Cytotoxicity to Five Cancer Cell Lines of the Respiratory Tract System and Anti-inflammatory Activity of Thai Traditional Remedy

Abstract

A Thai traditional remedy called Benchalokawichian (BLW) consists of 5 plant species, Ficus racemosa, Capparis micracantha, Clerodendrum petasites, Harrisonia perforata, and Tiliacora triandra. It has long been used in Thai traditional medicine to reduce fever in respiratory tract infection, but there is no report on either cytotoxicity against cancer cell lines of the respiratory tract system or anti-inflammatory effect. Thus, the objectives of this research were to investigate the cytotoxic activity of the ethanolic and water extracts of BLW, its single plant ingredients and its isolated compounds against 5 cancer cell lines of the respiratory tract, by SRB assay. Anti-inflammatory activity of all extracts and compounds was also tested by using lipopolysaccharide-induced nitric oxide (NO) in RAW 264.7 cells. The main compounds were isolated by high-performance liquid chromatography and compared with BLW and plant ingredients. A major compound of BLW and H. perforata ethanolic extracts is perforatic acid, which inhibited the growth of 2 lung cancer cell lines, A549 and H226, with IC₅₀ values of 6.7 and 13.2 µg/mL. The ethanolic extract of BLW and T. triandra showed cytotoxic activity against all cancer cell lines with IC₅₀ values in the range of 10.1 to 45.2 µg/mL. In contrast, all EtOH extracts showed moderate anti-inflammatory activity, but the water extract had no inhibitory effect on either activity. Pectolinarigenin and O-methyllaloptaeroxyrin, 2 minor compounds, exhibited NO inhibitory effect with IC₅₀ values of 7.1 and 7.9 µg/mL, respectively, whereas perforatic acid was inactive (>50 µg/mL). Moreover, pectolinarigenin showed high cytotoxic activity against all cancer cell lines of the respiratory system with IC₅₀ values in the range of 1.9 to 9.1 µg/mL. As a result, these 2 minor compounds can be used as markers for quality control of BLW for anti-inflammatory activity. Perforatic acid and pectolinarigenin are of interest for further study on their cytotoxic mechanism. Remarkably, T. triandra, one of the plant components of BLW, is possibly the source of the active cytotoxic compounds.

Cancer of the respiratory tract system (bronchus and lung cancer) is 1 of the top 3 cancers in both men and women. ¹ About 2 in 3 of the cases are linked to smoking, foods, behavior, environment, and lack of exercise. Although, several methods for cancer treatment are available, including chemotherapy, radiotherapy, and surgery, they all show various side effects such as fatigue, weight loss, nausea, vomiting, and bleeding. Nowadays, alternative medicines are used to treat cancer patients because they have fewer side effects and can increase the quality of life of the patients. Nitric oxide (NO) is an important biologically active molecule that plays a key part in the host defense against bacteria, protozoa, and tumor cells. ² It is also involved in non-specific (innate) host defense, and participates in the complex mechanism of tissue injury, acting as a major mediator of inflammatory processes and apoptosis. ³ The inflammatory process correlated with multiple cytotoxic effects is related to the ability to increase vascular permeability and edema. ⁴

Benchalokawichian (BLW), a Thai Traditional medicine, consists of the roots of 5 plant species: Ficus racemosa Linn., Capparis micracantha DC., Clerodendrum petasites S. Moore., Harrisonia perforata Merr., and Tiliacora triandra Diels. It is commonly used to reduce fever. ⁵ Folk doctors appropriately adjust the ratios of the plants according to the symptoms. For example, C. petasites is added in high ratio in the case of fever associated with nausea and vomiting; C. micracantha and H. perforata are added in the case of fever with blister rashes, and F. racemosa is added in the case of fever with diarrhea. The ethanolic extract of BLW has shown antipyretic, moderate antioxidant, and anti-inflammatory activities. ^6-8 An in vitro study of the antiallergic activity of the ethanolic extract, fractions, and pure constituents from BLW were studied, the result showed that pectolinarigenin and O-methylalloptaeroxylin were isolated from the chloroform fraction, had higher antiallergic activity than the others. ⁹ Pectolinarigenin was reported as a minor compound with a content of 0.18%, w/w. ¹⁰ The dominant peak on the chromatogram of BLW and H. perforata was identified as being perforatic acid. ¹¹ In the present investigation, we have investigated the cytotoxic and anti-inflammatory activity of the water and ethanol extracts, and isolated compounds of BLW.

