Phytochemical profiling and phase II enzyme-inducing properties of Thunbergia laurifolia Lindl. (RC) extracts

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Journal of Ethnopharmacology 114 (2007) 300–306

Phytochemical profiling and phase II enzyme-inducing properties of Thunbergia laurifolia Lindl. (RC) extracts Ratchadaporn Oonsivilai a,1 , Crystal Cheng b,2 , Joshua Bomser b,2 , Mario G. Ferruzzi c,3 , Suwayd Ningsanond a,∗ a

School of Food Technology, Suranaree University of Technology, 111 University Avenue, Muang, Nakhon Ratchasima 30000, Thailand b Department of Human Nutrition, The Ohio State University, 325 Campbell Hall, 1787 Neil Avenue, Columbus, OH 43210, United States c Department of Food Science, Purdue University, 745 Agriculture Mall Drive, West Lafayette, IN 47907, United States Received 19 March 2007; received in revised form 9 July 2007; accepted 3 August 2007 Available online 19 August 2007

Abstract Thunbergia laurifolia Lindl. (Acanthaceae) or Rang Chuet (RC) is described in traditional medicine for protection against dietary and environmental toxicants. This work, therefore, investigated RC’s phytochemical profile, antimutagenic activity, and xenobiotic detoxification potential in its extracts. RC extracts were prepared by infusion with water, ethanol, acetone and subsequently assayed for major phytochemical constituents. Total phenolic content was 24.33, 5.65, and 1.42 ␮g gallic acid equivalent (GAE) per mL for water, ethanol and acetone extract, respectively. HPLC analysis identified caffeic acid and apigenin as primary constituents of water extracts. Acetone and ethanol extracts contained primarily chlorophyll a and b, pheophorbide a, pheophytin a, and lutein. Treatment of Hepa 1C1C7 cells with standardized RC extracts resulted in a dose-dependent increase in QR specific activity for all extracts. Acetone extract (92 ␮g GAE/mL) increased QR activity 2.8-fold, while ethanol (120 ␮g GAE/mL) and water (1000 ␮g GAE/mL) extracts increased QR activity by 1.35- and 1.56-fold, respectively. The RC extracts were subsequently assayed for mutagen and antimutagenic activity by bacterial reverse mutagenesis assay. All three RC extracts exhibited strong dose-dependent antimutagenic activity inhibiting 2-aminoanthracene induced mutagenesis up to 87% in Salmonella typhimurium TA 98. These results support the traditional medicinal use of RC for detoxification and suggest the potential role of both phenolic acids and natural chlorophyll constituents in modulating these effects. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Phenolic acids; NQO1; Antimutagenicity; Flavonoids; Rang Chuet; HPLC

1. Introduction Thunbergia laurifolia Lindl. (Acanthaceae, Thai name: Rang Chuet (RC)) is a vine widely distributed in Southeast Asia belonging to the larger family of Acanthaceae. RC is a shrub with small oblong or ovate leaves and bluish-purple flowers. It can be divided into three types designated by flower color: white, yellow, or purple. Purple varieties are believed to pos∗ 1 2 3

Corresponding author. Tel.: +66 44224233; fax: +66 44224387. E-mail address: [email protected] (S. Ningsanond). Tel.: +66 44224233; fax: +66 44224387. Tel.: +1 614 2476622; fax: +1 614 2928880. Tel.: +1 765 4940625; fax: +1 765 4947953.

