Adult heavy smokers were recruited for enrollment in this study. The inclusion criteria were 18–65 years in age, smoker of more than 20 cigarettes per day, a smoking history exceeding 1 year, and no concurrent use, or use in the previous 3 months, of prescription medication or antioxidant vitamins. Those enrolled had to be willing to complete a 1-month trial. Those interested in participating were interviewed by an experienced clinical coordinator and selected for participation if they met eligibility criteria. The clinical coordinator interviewed all study participants and asked them to complete a demographic and health information questionnaire. Study participants were randomly assigned to a 118-mL placebo, 29.5-mL noni, or 118-mL noni dose groups. Both males and females were enrolled in equal proportions.
Ten milliliters of whole blood were drawn from each participant upon enrollment and again at completion of the intervention period. Blood samples were drawn into tubes containing heparin, which were then centrifuged at 1500g for 20 min to remove plasma. The remaining fractions were transferred to 50-mL tubes. Fifteen milliliters of red blood cell lysis buffer (150 mmol/L NH4Cl, 10 mmol/L sodium hydrogen carbonate, 1 mmol/L ethylenediamine tetraacetic acid, pH 7) was added, and the mixture was incubated at room temperature for 5 min. Samples were then centrifuged at 300g for 10 min at 4°C. The PBL pellet was washed with 5 mL of red blood cell lysis buffer, incubated at room temperature, and centrifuged at 300g for 10 min at 4°C. This procedure was repeated three times. The final PBL pellet was resuspended with 0.5 mL of buffer (150 mmol/L sodium chloride, 10 mmol/L EDTA, pH 8.0), and cells were lysed by vortex. DNA was then isolated with the FastDNA® kit (MP Biomedicals, Solon, OH). DNA samples were stored at −80°C until 32P-postlabeling.
DNA adducts were measured by the nuclease P1 procedure of the 32P-postlabeling assay (Reddy and Randerath 1986). Five to ten micrograms of DNA were digested with micrococcal endonuclease and spleen phosphodiesterase to produce 3′ mononucleotides. Enrichment of adducts involved dephosphorylation of unmodified normal nucleotides with nuclease P1 to prevent radiolabeling. Adducted nucleotides were then labeled with [γ-32P]ATP by T4 polynucleotide kinase (Wang and Liehr 1995a). Labeled nucleotides were purified by spotting nucleotide solutions onto polyethyleneimine-(PEI-)cellulose thin layer chromatography (TLC) plates, followed by removal of normal nucleotides by plate development with a sodium phosphate mobile phase (2.3 mol/L, pH 5.75) and Whatman 1 paper wick (Whatman Ltd., Maidstone, U.K.). Afterward, the chromatogram was cut into 1.0 × 2.4 cm strips beginning at 2.4 cm above the origin point. The central and furthest (upper) strips, which contained LOOH adducts (such as 4-hydroxy-2-nonenal) and MDA adducts, respectively, were retained. Adducts were transferred from each strip to fresh PEI-cellulose TLC sheets by a magnet transfer technique (Lu et al. 1986). Two-dimensional chromatography was completed with different mobile phases. For the first direction, a 3.6 mol/L lithium formate and 8.5 mol/L urea solution (pH 3.6) served as the mobile phase. The plate was then developed in a second direction, at a right angle to the first, with a mobile phase composed of 0.8 mol/L lithium chloride, 0.5 mol/L Tris–HCl, and 8.5 mol/L urea (pH 8.0). Adducts were detected by autoradiography at −80°C using Kodak X-OMAT (Eastman Kodak Co., Rochester, NY) film and DuPont Lightning Plus intensifying screens (DuPont, Wilmington, DE) (Wang and Liehr 1995a). Adduct spots were removed from chromatograms and transferred to scintillation vials. Counts per minute from each sample were measured in a scintillation counter. Adduct levels were expressed as relative adduct labeling (RAL), which is the ratio of counts per minute of adducted nucleotides and counts per minute of total nucleotide samples. A value of 1 RAL × 109 corresponds to 1 adduct in 109 bases (Wang and Lu 1990).
