Reference
Hodgin KS, Donovan EK, Kekes-Szabo S, et al. A placebo-controlled, pseudo-randomized, crossover trial of botanical agents for Gulf War illness: resveratrol (Polygonum cuspidatum), luteolin, and fisetin (Rhus succedanea). Int J Environ Res Public Health. 2021;18(5):2483.
Design
Pseudo-randomized, placebo-controlled clinical trial
Participants
Twenty-one male veterans aged 37 to 65 years present in the Persian Gulf region between August 1990 to August 1991 who also met the Kansas Gulf War Illness case criteria
Details
Fifty-six male Gulf War veterans consented to participate. Thirteen were excluded in screening, 1 was lost to follow-up after screening, and 3 self-withdrew before beginning any treatment. Thirty-nine remained eligible and were randomized to the botanical treatments. Fifteen were randomized to receive only botanicals not in this paper (Boswellia, Curcuma, Epimedium, maritime pine, Ganoderma, and Urtica), and 24 were randomized to receive at least 1 of the botanicals in this paper (resveratrol luteolin, and fisetin). Of the 24 in this report, 1 self-withdrew due to a swollen lymph node from a bacterial infection while taking fisetin, 1 had an adverse event unrelated to the study and self-withdrew, 1 was withdrawn for poor intervention adherence while on luteolin, and 1 was investigator-withdrawn for retrospective data reporting on resveratrol and luteolin.
Inclusion Criteria
Male, aged 37 to 65 years, present in the Persian Gulf between August 1990 to August 1991, fulfilled the Kansas Gulf War case criteria (KGW) for Gulf War Illness (GWI), well-controlled diabetes mellitus (glycated hemoglobin A1c [HbA1c] <9%), no diagnosis of cancer in the past 5 years, able to attend study site for 11 monthly visits, ability to provide a venous blood draw, successful completion of ≥80% of symptom reports during the baseline. Prophylactic aspirin at 81 mg/d was permitted.
Exclusion Criteria
Daily use of opioids or anti-inflammatory medications, use of nitroglycerine or lithium, history of anaphylaxis to any botanical in the study, hypotension (<90/60) history of cardiovascular disease, diagnosis of rheumatological or autoimmune disease, blood or clotting disorder, heart disease or stroke, current litigation of a worker’s compensation claim, inability to read or understand English, acute infection (temperature >100.4°F), positive rheumatoid factor, positive antinuclear antibody, erythrocyte sedimentation rate >40 mm/hr, C-reactive protein (CRP) >10.0 mg/L, bipolar, manic depression, schizophrenia, recent hospitalization for alcohol or drug dependence, depression or PTSD, severe depression score >16 on the Hospital Anxiety and Depression Scale (HADS), current posttraumatic stress disorder using the PTSD Checklist–Military Version (PCL-M) and the Clinician-Administered PTSD Scale (CAP), which reflected DSM-IV criteria.
Intervention
All treatments were given twice daily.
Resveratrol: low dose was 200 mg in the morning and 0 mg in the evening; high dose was 400 mg in the morning and 200 mg in the evening.
Luteolin and fisetin were evenly split between morning and evening doses. Luteolin was 200 mg for low dose and 400 mg for high dose. Fisetin was 200 mg for low dose and 800 mg for high dose.
Resveratrol was sourced from Pure Encapsulations (Sudbury, MA) to contain 20% trans-resveratrol; luteolin was sourced from API Solutions (Daphne, AL); and fisetin was sourced from VitaCost (Boca Raton, FL). Double Oak Mountain Pharmacy in Birmingham, AL, re-encapsulated the interventions in 0 or 00 opaque, blue gelatin capsules while microcrystalline cellulose was inside the same-colored placebo capsules. All capsules were placed in QUBE Weekly, 28-cavity, cold-seal-compliance blister packs by Pharmacy Automation Supplies (Romeville, IL).
The study protocol consisted of online and phone screening and, if accepted, in-person screening. Each subject was then given a placebo, low dose followed by a high dose, and then returned to a placebo before beginning the next treatment (each 28 days + 3 days). The participants tested up to 3 different botanical items in the same above dosage order.
