Effects of Botanical Microglia Modulators in Gulf War Illness
Phase: NA
Conditions: Gulf War Illness
Sponsor: University of Alabama at Birmingham

Effects of Botanical Microglia Modulators in Gulf War Illness 
 
 
Study Protocol & Statistical Analysis Plan 
 
 
NCT02909686 
 
 
10/11/2022 
 
 
Jarred Younger, Professor, Principal Investigator 
University of Alabama at Birmingham 
Birmingham, AL 35294 
 
 

A Placebo-Controlled, Pseudo-Randomized, Crossover Trial of Botanical Agents for Gulf 
War Illness: Curcumin (Curcuma longa), Boswellia (Boswellia serrata), and French 
Maritime Pine Bark (Pinus pinaster) 
 
2. Materials and Methods 
The full study was a pseudo-randomized, placebo-controlled, crossover clinical trial testing 
the effects of nine different anti-inflammatory botanical compounds on symptoms of GWI. The 
list of agents tested in the larger study were curcumin, boswellia, maritime pine, epimedium, 
fisetin, luteolin, reishi mushroom, resveratrol, and stinging nettle. Participants trialed the 
botanicals serially, with each participant receiving up to three botanical agents. Outcome measures 
were obtained daily throughout the entire duration of participation. The University of Alabama at 
Birmingham (UAB) Institutional Review Board first approved the study protocol (F150318011) 
on 30 June 2015, and the study was registered on ClinicalTrials.gov (NCT02909686) on 21 
September 2016. Participants were recruited to the study through public flyers and online postings. 
All participants were given a written description of the study and provided written informed 
consent. Participants were randomized to botanicals so that each condition was completed by at 
least 10 individuals. 
2.1. Participants 
Men were considered for inclusion in the study if they were aged 37–65 and able to attend 
up to 11 study visits every 30 +/− 3 days. Participants were required to meet Kansas GWI case 
definition criteria [36], with the exception that (1) participants were permitted to have a 
comorbid diagnosis of diabetes mellitus type 2 if controlled with medications and if having a 
hemoglobin A1C ≤ 9% and (2) one individual with a remote history of cancer (Hodgkin’s 
lymphoma in remission for 20 years) was included. Participants must have been present in the 
Persian Gulf between August 1990 and August 1991. 
Exclusionary criteria included current opioid, daily anti-inflammatory, nitroglycerine, or 
lithium medication use; history of anaphylaxis to study botanical compounds; presence of severe 
depressive symptoms as indicated by a Hospital Anxiety and Depression Scale (HADS [37]) 
depression subscale score ≥16; presence of a blood or clotting disorder; hypotension (under 
90/60 mmHg) or history of cardiovascular disease; diagnosed rheumatologic or autoimmune 
disease; and acute infection (body temperature over 100.4 °F). Baseline laboratory values of 
erythrocyte sedimentation rate (ESR) > 40 mmHr, positive rheumatoid factor (RF), CRP > 10.0 
mg/L, or positive antinuclear antibody (ANA) were also exclusionary. Participants could also not 
have current Posttraumatic Stress Disorder (PTSD). PTSD was initially screened with the PTSD 
Checklist—Military Version (PCL-M [38]), and individuals with PCL-M scores ≥50 were given 
the Clinician Administered PTSD Scale (CAPS-5 [39]) for a final determination. 
2.2. Botanicals 
The botanical compounds were sourced from a university-approved vendor, Pure 
Encapsulations (Sudbury, MA, USA), prior to being sent to a compounding pharmacy for re-
encapsulation. Botanical compounds were re-encapsulated in size 0 or size 00 blue gelatin 