The ethanolic extract of T. tiliacora (TTE) and the ethanolic extract of Benchalokawichian remedy (BLWE) exhibited the cytotoxic activity against respiratory system cancer cells, including 3 human lung cancer cell lines (A549, COR- L23, and NCI-H226), and oropharynx (KB) and larynx (Hep-2) cancer cell lines. At a concentration 50 µg/mL, BLWE showed inhibition of all cell lines by more than 80%. TTE exhibited significantly better cytotoxic activity against 3 types of cell lung cancer cell lines than the others in the screening test. BLWE showed more potent cytotoxic activity against oropharynx and larynx (KB and Hep-2) cancer cell lines than TTE. Nevertheless, TTE inhibited COR-L23 better than BLWE (IC₅₀ values of 25.7 and 33.7 µg/mL, respectively). However, both showed good cytotoxicity against NCI-H226 according to the NCI standard (<20 µg/mL).

Previous research reported that all ethanolic extracts showed moderate inhibition of lipopolysaccharide (LPS)-induced inflammation. Although, BLWE exhibited a better inhibitory effect on NO release than the others (IC₅₀ = 40.3 µg/mL), the positive control, indomethacin, showed an IC₅₀ value of 20.3 µg/mL. ⁸ None of the aqueous extracts inhibited cytotoxic and inflammatory activities in this study. Similarly, the water extract had been reported previously to have no anti-inflammatory activity, but an antiallergic effect. ^12,13 BLWE showed moderate antiallergic activity ⁹ and also showed good antibacterial activity against Staphylococcus pyogenes. ¹⁴ In addition, it also showed high cytotoxic activity against respiratory tract cancer cells and moderate anti-inflammatory activity. It is possible that BLWE can be used for respiratory tract diseases, such as rhinitis, which show allergic symptoms and inflammation, and also for the treatment of respiratory tract cancer, especially squamous carcinoma. Benchalokawichian is used to reduce fever in Thai Traditional medicine following the National List of Essential Medicine. Thus, it should be recommended for the treatment of patients who have a fever and respiratory tract diseases, including allergy, inflammation, and cancer. However, this is the first report of the cytotoxic activity of BLWE and its ingredients.

The 3 compounds isolated in this study shown in Figure 1 as perforatic acid, O-methyllaloptaeroxyrin, and pectolinarigenin, have been reported from H. perforata, ^11,15 but there is no research report on the cytotoxic and anti-inflammatory activities of perforatic acid and O-methyllaloptaeroxyrin. Pectolinarigenin has been isolated from several plants, for example, the stem and leaves of Clerodendron siphonenthus, ¹⁶ the flowers of Millingtonia hortensis ¹⁷ and Trifolium pratense ¹⁸ the leaves of Mentha and Ocimum spp., ^19-21 the branches of H. perforata, ^11,15 and the root of Clerodendrum indicum. ²² Pectolinarigenin isolated from Linaria reflexa showed strong cytotoxic activity against large cell carcinoma (COR-L23), and lung carcinoma (A549) with IC₅₀ values of 4.1 and 5.6 µM. ^23,24 In the same way, pectolinarigenin from BLWE showed good cytotoxic activity by Sulforhodamine B (SRB) assay against respiratory system cancers such as Hep2, KB, COR-L23, and A549 ( Table 1). Perforatic acid also exhibited cytotoxic activity against A549 and H226 lung cancer cell lines, with IC₅₀ values of 6.7 and 13.2 µg/mL, respectively. In contrast, O-methyllaloptaeroxyrin showed no cytotoxic activity (IC₅₀ >50 µg/mL). Two minor compounds isolated from BLW, pectolinarigenin and O-methyllaloptaeroxyrin, showed strong antiallergic activity in vitro. ⁹ Pectolinarigenin isolated from Cirsium chanroenicum (Compositae) showed a strong in vitro anti-inflammatory effect inhibiting COX2-catalyzed PGE2 production from LPS-treated RAW 264.7 cells and 5-LOX-catalyzed LT production from A23187-treated RBL-1 cells. ²⁵ O-Methyllaloptaeroxyrin (perforatin A or alloptaeroxylin methyl ether) has been isolated from many plants such as the heartwood of Cedrelopsis grevei, ²⁶ the bark of C. gracilis, ²⁷ the wood of Ptaeroxylon obliquum, ²⁸ the stem of Cneorum tricoccum, ²⁹ branches of H. perforata, ¹⁵ and Iranian Rosmarinus officinalis oil. ³⁰ It has been reported to possess antihypertensive, antiplasmodial, and antimycobacterial effects. ^15,28 Perforatic acid was isolated as the main compound of H. perforata a long time ago ^15,31,32 ; it had been reported to lack antiplasmodial and antimycobacterial activities. ¹⁵ It was also inactive in the NO anti-inflammatory assay in this study. Nevertheless, there is no report on the inhibitory effect on NO release of these 3 compounds. Both pectolinarigenin and O-methyllaloptaeroxyrin inhibited NO release from LPS-induced inflammation (IC₅₀ values 7.2 and 7.9 µg/mL, respectively), effects greater than that of indomethacin, whereas perforatic acid was not active (IC₅₀ >50 µg/mL). Pectolinarigenin showed a good anti-inflammatory effect, which has been reported for the ethanolic extract of C. petasites (CPE) ²² and the ethanolic extract of H. perforata (HPE). ^11,15 It is rational therefore HPE and CPE also showed moderate anti-inflammatory activity. As pectolinarigenin and O-methyllaloptaeroxyrin showed anti-inflammatory activity, BLWE demonstrated the best effect in the NO assay compared with the other ethanolic extracts. This is the first report on reduced LPS-induced NO production in RAW264.7 cell lines produced by perforatic acid, O-methyllaloptaeroxyrin, and pectolinarigenin.