0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.08.015

sess compounds that deliver health benefits particularly from materials of the stem, root and leaves (Faculty of Pharmacy, 1987). Various parts of this RC variety are utilized in preparation of aqueous extracts of fresh leaves, dried leaves, dried root and bark with applications in traditional Thai medicine. These extracts are reported to have detoxification, anti-inflammatory and antipyretic properties (Tejasen and Thongthapp, 1979; Ruengyutthakan, 1980; Charumanee et al., 1998; Chanawirat et al., 2000; Srida et al., 2002; Thongsaard and Marsden, 2002; Khunkitti et al., 2003). To date, limited information is available regarding the characterization of physiologically active components present in RC. Kanchanapoom et al. (2002) reported two iridoid glucosides, 8-epi-grandiforic and 3 -O-␤-glucopyranosyl-stibericoside iso-

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lated from the aerial part of RC along with seven known compounds benzyl ␤-glucopyranoside, benzyl ␤-2 O-␤-glucopyranosyl, glucopyranoside, grandifloric acid, E-2hexenyly ␤-glucopyeanosdie, hexanol ␤-glucopyranoside, 6-Cglucopyranosylapigenin and 6,8-di-C-glucopyranosylapigenin. Both flowers and leaf materials of RC have been found to contain other bioactive phenolic constituents including delphinidin 3:5-di-O-␤-d-glucopyranoside, apigenin, apigenin7-O-␤-d-glucopyranoside and chlorogenic acid (Purnima and Gupta, 1978; Thongsaard and Marsden, 2002). Limited information is also available regarding the characterization of the lipophilic (i.e. chlorophylls and carotenoids) components of RC. Considering the growing use of organic solvents to produce commercial extracts with higher yields, consideration of these lipophilic phytochemicals in dried RC leaf material is warranted. While carotenoids are well characterized as antioxidants and potential cancer preventative compounds (Bertram and Vine, 2005), chlorophyll derivatives have demonstrated bioactivity consistent with detoxification benefits attributed to RC. Specifically, chlorophyll derivatives including chlorophyll a and pheophorbide a can modulate xenobiotic detoxification pathways including induction of QR activity (Fahey et al., 2005). Furthermore, natural chlorophylls and chlorophyllin (more water soluble derivatives) have demonstrated appreciable antimutagenic activity against complex environmental and dietary mutagens (Sarkar et al., 1994; Dashwood 1997; Ferruzzi and Blakeslee, 2007). With increasing use of RC leaf materials in Thai medicine, it is critical to better understand the phytochemical profile and specific biological activities of different RC preparations. The specific objectives of this work were to quantify total phenolics, carotenoids and chlorophyll derivatives present in specified RC leaf extracts and to assay specific preparations for antimutagenic activity and their ability to modulate key markers of xenobiotic detoxification.

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2.2. Chemicals and standards Solvents including acetone, acetonitrile, ethanol, ethyl acetate and methanol (Mallinckrodt-Baker, Phillipsburg, NJ) were of certified HPLC and ACS grade. A 1.0-M ammonium acetate buffer solution (Fluka; Ronkonkoma, NY, USA) was prepared with double distilled (dd) water and adjusted to pH 4.6 with glacial acetic acid. The following standards were obtained: chlorophyll a, chlorophyll b, lutein, caffeic acid (Sigma–Aldrich, St. Louis, MO), and apigenin (Indofine Chemical Company, Inc., Hillsborough, NJ). Pheophytin a and b standards were synthesized from chlorophyll a and b as described previously (Schwartz et al., 1981; Ferruzzi et al., 2001). Briefly, 1 mg of chlorophyll a or chlorophyll b was dissolved in 10 mL of acetone. Four hundred microliter of 0.1N HCl was added into 5 mL of chlorophyll a or b solution. Complete conversion to pheophytins was followed by HPLC analysis as described below. Pheophytins were extracted with 5 mL of petroleum ether, dried under a stream of nitrogen and kept at −80 ◦ C until use. Prior to HPLC calibration, each standard was dissolved in appropriate solvent and filtered through a 0.45-␮m PTFE filter. 2-Aminoanthracene (2-AA), the test mutagen for the bacterial reverse mutagenicity assay, was purchased from Moltox (Boone, NC, USA). 2.3. Preparation of RC extracts Approximately 100 mg RC leaf powder was extracted with three 12-mL portion of boiling water, ethanol, or acetone in a shaking water bath at 25 ◦ C for 15 min. Samples were centrifuged at 3000 × g (Thermo IEC, Waltham, MA) for 3 min after which solvent layers were collected and filtered by vacuum. Filtrates were combined and final volume was adjusted volumetrically (with representative extraction solvent) to 50 mL. Aliquots of 2 mL were dried under vacuum (Rapid Vap® Vacuum Evaporation Systems, Labconco corporation, Kansas city, MO) and stored frozen at −20 ◦ C until use.