capsules by Double Oak Mountain Pharmacy in Birmingham, AL. Capsules were placed in 
standardized QUBE Weekly (28 cavity) Cold Seal Compliance Blister packs (Pharmacy 
Automation Supplies, Romeoville, IL, USA). 
Curcumin was obtained as Pure Encapsulation’s CurcumaSorb product (SKU#: MCU1), 
which contains the trademarked Meriva® turmeric phytosome (Indena, S.p.A., Milan, Italy). 
Curcumin was administered at 1000 mg/day (lower dosage condition) and 4000 mg/day (higher 
dosage condition). Boswellia was obtained as Pure Encapsulation’s Boswellia product (SKU# 
BW31) and was administered at 400 mg/day and 800 mg/day. Maritime pine bark extract was 
obtained as Pure Encapsulation’s Pycnogenol (pine bark extract) product (SKU# PY16) which 
contains the trademarked Pycnogenol (Horphag Research, Hoboken, NJ, USA). Maritime pine 
was administered at 200 mg/day and 400 mg/day. All botanicals were administered twice per day 
(morning and evening), with the total daily dosage being split evenly between the morning and 
evening doses. 
2.3. Study Protocol 
All participants were assessed for initial eligibility for the study using an online 
prescreening questionnaire, followed by a phone interview. Individuals who met initial inclusion 
criteria after the online screening and phone interview were given an in-person screening at 
UAB’s Center for Clinical and Translational Science Clinical Research Unit (CCTS CRU). 
At the in-person screening visit, participants provided written informed consent as well as 
blood samples to test for exclusionary lab values. Participants also completed baseline study 
measures on a tablet device and were loaned a tablet for completion of daily symptom reporting 
during the study. Following the screening visit, participants reported symptoms every day (in the 
evening) during a one-month (30 +/− 3 days) baseline period, which served as a habituation 
period and a means to assess whether participants would reliably complete their daily symptom 
reports. Participants were allowed to continue with study participation if they completed at least 
80% of baseline symptom reports. 
Participants were then pseudo-randomized to receive up to three out of the nine botanical 
compounds, in the design presented in Figure 1. Botanical assignments were pseudo-randomized 
so that (1) approximately equal numbers of participants would take each botanical and (2) to 
ensure there were no drug interactions that contraindicated the use of the botanicals assigned to 
participants. There were no contraindications used for curcumin or boswellia. Individuals with 
prediabetes, evidence of diabetes, or diabetic medications were excluded from taking maritime 
pine due to its lowering effects on blood glucose [40]. Contraindications were handled by a 
pharmacist with no other connections to the study so that all research personnel could remain 
blinded to the botanical being assigned. After completion of a full protocol of three botanical 
assignments, participants were offered the opportunity to re-enroll into the study protocol to 
receive up to three more botanicals. 

 
Figure 1. Study protocol. Each participant completed testing of up to three botanicals. Some 
participants opted to re-enroll in the study protocol after completion, resulting in a maximum of 
six botanical assignments. For each botanical, there was a placebo condition, followed by lower-
dose botanical and higher-dose botanical conditions. The period of time between visits was 30 +/− 
3 days. 
After the baseline period, participants returned once monthly (every 30 +/− 3 days) for up to 
10 additional visits to the CCTS CRU for dispensation of their placebo or botanical capsule kits. 
At visits 4, 7, and 10, participants received additional blood draws to monitor liver and kidney 
function (sodium, potassium, chloride, bicarbonate, anion gap, glucose, blood urea nitrogen 
(BUN), creatinine, calcium, phosphorus, albumin, total protein, total bilirubin, direct bilirubin, 
indirect bilirubin, alkaline phosphatase, aspartate aminotransferase (AST), and alanine 
aminotransferase (ALT)). Participants were compensated for each laboratory visit. 
For each botanical, participants followed the same protocol: one month (30 +/− 3 days) of 
placebo, one month (30 +/− 3 days) of lower-dose botanical, and one month (30 +/− 3 days) of 
higher-dose botanical. Study participants were blinded to both the assigned botanicals and 

administration protocol (placebo, lower-dose, higher-dose). This administration order was 
chosen for safety reasons, i.e., adverse effects could be detected in a lower-dose condition before 
a higher-dose, with possibly greater adverse effects, would be administered. Research personnel 
were blinded to the assigned botanicals but not to the administration protocol. The study 
pharmacist was unblinded to both administration protocol and assigned botanicals but had no 
contact with study participants. Adherence to botanicals was checked at each study visit when 
participants returned their used blister packs. If participants had missed doses, study staff 
reminded them of the importance of a regular dosing schedule. 
2.4. Screening Measures 
The Kansas GWI case definition [36] was used to determine if participants met criteria for 
GWI. Exclusionary criteria for the definition included certain chronic conditions (heart disease, 
stroke, lupus, multiple sclerosis, cancer (other than skin cancer), melanoma, and liver disease) 
not associated with service in the Gulf War, as well as certain conditions that could impact 
participants’ ability to report their symptoms (bipolar disorder or manic depression, 
schizophrenia, or recent hospitalization for alcohol or drug dependence, depression, or PTSD). 
Inclusionary criteria for the definition required that participants report presence of symptoms that 
began during or after service in the Gulf War. Each symptom was scored on a severity scale of 0 
to 3, with 0 = none and 3 = severe. Veterans with a score of 2 (at least one moderate symptom or 
two mild symptoms) or greater in at least three out of six symptom domains (fatigue, pain, 
neurological/cognitive/mood, skin, gastrointestinal, and respiratory) met criteria for GWI. 
The HADS [37] was used to screen for severe depressive symptoms. Potential participants 
completed the questionnaire via the Qualtrics Research Suite Online Application as part of the 
online prescreening process. The HADS consists of 14 items divided into two, seven-item 
subscales: Anxiety (HADS-A) and Depression (HADS-D). Respondents rate items on a 0 to 3 
scale, with higher ratings indicating greater presence of the symptoms. Five of the 14 items are 
reverse scored. The total score is calculated by summing all items and ranges from 0–42. Scores 
of 16 or greater on the HADS-D subscale during prescreening were exclusionary for 
participation in the study. 
To screen for severe PTSD symptoms, the PCL-M [38] was used. Potential participants 
completed this questionnaire via the Qualtrics Research Suite Online Application as part of the 
online prescreening procedures. The PCL-M corresponds to criteria according to the fourth 
edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV [41]) and consists 
of 17 questions referring to military experiences and symptoms of stress and trauma (e.g., 
repeated, disturbing memories, thoughts, or images of a stressful military experience) answered 
on a rating scale of 1 (not at all) to 5 (extremely). A score greater than or equal to 50 indicates a 
probable diagnosis of PTSD according to DSM-IV criteria [42]. 
If potential participants scored 50 or greater on the PCL-M during online prescreening, the 
past-month version of the CAPS-5 [39] was performed at the in-person screening visit by a 
member of the UAB Office of Psychiatric Clinical Research. The 30-item structured interview 
evaluates PTSD diagnostic status and symptom severity by assessing onset and duration of 
symptoms, levels of distress, changes in social and occupational functioning, response validity, 
and symptoms of the dissociative subtype of PTSD. Each CAPS-5 item is rated on frequency and 
intensity, which is then combined into a single severity score for that symptom. A severity rating 
of 2 (moderate/threshold) indicates a symptom that meets diagnostic threshold for current PTSD 
[43]. If a potential participant met criteria for current PTSD, they were excluded from the study. 

