Assessment of Anti-oxidant and Anti-inflammatory Properties of Green Tea in Critically Ill Patients with Pneumonia

Background: Systemic inflammatory response syndrome is common in critically ill patients and negatively affects clinical outcomes. Preventing oxidative damage, may suppress this systemic response. Previous studies confirmed that polyphenols in green tea have both antioxidant and immuno-stimulant effect, so there could be a probability of green tea clinical application. Two groups of 8 patients enrolled in this randomized, controlled clinical trial. Treatment group received Green Tea Extract (GTE), and placebo group received extract's solvent for 7 days. Blood samples taken 3 times/week and oxidative, physiologic and inflammatory markers were measured. Results: Differences between the baselines for each variable were not significant. FRAP value showed slower decrease in GTE group. Thiol level increased in GTE group and decreased in Placebo group. IL-6 and APACHE II score were lower in GTE group on day7. Leucocyte count showed overall increase in GTE group and decrease in placebo group. Differences between two groups in all aforementioned variables were statistically insignificant. Conclusion: As a conclusion green tea had no significant effect on all measured variables; but in GTE group despite better oxidative status in the first 3 days, more inflammation was observed on day7. Considering small sample size further investigations are needed.

syndrome (ARDS) are the main critical care syndromes leading to oxidative injury (1).
SIRS is a common problem and according to reports SIRS affects 33% of all hospitalized patients, more than the half of all ICU patients and more than 80% of surgical ICU patients (3). These patients develop redox imbalance because of low intake of anti-oxidant vitamins (less than 66% of recommended daily allowance), alterations in endogenous levels of substances with anti-oxidant capacity and some kinds of nursing procedures (e.g. suctioning, turning, enteral or parenteral nutrition, etc.) (4)(5)(6).
There is no suitable drug to cover different pathological pathways of oxidative imbalance in SIRS, so combination therapy is preferred to monotherapy and in theory the best regimen could be a combination of anti-oxidants and immunostimulant (7).
Tea catechins act better than popular anti-oxidants such as vitamin C, vitamin E and carotene in coronary artery disease. Tea polyphenols show anti-oxidant activity not only by scavenging superoxides but also act as immunostimulant by improving activity of detoxifying and anti-oxidant enzymes such (e.g. Glutathione peroxidase, Glutathione reductdase, Glutathione-S-transferase, Catalase and Quinine reductdase) (10). Because of unique anti-oxidant properties of green tea and the possibility of its effectiveness in SIRS, we conducted this study to examine green tea's effects on inflammatory and oxidative indices in critically ill patients.  The green tea leaves were grinded by MESH 16, extracted by hydro alcoholic solvent (ethanol 40%) for half an hour while shaking frequently and then dried, based on Rusak method (12).

Poly phenolic standardization
After every time of extraction, extract standardization based on poly phenolic content performed by Total phenolic assay with folin-ciocalteu reagent (Merck − 1.09001.0100) and based on Singleton study (13). The standardized dry extracts kept in glass bottles at 4 °C until administration. We randomly selected 16 patients according to inclusion and exclusion criteria. Block randomisation process performed by randomisation webpage. Hospitalization less than 7 days in general ICU, having SIRS, APACHE II score more than 10, pneumonia diagnosis, consent, age more than 18 and rule out of pregnancy considered as inclusion criteria and vice versa as exclusion criteria. After a complete explanation about the process, written consent was taken from patients or their first-degree relatives.

Patients and methods
Recommended daily amount of polyphenol intake is 300 to 400 mg (14). So in this study, we used 390 mg daily in three divided doses in GTE group.
Eight randomly selected patients were received standardized extract containing 130 milligrams of poly phenol dissolved in 10 milliliters of ethanol 10% (GTE group), three times a day at 8 AM, 2 PM and 8 PM for one week and the rest received 10 milliliters of ethanol 10% with the same regimen (placebo group). Physician and nurses were blinded from solution contents by using amber colored bottles. Blood samples withdrawn on day 0 before administration, day 3 and day 7 after five hours from the last administration. The Blood samples are collected in clot activating tubes, serum separated by centrifuge (4000 rpm, 10 minutes), total anti-oxidant capacity and total thiol of serum measured within 4.5 hours and the rest stored at -80 °C for IL-6 plasma level evaluation. Mean arterial pressure (MAP), respiratory rate (RR), leucocyte count, pulse rate (PR), APACHE II score were also recorded during the study.