2. Methodology 2.4. Instrumentation and chromatography 2.1. Plant materials The medicinal plant Thunbergia laurifolia Lindl. (Acanthaceae) was collected from December 2005 to February 2006 from local area in Nakhon Ratchasima Province, Thailand and identified by Assoc. Prof. Dr. Sompong Thammathaworn (School of Biology, Suranaree University of Technology), Herbarium voucher specimens (Ratchadaporn 001) were prepared and deposited at School of Food Technology, Suranaree University of Technology and the Forest Herbarium (BKF), Office of Forest and Plant Conservation Research, National Park, Wildlife and Plant Conservation Department, Thailand. Leaves were air dried at 60 ◦ C for 6 h, after which they were ground in a blender (National, MX-T2GN, Taiwan) to a fine powder and stored in vacuum package at 4 ◦ C until use. Powdered leaf material such as this is typical of raw material utilized for manufacture of extracts and herbal tea products from RC.

Chlorophyll and carotenoid analyses were achieved as described by Ferruzzi et al. (2001) with modification. Briefly, a Hewlett-Packard 1090A system equipped with a diode array detector was utilized. Separation was achieved using a GraceVydac 201TP54 reversed-phase (4.6 mm × 250 mm) polymeric C18 column with guard column containing the same stationary phase (Grace Vydac, Apple Valley, MN). A gradient elution profile was used based on a binary mobile phase system consisting of methanol:water:ammonium acetate (73:23:2, v/v/v) in reservoir A and ethyl acetate in reservoir B. A flow rate of 1.0 mL/min was applied with initial setting at 100% (A) with a linear gradient to 50:50 (A/B) over 10 min. The gradient was held for 10 min followed by a 5 min linear gradient back to 100% (A) and equilibration at the initial condition for 5 min for a total run time of 30 min. Detection and tentative identification of all chlorophyll derivatives and lutein were accomplished by comparison of retention times and on line-electronic absorp-

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tion spectra with that of authentic standards collected between 250 and 600 nm. Pheophorbide a was tentatively identified based on electronic absorption spectra (Fiedor et al., 2003) and early elution common to water soluble chlorophyll derivatives under these chromatographic conditions (Canjura and Schwartz, 1991). Quantification of chlorophylls, pheophytins, and lutein were accomplished using multilevel response curves constructed with authentic standards. Pheophorbide a levels were estimated based on the response of pheophytin a due to the lack of authentic standards for this chlorophyll derivatives. 2.5. Total phenolics Total soluble phenolic constituents of the extracts (water, ethanol, and acetone) were determined employing the method of Waterhouse (2002) using the Folin-Ciocalteu reagent with gallic acid as standard. Twenty microliter of freshly prepared RC extract was added into a 1.5 mL cuvette, to which 1.58 mL of dd water and 100 ␮l Folin-Ciocalteu reagent was added. The sample was thoroughly mixed and incubated for 5 min at room temperature. Following incubation 300 ␮l of the Na2 CO3 (2%, w/v) solution was added and the mixture was allowed to stand at room temperature for 2 h. Absorbance was measured at 765 nm. Results were expressed as gallic acid equivalents. 2.6. Antimutagenicity assay The modified microscreen method of Diehl et al. (2000) was utilized with modifications as described by Ferruzzi et al. (2002). Salmonella typhimurium TA98 (Moltox; Boone, NC, USA) was utilized as the test strain. Six-well plates pre-poured with 5 mL of Vogel-Bonner minimal glucose agar per well and top agar (2%) were purchased from Moltox (Boone, NC, USA). Bacterial stock cultures were inoculated into 10 mL of Oxoid#2 nutrient broth and inoculated at 37 ◦ C for 6 h until optical density at 600 nm of 0.8 was achieved. Mammalian microsomal activation system (S-9 mix) was prepared by diluting Aroclor 1254-induced rat liver microsomes (Moltox; Boone, NC, USA) into 1.0 mol L−1 glucose-6-phosphate-NADP solution at 4% (v/v). 2-AA stock solution (100 ␮g/mL) was prepared in sterile dimethyl sulfoxide (DMSO). Individual RC extracts was dissolved in DMSO, filter sterilized and diluted to concentrations between 0.01 and 0.40 mg GAE/mL for incorporation into mutagenicity assay. The mammalian S9 activation system (S-9 mix) was prepared by diluting Aroclor 1254-induced rat liver microsomes (Moltox, Boone, NC, USA) with glucose-6-phosphate-NADP at 5%. Microscreen assays were plated by incorporation of 25 ␮L of 2-AA (0.625 ␮g), 100 ␮L of S-9 mix, 25 ␮L of bacterial culture and 25 ␮L of RC extracts or DMSO into a 2 mL. Samples were preincubated for 20 min at 37 ◦ C, combined with 500 ␮L of molten top agar, mixed, poured onto the surface of wells and incubated for 48 h at 37 ◦ C. A separate set of experiments was conducted with RC extracts incorporated at the plating step instead of pre-incubation. Incorporation of RC extracts after metabolic activation of 2-AA was done to allow for tentative differentiation between inhibition of 2-AA activation or of the mutagenic metabolic end products of 2-AA. Following incu-

