), Sanguisorba officinalis. Linn. (“di yu”), Agrimonia pilosa. Ledeb. (“xian he cao”), and Paris polyphylla. Smith (“zhi hua tou”) are well-known traditional Chinese medicines. They are commonly used in traditional Chinese medicine formulae against cancer. In this study, the antioxidant and anticancer effects of water extracts of these herbs were investigated. In the antioxidant and anticancer studies, water extracts of di yu, xian he cao, and da huang were show to be the most antioxidative and had the highest growth inhibitory effect on human lung adenocarcinoma A549 cell and human breast cancer MCF-7 cell. By comparing their percentage free radical scavenging capacity (SR%) and percentage growth inhibition on A549 and MCF-7 cells, a positive linear relationship between antioxidant activity and anticancer effect of the five herbal water extracts was found. This suggested that the antioxidants of the herbal water extracts might contribute to their anticancer effects on A549 and MCF-7 cells.

Keywords:

• Anticancer activity
• antioxidant activity
• chinese medicinal herbs

Introduction

Cancer is an uncontrolled growth and spread of cells that may affect any tissues of the body. Currently, there is no universally effective treatment for cancer. It emerges as a major health problem in many developed countries. Many research interests are, therefore, focused on the potential role of traditional Chinese medicinal (TCM) herbs on the prevention and treatment of cancer (Powell et al., 2003; Schwarz et al., 2003). According to the World Health Report (World Health Organization, 2003), lung and breast cancer are the most common cancers in men and women, respectively. Preventing and treating these two cancer types with TCM herbs is one of the important areas of contemporary nutraceutical and pharmacological research.

Natural products, such as TCM herbs, have been used in the treatment of various chronic human pathologic conditions (Yan et al., 2006) because they contain high antioxidative ingredients that could reduce the risk of cancers, aging, atherosclerosis, and other oxidant-induced diseases (Singal et al., 1998; Hu et al., 2000; Noguchi et al., 2000; Bedda et al., 2003; Correa et al., 2005). Antioxidants can protect living organisms from DNA/protein damage and lipid peroxidation caused by reactive oxygen species, such as free radicals. Because of the high content of antioxidants, such as phenolics, in TCM herbs, they have great potential to prevent and treat various human degenerative diseases, such as cancer.

Although extensive research was done on the antioxidant effect and anticancer effect of TCM herbs (Hu et al., 2002; Cheng et al., 20042005), in the literature only a few studies explored the relationship between antioxidative and anticancer properties of TCM herbs. In this study, five TCM herbs, which are commonly found in TCM formulae prescribed for cancer, were selected for investigation (Table 1). They are Lobeliae chinensis. Herba (“ban bian lian”), Rheum officinale. Baill. (“da huang”), Sanguisorba officinalis. Linn. (“di yu”), Agrimonia pilosa. Ledeb. (“xian he cao”) and Paris polyphylla. Smith (“zhi hua tou”).

Table 1.. List of herbal extracts tested.

Chinese name	Scientific name	Family	Plant part(s)	Voucher number
Ban bian lian	Lobeliae chinensis. Herba	Campanulaceae	Dried aerial part	AC001
Da huang	Rheum officinale. Baill.	Polygonaceeae	Dried root and rhizome	AC002
Di yu	Sanguisorba officinalis. Linn.	Rosaceae	Dried root	AC003
Xian he cao	Agrimonia pilosa. Ledeb.	Rosaceae	Dried aerial part	AC004
Zhi hua tou	Paris polyphylla. Smith	Trilliaceae	Dried rhizome	AC005

Apart from the anticancer effect, they have other pharmacological effects. For example, ban bian lian has been widely used to treat anasarca and ascites, carbuncles, boils and snake or insect bite, and ascites in late schistomiasis; da huang has been used as a laxative, antiphlogistic and hemostatic in the treatment of obstipation, gastrointestinal indigestion, diarrhea, and jaundice (Tang et al., 1992; Huang et al., 1993; Newall et al., 1996) di yu has been used for the treatment of hemostasis and inflammation (Liu et al., 2005); xian he cao has been used to stop bleeding, inhibit Gram-positive bacteria, and get rid of tapeworms; and zhi hua tou has been used to alleviate pain, cough, and dyspnea, remove blood stasis, and stop bleeding.

In order to examine the relationship between antioxidant and anticancer activities, the five selected TCM herbs were extracted with water to mimic a TCM decoction, which is produced by boiling a combination of herbs in water. Their antioxidative powers were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and Folin-Ciocalteu assay, and their anticancer effect was monitored by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay on growth inhibition on human lung adenocarinoma A549 cells and human breast cancer MCF-7 cells to investigate the relationship between these two important parameters.

Materials and Methods

Selection of Chinese medicinal herbs

A total of five Chinese medicinal herbs were selected for this study that are commonly found in traditional Chinese medicine formulae prescribed for cancer. All the herbs were purchased from Hip Shing Hong Ltd. (Hong Kong). The voucher samples of herbs were stored in the laboratory of Dr. Peter H.F. Yu. Their Chinese names (“pin yin”), scientific names, and families are listed in Table 1.