2.5. Main Outcome Measures 
The Qualtrics Research Suite Offline Application (Qualtrics, Provo, UT, USA) was used for 
daily symptom reporting. As part of the daily report, participants scored symptoms on a 0–100 
digital visual analog scale (VAS). GWI symptom severity, the primary outcome variable of 
interest, was assessed by asking, “Overall, how severe have your symptoms been today?” 
anchored on the left by, “No symptoms at all,” and on the right by, “Severe symptoms.” The 
single-item, overall GWI severity measure was chosen as the primary outcome, because of the 
multisymptom and idiosyncratic nature of the condition’s manifestation in affected individuals. 
Veterans living with GWI may primarily experience pain, fatigue, gastrointestinal distress, or 
any of several other common symptoms. The overall severity measure was determined to be a 
way to provide the most universal assessment of GWI symptom severity across participants with 
varying symptom presentations. Participants were instructed to use the GWI symptom severity 
measure to include whichever specific symptoms they attributed to GWI. 
2.6. Secondary Outcome Measures 
Several specific symptoms were assessed with similar one-item measures. These symptoms 
included pain, fatigue, cognitive dysfunction, depressed mood, dermatologic complaints, 
respiratory problems, and gastrointestinal distress. Of these, two (pain and fatigue) were 
endorsed by a sufficient number of participants to allow for statistical analysis. These two 
symptoms were measured with two separate 0–100 VAS items. Pain was assessed by asking, 
“Overall, how severe is your pain?” anchored on the left by, “No pain at all,” and on the right by, 
“Severe pain.” Fatigue was assessed by asking, “How fatigued have you felt today?” anchored 
on the left by, “Not fatigued at all,” and on the right by, “Severely fatigued.” 
 
2.7. Statistical Analyses 
Analyses were conducted using SPSS Statistics for Windows, version 24 (IBM Corp., 
Armonk, NY, USA). Effects of botanicals on symptom severity were tested using separate two-
level linear mixed models (LMM). LMMs are designed for nested data structures and allow for 
the dependence between observations typically seen with repeatedly measured variables. In this 
study, longitudinal outcome assessments are nested within individuals and conditions, violating 
the assumption of independence between observations. For each botanical, three models were built 
with condition (baseline, placebo, lower-dose, and higher-dose) entered as a fixed factor and 
symptom severity (GWI, pain, or fatigue) as the dependent variable, resulting in a total of nine 
models. Subject ID was used as the subject identifier, and the day in the study was the repeated 
measures index. A compound symmetry repeated measures covariance structure was selected, as 
it improved the Bayesian information criterion (BIC) and Akaike information criterion (AIC) for 
model performance beyond the AR (1) autoregressive covariance structure. For main analyses, 
daily GWI symptom severity was entered as the dependent variable. For all analyses, the outcome 
variable was derived by taking a mean of the last 14 days of daily symptom severity reports for 
each participant during each condition (placebo, lower dose, higher dose). This was consistent with 
the registered analysis plan, in order to allow the botanicals time to exert clinical effects. A 