bation his+ revertant colonies were counted and corrected for spontaneous mutations determined by DMSO controls. 2.7. QR assay The QR assay was performed as described by Bomser et al. (1996). Hepa 1c1c7 cells (ATCC CRL 2026) were seeded in 96-well dishes and treated for 48 h with control (basal media) and test media containing dried RC extract dissolved in ethanol and diluted to concentration between 25 and 200 ␮g per mL with basal media. Following 48 h of treatment the media was decanted and cells lysed by agitating the plates on an orbital shaker in the presence of 50 ␮L/well of 0.8% digitonin and 2 mM EDTA, pH 7.8 for 10 min. Before the plates were assayed for QR activity, a cocktail containing 25 mM Tris–Cl (pH 7.4), 0.67 mg/mL bovine serum albumin, 0.01% Tween-20, 5 ␮M FAD, 30 ␮M NADP+, 1 mM glucose 6-phosphate, 2 U/mL bakers yeast glucose-6-phosphate dehydrogenase, and 0.3 mg/mL MTT [3-(4,5-dimethylthiazo-2yl)-2,5-diphenyltetrazolium bromide was prepared for all plates to be assayed (25 mL/plates). Shortly before each plate was scanned, 25 ␮L of 50 mM menadione dissolved in acetonitrile was mixed with 25 mL of assay cocktail. After 200 ␮L/well of the resulting solution was added to the plates, the plates were placed in an UVmax microtiter plate scanner (Molecular Devices, Menlo Park, CA, USA), and the rate of formation of the formazan dye was quantitated at 610 nm for 2 min. The dicoumarol-inhibitable rate of MTT reduction was measured by rescanning the plates after the addition of 50 ␮L/well of 0.3 mM dicoumarol dissolved in 0.5% DMSO and 5 mM potassium phosphate (pH 7.4). The QR-inducing activity of all fractions was normalized based on the total phenolic content. The concentration of the fractions, expressed as ␮g GAE per mL required to significantly induce QR-specific activity relative to controls, was used as an indicator for induction potency. 2.8. Statistical analyses Descriptive statistics including mean and standard error of mean (S.E.M.) were calculated for phytochemical constituents of RC extracts (n = 5). QR induction data was expressed as fold-induction of QR specific activity (n = 8) in treated versus control cells receiving no RC treatment. The reduction of total his+ revertants relative to 2-AA controls, corrected for spontaneous mutations, is defined as %inhibition. Descriptive statistics including mean and standard error of mean (S.E.M.) were calculated for each extracts fold induction of QR, and %inhibition. Group differences were determined by analysis of variance with Tukey–Kramer post hoc test (α < 0.05). 3. Results and discussion 3.1. Chlorophyll and carotenoid profile of RC extracts Extraction of dried RC leaves by hot water, ethanol, and acetone provided dried extracts with yields of 32.6 ± 0.2, 23.2 ± 0.1, and 36.6 ± 0.2 weight by weight, respectively. Sub-