Figure 1

Perforatic acid (1), O-methyllaloptaeroxyrin (2), and pectolinarigenin (3).

Table 1

IC₅₀ Values of Cytotoxic Activity Against 2 Types of Larynx and 3 Types of Lung Cancer Cell Lines, and Anti-Inflammatory Activity (μg/mL ± SEM, N = 3).

Sample *	Cytotoxic activity, IC50 (µg/mL)± SEM, µM					Anti-inflammatory activity, IC50 (µg/mL)± SEM, µM
	KB	Hep2	A549	COR-L23	NCI-H226	RAW264.7
H. perforata, HPE	>50	>50	>50	>50	>50	53.2 ± 5.2
H. perforata, HPW	>50	>50	>50	>50	>50	>100
C. micracantha, CME	>50	>50	>50	>50	>50	61.1 ± 4.3
C. micracantha, CMW	>50	>50	>50	>50	>50	>100
C. petasites , CPE	>50	>50	>50	>50	>50	46.5 ± 0.9
C. petasites, CPW	>50	>50	>50	>50	>50	>100
F. racemosa, FRE	>50	>50	>50	>50	>50	>100
F. racemosa, FRW	>50	>50	>50	>50	>50	>100
T. triandra, TTE	42.1 ± 4.5	45.2 ± 7.1	33.6 ± 0.6	25.7 ± 7.9	19.5 ± 1.4	54.6 ± 5.3
T. triandra, TTW	>50	>50	>50	>50	>50	>100
Benchalokawichian, BLWE	32.0 ± 0.02	32.7 ± 2.1	32.3 ± 0.7	33.6 ± 0.9	10.1 ± 2.8	40.4 ± 2.0
Benchalokawichian, BLWW	>50	>50	>50	>50	>50	>100
Perforatic acid	NT	NT	6.7 ± 0.04 (22.1)	NT	13.2 ± 0.64 (43.8)	>50
O-Methyllaloptaeroxyrin	>50	>50	>50	>50	>50	7.9 ± 0.72 (28.9)
Pectolinarigenin	6.2 ± 1.7 (19.7)	1.9 ± 0.6 (6.1)	7.8 ± 0.2 (24.7)	9.1 ± 0.7 (29.0)	NT	7.1 ± 0.40 (22.8)
Indomethacin a	NT	NT	NT	NT	NT	20.3 ± 3.7

^aPositive control for anti-inflammatory activity.

*Name code provided by their initial scientific name combine with the type of extract, as E represented the ethanolic extract and W represented the water extract

Only the ethanolic extract of Tiliacora triandra and BLW showed cytotoxic activity. The dominant peak in the chromatogram of H. perforata at a retention time of 26.4 minutes was identified as perforatic acid. This compound showed in vitro cytotoxic activity against 2 lung cancer cell lines, while it had no effect on NO production in activated RAW 264.7 cells. Pectolinarigenin and O-methyllaloptaeroxyrin showed strong anti-inflammatory activity, and, moreover, pectolinarigenin also showed cytotoxic activity. The results suggest that these 2 compounds can serve as biomarkers for anti-inflammatory and cytotoxic activities of BLW. However, the mechanisms of perforatic acid and pectolinarigenin as cytotoxic compounds should be further studied.