restricted maximum likelihood (REML) estimation approach was used. Post hoc contrasts were 
carried out with the least-squares differences method. Significance for all tests was set at p < 0.05. 
Sample size was determined with a-priori power analyses conducted in G*Power 3 [44]. The 
botanical trials were powered to detect a medium effect (Cohen’s d = 0.5) with 0.99 power at a p = 
0.05 level of significance. Ten individuals were needed to reach 0.99 power, due to the large 
number of repeated outcome assessments (56 per participant, repeated measures correlation of 
0.5). The trials were not powered to detect small (Cohen’s d = 0.25) effects, with only 0.42 
predicted power. Small effects, however, were not expected to have important clinical impacts on 
GWI. 
 
 
 

A Placebo-Controlled, Pseudo-Randomized, Crossover Trial of Botanical Agents for Gulf 
War Illness: Resveratrol (Polygonum cuspidatum), Luteolin, and Fisetin (Rhus 
succedanea) 
 
2. Materials and Methods 
The three agents discussed in this report were part of a larger program of study that 
investigated the effects of nine anti-inflammatory botanicals on GWI symptoms. The study 
protocol was approved by the University of Alabama at Birmingham (UAB) Institutional Review 
Board on 30 June 2015 (F150318011), and this study was registered on ClinicalTrials.gov on 21 
September 2016 (NCT02909686). Individuals were recruited via radio, print, and online 
advertisements. All participants provided written informed consent. Participants were randomized 
to receive up to three botanical compounds in total over the course of the study, such that each 
botanical was trialed by at least ten individuals. Participants recorded symptoms daily throughout 
the baseline, placebo, and treatment periods. 
2.1. Participants 
Inclusion criteria included the following: male sex; age 37–65; presence in the Persian Gulf 
region between August 1990 and August 1991; ability to come to the study site for 11 monthly 
visits; ability to receive a venous blood draw; and successful completion of ≥80% of daily 
symptom reports over the baseline period. All participants had to fulfill Kansas Gulf War Illness 
case criteria [33], with an exception for well-controlled diabetes mellitus type 2 (A1C ≤ 9%) and 
in one case for an individual with a history of cancer outside of the last 5 years (Hodgkin’s 
lymphoma in remission for 20 years). In such cases of unclear decisions for inclusion, the author 
of the Kansas GWI case criteria was consulted for guidance. 
Individuals were excluded from study participation for the following: current daily use of 
opioid or anti-inflammatory medications; use of nitroglycerine or lithium; history of anaphylaxis 
to any botanical used in the study; hypotension (<90/60 mmHg) or history of cardiovascular 
disease; diagnosis of rheumatologic or autoimmune disease; blood or clotting disorder; current 
litigation of worker’s compensation claim; and inability to read and understand English. Baseline 
exclusionary criteria also included acute infection (body temperature above 100.4 °F), positive 
rheumatoid factor (RF), positive antinuclear antibody (ANA), erythrocyte sedimentation rate 
(ESR) > 40 mm/hr, and CRP > 10.0 mg/L. Individuals with current severe depressive symptoms 
as suggested by a depression subscale score ≥ 16 on the Hospital Anxiety and Depression Scale 
(HADS; [34]) were excluded. Current Posttraumatic Stress Disorder (PTSD) was also 
exclusionary. Individuals with scores ≥ 50 on the PTSD Checklist–Military Version (PCL-M; 
[35]) were assessed for the presence of current PTSD with the Clinician Administered PTSD 
Scale (CAPS-5; [36]). 
2.2. Treatments 
Resveratrol was sourced from Pure Encapsulations (Sudbury, MA, USA) and was 
standardized to contain 20% trans resveratrol. Luteolin was obtained from A.P.I. Solutions 
(Daphne, AL, USA), and fisetin was sourced from VitaCost (Boca Raton, FL, USA). All 

