The overall antioxidant potential of the greens+ expressed as trolox equivalent was estimated using the ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) method [16]. Results are expressed in terms of nmol/L trolox equivalent.

Multilamellar liposome membranes were prepared using 100 mg egg phosphatidyl choline in 20 mL of 2 mM phosphate buffer using the method as described by Balachandran and Rao [17]. They were used as a model system to simulate biological cell membranes to measure antioxidant properties of greens+. In 1 mL of Liposomal solution, lipid peroxidation was initiated by adding 100 μL of 1 mM FeSO₄ in the presence of 100 μL of greens+ solution at various concentrations (0.5%, 1%, 1.5%, 2% and 2.5%). The final concentrations of liposome and FeSO₄ in the incubation mixture were 5 mg/mL and 0.09 mM respectively. Lipid oxidation was measured by the thiobarbituric acid (TBA)-malondialdehyde (MDA) assay [18].

One gram of greens+ powder was extracted with 20 mL 50% acetone and hydrolyzed with 5 mL of 6 M HCl for 1 h. The hydrolysate was cooled and centrifuged at 3,000 rpm for 15 min at room temperature. The supernatant was then neutralized with 10 N NaOH to pH 6.6 and diluted with distilled water. The diluted extract was passed through Sep-Pak C18 (Waters Corporation, Milford, MA, USA) cartridge column for solid phase extraction. The column was then eluted with 100% acetone.

The analysis was carried out according to the method of Hollman et al. [19]. Chromatographic separations were carried out on a μ-Bondapak C18 (Waters Corporation, Milford, MA, USA) column (3.9 × 150 mm, 10 μm) at 35 °C. A UV/Visible detector (Shimadzu, Kyoto, Japan) was used to detect polyphenols. The mobile phase consisted of solution A: 0.1 M NH₄H₂PO₄, pH 4.4 and solution B: 0.1 M NH₄H₂PO₄, pH 4.4 (20%), acetonitrile (60%), methanol (10%) and water (10%) with gradient 0–15% B, 0–5 min; 60% B, 5–30 min; 100% B, 30–50 min. The flow rate of the mobile phase was 1 mL/min. Ten microlitre was injected onto the column. The external standards of flavonoids such as quercetin, kaempferol, myricitin, luteolin and apigenin (Sigma Chemical Co., St. Louis, MO, USA) were used as reference standards.

For the analysis of polyphenols in plasma, the method as described previously for greens+ herbal preparation was used.

Greens+ is a commercially available herbal supplement containing several botanical products that are considered as good sources of the polyphenols having antioxidant properties. The herbal preparation itself was never tested for its antioxidant properties. Results obtained in this study are shown in Figure 2. A dose-dependent linear antioxidant effect was observed with up to 1 mg of greens+ extract.

Based on this observation, the antioxidant potential of greens+ was evaluated in a liposomal lipid peroxidation model. Liposomes are commonly used to simulate cell membranes. As shown in Figure 3, a significant (p < 0.05) reduction in the production of malondialdehyde (MDA), which is a measure of lipid peroxidation, was observed at higher concentrations (2% and 2.5%) of greens+ compared to lower concentrations (0.5%, 1% and 1.5%). The reduction of lipid peroxidation in the liposomes, in the presence of higher concentrations of greens+, suggest the ability of the polyphenols present in the herbal preparation to protect membrane lipids form oxidation.

HPLC procedure was used to detect the presence of polyphenols present in greens+ and to quantify them. Polyphenol composition of the herbal preparation is shown in Table 2. Quercetin, apigenin, kaempferol and luteolin were the four major polyphenols detected. This study was the first to show the presence of these polyphenols in greens+.

No changes were observed in the body weights, BMI and blood pressure of the subjects after oral supplementation of greens+ at two different doses for four weeks. These observations suggest no adverse effects of ingesting greens+ even up to six teaspoons per day for four weeks.

Results are shown in Figure 4. Although, the consumption of three teaspoons of greens+ per day for four weeks increased the total antioxidant capacity, it was not statistically different from the baseline. However, maximum antioxidant capacity was observed after six teaspoons of ingestion per day for four weeks. This increase from the baseline was statistically significant (p < 0.05).

