Hematologic and Serological Investigation of Effect on Gyeongokgo in Healthy Individuals : a Randomized, Subject-assessor-blind, Placebo-controlled, Single-center Pilot Study

Abstract

There are no published data on Gyeongokgo (GOK) safety or efficacy despite being commonly use. The Gyeongokgo (GOK) is commonly used in traditional Korean medicine to promote a health qi and blood, but their objective data was not sufficient in clinical field. To investigate the safety and efficacy of GOK with hematologic and serologic testing and the change of the quality of life in healthy individuals. Randomized, subject-assessor-blind, placebo-controlled, single-center pilot study Participants and Interventions 29 healthy volunteer subjects were randomly placed into the GOK group (n = 20) or placebo control group (n = 9) and instructed to take one treatment packet (GOK or placebo) twice daily for 4 weeks. Subjects were assessed using the Fatigue Severity Scale (FSS) and Short Form 36 Health Survey (SF-36) and underwent hematologic and serologic tests and body composition analysis. The FSS total score (p = 0.093) and SF-36 general health index (p = 0.002) were improved following treatment in the GOK group. Post-treatment thyroid-stimulating hormone levels were increased in the GOK group compared with pre-treatment levels (p = 0.0042). C-reactive protein levels decreased (p = 0.0256) in the GOK group compared with that the placebo group. In time-series tests, GOK did not affect post-prandial serum triglycerides, glucose, insulin, or C-peptide levels. Notably, elevations in serum fasting triglycerides at 2- (p = 0.0333) and 4-hours (p = 0.0414) post-prandial were lower than those in the placebo group. GOK reduced fatigue levels and did not significantly affect laboratory test results performed to measure safety, serum glucose, and lipid profiles. Post-meal triglyceride levels were effectively reduced with treatment.

Introduction

Gyeongokgo (GOK) is a prescription recorded in the Donguibogam that consists of four ingredients: ginseng (Panax ginseng CA Meyer, root), poria (Poria cocos [Schw.] Wolf), Rehmannia glutinosa (Rehmannia glutinosa var. purpurea), and honey (Apis indica Radoszkowski). Clinically, GOK is widely used in patients who have been weakened by surgery or have reduced immune function and as a health restorative by students, office workers, and the elderly. Several experimental studies have reported its effects on preventing osteoporosis ¹⁾ and improving atopic symptoms ²⁾ , as well as its anti-inflammatory ³⁾ and anti-cancer effects ⁴⁾ . To date, the only studies on GOK have been conducted on restricted subjects or variables, including reports of its beneficial effects on athletes ^5,6) . There are no published data on GOK safety or mechanism of action. In South Korea, Western medicine and Traditional Korean medicine are dichotomized, and patients often take both kinds of medicine simultaneously. This means there is a particular need for basic data on the pharmacologic activity and safety of traditional Korean medicines. We conducted the randomized clinical trial including 29 healthy 20-year-old to 40-year-old subjects using hematologic and serologic tests, urinalysis, body analysis and questionnaires, before and after 4 weeks of GOK or placebo treatment. The aim of this study was to acquire objective data about the hematologic and serological test and questionnaires of this representative restorative used in traditional Korean medicine.

Materials and Methods

1. Study subjects

This study was a 4-week-long, single-center, subject-assessor-blind, placebo-controlled, parallel, randomized clinical trial. Before starting the study, approval was granted by the Catholic Kwandong University International St. Mary’s Hospital Institutional Review Board(IRB) of the under project number IS14TISF0016. This study was also registered as KCT0002652 in Clinical Research inform action Service(CRIS). Subjects were physically and mentally healthy males and females aged 20 to 40 who had seen one of the clinical trial flyers posted in the hospital between March and April 2015 and had willfully consented to participate(Fig. 1). The following exclusion criteria were applied: ¹⁾ underlying diseases including hypertension, hepatitis, or tuberculosis; ²⁾ a history of endocrinologic or hormone disorders; ³⁾ a history of hepato-pancreato-biliary or renal disorders; ⁴⁾ a history of impaired blood glucose regulation (i.e., impaired fasting glucose, impaired glucose tolerance, or diabetes); ⁵⁾ steroid or hormonal drug use less than 1 month before the start of the study; ⁶⁾ pregnancy or breastfeeding; and ⁷⁾ any abnormal findings that might possibly have impacted the study results. The researchers first confirmed any matters that contravened the clinical trial conditions via a phone call or visit to each subject. Following this, a total of 32 subjects (16 male and 16 female) underwent screening including hematologic tests, urinalysis, electrocardiography, and a chest x-ray. The 29 subjects who passed the screening were randomly allocated by the random number table to a GOK treatment group of 20 subjects (9 males and 11 females), or a placebo control group of 9 subjects (4 males and 5 females). The probability of random allocation to the GOK group was set at 2:1 to encourage participation. Later, the subjects were gathered at a pre-designated location; provided with identical meals; and subjected to a questionnaire, hematologic tests, urinalysis, and body composition analysis. Subsequently, the subjects were asked to complete a daily medication diary to assess compliance, use of other medicines, and alcohol consumption. At the end of the 4-week treatment, the subjects were again gathered, provided with identical meals, and subjected to the same questionnaire and tests as at the first visit.