botanicals were sent to Double Oak Mountain Pharmacy in Birmingham, AL, USA, where they 
were re-encapsulated in size 0 or 00 opaque blue gelatin capsules. Microcrystalline cellulose was 
used as filler for the placebo treatment, with the same blue covering capsules as the botanical 
compounds, so that all treatments appeared identical. All capsules were placed in standardized 
QUBE Weekly (28 cavity) Cold Seal Compliance Blister Packs (Pharmacy Automation Supplies, 
Romeoville, IL, USA). 
Treatments were administered twice a day. The lower dose for resveratrol was 200 mg/day 
(200 mg in morning and 0 mg in evening), and the higher dose for resveratrol was 600 mg/day 
(400 mg in morning; 200 mg in evening). For luteolin, the lower dose was 200 mg/day, and the 
higher dose was 400 mg/day. Fisetin was administered at 200 mg/day for the lower dose and 800 
mg/day for the higher dose. For both luteolin and fisetin, total dosage was evenly split between 
morning and night doses. 
2.3. Study Protocol 
The study protocol is depicted in Figure 1. Interested individuals completed an online pre-
screening questionnaire prior to a phone interview with research personnel in order to assess for 
initial eligibility. For individuals who met initial inclusion criteria, an in-person screening visit 
was held at the UAB Center for Clinical and Translational Science Clinical Research Unit 
(CRU). All participants provided written informed consent at the in-person screening. Baseline 
study questionnaires were administered on a tablet device, and vital signs and venous blood 
samples were collected by CRU staff to assess for exclusionary laboratory values. Participants 
were also provided with computer tablet devices to take home with them in order to complete 
once daily symptom reports during the entire study period. A one-month baseline symptom 
reporting period began immediately following the in-person screening visit. Participants were 
required to complete at least 80% of daily symptom reports during this period to remain eligible 
for the study. 

 
Figure 1. Study protocol. Each participant completed testing of up to three botanicals. For each 
botanical, there was a placebo condition, followed by lower-dose botanical and higher-dose 
botanical conditions. The period of time between visits was 30 ± 3 days. Some participants re-
enrolled in the study after completion, receiving up to a maximum of six botanicals. 
Pseudo-randomization was then performed so that participants were assigned to receive up 
to three out of the nine botanical compounds, as shown in Figure 1. Pseudo-randomization was 
done in such a way to prevent drug interactions that contraindicated the use of the botanical in a 
given participant. There were no contraindications for the use of luteolin. Because blood glucose 
may be lowered by resveratrol [37] and fisetin [38], individuals taking medications for diabetes, 
or with evidence of prediabetes or diabetes, were excluded from taking these agents. 
Additionally, individuals with clotting disorders, hypotension, or those taking anticoagulants or 
antihypertensive medications were excluded from taking resveratrol, due to its effects on blood 
pressure [39,40]. Prophylactic use of 81 mg aspirin daily was permitted. 
All study visits were held at the CRU and were conducted once a month for 10 months after 
completion of the baseline period. Participants received kits at each visit containing a one-month 

supply of either placebo or botanical capsules. Kidney and liver function were monitored with 
blood draws conducted at visits 4, 7, and 10. The following laboratory values were tested at 
those visits: sodium, potassium, chloride, bicarbonate, anion gap, glucose, blood urea nitrogen, 
creatinine, calcium, phosphorus, albumin, total protein, total bilirubin, direct bilirubin, indirect 
bilirubin, alkaline phosphatase, aspartate aminotransferase (AST), and alanine aminotransferase 
(ALT). 
The same protocol (one month of placebo, one month of lower-dose botanical, and one 
month of higher-dose botanical) was completed for each botanical. For safety reasons, lower-
dose conditions always preceded higher-dose conditions, allowing adverse effects to be detected 
before a higher dose of the agent was used. Only the study pharmacist was unblinded to the 
assigned botanicals. All other study staff were aware of the design condition order, but were 
blinded to the specific botanical taken by each participant. Participants were blinded to both the 
administration order (placebo, lower-dose, and higher-dose) and the botanical compounds. 
Treatment adherence was monitored at each study visit. Each month, participants were 
instructed to return their blister packs. Study staff checked for any missed doses and discussed 
with participants the importance of adhering to the administration regimen. Participants were 
provided with the option of re-enrolling in the study after completing the protocol, such that they 
could be assigned up to a total of six out of the nine compounds. 
2.4. Screening Measures 
Participants were screened for GWI using the Kansas Gulf War Illness (GWI) case 
definition [33], in which symptoms are rated in severity from 0 (none) to 3 (severe). Inclusion 
criteria were met if an individual obtained a score of 2 or greater (indicating at least one 
moderate symptom or two mild symptoms) across three or more of six symptom domains (pain, 
fatigue, neurological/cognitive/mood, gastrointestinal, respiratory, and skin). Symptoms were 
required to have begun during or after Gulf War service. Chronic medical conditions not 
associated with Gulf War military service (including heart disease, stroke, lupus, multiple 
sclerosis, cancer [other than skin cancer], melanoma, and liver disease) were exclusionary for 
case definition. Due to the possibility of impacting a participant’s ability to report symptoms, the 
following other conditions were exclusionary per case criteria: bipolar or manic depression, 
schizophrenia, and recent hospitalization for alcohol or drug dependence, depression, or PTSD. 
Participants were screened for symptoms of severe depression with the HADS [34]. This 
questionnaire was part of the online pre-screening process and was completed using the Qualtrics 
Research Suite Online Application. The 14 items of the HADS contain subscales for Anxiety 
(HADS-A) and Depression (HADS-D), each with 7 items. Items are rated from 0 to 3, and 
reverse scoring is used for 5 of 14 total items. Higher ratings suggest greater symptomatology. 
All ratings are summed for a total score ranging from 0 to 42. 
Participants were also screened for severe symptoms of PTSD using the PCL-M [35]. Like 
the HADS, the PCL-M was completed during the online pre-screening process using the 
Qualtrics Research Suite Application. The 17-item PCL-M reflects PTSD diagnostic criteria of 
the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV; [41]). 
Each item inquires about trauma and stress symptoms associated with military experiences (e.g., 
Repeated, disturbing memories, thoughts, or images of a stressful military experience?) and is 
rated on a Likert scale from 1 (not at all) to 5 (extremely). Scores of 50 or greater suggest an 
increased likelihood of meeting DSM-IV diagnostic criteria for PTSD [42]. 