Extraction

Each dried Chinese medicinal herb was blended into powder. One gram of each Chinese medicinal herb powder was mixed with 10 mL distilled water in a universal bottle and was shaken in a 37°C shaker at 300 rpm for 2 h. After 2 h, the solution was collected after centrifuged at 4000 rpm for 3 min. The remaining residues were extracted by the above method two more times. Finally, all the solution was poured together and filtered by suction filtration and lyophilized for about 1 week. After lyophilization, the Chinese medicinal herb powders (herbal water extracts) were preserved in a refrigerator at −20°C until further use.

Chemicals

DPPH powder was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Ascorbic acid was obtained from Panreac Quimica Sa (Madrid, Spain), E.U. Celltiter 96 aqueous MTS reagent powder was obtained from Promega Chemicals Co. (Madison, WI, USA). Dulbecco's modified Eagle medium (DMEM) and fetal bovine serum were obtained from Gibco, Invitrogen Co. (Grand Island, NY, USA). Gallic acid was obtained from Advanced Technology and Industrial Co., Ltd. (Hong Kong). Folin-Ciocalteu phenol reagents were obtained from Merck (Darmstadt, Germany). All the other chemicals used were of analytical grade.

Preparation of extracts and standards

For antioxidant experiments, extracts were dissolved in distilled water to give a final concentration of 1 mg/mL. Solutions of ascorbic acid and gallic acid, which were used as control and standards, respectively, were dissolved in distilled water. For in vitro. cytotoxicity experiment, the extracts were prepared to give a concentration of 1 mg/mL in DMEM. The solutions were serially diluted with respective solvent to various working concentrations.

DPPH method

The antioxidant activities of the herbal water extracts and standards were measured by the DPPH assay, based on the free radical scavenging capacity of extracts on the stable DPPH free radical. Briefly, methanol was used to set blank at 515 nm. The absorbance value (A._control) of DPPH (0.024 mg/mL) in methanol was measured and recorded as control at 515 nm. Then, each extract solution (1 mg/mL) was mixed with the control and the change in absorbance (A._sample) was recorded until the absorbance change was equal to or smaller than 0.003 absorbance units per minute (Schlesier et al., 2002). Finally, the absorbance (A._original) of herbal water extract or standard in methanol was measured to eliminate the color interference of the herbal water extract. The percentage free radical scavenging capacity (SR%) was calculated using the following equation:

Folin-Ciocalteu method

The total phenolics compounds in the herbal water extracts were measured by Folin-Ciocateu method, based on the conversion of Folin-Ciocalteu reagent. Each herbal water extract was dissolved in distilled water to give a final concentration of 1 mg/mL. Sample was mixed with Folin-Ciocalteu reagent and sodium carbonate. After standing for 2 h at room temperature, the absorbance of each sample was measured at 750 nm using a spectrophotometer, and 100 µL distilled water instead of sample was used as blank (Schlesier et al., 2002). The total phenolics content in 1 mg/mL of sample was expressed as gallic acid equivalents. The estimation of phenolic compounds in the samples was carried out in triplicate.

Cell culture

Human cancer cell lines including human breast cancer MCF-7 and human lung adenocarcinoma A549 were obtained from ATCC (Baltimore, MD, USA). They were maintained in low-glucose DMEM supplemented with 10% fetal bovine serum in a 5% carbon dioxide incubator at 37°C.

MTS assay

The effects on inhibition of cell growth were measured by MTS growth inhibition assay, based on the ability of living cells to convert tetrazolium salt into purple formazan dye. MTS growth inhibition assay was performed according to the instructions provided by Promega Co. Briefly, cells were seeded in a 96-well plate on day 1. On day 2, the cells were treated without or with 1 mg/mL of each herbal water extract for 3 days more. Afterward, MTS solution was added and the optical density was recorded at 490 nm using a microplate reader (model 550; Bio-Rad; Hercules, CA). Results are expressed as the percentage growth inhibition with respect to untreated cells.

Statistical analysis

Data were expressed as means ±SEM, and n denotes the number of replications for each data point. Comparisons of parameters among different groups were made with one-way analysis of variance, followed by Newman-Keul's test for multiple comparisons among means. In all cases, differences between treatment groups were considered significant when p < 0.05. All statistical analysis tests were performed by using GraphPad Prism 4.02 for Windows (GraphPad Software, San Diego, CA, USA).

Results

Measurement of free radical scavenging capacity

The antioxidant activity of herbal water extracts was measured in terms of the percentage free radical scavenging capacity (SR%). Among the five herbal water extracts, the SR% of di yu (92.53 ± 0.356%), xian he cao (90.44 ± 2.237%), and da huang (89.06 ± 1.244%) were comparable with that of ascorbic acid (96.03 ± 0.022%) (p > 0.05). The free radical scavenging capacity of ban bian lian and zhi hua tou water extracts were extremely low with their SR% significantly lower than ascorbic acid (p < 0.001) (Fig. 1).

Figure 1 Percentage free radical scavenging capacity (SR%) of the five herbal water extracts. Data are expressed as mean ±SEM, n = 3.

