The trial was conducted according to the Declaration of Helsinki of 1975 and ICH-Good Clinical Practice (GCP) guidelines, revised in 2013; approved by the University Institutional Review Board for Human Subjects Research (protocol #PRO-FY2021-416) at the University of Memphis and registered on clinicaltrials.gov (NCT05162820). The trial was conducted as a randomized, placebo-controlled double-blind clinical trial, between October 2021 and June 2023 at a single site in Memphis, TN, USA. For this exploratory pilot study, no power analysis was performed but a convenience sample of 14 men and 36 women (completers), based on prior unpublished pilot work, were recruited and completed all phases of this study, including 16 active smokers and 34 non-smokers. See Table 1 for subject descriptive characteristics.

Table 1. Baseline subject characteristics by condition [mean (SD) or n (%)].

Subjects were required to be 30 - 60 years old, with a body mass index (BMI) between 18 - 39.9 kg/m² (not morbidly obese). Smokers were initially required to smoke a minimum of 5 cigarettes daily and experience occasional mild skin irritations such as rash, dryness, redness, or itching. Due to challenges with subject recruitment for smokers, the criteria were broadened to include individuals who smoke cigarettes or vape regularly, for the past 12 months, and experience skin irritations. Non-smokers could not have used tobacco products or any smoked product (e.g., marijuana, cigar, pipe) in the past 12 months and could not be exposed to regular second-hand smoke (e.g., live with or work in close proximity to a smoker). Additional exclusion criteria included alcohol consumption within 48 hours of testing, caffeine consumption within 24 hours of testing, active infections or illnesses, pregnancy, and the use of medications or supplements that might impact the study outcomes including immune function or antioxidant nutritional supplements, over the counter medications such as vitamin-C, elderberry, ginger, turmeric, quercetin, supplements with “Immun” or have immunity on their label, immunosuppressives: prednisolone, azathioprine, mycophenolate mofetil, monoclonal antibodies—of which there are many ending in “mab”, such as bevacizumab, rituximab and trastuzumab, anti-TNF drugs such as etanercept, infliximab, adalimumab, certolizumab and golimumab, methotrexate, ciclosporin, tacrolimus, sirolimus, cyclophosphamide, leflunomide, immunotherapies: immune checkpoint inhibitors, adoptive cell therapies, monoclonal antibodies, oncolytic virus therapy, immune system modulators, etc.

All participants were informed of the study procedures, risks, and benefits through verbal and written forms. During the screening visit, participants completed health history, medication and dietary supplement usage, and physical activity questionnaires. Measurements of heart rate, blood pressure, height, weight, waist, and hip circumference were taken. Women self-administered a pregnancy test. Eligible subjects were then assigned a subject number for privacy and scheduled for their initial testing visit. Menstruating women began the study within the first 5 days of their menstrual cycle to control for hormonal fluctuations. Women with regular menstrual cycles would have reported for the day 30 and day 60 visit at a similar (although not identical) time of month.

This exploratory study used a randomized, double-blind placebo-controlled design. Placebo and experimental capsules were produced according to Good Manufacturing Practices at Deerland Probiotics and Enzymes, Kennesaw, GA. The experimental capsules contained 100 mg of Solarplast^® (enzymatically treated, non-genetically modified organism spinach extract), rice dextrin as needed, microcrystalline cellulose as needed and 1.2% medium chain triglycerides. Characterization of Solarplast’s antioxidant capacity and phenolic component makeup have previously been reported [18]. The placebo capsules contained only rice dextrin, microcrystalline cellulose, and 1.2% medium chain triglycerides. Participants were instructed to take their assigned capsules daily with breakfast for 60 days.

Subjects reported to the lab in the morning following an overnight fast (>10 hours) where they filled out general health and well-being questionnaires (e.g., Warwick-Edinburgh Mental Wellbeing Scale [WEMWBS]) and an in-house skin health questionnaire in which subjects were instructed to indicate how they felt about their skin over the past week for each listed characteristic compared to how it normally is, using a discreet scale from 1 (very bad) to 5 (very good). These questionnaires were then filled out weekly during the study intervention. A skin analysis was performed using a Pear 3D Plus Skin Analysis system (DermaQuip, Marietta, GA), with a blood pressure and heart rate assessment being conducted after a 10-minute quiet rest. Thereafter, a blood sample was taken. This procedure was repeated at baseline (pre), 30 days, and 60 days.