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Fig. 1. HPLC separation of lutein and chlorophyll derivatives from RC acetone extract. Peak identifications: (1) pheophorbide a; (2) lutein; (3) chlorophyll b; (4) chlorophyll a; (5) pheophytin b; (6) pheophytin b ; (7) pheophytin a; (8) pheophytin a . Online electronic absorption spectra were collected from 250 to 600 nm. Responses at 400 and 450 nm are shown.

sequent HPLC analysis of acetone and ethanol extracts found seven chlorophyll derivatives and one major carotenoid, lutein (Fig. 1). Chlorophyll and carotenoid pigments were not detected in hot water extract of RC. Lutein and major chlorophyll derivatives including, chlorophyll a, chlorophyll b, pheophytin a and b were identified based on-line UV–vis spectra and co-chromatography with authentic standards. Due to the lack of an authentic standard for pheophorbide a this derivative was tentatively identified by comparison of online electronic absorption spectra and of previous separation of water soluble chlorophyll derivatives by this method (Canjura and Schwartz, 1991).

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Total chlorophyll content was determined to be 5.5 and 6.6 ␮g per mL for ethanol and acetone extracts, respectively (Table 1). Of the chlorophyll derivatives found in RC extracts, pheophytin a (1.5 and 1.6 ␮g/mL) and pheophorbide a (1.1 and 2.0 ␮g/mL) were most abundant. Chlorophyll a, chlorophyll b, and pheophytin a were determined to be 0.5, 1.3, 1.5 and 0.6, 1.4, 1.6 ␮g/mL for ethanol and acetone extract, respectively. Pheophytin a , an epimer of pheophytin a, was present at 1.1 and 1.0 ␮g/mL in ethanol and acetone extracts, respectively. Pheophytin b was not detected in acetone and ethanol extract. Lutein content of ethanol and acetone RC extracts was determined to be 0.02 ␮g/mL and 0.04 ␮g/mL for ethanol and acetone extracts, respectively. These results indicate that chlorophyll derivatives are the predominant lipophilic phytochemicals present in ethanol and acetone extracts of RC and are efficiently extracted by the conditions utilized in these preparations. Furthermore, the presence of significant amounts of chlorophyll degradation products (pheophytins and pheophorbides) is likely a result of the post-harvest handling of leaves prior to extract preparation. Any significant lag between harvest and process would allow for endogeneous chlorophyllase enzymes to begin the process of conversion to chlorophyllides and ultimately pheophorbides (Koca et al., 2006). Furthermore, hot air drying and milling of the RC leaves during raw material preparation, similar to the method generally used by local people, would further expose leaf material to conditions favorable to degradation of chlorophyll. The extreme sensitivity of chlorophyll to heat is known to generate metal free pheophytin derivative (Koca et al., 2006). Therefore, the final chlorophyll composition of dried RC leaves and resulting extracts will be indicative of natural and process induced chlorophyll degradation with minimal amounts of native chlorophyll remaining. Chlorophylls possess both antimutagenic and antigenotoxic activities against several dietary and environmental mutagens (Dashwood, 1997; Ferruzzi and Blakeslee, 2007) The presence of these compounds in leaf extracts of RC further support the

Table 1 Extraction yield and phytochemical content of RC preparations Extraction solvent Water

Ethanol

Acetone

%Yield (w/w × 100)

