A botanical product containing cistanche and ginkgo extracts improves chronic fatigue syndrome: a randomized, double-blind, placebo-controlled study

Background Dietary therapy may be benecial in alleviating symptoms of chronic fatigue syndrome (CFS), a disorder characterized by extreme fatigue with no underlying medical condition. The aim of this study is to evaluate the protective effect of a botanical product containing cistanche (Cistanche tubulosa (Schenk) Wight) and ginkgo (Ginkgo L.) extracts on adults with CFS in a randomized, double-blind, placebo-controlled clinical trial.

We previously developed a botanical product containing the extracts of cistanche and ginkgo and proved the health claim of improving memory in healthy subjects [8,9]. In this randomized, double-blind, placebocontrolled study, we tried to test the protective effect of the product in subjects with CFS.

Trial design
This randomized, double-blind, placebo-controlled clinical trial was conducted in Community Hospital of Baoshan District, Shanghai, China. The subjects were identi ed from a hospital-owned database. A total of 190 subjects were initially enrolled in the study at baseline, and strati ed by age and genders, either 1) males aged 35-50 years old, females aged 35-40 years old or 2) males aged 51-60 years old, females aged 41-60 years old. The subjects were randomly assigned to 3 groups (group 1: placebo; group 2: low dose of the product; group 3: high dose of the product). The subjects consumed their designated products once daily for 60 days. During the study, 15 subjects withdrew, leaving 175 subjects in the per protocol (PP) analysis (Fig. 1). The total dropout rate was 7.9%. Blood samples and questionnaires of Chalder fatigue, quality of life (QOL), and sexual life quality (SLQ) were collected at baseline and postintervention (day 60). Informed consent was obtained from all subjects. This study was approved by the Institutional Review Board (IRB) of Shanghai Nutrition Society and registered at ClinicalTrials.gov (NCT02807649).

Intervention
The test products were tablets containing cistanche extract (300 mg for low dose group, 450 mg for high dose group; standardized with 7.55% echinacoside) and ginkgo extract (120 mg for low dose group, 180 mg for high dose group; standardized with 2.85% total avanol glycosides). All the test products and placebo were manufactured in a Good Manufacturing Practice pilot plant (Nutrilite) under quality assurance considering the presence of microorganisms, heavy metals, and pesticide residue.

Inclusion/exclusion criteria
The inclusion criteria were as follows: 35-60 years old male or female volunteers, who were diagnosed as CFS by study physician. More details on the assessment of CFS are provided in the following section. Subjects were excluded if their body mass index was ≥ 28; they had u/symptoms of viral infections within three months before the rst visit to the site; had history of or be diagnosed of any of the following diseases that might affect the study results: gastrointestinal disorders, skeletal muscle dysfunction, hepatopathy, nephropathy, endocrine disease, blood disorders, respiratory and cardiovascular diseases; were currently taking medicine for cardiovascular or metabolic disease; were current smoker; were current or previous alcohol abuser; were pregnant or lactating; were currently having or had any medical or nutritious therapies, including taking protein supplements or nutrients that promoted exercise capacity within 3 months before screening; had lost or gained weight over 5 kilograms within 3 months before screening; had hospitalizations within 3 months before screening; had participated in similar clinical trials within 6 months before screening; or were not willing to comply with the study procedures.

CFS assessment
The CFS was assessed at the enrollment in accordance with the diagnosis criteria published by U.S. Centers for Disease Control (CDC) [2]. It was diagnosed as CFS when 1) the continuous or recurrent attacks of unexplained severe fatigue lasted for more than 6 months and it could not be alleviated after su cient rest, resulting in substantial reduction in previous levels of occupational, educational, social, or personal activities; 2) Four or more of the following symptoms were concurrently present for over 6 months: impaired memory or concentration; sore throat; tender cervical or axillary lymph nodes; muscle pain; multiple joint pain; new headaches; unrefreshing sleep; and post-exertional malaise. After the intervention, each of above symptoms was assessed again for evaluation of the product e cacy. The overall effective evaluation was based on the sum of cured cases (all symptoms were relieved) and relived cases (some symptoms were relieved).