Experimental

Materials and Instruments

The 5 plants found in BLW were extracted by decoction and maceration with 95% EtOH to obtain water and ethanolic extracts. O-Methyllaloptaeroxyrin and pectolinarigenin were isolated from BLW ethanolic extract as previous reported. ⁹ All cell lines were purchased from the American-type culture collection (ATCC). Minimum Essential Media (MEM) and Roswell Park Memorial Institute (RPMI) culture media were purchased from Biochrom, and other reagents and supplements from Costar Corning. NMR spectra were recorded using a Bruker Avance 600 MHz spectrometer. ESIMS were obtained on a Bruker-Hewlett-Packard 1100 Esquire-LC system mass spectrometer, and IR spectra were recorded using a Perkin-Elmer FTS FT-IR spectrophotometer.

RP-HPLC Analysis

Chromatographic fingerprints of BLW extract and its components were obtained using a high-performance liquid chromatographic (HPLC) method according to Sakpakdeechareon et al. ¹⁰ The HPLC instrument (Agilent^® LC 1100/1200 system) consisted of a quaternary pump (model G1311A), an automatic injector (model G1329A), and a photodiode array detector (model G1315D). A chromatographic fingerprint was constructed using a reverse-phase C18 column (Phenomenex^® Luna, 5 µm, 250 mm × 4.6 mm). The mobile phase was composed of 0.1% ortho phosphoric acid (A) and acetonitrile (B). Constituents in the sample were gradient-eluted with a flow rate of 1.0 mL/min using the following program: 0 to 30 minutes, 95%A; 30 to 35 minutes, 5%A; and 35 to 40 minutes, 95%A. A concentration of 10 mg/mL of extracts and 1 mg/mL of the 3 pure compounds were prepared. Ten µL of sample was injected and the detector was set at a wavelength of 331 nm. Data were analyzed by ChemStation^® software. The HPLC chromatogram of BLW extract presented peaks for perforatic acid, O-methyllaloptaeroxyrin, and pectolinarigenin at retention times of 16.3, 20.7, and 22.5 minutes, respectively.

The ethanolic extract of H. perforata was separated by vacuum liquid chromatography to give 5 fractions (A-E). All were investigated by HPLC. The dominant peak at a retention time of 26.4 minutes was collected following the HPLC method, and then further purified with an increasing polarity of dichloromethane: methanol by CC to give a yellow wax, 6.3 mg. ¹H and ¹³C NMR spectroscopic data were identical to those of perforatic acid ¹¹ ; this identity was confirmed by IR and ESIMS.

Preparation of Sample

The ethanolic extracts and compounds dissolved in sterile dimethyl-sulfoxide, and the water extracts in sterile water were filtered through 0.22 µm membranes. The stock samples for cytotoxic activity were prepared to a concentration of 10 mg/mL. For anti-inflammatory activity, the concentration of 50 mg/mL of ethanolic extract and 10 mg/mL of water extract were stocked. A concentration of 50 µg/mL of sample was used for the screening test. For any sample which showed a % of inhibition more than 50%, an IC₅₀ value was determined.

Determination of Cytotoxic Activity by SRB Assay

Five types of cancer cell lines of the respiratory system (human lung carcinoma cell line COR-L23 (ECACC^® 92031919^™), human lung adenocarcinoma cell line A549 (ATCC^®CCL-185^™), human lung squamous carcinoma cell line NCI-H226 (ATCC^®CRL-5826^™), human laryngeal carcinoma Hep-2 (ATCC^® CCL-23^™), and human epidermoid carcinoma cell line KB (ATCC^® CCL-17^™) were used to determine cytotoxic activity. The estimated cell number was determined by staining the total cellular proteins with SRB dye ³³ following the procedure of Itharat et al. ³⁴ The dyed cells were detected at a wavelength of 492 nm. The percentage of inhibition was calculated by this formula: [(Abs. Control - Abs. Sample)/Abs. Control] × 100

Determination of Anti-Inflammatory Activity by NO-Inhibitory Effect

Mouse macrophage leukemia-like (RAW264.7, ATCC TIB-71^TM) cells were used as immune cells, which were induced by LPS to release inflammatory mediators, including NO. ³⁵ Griess reagent was used to determine nitrite, which is a stable end product of NO in cell culture supernatants. ³⁶ The inhibitory effect on NO production was evaluated using a modified method, as previously reported. ³⁷ Cytotoxicity was also determined using the yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay. The color was detected at a wavelength of 570 nm. Indomethacin was used as positive control. The percentage of inhibition was calculated by this formula:

% Inhibition = [(A – B)/A– C)] × 100

Control (A) : LPS (+), Test sample (–)

Sample (B) : LPS (+), Test sample (+)

Blank (C) : LPS (–), Test sample (–)

The color of the cells supernatant was detected by spectrophotometry. The % inhibition was calculated from cell viability. Various % inhibition of concentrations provided IC₅₀ values by PRISM program. All experiments were carried out in triplicate and presented as means ± SEM.