If a PCL-M score of 50 or above was obtained, potential participants were evaluated further 
for current PTSD symptoms using the last month version of the CAPS-5 [36]. Personnel of the 
UAB Office of Psychiatric Clinical Research administered the CAPS-5 at the CRU during the in-
person screening visit. The CAPS-5 is a structured interview consisting of 30 items assessing 
PTSD symptom severity and diagnostic status (symptom duration and onset, distress levels, 
impact on social and occupational functioning, response validity, and dissociative subtype 
symptoms). Items are rated with a severity score based on frequency and intensity of symptoms. 
A symptom that meets the threshold for diagnosis of current PTSD is suggested by a severity 
rating of 2 (“Moderate/threshold”). 
2.5. Main Outcome Measures 
Participants reported their symptoms once daily in the evening via the Qualtrics Research 
Suite Offline Application (Qualtrics, Provo, UT, USA). The reports consisted of visual analog 
scale (VAS) ratings of symptoms scored from 0 to 100. Given that GWI consists of multiple 
idiosyncratic symptoms, a single-item severity score was chosen as the primary outcome to 
provide a common measure for GWI symptom severity. The main outcome was assessed with 
the following question, “Overall, how severe have your symptoms been today?” with “Not 
severe at all” fixed on the far left and “Extremely severe” fixed on the far right. Research 
personnel instructed participants to rate their daily overall GWI symptom severity based on their 
particular GWI symptoms, encompassing all possible six GWI domains. 
2.6. Secondary Outcome Measures 
In addition to the GWI symptom severity item, participants responded to several other daily 
single-item measures. These items assessed specific symptoms including pain, fatigue, cognitive 
dysfunction, depressed mood, skin problems, respiratory complaints, and gastrointestinal issues. 
A sufficient number of participants endorsed the pain and fatigue items so that statistical 
analyses could be applied. Participants responded to pain and fatigue questions using two 
separate 0–100 VAS ratings. To assess pain, participants were asked, “Overall, how severe is 
your pain?”, with “No pain at all” fixed on the left and “Severe pain” on the right. The item 
regarding fatigue asked, “How fatigued have you felt today?”, with “Not fatigued at all” fixed on 
the left and “Severely fatigued” on the right. 
 
2.7. Statistical Analyses 
SPSS Statistics for Windows, version 24 (IBM Corp., Armonk, NY, USA) was used to 
conduct analyses. Linear mixed models (LMM) were utilized to test differences in self-reported 
symptom severity between the four conditions. A separate model was tested for each of the three 
botanicals. Subject ID was entered as the subject identifier, with all individual longitudinal data 
nested within-person. The participant’s day in the study was entered as the index variable for 
repeated measures. Compound symmetry was selected as the repeated measures covariance type. 
The AR (1) autoregressive covariance type was also considered, but it did not improve the Akaike 
information criterion (AIC) or Bayesian information criterion (BIC) for model performance. The 
dependent variable was daily GWI symptom severity (0–100). Treatment condition (baseline, 

placebo, lower-dose, and higher-dose) was entered as a fixed factor. The last 14 days of each 
condition were selected for analyses to permit time for clinical effects of the treatments to occur. 
A restricted maximum likelihood (REML) estimation approach was used. Statistical significance 
was set at p < 0.05 for all tests. The secondary analyses were conducted using the same approach. 
We performed a-priori power analyses using G*Power 3 [43] to estimate sample size. Each 
of the nine botanical trials was powered to detect a medium effect (Cohen’s d = 0.5) at 0.99 power 
with p = 0.05 threshold for significance. Given 56 repeated outcome measurements per participant 
(with a repeated measures correlation of 0.5), each trial required 10 individuals to obtain 0.99 
power. Our studies were not powered (0.42 predicted power) to detect small effects (Cohen’s d = 
0.25). 
 