Chalder fatigue questionnaire
The Chalder fatigue questionnaire has been widely used to measure the extent and severity of physical and mental fatigue in clinical and epidemiological populations. The original Chalder fatigue scale consisted of 14 items designed to measure fatigue severity over the past 3 months [10]. In the following studies, 3 items were dropped from the 14-item version, resulting in a revised 11-item version [11]. For the 11 items, there are two subscales to evaluate two types of fatigue: physical and mental. The score was rated on a four-point Likert scale (0 = not at all; 1 = the same as usual; 2 = more than usual; 3 = much more than usual). Higher score indicates a greater fatigue.

SLQ questionnaire
The SLQ questionnaire was previously developed to evaluate the quality of sexual life of the subjects [14].
The original 10 item scores (ranging from − 4 to + 4 in value) were converted to a 0 to 8 scale score by adding 4 to each recorded response. The 0 to 8 scale score was converted to a standardized score by dividing that score by 8 and multiplying the result by 100. The SLQ scale score for a respondent was computed as the mean of the 10 standardized item scores.

Biochemical analysis
Blood ammonia, glucose, free fatty acid, creatine kinase, C-reactive protein, lactic acid, estradiol (only for female), and testosterone (only for male) were determined using commercially available kits according to the manufacture's instruction (Jiancheng, Nanjing, China).

Sample size calculation
Sample size was chosen based on a signi cant level of 0.016 (0.05 after adjustment for three multiple comparisons), 80% power and expected proportion of responders of 0.5 and 0.8 for placebo and intervention groups, respectively. Sixty-three participants for each group was to give 53 completed participants with accounting for 15% dropout rate. A total number of 190 subjects were enrolled to ensure at least 159 subjects in completing the study.

Statistical analysis
Statistical analyses were completed using SAS 9.4 (SAS Institute Inc, Cary, NC, USA). All statistical tests of our hypothesis were 2-sided and performed at the 0.05 signi cance level. Means and standard deviations were summarized for normally distributed continuous variables, medians were provided for non-normal variables, and frequencies and percentages were provided for categorical variables. Evaluations of product effect were performed using chi-square test or analysis of covariance (ANCOVA) followed by pair-wise group comparison for the variables with signi cant group difference.

Characteristics of subjects
Summary statistics of baseline subject characteristics were shown in Table 1. No group difference was observed at baseline among groups in demographic data, including gender, age, body weight, height, body mass index, body temperature, and blood pressure.

CFS assessment
The frequency and percent of subjects with cured or relieved CFS, as well as the total effective cases after 60 days of product intervention were shown in Table 2. Nine (15.5%) subjects with CFS were cured in low dose group, 11 (18.6%) in high dose group, while 0% in placebo group. Some of the symptoms were relieved in 33 (56.9%), 37 (62.7%) and 16 (27.6%) subjects in low dose, high dose, and placebo group, respectively. These resulted in an overall effectiveness of 72.4% in low dose group and 81.4% in high dose group (P < 0.001 compared to 27.6% in the placebo group). Among all 8 individual symptoms, impaired memory or concentration, muscle pain, unrefreshing sleep, and post-exertional malaise were signi cantly (P < 0.001) relieved in the subjects of low dose and high dose group compared to those in the placebo group after product intervention (Supplementary Table 1).

Chalder fatigue questionnaire
The score of each question in the 11 item-Chalder fatigue questionnaire and the total physical and mental fatigue scores were summarized in Table 3. After 60 days of product intervention, both low dose and high dose groups had signi cantly lower fatigue scores compared to the placebo group for all physical and mental fatigue questions (P < 0.001). Signi cant difference was also observed for low dose group versus high dose group in total physical fatigue score (P < 0.01) and Q3-sleepy or drowsy feeling score (P < 0.01), suggesting superior effect of high dose of the product.