The iridoid content, inclusive of deacetylasperulosidic acid (DAA) and asperulosidic acid (AA), was determined by HPLC, according to a previously reported method (Deng et al. 2011). Other significant secondary metabolites, such as scopoletin, rutin, and quercetin, were also determined by HPLC.
HPLC grade acetonitrile (MeCN), methanol (MeOH), and water were obtained from Sigma-Aldrich (St. Louis, MO). Analytical grade formic acid was purchased from Spectrum Chemical Mfg. Corp. (New Brunswick, NJ). DAA and AA standards were isolated from authentic noni fruit in our laboratory. Their identification and purities were determined by HPLC, mass spectrometry, and nuclear magnetic resonance to be higher than 99%. They were accurately weighed and then dissolved in an appropriate volume of MeOH to produce corresponding stock solutions. The working standard solutions of DAA and AA for the calibration curve were prepared by diluting stock solutions with MeOH in seven concentration increments ranging from 0.00174–1.74 and 0.0016–0.80 mg/mL, respectively. All stock and working solutions were maintained at 0°C. The calibration curves of the standards were plotted after linear regression of the peak areas versus concentrations.
For iridoid analyses, samples of noni juice and placebo were diluted with MeOH-H2O (1:1) and then filtered through a 0.45-μm nylon membrane filter. Chromatographic separation was performed on a Waters 2690 separations module coupled with 996 photodiode array (PDA) detectors, equipped with a C18 column (4.6 × 250 mm, 5 μm; Waters Corporation, Milford, MA). The pump was connected to two mobile phases: (A) MeCN and (B) 0.1% formic acid in H2O (v/v), and eluted at a flow rate of 0.8 mL/min. The mobile phase was programmed consecutively in linear gradients as follows: 0–5 min, 0% A; and 40 min, 30% A. The PDA detector was monitored in the range of 210–400 nm. The injection volume was 10 μL for each of the sample solutions. The column temperature was maintained at 25°C. Data collection and integration were performed using Waters Millennium software revision 32 (Waters Corp., Milford, MA).
Analyses of scopoletin, rutin, quercetin, and chlorogenic acid were also performed by HPLC, according to a previously reported method (Deng et al. 2010). Chemical standards were accurately weighed and then dissolved in an appropriate volume of MeOH/MeCN to produce corresponding stock and working standard solutions. Chromatographic separation was performed on a Waters 2690 separations module coupled with a 996 PDA detector, and equipped with a C18 column. The mobile phase system was composed of three solvents: (A) MeCN; (B) MeOH; and (C) 0.1% TFA in H2O (v/v). The mobile phase was programmed consecutively in linear gradients as follows: 0 min, 10% A, 10% B, and 80% C; 15 min, 20% A, 20% B, and 60% C; 26 min, 40% A, 40% B, and 20% C; 28–39 min, 50% A, 50% B, and 0% C; and 40–45 min, 10% A, 10% B, and 80% C. The elution was run at a flow rate of 1.0 mL/min. The UV spectra were quantified at 365 nm.
Total polyphenols were determined by the Folin–Ciocalteu method. Samples were centrifuged and diluted 1:10 with deionized water. The diluted samples (10 μL) were mixed with 800 μL deionized water and 50 μL Folin–Ciocalteu (2N). Following incubation at room temperature for a few minutes, 150 μL Na2CO3 (saturated) was added, and sample tubes were shaken and allowed to incubate at room temperature for 2 h. Vehicle blanks and gallic acid standards were prepared in the same manner. Following incubation, the absorbance of the blanks, standards, and samples were measured at 765 nm in a microplate reader. Absorbance versus gallic acid concentration was used to create a calibration curve. This curve was used to determine the total phenol content of the samples. As noni fruit is a source of vitamin C (West et al. 2011), concentrations of this vitamin in both the placebo and noni juice product were also measured after pasteurization and bottling.