Study Parameters assessed
Kansas Gulf War Illness case criteria, HADS, CAP, and Qualtrics Research Suite Offline Application (Qualtrics, Provo, UT) to rate daily GWI symptoms once each evening. Daily single-item measures of pain, fatigue, cognitive dysfunction, depressed mood, skin problems, respiratory complaints, and gastrointestinal issues were also rated.
Data analysis was done on SPSS version 24. Linear mixed models, test differences in self-reported symptoms severity between the 4 treatments, with the last 14 days selected for analyses to permit time for clinical effects of the treatment to occur. A restricted maximum likelihood estimation approach was used. Statistical significance was set at P<0.05 for all tests. A-priori power analysis was done using G*Power 3 to estimate sample size.
Results
The final analysis included 21 male veterans, aged 46 to 57 years. Two were assigned all 3 botanicals, 7 were assigned 2 of the botanicals, and 12 were assigned 1 of the botanicals. Nineteen (90.5%) self-identified as white, non-Hispanic, and 2 (9.5%) self-identified as black, non-Hispanic. All except for the withdrawn participants completed placebo, low dose, and high dose. One completed placebo and low-dose fisetin but discontinued after 1 week of high-dose fisetin due to nausea and gastroesophageal reflux (GERD) that resolved 1 week after discontinuation. One participant who received all 3 substances rated all GWI symptom severity as 0 and could not be included in the analysis for the main outcome question.
Of the 9 included in resveratrol analysis, baseline GWI severity was 31.8, placebo 28.3, low dose 23.2, and high dose 22.1, with the latter 2 being significantly lower than baseline, P=0.003, P=0.035, respectively. Placebo was similar to baseline, P=0.606, and there was no significant difference between low- and high-dose resveratrol, P=0.444.
Of the 10 included in the luteolin analysis, baseline GWI severity was 31.2, placebo 25.3, low dose 26.0, and high dose 24.6. All conditions were lower than baseline with placebo P=0.0001, low dose P=0.0001, and high dose P=0.0001, and low dose (P=0.718) and high dose (P=0.492) were not significantly different than placebo.
Of the 10 included in the fisetin analysis, baseline GWI severity was 41.9, placebo 41.0, low dose 40.3, and high dose 39.5 with no significant effect for fisetin (P=0.913). Neither placebo (P=0.794), nor low-dose (P=0.504), nor high-dose fisetin (P=0.616) was significantly different from baseline.
Self-reported pain severity was significantly reduced for both doses of resveratrol compared to placebo, but there was no effect on fatigue severity. There was no effect of luteolin and fisetin compared to placebo on pain or fatigue severity.
Fourteen of the 21 participants reported mild-to-moderate adverse events. Aggravation of GERD was the most common side effect, followed by migraine headache in 2 participants during the placebo, and worsening fatigue in 2 participants during high-dose fisetin. Other events included elevated creatine during fisetin, flushing during luteolin, elevated glucose during luteolin, anxiety during placebo, and headache and nausea during fisetin.
Key Findings
Resveratrol, luteolin, and fisetin were examined in low and high doses and compared to baseline and placebo in 21 Gulf War veterans who met inclusion criteria and Kansas Gulf War case criteria. Of the 3 interventions, only resveratrol in both low dose (200 mg) and high dose (600 mg) had any significant effect on GWI severity and pain. None of the interventions had any effect on GWI fatigue.