32.6 ± 0.2

23.2 ± 0.1

36.6 ± 0.2

Phytochemical content Lutein (␮g/mL)a Chlorophyll a (␮g/mL)a Chlorophyll b (␮g/mL)a Pheophytin a (␮g/mL)a Pheophytin a (␮g/mL)a Pheophorbide a (␮g/mL)a,c

ND ND ND ND ND ND

0.02 0.50 1.30 1.50 1.10 1.10

ND 24.34 ± 57.7

5.52 ± 0.1 5.65 ± 7.9

Total Chlorophylls (␮g/mL) Total Phenolics (␮g GAE/mL)b a

± ± ± ± ± ±

0.0 0.0 0.0 0.0 0.1 0.0

0.04 0.60 1.40 1.60 1.00 2.00

± ± ± ± ± ±

0.0 0.1 0.1 0.1 0.0 0.2

6.64 ± 2.7 1.42 ± 0.8

Determined by HPLC as described in Section 2. Determined by Folin-Cicalteau colorimetric assay as described in Section 2. c Due to a lack of an authentic pheophorbide a standard, levels were estimated based on the response factor for pheophytin a which maintains similar electronic absorption spectra to pheophorbide a (Fiedor et al., 2003). b

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traditional use of this extract as a protective agent against environmental toxicants. 3.2. Phenolic acid content of RC extracts All RC extracts contained appreciable levels of phenolic compounds as assayed by the Folin-Cicalteau method. Water extracts of RC maintained the highest phenolic contents at 24.34 ␮g GAE/mL followed by ethanol and acetone RC at 5.65 and 1.42 ␮g GAE/mL, respectively. Both flower and leaf materials of RC have been previously found to contain several bioactive phenolic constituents including delphinidin 3:5-di-O-␤-d-glucopyranoside, apigenin and apigenin-7-O-␤-d-glucopyranoside and chlorogenic acid (Purnima and Gupta, 1978; Kanchanapoom et al., 2002). A preliminary phenolic profiling of RC water extract detected the presence of apigenin and apigenin glucosides as well as phenolic acids such as caffeic acid, gallic acid and protocatechuic acid (data not shown). However, quantification of individual phenolic compounds was hampered by the complexity of the RC profile resulting in several co-eluting peaks. Resolution of the RC phenolic fraction will be the subject of future work by our group. While providing an appreciable level of phenolics, hot water extracts of RC leaf powder deliver significant less phenolic constituents than other common beverages such as coffee and tea. For a standard 250 mL serving RC hot water extract would provide ∼6.1 mg of total phenolics compared to ∼150 mg for tea (Lee et al., 2003) and ∼240 mg for coffee (Sanchez-Gonzalez et al., 2005). While not achieving the levels of these commonly consumed beverages, water extracts of RC could serve as an additional dietary source of phenolic antioxidants. 3.3. Antimutagenic activity of RC extract RC extracts were screened for their ability to inhibit the mutagenicity of 2-AA in a Salmonella typhimurium TA 98 microscreen assay. Extracts were dissolved in sterile DMSO at concentrations between 0.01 and 0.40 mg GAE/mL. RC extracts were either mixed with 2-AA and S-9 fraction (pre-incubated), or added just prior to combination (non pre-incubated) with top agar and plated. Plates were incubated for 48 h at 37 ◦ C after which his+ revertant colonies were counted. Assays were conducted with 0.625 ␮g per well of 2-AA providing 285 ± 4.3 his+ revertants per well corrected for spontaneous background revertants. All RC extracts demonstrated pronounced dose-dependent inhibition of the mutagenic activity of 2-AA (Fig. 2). Acetone extracts of RC were found to inhibit a maximum of 81.1 ± 12.2% of 2-AA induced mutagenicity at its highest experimental concentration of 0.17 mg GAE/mL (0.004 mg GAE per well) while ethanol and water extracts were found to inhibit 86.6 ± 9.8% and 71.2 ± 5.3% at concentrations of 0.15 and 0.2 mg GAE/mL, respectively (0.003 and 0.005 mg GAE per well). Interestingly, RC water extracts demonstrated evidence of cytotoxic activity. This was observed as thinning of background lawn accompanied by a decrease in the total number of colonies but increasing in relative size of revertant colonies (data not shown). It is there-