QOL and SLQ questionnaires
The individual and area (PHYS, PSYCH, SOCIL, ENVIR) scores of the QOL questionnaire were shown in Table 4. After 60 days of product intervention, signi cant group difference was observed in Q1-overall rating of quality of life, Q2-overall satisfaction of health; Q3, 10, 16 and 17 under the PHYS area, as well as the area score; Q7 and 26 under the PSYCH area; and Q21 under the SOCIL area.
The individual and scale score of the SLQ questionnaire with strati cation of gender were shown in Table  5. For male subjects, there were signi cant group difference in all 10 items and scale score after the product intervention. For female subjects, signi cant group difference was observed in all items except ease of insertion and partner's overall pleasure of lovemaking.

Blood biomarkers
The concentrations of blood biomarkers were comparable at baseline (Table 6). After the product intervention, both low dose group and high dose group had signi cantly lower blood ammonia (P < 0.01 and P < 0.05, respectively) and blood lactic acid concentration (P < 0.05 and P < 0.01, respectively) than the placebo group. The intervention of product led to a trend of increase in testosterone, although the difference did not reach statistical signi cance (P = 0.0743).
Logistic regression was used to evaluate the effect of the change in blood biomarkers on the cure and relief of CFS symptoms (overall effectiveness). The results were shown in Table 7. The change in blood glucose and lactic acid concentration were signi cantly associated with the effectiveness for CFS symptoms in both univariate analysis (P = 0.0058 and 0.0061, respectively) and multivariate analysis (P = 0.0075 and 0.0108, respectively). Pearson correlation coe cient and signi cance test were further used to evaluate the correlation between the changes of blood biomarkers and the changes of the Chadler fatigue questionnaire scores (Supplementary Table 2). The change in blood lactic acid concentration was signi cantly correlated to the total physical fatigue score (R = 0.1639, P = 0.0302), Q1-problems of tiredness (R = 0.1886, P = 0.0166), and Q2-need to rest more (R = 0.2254, P = 0.0027).

Discussion
The etiology of CFS is still unknown, therefore, the diagnosis of CFS is made in accordance with symptom-speci c criteria [5]. The CDC criteria (1994) is now the most frequently used case de nition for CFS [2]. In the current study, we followed the CDC criteria to enroll CFS subjects, and after the intervention of our product, there was signi cantly higher effective (cure and relief) rate in the product group compared to the placebo. Among all 8 mentioned symptoms in the CDC criteria, impaired memory or concentration, muscle pain, unrefreshing sleep, and post-exertional malaise were signi cantly relieved in the subjects consuming our product. In order to quantitively measure the fatigue, many questionnaires, such as Chalder fatigue questionnaire, checklist individual strength, fatigue severity scale, multidimensional fatigue inventory, and fatigue index symptom, were reported previously [15]. Among them, Chalder fatigue questionnaire is the most intensively used [10,11]. By using the Chalder fatigue questionnaire, we observed signi cantly improved physical and mental fatigue scores in the product group after the intervention.
QOL is an important outcome evaluated in the CFS research [5]. In the currently used WHO-QOL questionnaire, 26 items are divided into four sections, PHYS, PSYCH, SOCIL, ENVIR [12,13]. Our product could improve 4 items (Q3, 10, 16, and 17) in PHYS, 2 items (Q7 and 26) in PSYCH, 1 item (Q21) in SOCIL, but no item in ENVIR. Among all the items, Q3 was related to muscle pain, Q7 was related to impaired memory or concentration, and Q16 was related to unrefreshing sleep. These data were consistent with those from the CFS assessment. Poor sleep quality was widely reported in patients with CFS [16], and nocturia was associated with poor sleep quality [17,18]. In our study, we observed a signi cantly (P < 0.01) improved frequency of nocturia in the subjects of the product group (data not shown). The bene cial effect of our product on the nocturia mainly contributed to the cistanche, a well-known kidney yang-tonifying herb of TCM [7,19]. Improving memory is the original health claim of our product [8,9]. In the current study, we con rmed the bene cial effect on the memory and concentration, which largely depended on the ginkgo [20, 21]. Muscle pain was another improved symptom in this study, and it was the rst report to show the protective effect of cistanche and ginkgo on the muscle pain.
Considering the kidney yang-tonifying effect of cistanche, and kidney is a critical organ related with sexual function from the TCM perspective [22], we additionally collected SLQ questionnaire in the current study. Different from the male subjects who achieved all items improved, female subjects failed 2 items, which were ease of insertion and partner's overall pleasure of lovemaking. These interesting results suggested that our product was possibly more effective for men, especially the erectile function. We further determined the sexual hormones and observed a trend of increase in testosterone after the intervention, instead, no signi cance was found in estradiol. These data were consistent with abundant of previously reported in vivo data, which showed that the cistanche extract increased testosterone level and improved reproductive dysfunction in rats [23][24][25][26]. No report about the effect of cistanche on estradiol for now.
Lactic acid is formed and accumulated in muscle under the condition of high energy demand and insu cient oxygen supply [27]. The accumulation of lactic acid is very common in both CFS patients and animals [28,29]. It was previously reported that cistanche and ginkgo extracts could reduce the blood lactic acid level in mice and rats [30,31]. In our RCT, we also observed a signi cant decrease of blood lactic acid level in the product group. Furthermore, the change in lactic acid concentration was signi cantly associated with the effectiveness for CFS symptoms in both univariate analysis and multivariate analysis. Pearson correlation coe cient and signi cance test also showed that the change in lactic acid concentration was signi cantly correlated to that in total physical fatigue score of the Chalder fatigue questionnaire. All data suggested that blood lactic acid could be a potential biomarker to predict the prognosis of CFS.
The limitation of is the study is the deterministic nature of formula research, making it di cult to attribute bene cial effects on CFS to any speci c constituent, therefore, we cannot estimate the additional improvement that the combination provided over cistanche or ginkgo alone. In addition, the assessment of CFS and life quality was performed by subjective questionnaires, instead of objective tests, which will be needed to con rm the conclusion in the future study.