Practice Implications
The Gulf War began after Iraq invaded Kuwait in August 1990. What followed was a coalition of 35 countries that for 42 days initiated relentless air attacks followed by 100 hours of on-the-ground combat. Some 4,600 Canadian forces were involved with no casualties; 148 American casualties occurred in combat, and 145 occurred from disease and misadventure. In January 1992, beginning in Indiana, an unusual illness was reported that became labeled as “Gulf War Syndrome,” now known as Gulf War Illness.1 While symptoms varied, poor memory, fatigue, muscle and joint aches, rashes, and diarrhea tended to predominate.1 Most, if not all, of the members of the coalition reported veterans who suffered from GWI. Of the 697,000 American troops involved in the conflict, 175,000 to 250,000 veterans continue to be affected by GWI. Possible causes included oil well fires, enemy chemical weapons destroyed near Khamisiyah (sarin nerve gas), various diseases and microbes (viruses, streptococcal species, microsporidia, leishmania, mycoplasma, aflatoxin), vaccinations against anthrax and botulism including their adjuvants, and psychological and cultural factors associated with the after-effect of combat and war.1
A 1997 report on 20,000 American Gulf War veterans found 18.6% had musculoskeletal disorders, 18.3% psychiatric disorders, 17.8% fatigue and insomnia, 31% joint pain, 19% tension headaches, 17% dysthymia, and 80% had conditions commonly found in a similarly aged adult population.2 Fortunately, GWI does not increase mortality. It does, however, increase hospitalizations.1 No serious illnesses were reported 8 years after the war, and no increased incidence of birth defects was found in a large study of Gulf War veterans.3 Other findings include increased asthma, depression, PTSD, alcohol abuse, anxiety, and sexual discomfort.4 Factor analysis did not confirm the uniqueness of Gulf War syndrome compared to non–Gulf War military veterans.5,6 War is stressful, thus the concepts of memes, where a psychosomatic illness can be transmitted from one psychologically susceptible individual to another, may also be a factor.7,8
Of the 697,000 American troops involved in the conflict, 175,000 to 250,000 veterans continue to be affected by GWI.
Resveratrol, a polyphenol stilbene, is found in over 70 plant species, acts against bacteria and fungi, and possesses antioxidant and antitumor properties, as well as anti-inflammatory, cardioprotective, vasorelaxant, phytoestrogen (agonist), and neuroprotective effects.9 The trans form is more prevalent in nature, more bioactive, and has greater anticancer properties. Bioavailability is low, but nanoformulations can improve this by a factor of 3.5, and methylated-beta-cyclodextrins improve its solubility by a factor of 400.9 As an anti-inflammatory, resveratrol can target COX, 5-LOX, protein kinase B, COX-1 and COX-2, nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6), and macrophage inflammatory protein-2, as well as other inflammatory pathways.9 In high doses, it may interact with CYP3A4, P-glycoprotein, multidrug resistance-associated protein-2, and organic anion transporters.9
Luteolin, a flavonoid often found in the glycoside form in many fruits, vegetables, and plants, possesses antioxidant, anticancer, antimicrobial, neuroprotective, cardioprotective, antiviral (including SARS-CoV-2), and anti-inflammatory effects, but has low bioavailability.10
It is also a selective antagonist of the vasopressin receptor and dopamine D4 receptor, as well as potentiation of GABA-A with potential for ameliorating neurodegenerative pathologies.10,11 In humans it undergoes conjugation and sulphation. Nanoformulations and microemulsion with phospholipid matrix enhance solubility, bioavailability, and efficacy.10
Fisetin, a flavonoid found in fruits and vegetables (strawberry, apple, persimmon, grape, onion, and cucumber), has neurotrophic, antioxidant, anticancer, anti-inflammatory effects, and an average daily intake of 0.4 mg.11 It can increase intracellular glutathione, inhibit matrix metalloproteinase-1, and induce apoptosis in lung, colon, prostate, pancreas, and melanoma cell lines.11
The interventions chosen for this trial were biochemically related as polyphenols. Polyphenols include hydroxybenzoic acids, hydroxycinnamic acids, lignans, flavonols, flavones, isoflavones, flavanones, anthocyanidins, flavanols, stilbenes, and flavonoids, with similar properties such as neuroprotective, anti-inflammatory, anticancer, and antioxidant effects.12 Polyphenol content in the average North American diet is low, ranging from less than 1 mg to perhaps 25 mg/day, but in European countries can exceed 200 mg/day.12 Polyphenol content varies widely within a single food item depending on varietal type, environmental factors during growth and maturation, time of harvest, and handling, processing, and storage methods (oxidation enhances black tea polyphenols, browning reduces polyphenols, peeling removes most polyphenols).12