Fig. 2. Inhibition of 2-AA mutagenicity by RC extracts in Salmonella typhimurium TA 98 assay, ethanol extract (panel A); acetone extract (panel B); water extract (panel C). Data represents percent inhibition (n = 5) of mutagenicity induced by 0.625 ␮g of 2-AA per well resulting in 285 ± 4.3 his+ revertants per well corrected for spontaneous background revertants. Pre-incubation experiments (䊉) were conducted with RC extract addition with 2-AA and S-9 microsomal preparation in the preincubation step. Experiments with RC addition post incubation of 2-AA with S-9 microsomal preparation are depicted with ().

fore possible that a portion of the antimutagenic activity of water extracts could in fact be due to cytotoxic behavior. Acetone and ethanol RC extracts did not exhibit evidence of cytotoxic activity in the Salmonella assay. Several phytochemical constituents in RC identified in this study are known to be antimutagenic agents. Phenolic constituents, including caffeic acid, have been reported to have antimutagenic activity (Moridani et al., 2002). Both lipophilic and water-soluble chlorophyll derivatives abundant in ethanol and acetone RC extracts have previously demonstrated pow-

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erful antimutagenic activity in bacterial mutagenicity assays (Yoshikawa et al., 1996; Dashwood, 1997; Matile et al., 1999; Ferruzzi et al., 2002). In this study, RC ethanol and acetone extracts delivered a maximum of 0.3 and 0.2 nmol of total chlorophyll per well at the highest doses assessed. Reduction of 2-AA induced reversion by 86.6 ± 9.8% and 81.1 ± 12.2% for ethanol and acetone, respectively by these extracts is likely due, in part, to the several chlorophyll derivatives characterized in these extracts.

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2-AA is an indirect mutagen requiring metabolic activation by Phase 1 cytochrome P450 enzymes present in the S-9 fraction. Exclusion of RC extracts during the preincubation step resulted in a loss of the antimutagenic activity against 2-AA (Fig. 2). Only hot water extract showed partial decrease in revertant colonies. However, this was again due in large part to the partial killing of Salmonella typhimurium TA 98 rather than by true antimutagenic activity. Differences in antimutagenic activity of RC extracts incubated together with 2-AA and S-9 during metabolic activation and RC extracts added after cells are incubated with 2-AA and S-9 raise the possibility that RC extracts may inhibit transformation of 2-AA and/or specific components present in RC extracts require metabolic activation by Phase 1 enzymes in order to provide protective effects. 3.4. Induction of QR activity The ability of RC extracts to modulate QR activity was investigated in cell culture. Dried extracts were solubilized in ethanol, diluted in basal media and applied to 80–90% confluent cultures of Hepa 1c1c7 cells. QR activity was assessed after 48 h of treatment with RC extracts. Results are expressed as concentration of RC extracts required to increase QR activity, compared to vehicle controls. QR activity was increased upon treatment of Hepa 1c1c7 cells with RC extracts (Fig. 3). Acetone RC extracts showed the highest induction potency followed by ethanol and water extracts, respectively. Acetone extract at its highest concentration (140 ␮g GAE/mL) increased QR activity 2.8-fold compared with controls, while ethanol (120 ␮g GAE/mL) and water (1000 ␮g GAE/mL) extracts increased QR activity by 1.4- and 1.6-fold, respectively. As with antimutagenicity assays, water extracts of RC were cytotoxic above 1000 ␮g GAE/mL. The higher activity of acetone and ethanol extracts may be due, in part, to the chlorophyll derivatives present in this RC extract. Recently, Fahey et al. (2005) demonstrated that chlorophyll derivatives are potent inducers of QR in murine hepatoma cells. The chlorophyll content of acetone and ethanol extracts would thus explain, in part, the observed activity of these extracts. Although water extract showed minimal QR inducing activity, cytotoxicity of this extract was observed (data not shown). The observed cytotoxicity of RC water extracts at higher dosages is interesting in light of the traditional consumption of RC as hot water infusions. The specific mechanisms and components of RC water extract responsible for this cytotoxicity are not known. 4. Conclusions

Fig. 3. Induction of QR specific activity in Hepa 1c1c7 cells treated with RC water extract (panel A); ethanol extract (panel B); acetone extract (panel C). Values represent mean ± S.E.M. for eight samples. Different letter represent significant differences in induction of QR.