Assessment of total antioxidant capacity
Ferric Reducing Ability of Plasma (FRAP) was considered as our major antioxidant assessing method because of lower cost and ease of use. In this method Fe 3+ make a complex with reagent TPTZ (2,4,6-tripyridyl-s-triazine), when this complex become expose to antioxidants in serum, Fe 3+ will reduce to Fe 2+ and lead to blue color formation with maximum spectrophotometric absorption at 593 nm (15). The only limitation of this method is inability to measure SH-containing antioxidants, so there is need to use another test for detecting this group of antioxidants (16). We performed the tests as explained by Benzie et al. (17) study, while adjusting the process based on our equipment and limitations.
In order to make reactant solution, 25 mL of acetate buffer 300 mmol/L (pH 3.6), 2.5 mL of TPTZ containing 300µL fresh warmed reactant, 10µL of Calibration solution and 30µL distilled was read at 593 nm wavelength after 4 minutes of mixing for 3 times with 15 seconds intervals. Average of three absorptions was recorded and calibration curve was established. FRAP assessment Process of serum was the same as calibration but calibration solution substitute with serum samples.

Assessment of Serum Total Thiol
SH-groups are protein dependent antioxidants and using Ellman reagent is the most common method for their assessment. Elman reagent is 2,2′-Dinitro-5,5′-dithiodibenzoic acid (DTNB) which can binds to sulfhydryl groups and forms yellow color with maximum absorption at 412 nm wavelength (18)(19)(20).
This test adjunct to FRAP test would complete serum antioxidant capability coverage. Total thiol groups were calculated based on formula reported by Hu (18) : For serum analysis, Process was the same as calibration but calibration solution substitute with serum samples.

IL-6 plasma level evaluation
Samples kept in -80 ˚c refrigerator until end of the study, then IL-6 assessed by Human IL-6 Platinum ELISA (e Bioscience, Affymetrix LOT# 106832061) based on manufacture instructions.

Statistical analysis
The results were analyzed by statistical program SPSS version 24. P-value of less than 0.05 was considered statistically significant.

Demographic and baseline characteristics of patients
Demographic and baseline characteristics of the patients are presented in Table1.  (Fig. 1)

Serum total thiols analysis
Glutathione baseline was 0.055 ± 0.017 in GTE group and 0.053 ± 0.024 mg/L in placebo group (p = 0.623). On day 3 increase in serum glutathione levels in both groups (GTE 0.063 ± 0.028, placebo 0.075 ± 0.056 mg/L) was observed and on day 7 the level in GTE group was higher than placebo (GTE 0.073 ± 0.031, placebo 0.049 ± 0.008 mg/L), but no significant difference was seen (p = 0.20). (Fig. 2)

Mean Arterial Pressure and pulse rate
Map and PR in both group decreased during the study and no significant differences were seen (Pvalue > 0.05). (Fig. 5-Fig. 6)