Conclusions
We carried out a randomized, double-blind, placebo-controlled study using a botanical product containing cistanche and ginkgo extracts, and demonstrated that the product could relieve the symptoms of CFS, as well as improve the scores of Chalder fatigue, QOL and SLQ questionnaires. These ndings suggest a nutritional supplementation approach with botanical extracts as an alternative strategy in mitigating CFS. The study was conducted according to the guidelines laid down in the 1964 Declaration of Helsinki and its later amendments. All procedures involving human subjects were reviewed and approved by the IRB of Shanghai Nutrition Society. Written informed consent was obtained from all participants.

Consent for publication
Not applicable.

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.

Competing interests
The authors declare that they have no competing interests.

Funding
Not applicable.
Authors' contributions SL and JD designed research; CH, DV and MM conducted research; CW and AY provided resources; JK and BL analyzed data; LC and SL validated the analysis; JK and CH wrote the paper; JD had primary responsibility for nal content. All authors read and approved the nal manuscript. Data are frequency (%) or mean ± standard deviation (SD). Group differences were evaluated using a chi-square test or one-way analysis of variance (ANOVA). N/A, not applicable.  Data are frequency (%). Product effect was evaluated by chi-square test. Post-hoc test with Bonferroni adjustment was applied to variables with significant group difference for further pair-wise group comparison. ** P < 0.01, *** P < 0.001 compared with placebo group. Data are mean ± SD. Product effect was evaluated by analysis of covariance (ANCOVA). Post-hoc test with Bonferroni adjustment was applied to variables with significant group difference for further pair-wise group comparison at day 60. *** P < 0.001 compared with placebo group, ## P < 0.01 compared with low dose group. Data are mean ± SD. Product effect was evaluated by ANCOVA. Post-hoc test with Bonferroni adjustment was applied to variables with significant group difference for further pair-wise group comparison at day 60. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with placebo group.
The item score of Q3, Q4 and Q26 were reversed (calculated as 6 minus raw item score).  Data are mean ± SD. Product effect was evaluated by ANCOVA. Post-hoc test with Bonferroni adjustment was applied to variables with significant group difference for further pair-wise group comparison at day 60. * P < 0.05, ** P < 0.01 compared with placebo group.
Estradiol concentration data was highly skewed. Median was reported and non-parametric method was used for group comparison. Logistic regression was used to evaluate the associations between product effectiveness (cured and relieved) and the changes in blood biomarkers after the intervention. OR, odds ratio; CI, confidence interval; N/A, not applicable. Figure 1 Flowchart of the clinical trial

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