2.4.1. Blood Collection

Single venipunctures were performed observing universal precautions to collect a 10 mL heparinized blood sample (BD Vacutainer REF367874, BD, Franklin Lakes, NJ) from subjects at baseline, 30 day, and 60 day test visits. These samples were analyzed for white blood cell counts and distribution (VetScan HM5, Abaxis, Union City, CA), protein oxidation (OxiSelect AOPP Assay STA-318, Cell Biolabs, Inc., San Diego, CA), lipid peroxidation biomarkers (Malondialdehyde Assay NWK-MDA01, Northwest Life Science Specialties, Vancouver, WA), and cytokines: TNF-α, IL-6, IL-10, and IL-1β (Milliplex HCYTA-60K-04 Human Cyto/Chem/GF Panel A, Millipore Sigma, Burlington, MA). For ex vivo cytokine analyses, blood samples were challenged with 250 ng/mL lipopolysaccharide (LPS) stimulation or control [19]. The blood borne variables were of the highest priority and viewed as our primary outcomes.

2.4.2. Pear 3D Plus Skin Analysis System by DermaQuip

The Pear 3D Plus system was used for assessing topographical, pigmentation and color, via photographical image analysis of the subjects’ facial skin [20]. Photographic analyses like the Pear 3D Plus system have been developed for clinical use for monitoring skin health [21] and this tool was utilized in the present study for systematic analysis of each subject’s skin for a variety of parameters including pores, wrinkles, elasticity, surface spots, UV spots, skin tone, moisture, and oil.

Images and analyses were performed by a trained investigator. Subjects placed their chin on the chin support before the system imaged their facial skin (~1 min) and then analyzed the images (<5 min) for the different skin variables. The analyses were used to monitor changes in predicted facial skin age for each individual skin variable at each visit.

2.4.3. Dietary Intake and Physical Activity

Subjects followed their usual activity patterns over the course of the study period but refrained from strenuous activity for the 48 hours preceding each test visit. Subjects also recorded their dietary intake on food logs for five days prior to each test visit, and data were analyzed using Food Processor Pro (Esha Research). Subjects were instructed to keep both their dietary intake and physical activity levels consistent over the 60-day study period.

Descriptive statistics were used to outline baseline characteristics (means, standard deviations, frequencies and percentages). The assumptions of outliers and normal distribution of the data were inspected by using box-plots, normal probability plots, and the Shapiro-Wilk test of normality. Two-way mixed analyses of variance (ANOVA) were conducted to evaluate any differences in dietary intake variables between treatment groups at pre-treatment, 30 days, and 60 days. In addition, two-way ANOVAs were conducted to examine the effects of smoking status and treatment (Solarplast^® vs. placebo) on biological variables, plasma cytokine variables, skin imaging, and self-reported feelings of health and well-being at baseline and two timepoints (e.g., timepoint 1 = 30 day minus baseline; timepoint 2 = 60 day minus baseline). Because the primary objective was to evaluate change from baseline, analyses were performed on change scores (30-day minus baseline and 60-day minus baseline), with baseline values incorporated into the outcome measure, rather than included as a separate covariate. If interaction effects were not significant, follow-up analyses were conducted by examining the main effects of each independent variable separately using one-way ANOVAs or independent t-tests, as appropriate. In cases where assumptions of normality were violated, non-parametric alternatives (e.g., Mann-Whitney U tests or Kruskal-Wallis tests) were employed. Statistical analyses were conducted using a per-protocol (complete-case) approach. Of the 59 participants randomized, 50 completed the study and contributed data to the primary analyses. Participants who withdrew before study completion were excluded from the analyses, and missing data resulting from participant withdrawal were not imputed. For individual outcome measures, analyses were conducted using all available data; therefore, sample sizes varied slightly across outcomes because of occasional missing measurements. All analyses were performed using the Statistical Package for the Social Sciences (SPSS for Windows, version 28; SPSS, Chicago, IL). All statistical tests were two-tailed with the significance level represented by p < 0.05.

Fifty subjects completed the 60-day intervention. In addition to the individuals that completed the study, 32 additional individuals provided informed consent, but did not complete all aspects of the study for the following reasons: 21 for scheduling issues; 2 due to illness unrelated to treatment; 1 due to problems with blood collection; 8 subjects missed visits and stopped communicating with the research staff. Detailed characteristics are in Table 1 and the CONSORT diagram is in Figure 1. Of the 16 smokers that completed the study, 62.5% reported solely smoking traditional cigarettes, while the remainder used a combination or solely vaped. All smokers regardless of use type were analyzed together. Compliance was determined via capsule counts and found to be similar between treatments with 94.6% for Solarplast^® and 93.5% for placebo.

Figure 1. CONSORT Diagram specific to the impact of Solarplast^® on oxidative stress, immune function, and skin appearance in active smokers and non-smokers.

There were no statistically significant changes to subjects’ self-reported behavior related to exercise, sleep, alcohol consumption, or water consumption throughout the course of the study, (Table 2; p > 0.05). Additionally, subjects’ nutrition remained consistent (Table 3; p > 0.05), suggesting these effects should not have influenced the outcomes.