Discussion
Oxygen is a necessary agent for cell respiratory and energy production. As a result of reduction and oxidation reactions, active radical species will produced, which have some benefits such as foreign showed beta carotene-rich beverage such as tomato juice (500 ml/day) is as powerful anti-oxidant as high dose vitamin E (800U/day) in promotion of LDL resistance to oxidation (14). So there is a potential of clinical application of natural anti-oxidants.
The best combination for anti-oxidant therapy would be a radical scavenger with immunostimulant properties (7,10), so in this study we used green tea which is immunostimulant and powerful antioxidant at the same time. Health effects of anti-oxidant consumption for a short time remains even for months while single dose consumption only have effect for a few hours (24), thus it's better to use them in long term to see their effects. In this study we administered polyphenolic supplement for 7days.
Many studies evaluated maximum tolerable dose (MTD) of green tea (25), but expression of MTD base on weight of green tea leaves, milligram of extract achieved by different solvents or milligram of polyphenols, makes it hard to compare the results. We consider regular recommended dose of green tea which there was no report of complications by this dose (14).
Comparing two groups of patients after treatment, shows no statistically significant difference for ferric reducing ability of serum as an anti-oxidant capacity marker. Moreover, measured Glutathione levels in GTE and placebo groups showed non-significant elevation in GTE group's serum level.
Several in-vitro studies showed green tea polyphenols chemical structures have higher anti-oxidant activity compare to ascorbate and α-Tocopherol. Pharmacokinetic investigations on green tea polyphenols have shown that tea catechins rapidly and extensively metabolized by glucoronidation, sulfation and methylation process, thus these catechins have low bioavailability. Even high doses of green tea lead to low serum concentrations and low anti-oxidant activity in contrast to in-vitro studies (26).
In this study serum IL-6 level and APACHE II score were lower in GTE group but it did not reach statistical difference. According to studies hypoxia-induced reactive oxygen species are signaling agents for pro-inflammatory cytokines production and endothelial permeability alteration (27). Antioxidants are different in strength of ROS inhibition. They can decrease transcription of proinflammatory cytokines, so antioxidant therapy may prevent inflammatory-induced disease and septic shock (28). IL-6 and APACHE II score have a valid role in mortality prediction in sever sepsis patients (29,30). One study assessed the effect of anti-oxidant therapy in surgical ICU patients by measuring α-Tocopherol and ascorbate serum levels and their relation with pro-inflammatory cytokines such as TNFα, IL-1, IL-6, and IL-8. Reductions in cytokines levels, organ failure incidence and length of ICU stay were observed in the anti-oxidant group at the end of therapy. These data demonstrate the benefits of early anti-oxidant therapy in these patients (21).
Both MAP and pulse rate decreased during the study in two groups, but they did not reach statistical significance. A clinical trial which enrolled 1507 individuals reported that regular tea consumption for at least 1 year, decrease the risk of developing hypertension in a dose dependent manner and intake of tea drink more than 600 ml/day reduce this risk by 65% (31), however in our study green tea administration in critically ill patients didn't have negative effect on patients tissue perfusion.
Slower increase in WBC counts and better response to antibiotic therapy was seen in GTE group on the first three days but unexpectedly, leukocyte count increased in GTE group and decreased in placebo group by continuing the therapy. Application of tea as an anti-microbial for topical treatment of surface infections is preferable, because systemic intake of tea can't reach Minimum Inhibitory Concentration (MIC) as a consequence of low bioavailability (8, 26). Green tea leaves have immunostimulating effect depend on catechin content and this effect can be potentiated by formation of a complex of catechins and tea polysaccharides. High concentration of both catechins and polysaccharides can be detected in buds and first leaves (32). In this study we used first leaves thus high content of polysaccharides was expected. A pharmacological study reported an increase in white blood cells counts in dogs which received green tea, increase in neutrophil counts more than 1.7 fold, monocyte counts more than 2.7 fold and eosinophil counts more than 3 fold over vehicle control were seen (33). But in another experimental study on rats leukocytes counts reduced (34). Because of different results in each species, animal data cannot be extrapolated to human and no clinical trial specifically evaluated relation between green tea intake and leukocyte counts yet.

Conclusion
In this study regarding slower reduction in FRAP, increase in serum glutathione, lower serum IL-6, better anti-oxidant defense and lower inflammation trend in first three days were observed but worse oxidative stress and higher inflammation in later days of therapy in GTE group compare to placebo, which could possibly be explained that immuno-stimulant effects may overcome antioxidant effects by continuing intake of polyphenols after 3 days; Although this results didn't reach to the level of significance. Small sample size was the main limitation of this study, so further investigations with greater sample size is recommended and further evaluations to find green tea's MTD and effective dose and duration is needed.

-Competing interests:
We have no conflicts of interest to disclose.
-Funding: This study is performed by authors personal funding.  Comparison the effect of therapy between two groups shows statistically non-significance difference (P-value>0.05).

Figure 2
Level of serum thiol at the end of treatment duration in GTE group was higher than placebo group, but this difference was not significant (P-value>0.05).

Figure 4
Comparison of APACHE II score between groups. After 7 days treatment, insignificant lower APACHE II score was observed in GTE group (P-value>0.05).

Figure 5
MAP comparison between two groups. No significant difference was seen (P-value>0.05) Figure 6 Effect of treatment on Pulse Rate have shown non-significant difference between two groups (P-value>0.05).

Figure 7
Leukocyte count measured 3 times. After 7 days treatment, GTE group showed higher level than placebo group (P-value>0.05)