Absorption occurs in the small intestine and then the liver by methylation, sulfation, and glucuronidation with elimination via the kidneys.12 Circulating polyphenols are highly bound to albumin, can penetrate various tissues (a process poorly understood), and then are eliminated in urine and bile.12 The role of one’s microbiome on these processes is also poorly understood, but likely very important, and poorly studied except for conversion of daidzein to equol in Japanese men.12,13 Polyphenol combinations may increase intestinal absorption and bioavailability, with much of the focus on quercetin-resveratrol-curcumin combinations with and without piperine.14
The investigators did not justify the low dose and high dose for the 3 polyphenols used in this trial, did not supply references to any previous clinical trial data or studies, or explain why they selected these 3 polyphenols compared to the many other polyphenols with similar properties. Their brief review notes that all 3 are anti-inflammatory, antioxidant, and neuroprotective and, thus, potentially beneficial to Gulf War veterans. In prior studies, resveratrol has been used at 2 to 3 g/d for 2 to 6 months with possible gastrointestinal upset.15 Luteolin was given to children with autism spectrum disorders at 100 mg/10 kg for 26 weeks with food.16 The Mayo Clinic has a phase 2 trial, ending in 2023, where women are receiving 20 mg/kg/d of luteolin for 3 days, repeated every 28 days over 20 weeks.17 The arbitrary doses in this GWI trial may add little to our knowledge on effective polyphenol dosing, especially given that Gulf War Illness is still a poorly defined condition with an unknown etiology and lacks standardized criteria for diagnosis.1,2
What this trial did reveal is that resveratrol at 200 mg and 600 mg significantly reduced pain compared to baseline and placebo. Luteolin at 200 mg and 400 mg, and fisetin at 200 mg and 800 mg, had no effect on GWI pain or fatigue and resulted in no improvement in daily symptoms compared to baseline or placebo. Although adverse events were mild to moderate, 6 occurred with resveratrol, 7 with luteolin, and 15 with fisetin. A review of the named commercial sources for luteolin and fisetin yielded no information on the biological source of the luteolin and fisetin, respectively. The study’s title suggests fisetin was from Rhus succedanea, but a review of the literature found fisetin primarily in Rhus verniciflua and few other Anacardiaceae Rhus species.18 Plants in the Anacardiacea family are a common allergen to large numbers of the human population; was this a factor in the adverse events? Pure Encapsulations stated on their website that their 100-mg trans-resveratrol is from red wine, Vitis vinifera, polyphenols, and grape seed (Vitis vinifera) extract, and Polygonum cuspidatum root, all standardized to trans-resveratrol. Why the authors cited only Polygonum cuspidatum in the title is unknown. In my review of their parallel paper on Curcuma longa, Boswellia serrata, and Pinus pinaster extracts for GWI, the authors wrote in the limitations section that “because of the lack of evidence indicating which dosages of these botanical compounds might be effective to provide symptom relief, we tested 2 different dosages for each compound, following best practices for naturopathic medicine.”19 No reference was provided for this and related statements. Their literature review on dosing of these latter 3 botanicals was as scant as in the paper reviewed in this report.
The article was well-written, but exclusion criteria were found in multiple portions of the paper. Justification of both dosages and selection of these 3 polyphenols was absent. The exact botanical sources of the compounds in the title do not correlate with the identified commercial sources. Purity analysis of the botanical compounds, critical for study duplication and additional research and a National Institutes of Health requirement, was absent in this paper. The research team is from the Department of Psychology, University of Alabama, Birmingham, who partnered with a community pharmacy that provided compounding services for the study preparations; a botanical expert was not part of the team. The questions around the cause of GWI and its effective treatment remain unanswered and were not addressed in this paper or the latter paper.
Summary
Resveratrol, luteolin, and fisetin, common food polyphenols, were examined in low and high doses and compared to baseline and placebo in 21 Gulf War veterans who completed the Kansas Gulf War case criteria. Of the 3 interventions, only resveratrol in daily low dose (200 mg) and daily high dose (600 mg) for 28 days had a significant effect on GWI symptom severity and pain. Luteolin at 200 mg and 400 mg daily and fisetin at 200 mg and 800 mg had no significant effect on GWI severity after 28 days each. None of the 3 interventions had any effect on GWI fatigue. A solution to Gulf War Illness, both diagnostic and therapeutic, remains to be found.