 

A Placebo-Controlled, Pseudo-Randomized, Crossover Trial of Botanical Agents for Gulf 
War Illness: Reishi Mushroom (Ganoderma lucidum), Stinging Nettle (Urtica dioica), and 
Epimedium (Epimedium sagittatum) 
 
2. Materials and Methods 
This report is part of a larger clinical trial in which nine botanical agents were tested for 
effects in GWI. A pseudo-randomized, placebo-controlled, crossover design was used. Curcumin, 
boswellia, maritime pine, epimedium, fisetin, luteolin, reishi, resveratrol, and stinging nettle were 
tested in the larger study. In this paper, the results of reishi, stinging nettle, and epimedium are 
discussed. All procedures were approved by the Institutional Review Board of the University of 
Alabama at Birmingham (UAB: F150318011) in June 2015, and the trial was registered 
on ClinicalTrials.gov (NCT02909686) accessed on 21 September 2016. 
2.1. Participants 
Individuals screened for the study were men between the ages of 44 and 65 who met the 
Kansas GWI case criteria [34] and were deployed to the Persian Gulf region between 1990 and 
August 1991. As participants must have been at least 18 years old during the Persian Gulf War, 
the youngest possible age at the study start in 2016 was 44 years. All inclusion and exclusion 
criteria in the Kansas GWI criteria were utilized, with the exception that individuals with 
diabetes type 2 could participate. Individuals with diabetes could participate only if the disease 
was medically controlled, with hemoglobin A1C below 9%. 
Individuals were excluded from participation if they were taking opioid analgesics, anti-
inflammatory medications, nitroglycerine, or lithium, or if they reported allergies to any study 
compounds. Participants could not have blood/clotting disorders, hypotension (below 90/60 
mmHg), cardiovascular disease, rheumatologic disorders, autoimmune conditions, or acute 
infection with a body temperature over 100.4°F. Individuals could not participate if their 
laboratory blood values showed an erythrocyte sedimentation rate greater than 40.0 mm/Hr, a C-
reactive protein value greater than 10.0 mg/L, or positive rheumatoid factor or antinuclear 
antibody. 
Individuals were also excluded if they scored 16 or greater on the Hospital Anxiety and 
Depression Scale (HADS; [35]) depressive subscale by summing the depression items rated on a 
0 to 3 scale. Significant posttraumatic stress disorder (PTSD) was identified if individuals scored 
equal or greater than 50 on the 17-item PTSD Checklist—Military Version (PCL-M; [36]). 
Individuals with possible ongoing PTSD were subsequently assessed by a member of UAB’s 
Office of Psychiatric Clinical Research, using the Clinician Administered PTSD Scale (CAPS-5; 
[37]). The 30-item structured interview was used to make a final determination of current PTSD, 
using established criteria [38]. Individuals meeting criteria for current PTSD were excluded from 
the study. 
2.2. Botanicals 
Reishi was sourced from JHS Natural Products, Mushroom Science (Eugene, OR, USA) as 
a hot water and alcohol extract with 12% polysaccharides and 4% triterpene. Stinging nettle was 

procured from Nature’s Way (Green Bay, WI, USA) as pure nettle leaf. Epimedium was from 
Barlowe’s Herbal Elixirs (Palm Beach, FL, USA) as a 20% icariin extract. Procured agents were 
sent to a compounding pharmacy (Double Oak Mountain Pharmacy, Birmingham, AL, USA) for 
re-encapsulation. Materials were put in size 0 or 00 blue gelatin capsules and organized in 
weekly Cold Seal Compliance blister packs (Pharmacy Automation Supplies, Romeoville, IL, 
USA). Participants and research personnel were blinded to the botanical agent assignments. 
Reishi was administered at 1600 mg (lower dosage) and 3200 mg (higher dosage) per day. 
Epimedium was given at 500 mg and 1000 mg per day (100 mg and 200 mg of icariin, 
respectively). Stinging nettle was given at 435 mg and 1305 mg per day. All botanicals were 
administered twice per day (morning and evening), with the total daily dosage being split evenly 
between the morning and evening doses. Placebo was administered in identical gelatin capsules 
with microcrystalline cellulose filler. 
Individuals on any antihypertensive medications were not allowed to take reishi or 
epimedium, even if their blood pressure was well-controlled. Individuals were not assigned to 
take stinging nettle if they had any signs of diabetes, pre-diabetes, or were taking medications for 
diabetes. Individuals could participate in the study despite being excluded from taking one or 
more of the botanicals, as the entire list of nine botanicals had several contraindications. As 
many individuals with GWI also suffer from conditions such as high blood pressure and 
diabetes, excluding these individuals would have created a sample that is not representative of 
the general patient population. 
2.3. Study Protocol 
Individuals were screened via an online questionnaire and phone interview, followed by an 
in-person screening conducted at UAB’s Clinical Research Unit (CRU). Informed consent was 
obtained at the in-person visit, and blood samples were collected for further screening. As the 
screening involved blood tests, individuals provided informed consent before being determined 
eligible for participation. Therefore, several individuals who provided consent were not eligible 
to participate in the study. Consented individuals received a tablet device for completing daily 
symptom reports each evening for the duration of their study participation. 
Before beginning capsules, all participants completed a baseline period of 30 days. This 
period served as the baseline for all botanicals taken by the participant. Participants then were 
pseudo-randomized to receive up to three of the nine study compounds, as seen in Figure 1. A 
pseudo-randomized procedure was used to avoid contraindications with the botanicals. 
Randomization was conducted by a pharmacist. The pharmacist in charge of blinding and 
randomization was not otherwise involved in the study. The research personnel were blinded to 
the botanicals assigned to each participant. For each botanical, participants completed 30 days of 
placebo capsules, 30 days of lower-dose botanical, and 30 days of higher-dose botanical. 
Participants were blinded to all aspects of the study (the protocol and botanicals assigned). 
Research personnel were blinded to the botanicals assigned, though they were aware that placebo 
occurred first, followed by lower-dose and higher-dose treatment. 