RC extracts produced from common solvents (hot water, acetone and ethanol) were analyzed for major phytochemicals. Acetone and ethanol extracts of RC were found to be good sources of chlorophyll derivatives including native chlorophylls and pheophytins. Additionally, a water-soluble derivative was tentatively identified as pheophorbide a in RC chlorophyll fraction. Further analytical characterization will be required to confirm the identity and accurately quantify pheophorbide a lev-

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els in RC extracts. RC water extract has highest content of total phenolic acids. Chlorophyll rich acetone and ethanol extracts demonstrated significant antimutagenic activity by inhibiting the 2-AA induced mutagenicity in a Salmonella typhimurium model. Furthermore, RC extracts demonstrated the ability to induce the phase II detoxification enzyme QR in cultured Hepa 1c1c7 cells. These results combined with those of Pramyothin et al. (2005) reporting hepatoprotective activity of RC aqueous extract and Fahey et al. (2005) demonstrating the ability of chlorophyll derivatives to induce QR, support the traditional medicinal use of RC for detoxification and suggest the potential role of both phenolic acids and natural chlorophyll constituents in modulating these effects. Acknowledgment This research work is supported by personnel development project, Commission on Higher Education, Ministry of Education, Thailand under a consortium program with Food Science Department, Purdue University, USA. References Bertram, J.S., Vine, A.L., 2005. Cancer prevention by retinoids and carotenoids: independent action on a common target. Biochemica et Biophysica Acta 1740, 170–178. Bomser, J., Madahvi, D.L., Singletary, K., Smith, M.A.L., 1996. In vitro anticancer activity of fruit extracts from Vaccinium species. Planta Medica 62, 212–216. Canjura, F.L., Schwartz, S.J., 1991. Separation of chlorophyll compounds and their polar derivatives by high performance liquid chromatography. Journal of Agricultural and Food Chemistry 39, 1102–1105. Chanawirat, A., Toshulkao, C., Temcharoen, P., Glinsukon, T., 2000. Protective effect of Thunbergia laurifolia extract on ethanol-induced hepatotaxicity in mice. Thesis, Faculty of Graduate Studies, Mahidol University, Bangkok, Thailand. Charumanee, S., Vejabhikul, S., Taesotikul, T., Netsingha, W., Sirisaad, P., Leelapornpisit, P., 1998. Development of Topical Anti-inflammatory Preparations from Thunbergia laurifolia Linn. Phase 1 Research Report, Faculty of Pharmacy. Chiangmai University, Chiangmai, Thailand. Dashwood, R.H., 1997. Chlorophylls as anticarcinogens (review). International Journal of Oncology 10, 721–727. Diehl, M.S., Wilaby, S.L., Snyder, R.D., 2000. Comparison of the results of a modified microscreen and standard bacterial reverse mutagenicity assays. Environmental and molecular mutagenesis 35, 72–77. Faculty of Pharmacy, 1987. Sirirukachat Queen Sirikit. Mahidol University, Thailand. Fahey, J.W., Stephenson, K.K., Dinkova-Kostova, A.T., Egner, P.A., Kensler, T., Talalay, P., 2005. Chlorophyll, chlorophyllin and related tetrapyroles are significant inducers of mammalian phase 2 cytoprotective genes. Carcinogenesis 26, 1247–1255. Ferruzzi, M.G., Blakeslee, J., 2007. Chlorophyll bioavailability and physiological relevance as dietary phytochemicals. A Review. Nutrition Research 27, 1–12.

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