Measurement of total phenolic content

Among the five herbal water extracts, di yu (0.35 ± 0.003 mg/mL), xian he cao (0.20 ± 0.010 mg/mL), and da huang (0.15 ± 0.007 mg/mL) had total phenolic equivalent higher than 0.10 mg/mL of gallic acid, whereas ban bian lian and zhi hua tou each had very low phenolic content that was less than one-twentieth phenolic content (>0.02 mg/mL) of the others (Fig. 2).

Figure 2 The total amount of phenolics of the five herbal water extracts. Phenolic contents are expressed as mg/mL of gallic acid. Data are expressed as mean±SEM, n = 3.

Effect of water extracts on the proliferation of A549 and MCF-7 cells

Any change in growth rate by the herbal water extracts on A549 and MCF-7 cells after 72 h was measured by MTS assay. All five herbal water extracts showed growth inhibitory effect on both A549 and MCF-7 cells (Table 2). The water extracts of xian he cao, di yu, and da huang showed obvious growth inhibitory effect.

Table 2.. Percentage inhibition of the five herbal water extracts on MCF-7 cell and A549 cell.

	Percentage growth inhibition (%)
Water extract (1 mg/mL)	MCF-7 cell	A549 cell
Ban bian lian	5.308 ± 0.905	1.67 ± 0.811
Da huang	73.71 ± 1.235	76.27 ± 2.533
Di yu	97.95 ± 0.441	98.02 ± 0.285
Xian he cao	77.52 ± 0.240	82.49 ± 0.627
Zhi hua tou	50.98 ± 1.731	48.35 ± 4.151

Data are expressed as means ±SEM, n = 3.

Relationship between the antioxidant activity and the anticancer effect

In order to investigate whether there was any correlation between antioxidant activity and anticancer effect, scatterplots between SR% and growth inhibition percentage on both cancer cell lines were established (Fig. 3). It was found that SR% was positively correlated with growth inhibition percentage on A549 cell (R² = 0.7560, p < 0.001) and on MCF-7 cell (R² = 0.7309, p < 0.001).

Figure 3 Relations of SR% and growth inhibition percentage of the five herbal water extracts on (A) A549 cells, (B) MCF-7 cells.

Discussion

TCM herbs have been used among Chinese communities throughout the world for a long period of time. There is abundant information about their usage, but the scientific evidence is still limited. In this study, a systematic approach was adopted to examine the antioxidant activity and growth inhibitory effect of five selected TCM herbs that are commonly found in TCM formulae prescribed to cancer patients.

As shown in Figs. 1 and 2, water extracts of di yu, xian he cao, and da huang are good free radical scavengers. They contain high phenolic content. Ban bian lian and zhi hua tou water extracts have low free radical scavenging powers and phenolic content. Phenolics are one of the antioxidants commonly found in most plants. In the current study, herbal water extracts that had high antioxidant activities also had high total phenolic content. This indicated that the antioxidant activity of the tested herbal water extracts is contributed mainly by their phenolic part.

By investigating the anticancer effect of the five herbal water extracts, it was found that all of them showed growth inhibition on human lung adenocarcinoma A549 cell and human breast cancer MCF-7 cell (Table 2). Among them, water extracts of di yu, xian he cao, and da huang had the highest growth inhibitory effect on A549 cells and MCF-7 cells. It suggested that they are effective for treating human lung and breast cancers. In order to investigate the relationship of anticancer and antioxidative effects, the result of the anticancer assay was correlated with the result of the antioxidant assay. This is the first study showing that the herbal water extracts with high antioxidant activity also had high growth inhibitory effect on A549 and MCF-7 cells. By plotting graphs of SR% versus growth inhibition percentage on A549 cells and MCF-7 cells (Figs. 3A and 3B), a high positive relationship between the antioxidant activity and growth inhibitory effects of five herbal water extracts was found. It suggested that the anticancer effect of the herbal water extracts on A549 and MCF-7 cells might be related to their antioxidant activity. In addition, the correlation coefficient is higher in A549 (R² = 0.7560) than in MCF-7 (R² = 0.7309) cells. It indicated that the antioxidants might somehow contribute to growth inhibition in A549 cells more than in MCF-7 cells. Further studies on the role of the antioxidants' growth inhibitory effect on both cell lines may provide insight for cancer therapy. It is also important to dissect the mechanistic functions between antioxidant power and anticancer activity of TCM herbs.

From the current study, water extracts of di yu, xian he cao, and da huang had the highest antioxidant activity and growth inhibitory effect on A549 and MCF-7 cells. They have high potential to be developed as a health product to enhance people's health for their high phenolic content, which could reduce the incidence rate of chronic degenerative diseases. Interestingly, these three TCM herbs also demonstrated an ability to inhibit cancer progression. This suggested that they could help patients with lung or breast cancer. Di yu, xian he cao, and da huang have both good protective and therapeutic effects on cancer. The dual characteristics of these herbs on chronic disease is in line with the philosophy of Chinese medicine, which is based on a holistic and spatial perception of the individual in the environment rather than upon the simplified cause and effect principles.