2. GOK and placebo preparation

GOK was manufactured according to methods recorded in the Donguibogam ⁷⁾ under the management and supervision of a specialist in traditional Korean pharmacology at the external manufacturer of traditional medicines associated with our hospital. After pulverizing 200 g poria and 100 g ginseng, these were thoroughly mixed with 1056 g Rehmannia glutinosa juice and 704 g honey. This mixture was poured into an earthenware jar, and the opening was sealed with five layers of oiled paper and one layer of silk fabric. The pot was heated for 72 hours using the bain-marie method in a copper pot over a mulberry wood fire and then cooled for 24 hours in cold well water. It was then heated for another 24 hours using the same method, and cooled for 24 hours before being made into its final, solid form and packed into a small earthenware jar. Finally, 1100 g GOK mixture was added to 2600 mL distilled water and heated for 10 minutes in a slow boiler used to manufacture herbal medicines. The 3600-mL volume of GOK liquid obtained was divided into 30 packets of 120 mL each, and the packets were sealed.

The placebo was prepared by adding 4000 mL distilled water to 800 g black rice powder and boiling for 30 minutes while mixing thoroughly. Disaccharide (300 g) was added, and the mixture was reheated while stirring thoroughly for another 5 minutes. The resulting placebo was divided into 30 120-mL packets that were then sealed.

3. Administration method

Subjects in each group drank one individually sealed 120-mL packet of liquid GOK (GOK group) or liquid placebo (control group) twice a day after breakfast and dinner, for 4 weeks.

5. Outcome measurements

1) Questionnaires

The Fatigue Severity Scale (FSS) is an instrument for assessing fatigue that was developed by Krupp et al 8) in 1989. It consists of nine items each scored on a 7-point Likert scale. It can easily be used in medical consultations and has shown strong validity and reliability in various fatigue-related disorders ^9,10) .

The 36-Item Short Form Survey (SF-36) is a representative instrument for assessing quality of life (QOL) comprised of 36 questions, divided into the three domains of functional status, well-being, and overall evaluation of health ¹⁰⁾ . Study subject QOL was assessed based on FSS and SF-36 scores before and after the study period. Both questionnaires can be viewed in the Appendix. We used the Korean versions of the assessments.

2) Laboratory tests for hematologic and immunologicresponses

Blood samples were obtained from subjects’ antecubital veins after a 12-hour overnight fast, centrifuged, and stored at -80℃. Blood samples were analyzed with an automated blood cell counter (Sysmex XN (XN) modular system, Sysmex, Kobe, Japan), Beckman Coulter AU5800 chemistry analyzers (Beckman Coulter Inc., Brea, CA, USA), and Unicel DXI 800 chemiluminescent enzyme immunoassays (Beckman Coulter Inc.). Glycosylated hemoglobin (HbA1C) levels were measured by high-performance liquid chromatography using a HLC-723G8 analyzer (Tosoh Co., Tokyo, Japan). Routine urinalysis was performed on a US-3100Rplus analyzer (Eiken Chemical Co. Ltd., Tokyo, Japan) in chronological order, and urine sediments were subjected to automated morphologic examination using a laser-based fluorescent flow cytometer, UF-1000i (Sysmex Co., Hyogo, Japan). For cytokine analysis, plasma samples were collected using EDTA, centrifuged, and stored at -8 0℃. Plasma cytokine levels including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, interferon (IFN)-r, IL-2, and IL-10 were determined using a Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel (Millipore Corp., Billerica, MA, USA) following the manufacturer’s kit-specific protocols. All samples were assayed in triplicate, and the mean absorbances were calculated from standard curves.

3) Body composition tests

The participants’ body compositions were measured using the Inbody 770 analyzer (Inbody Co., Ltd., Seoul, Korea) after vital sign stabilization.