Figure 5 shows the results of ingesting greens+ on lipid oxidation. As can be seen, a statistically significant reduction in serum MDA was observed after four weeks of treatment at the consumption level of six teaspoons. Although consumption of three teaspoons of greens+ for four weeks showed a minor increase in serum lipid MDA, this change was not statistically different from the baseline. This may have been due to the small number of subjects and a relatively shorter period of intake.

Results are shown in Figure 6. Similar to the effects on lipid oxidation, consuming Greens+ at the level of six teaspoons per day for four weeks significantly increased the levels of protein thiols indicating a significant reduction in protein oxidation. Although the levels of protein thiols for three-teaspoon consumption showed an increase over the baseline, it was not statistically significant.

Results are shown in Figure 7. A statistically significant increase in the activity of erythrocyte glutathione peroxidase was observed after consuming the herbal preparation for four weeks at both three and six teaspoons per day.

A study was undertaken to investigate the in vitro and in vivo antioxidant properties of the herbal preparation greens+. The liposome model was used in the in vitro studies to simulate a biological cell membrane. Greens+ showed a dose-dependent trolox equivalent antioxidant capacity under the in vitro conditions. A similar effect on the total antioxidant potential was also seen in the serum of human subjects consuming greens+ for a period of four weeks. HPLC analysis of greens+ showed the presence of four major polyphenols: quercetin, apigenin, kaempferol and luteolin. Since the powdered greens+ preparation contains several plant-derived products that are evidently significant sources of antioxidant polyphenols, the increase in the serum antioxidant capacity, following greens+ consumption, could be related to the absorption of the polyphenols. Kaempferol was found to be the only major polyphenol present in the human plasma at higher concentration (six teaspoons) after four weeks of consumption. Some of the other polyphenols found in greens+ were undetected in the plasma of subjects. This may be due to the accumulation of flavonoids mainly as glucoronide and sulfate conjugates in blood plasma [24]. Nevertheless, the conjugated metabolites of flavonoids may play a role in the antioxidant defense of blood plasma. Terao [24] found that oral administration of epicatechin and quercetin resulted in the accumulation of the glucoronide and sulphate conjugates in rat blood plasma.

The liposome model system used in this study also showed a significant antioxidant capacity at higher concentrations of ingestion. Cao et al. [25] demonstrated that the consumption of strawberries, spinach, red wine or vitamin C increased the antioxidant capacity of serum in elderly women. Other studies have shown that many flavonoids present in plants have the ability to scavenge peroxyl, alkyl-peroxy radicals, superoxide hydroxyl radicals and peroxynitrile in aqueous and organic environment.

The consumption of greens+ by healthy human subjects showed a significant increase in the levels of reduced thiols indicating a decrease in the level of oxidized proteins in the serum. The damage caused to enzymes via protein oxidation is significant in discussions regarding human health. The activity of metabolic enzymes that otherwise play a crucial role in the detoxification of carcinogens and repair the damaged DNA may be inhibited, the net result being a contribution to metabolic disorders and increased risk of chronic disease [26–29]. Also, oxidation of proteins present in cell membrane will alter both the structure and functional characteristics of the cells.

Serum MDA, indication of lipid peroxidation, was reduced significantly with the consumption of greens+. This effect is likely related to the presence of polyphenols. Plant phenolic compounds trap chain-initiating radicals at the interface of the membrane, thus preventing the progression of the radical chain reaction. Additionally, the phytochemicals such as the polyphenols can chelate the transition metal ions responsible for the generation of reactive oxygen species and therefore inhibit the initiation of the lipoxygenase reaction [30].

Greens+ administration did not change LDL oxidation as measured by LDL malondialdehyde. This could be ascribed to the presence of more water-soluble polyphenols that might have been absorbed by the protein-rich HDL fraction. Polyphenols have been shown to have high affinity for proteins [31]. Other studies have also shown similar lack of protection against LDL oxidation by the consumption of grape extract [32]and tea [33–35].

Results obtained in the present study also showed an increase in the erythrocyte glutathione peroxidase activity during greens+ supplementation.