Table 2. Psychosocial measures of participants pre-treatment, 30 day, and 60 day and participants’ significance of change outcomes.

*p-value < 0.05. ¹Averaged value of negative feelings from 1 (none of the time) to 5 (all of the time). ²Averaged value of experiencing negative health characteristics from 1 (None) to 5 (severe). ³T1 = 30 day − pre; T2 = 60 day − pre. ⁴Interaction is smoking status x treatment at each timepoint; Treatment is outcomes of Solarplast^® vs. placebo across time; Smoking Status is outcomes of smoker vs non-smoker across time.

Table 3. Dietary intake of participants pre-treatment, 30 day and 60 day.

Solarplast^® was well-tolerated, with no reported complaints received from subjects. No adverse events were noted, suggesting high tolerability.

There were no statistically significant interactions between treatment groups and smoking status for the change scores for systolic blood pressure (SBP), diastolic blood pressure (DBP), or heart rate. While there were statistically significant main effects of treatment for SBP change scores (p = 0.022) and DBP change scores (p = 0.013) at timepoint 2, the differences are within normal variable values and not clinically relevant (Table 4).

Table 4. Blood pressure, heart rate, and WBC measures of participants pre-treatment, 30 day and 60 day and participants’ significance change outcomes.

*p-value < 0.05. ¹T1 = 30 day − pre; T2 = 60 day − pre. ²Interaction is smoking status × treatment at each timepoint; Treatment is outcomes of Solarplast^® vs. placebo across time; Smoking Status is outcomes of smoker vs non-smoker across time (SBP: systolic blood pressure, DBP: diastolic blood pressure, WBC: white blood cells, LYM: lymphocytes, MON: monocytes, NEU: neutrophils, LY% % lymphocytes, MO%: percentage monocytes, NEU%: percentage neutrophils).

No statistically significant interactions between treatment groups and smoking status at both timepoints were found for WBC, LYM, MON, NEU, LY%, MO%, or NEU%, nor were there any statistically significant main effects for treatment (Table 4).

Neither smoking nor daily Solarplast^® administration significantly influenced Advanced Oxidative Protein Products (AOPP) or malondialdehyde (MDA), however AOPP and MDA were reduced by 13% and 15%, respectively, in smokers receiving Solarplast^®, but these did not reach significance (p > 0.05) as determined by two-way ANOVA (Table 5).

Table 5. Plasma cytokine measures of participants pre-treatment, 30 day and 60 day and participants’ significance of change outcomes.

*p-value < 0.05. ¹T1 = 30 day − pre; T2 = 60 day − pre. ²Interaction is smoking status x treatment at each timepoint; Treatment is outcomes of Solarplast^® vs. placebo across time; Smoking Status is outcomes of smoker vs non-smoker across time (IL-1β, 6, 10: Interleukins 1β, 6, 10, TNFα: Tumor Necrosis Factor α, AOPP: Advanced Oxidation Protein Product, MDA: malondialdehyde).

There was a significant finding observed for only one of the measured variables assessed by the Pear 3D Plus system (p < 0.05) in Table 6, specific to treatment. For all endpoints, there was no significant interaction between intervention group and smoking status. There was a significant improvement in moisture at 60 days in the Solarplast group compared to placebo, regardless of smoking status (F(1, 46) = 4.56, p = 0.038, partial η² = 0.090). Despite the significant finding for moisture levels, no other skin variables were impacted significantly and more importantly, overall skin age did not experience a significant change.

Subjective skin health (Table 7) was also assessed using a 5-point Likert scale with 1 indicating “very bad” and 5 indicating “very good” for various skin characteristics (acne, skin appearance, blemishes, brightness, dryness, elasticity, firmness, itchiness, rash, redness, and wrinkles, and overall skin health). There were no statistically significant differences in any of the subjective skin health endpoints, with the exception of “brightness.” There was a significant reduction in perceived brightness at day 60 in the Solarplast group compared to placebo, regardless of smoking status (F(1, 45) = 5.43, p = 0.024, partial η² = 0.108).

Table 6. Skin imaging outcomes of participants pre-treatment, 30 day and 60 day and participants’ significance of change outcomes.

*p-value < 0.05. ¹T1 = 30 day − pre; T2 = 60 day − pre. ²Interaction is smoking status × treatment at each timepoint; Treatment is outcomes of Solarplast^® vs. placebo across time; Smoking Status is outcomes of smoker vs non-smoker across time.

Table 7. Self-perceived skin outcomes of participants pre-treatment, 30 day and 60 day and participants’ significance of change outcomes.

*p-value < 0.05. ¹T1 = 30 day − pre; T2 = 60 day − pre. ²Interaction is smoking status × treatment at each timepoint; Treatment is outcomes of Solarplast^® vs. placebo across time; Smoking Status is outcomes of smoker vs non-smoker across time.