 
Figure 1. Study event flow. Participants took three of the tested compounds in the following order: 
placebo, lower-dose botanical, and higher-dose botanical. 
Throughout their involvement with the study, participants returned to the CRU every 
month. At these visits, participants would obtain a new supply of study capsules. Blood samples 
were also obtained at visits 4, 7, and 10 to conduct safety tests. Standard clinical renal and 
hepatic panels were performed. Participants first took the lower dosage and then the higher 
dosage of the botanical so that any renal or hepatic issues could be detected before the participant 
took the larger dosage. Participants received compensation during each visit. The participants 
received a total of $1500 for attending all 11 of the study visits and completing the protocol. 
Participants who successfully completed all three botanical assignments were allowed to enroll 
in the study a second time, where they would be assigned new botanicals. 
2.4. Outcome Measures 
Participants completed self-reported symptom severity measures every evening during the 
study, using the Qualtrics Research Suite (Qualtrics, Provo, UT, USA) on a tablet. The primary 

outcome used in this clinical trial was a 0–100 item assessing overall GWI severity. The item 
read, “Overall, how severe have your symptoms been today?” The zero response was anchored 
by “No symptoms at all” and 100 was labelled as “Severe symptoms”. This generic symptom 
response was chosen because GWI sufferers can have a wide range of principal complaints. 
2.5. Secondary Outcome Measures 
To further explore the impact of botanicals on common GWI complaints, we also measured 
changes in pain and fatigue severity. Both outcomes were measured on a 0–100 scale. Pain was 
measured by the item, “Overall how severe is your pain?” from “No pain at all” to “Severe 
pain”. Fatigue was measured by “How fatigued have you felt today?” from “Not fatigued at all” 
to “Severely fatigued”. Other GWI symptom domains such as respiratory and skin issues were 
rarely endorsed by participants and were not analyzed. 
 
2.6. Statistical Analyses 
All analyses were conducted in SPSS version 24 (IBM Corp., Armonk, NY, USA). For each 
botanical, a linear mixed model (LMM) was created to test changes in the primary outcome. LMMs 
were used to accommodate the repeated outcome assessments in each condition, nested in each 
participant. Subject ID was the repeated-measures nesting variable, the repeated-measures index 
was the day in the study, and the repeated measures covariance type was compound symmetry. 
The predictor was study condition, which could take four values (baseline, placebo, lower-dose, 
and higher dose botanical). The last 14 days of each condition were included in the models. The 
final 14 days of the baseline condition at the beginning of participation were used as the baseline 
for all tested botanicals. Post hoc tests were conducted using least-squares differences. A p < 0.05 
was used for all tests. The same statistical approach was used to test the secondary outcomes of 
pain and fatigue. Nine total models are presented in this report (three botanicals tested for the 
outcomes of GWI severity, pain and fatigue). 
If a participant did not complete the protocol, any valid data were still used in statistical tests. 
However, data imputation of missing values was not used, and participants were not included if 
they never took the assigned botanical. Therefore, we did not use a complete intent-to-treat method 
for analysis. 
The three trials were powered to detect within-person fixed effects for study condition 
(baseline, placebo, lower dosage and higher dosage). Detection of a medium effect size (Cohen’s 
d = 0.5) was targeted at a threshold of p < 0.05. With 56 repeated outcome assessments per 
participant, and a repeated measures correlation of 0.5, it would require 10 individuals to achieve 
0.99 power for the main effect test.