4) Adverse event monitoring

Adverse events during the trial were assessed through safety assessments and laboratory tests. All adverse reactions that occurred throughout the intervention and follow-up periods were evaluated as World Health Organization Adverse Reaction Terminology, and classified in 1-5 grades according to the Common Terminology Criteria for adverse Events (version 4.03).

5) Measurement of compliance and T

he subject who was less than 70% with compliance were dropped out.

6) Assessment of blinding

There was no assessment on blinding success.

7) Statistical analysis

Statistical analyses were performed by a statistician who was blinded to the subjects’ status. We used a paired t-test or Wilcoxon signed-rank test for changes within the groups and an independent two-sample t-test or Mann-Whitney U test (Wilcoxon rank sum test) for inter-group differences following treatment. All statistical analyses were performed using SAS v9.1.3 (SAS Institute Inc., Cary, NC, USA). P values less than 0.05 were considered statistically significant.

Fig. 1. Study flow chart (ITT, intention to treat; PP, per protocol).

Results

Of the 32 subjects originally enrolled in this study, 3 were excluded due to abnormal hematologic findings during screening, and the remaining 29 subjects were included in the final analysis. There were no significant differences in general characteristics between the two groups(Table 1).

Table 1. The General Characteristic of the study

The FSS questionnaire results showed no significant difference in scores between the GOK and control groups following the treatment period. However, after the treatment period, the GOK group did show a statistically significant decrease in overall score, as well as for the following specific items: 1) “I lose motivation when I am fatigued,” 2) “I become tired when I exercise,” 3) “I become tired easily,” 4) “I engage in less physical activity because of fatigue,” and 5) “I often experience problems due to fatigue”(Table 2).

Table 2. Fatigue severity scale results after the duration of the clinical trial

The SF-36 was administered to assess QOL, and both intra- and inter-group analyses were performed. Ten different items were evaluated, consisting of the eight individual items: PF (physical functioning), RP (rolelimitations – physical), BP (bodily pain), GH (general health), VT (vitality), SF (social functioning), RE (role limits – emotional), and MH (mental health), in addition to the physical component summary (PCS = PF + RP + BP + GH) and mental component summary (MCS = VT + SF+ RE + MH). Apart from general health, which significantly improved after GOK treatment, there were no significant differences(Table 3).

Table 3. The 36-Item Short Form Survey Results after the Duration of clinical trial

Table 4 summarizes the inter- and intra-group differences in hematologic and serologic findings following treatment. There was no statistical difference between the two groups except that the GOK group had lower C-reactive protein (CRP) levels after treatment (P = 0.0256). In the intra-group comparison, the GOK group showed significant decreases in white blood cell and albumin levels (P = 0.049 and P = 0.023, respectively), and significant increases in creatinine and thyroid-stimulating hormone (TSH) levels (P = 0.006, and P = 0.004, respectively). The control group showed a significant decrease in albumin (P = 0.042) and a significant increase in free thyroxine (T4) and TSH (P = 0.014 and P = 0.039, respectively). However, in the individual data, five participants (four and one in the GOK and control groups, respectively) showed values higher than the reference ranges in liver function tests for aspartate aminotransferase (AST), alanine aminotransferase (ALT), or gamma-glutamyl transferase (GGT).

Table 4. The 36-Item Short Form Survey Results after the Duration of clinical trial

Regarding post-prandial laboratory findings for serum glucose, triglycerides (TG), C-peptide, and insulin levels following the treatment period, the GOK group showed a significant decrease in TG at 2- and 4-hours post-prandial. There were also significant inter- and intra-group differences regarding the change at each measurementtime point from before to after the trial. Among the five subjects who had a reduction in TG of at least 50 mg/dl, all five had a decrease in TG 4 hours after eating, three had a decrease at 2 hours, and one had a decrease at fasting (Table 5).

Table 5. The laboratory findings for serum glucose, TG, C-peptide, and insulin level after meal after the duration of the clinical trial

Additionally, we investigated the regulatory effect of GOK on inflammatory-related cytokine levels but found no significant differences within or between groups (Table 6). The same was true for the body composition analysis for total body, body fat, and fat-free masses (Table 7).

Table 6. Serum cytokine levels after the duration of the clinical trial

Table 7. Body composition findings after the duration of the clinical trial

Finally, There was no adverse events during the trial. Every subjects was more than 70% with compliance.