In this study, mental wellbeing was assessed using the Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS) with a score range of 14 - 70, where positive wellbeing is associated with higher scores. While no two-way interactions were observed between treatment and smoking status, there was a statistically significant main effect of treatment for WEMWBS T2 change scores (60 day − pre), F(1, 45) = 9.89, p = 0.003, partial η² = 0.180 indicating that WEMWBS scores were statistically lower at 60 day for the Solarplast^® group as compared to placebo regardless of smoking status, when considering the change relative to each group’s baseline value.

Antioxidants are thought to ameliorate hypertension by reducing oxidative stress [22]. Dietary antioxidants such as those in fruits and vegetables have been found to reduce both SBP and DBP, however there is insufficient evidence that dietary antioxidant supplements provide a similar benefit [22][23]. Despite previous studies suggesting antioxidants can reduce blood pressure, no favorable effects were observed in this study, likely due to the normotensive status of most subjects at baseline (Table 1). Any minor changes observed do not appear to have clinical relevance.

White blood cell counts and distributions are altered with inflammation and disease [24]. Smoking is associated with elevated WBC counts and leads to alterations in both innate and adaptive immune responses [25]. In the present study we found no interactions for any measured parameter specific to white blood cells.

To evaluate the effects of Solarplast^® on oxidative stress, AOPP and MDA were measured. While Solarplast^® slightly lowered AOPP and MDA levels, these changes were not statistically significant.

Proinflammatory cytokines such as IL-1β and TNF-α increase due to oxidative stress [2]. Smokers experience more oxidative stress and therefore express higher levels of some inflammatory cytokines [26]. However, in this study no 3-way or 2-way interactions were statistically significant for any of the measured cytokines: IL-1β, IL-6, IL-10, and TNF-α (Table 5).

Lipopolysaccharide (LPS) can induce ROS and proinflammatory cytokines [13]. Antioxidants present in spinach may provide some protection against LPS actions [16]. To determine whether Solarplast^® offered any protection, heparinized whole blood was cultured with LPS or no LPS for 6 hours and cytokine levels were measured in the culture supernatants. Normalized value for cytokines were determined by dividing the LPS stimulated levels by the non-stimulated levels. Values for normalized IL-1β were lower by approximately 30% at day 30 (p = 0.008) and day 60 (p = 0.026) with Solarplast^® treatment as compared to placebo, when considering the change relative to each group’s baseline value and was more pronounced for non-smokers. Values for normalized IL-6 were also lower at day 30 (p = 0.02) and day 60 (p = 0.073) with Solarplast^® treatment as compared to placebo, when considering the change relative to each group’s baseline value.

Lipopolysaccharide Antioxidants provide multiple benefits to skin health and appearance [27]. Smokers experience more oxidative stress particularly to areas exposed to smoke including the face [28]. Smoking causes a decrease in blood flow to skin and dryness, along with other adverse effects that can prematurely age skin [29][30]. Smoking causes a decrease in blood flow to skin, along with other adverse effects that can prematurely age the skin [29]. Therefore, we hypothesized that the skin of smokers was likely to benefit more from antioxidant supplementation than the skin of non-smokers. The present study sought to extend these findings using both analytical and opinion-based measures among additional smokers and non-smokers.

As reviewed in Table 7, no significant statistical differences of interest were found for any of the self-assessed skin factors. However, a difference was detected via image analysis from the Pear 3D plus system (Table 6) for moisture levels. We noted no improvement in wrinkles with treatment and our baseline wrinkle data disagree with previous findings showing that smokers have more skin damage/wrinkles than non-smokers [31]. This may be attributed to the lower pooled average age of smokers (40.4 ± 10.4 years) versus non-smokers (46.6 ± 9.7 years) in the present study. Considering all skin outcomes, it should be noted that subjects had good overall skin health at the start of the study, with values withing normal ranges, and therefore, had less room for improvement in response to the Solarplast^® treatment.

Cognitive and mental health can be affected by oxidative stress, which can be reduced via antioxidant supplementation [32][33]. Individuals exposed to high levels of oxidants and who are in a chronic state of oxidative stress (e.g., smokers) may be more susceptible to impaired mental health [14]. In this study, mental wellbeing was assessed using the Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS) with a score range of 14 - 70, where positive wellbeing is associated with higher scores. Based on changes in scores from pre-treatment, Solarplast^® resulted in slightly lower scores at both the first and second visit, as compared to pre, with scores statistically lower at day 60 for the Solarplast^® group as compared to placebo group regardless of smoking status. The scores for both placebo and Solarplast^® were within the average wellbeing range (42 - 60) as determined by research in the UK and therefore likely do not indicate any clinically valuable differences [22][23].