Discussion

The FSS questionnaire results for the GOK group revealed significant decreases in scores for the following items after taking GOK: “I lose motivation when I am fatigued,” “I become tired when I exercise,” “I become tired easily,” “I engage in less physical activity because of fatigue,” and “I often experience problems due to fatigue.” In the SF-36, there was a significant increase in general health scores in the GOK group. Collectively, these findings suggest that GOK improved subjective fatigue, which is in line with previous reports that GOK ingestion can have beneficial effects on certain physical functions ^5,6) . In addition, an analysis of GOK composition revealed that it contains various amino acids and minerals with antioxidant functions, and the authors suggested that this is the mechanism of action by which it relieves fatigue ¹¹⁾ . There was no notable difference in questionnaire results between the two groups, even though there was a significant difference after treatment in the GOK group.

Oral GOK administration did not significantly affect the laboratory findings. There were no significant intra- or inter-group differences regarding glucose metabolism. There are two likely explanations for this. First, the degree of perceived change was not great because the subjects in both groups were healthy adults with normal insulin tolerance before treatment. Second, the saccharide and black rice in the placebo are nutritious ingredients commonly used in traditional Korean medicine to nourish the body.

Interestingly, we observed GOK-related improvements in post-prandial TG levels. This was similar to the findings of a previous mouse study, in which serum total cholesterol and TG levels were decreased, while high-density lipoprotein levels were increased following treatment with GOK and modified GOK ¹²⁾ .

CRP, a major inflammatory markers, was significantly lowered in the GOK group after the treatment period. Elevated CRP and high fasting TG are common hematologic findings in obese individuals, indicating that obesity is closely connected with both lipid metabolism abnormalities and vascular health ¹³⁾ . Previous research revealed that GOK reduces inflammatory cytokine secretion and effectively decreases the range of infarction in mice with middle cerebral artery (MCA) occlusion 14) . Considering that elevated CRP can be used as an important indicator of obesity ¹⁵⁾ , GOK might exert an effect on vascular health, which is in turn related to obesity and lipid metabolism. Furthermore, CRP can be used as an indicator of fatigue ¹⁶⁾ , and the results of our study are consistent with a report describing a correlation between decreasing CRP and indicators of actual QOL 17-¹⁹⁾ . Also, our GOK group exhibited a significant increase in TSH. This is consistent with a report stating that, although not statistically significant, the TSH concentration was higher in a group of GOK-treated mice with induced growth disorder ²⁰⁾ . Given the results of a study on individuals with normal thyroid function, in which people with lower normal-range TSH values felt more fatigue ²¹⁾ , our study suggests a possible mechanism for reduced fatigue after taking GOK. However, a biological false positive have to be considered in the case of CRP and TSH.

Five participants (four in the GOK group and one in the control group) exhibited levels of AST, ALT, GGT, or total bilirubin that were above the standard range after the treatment period. We found that these participants had above average alcohol intake during the study. Hence, they may have experienced adverse reactions unrelated to GOK.

Numerous traditional Korean medicines including GOK have been shown to affect levels of various cytokines ^22-25) , and our earlier study demonstrated that GOK significantly reduced TNF-α and IL-1β levels compared to a control in induced MCA infarction mice ¹⁵⁾ . We anticipated that GOK treatment would influence blood cytokine levels in humans, but possibly because the subjects in this study were healthy individuals with baseline cytokine levels within normal ranges, there were no statistically significant changes. Conversely, subjects in the previous studies had blood cytokine levels that were already altered by various pathologic disease states, which might have predisposed them to a greater response to GOK.

Many people worry that taking a restorative will make them put on weight, and because traditional Koreanmedicines are not zero-calorie, we measured subjects’ total body, body fat, and fat-free masses. There was no significant increase in total body weight for either group; indeed, a slight decrease in body weight was observed, although this was not statistically significant.

Our study had several limitations. First, the subjects’ lifestyle habits were not controlled during the trial period. There have been differences in daily habits such as diet and exercise, but also, because most of the subjects were young office workers, alcohol consumption and excessive work were frequent. The second limitation was the choice of placebo. The black rice and saccharide may have impacted glucose load and imparted generic restorative benefits to the control group subjects. Third, we only included young, healthy adults in whom any measurable effects were below the level of evidence required for clinical application. Finally, traditional Korean medicine prescriptions should be made according to the patient’s medical symptoms, a practice that was not incorporated into the study design and might have reduced the precision of the results. Future studies should include further investigation into CRP and TSH levels and identify objective indices that can be applied to explain the clinical effects of GOK and investigate its mechanism(s) of action.

Conclusion

Hematologic and serologic test results and QOL questionnaires revealed that in healthy 20- to 40-year-old adults, a 4-week GOK regimen did not cause any hepatotoxicity or nephrotoxicity; had a beneficial effect on some items of perceived QOL; and was associated with elevated TSH, reduced CRP, and reduced fasting TG levels.