The American Journal of Chinese Medicine, Vol. 31, No.

4, 623–628
© 2003 World Scientific Publishing Company
& Institute for Advanced Research in Asian Science and Medicine

Effects of In Vitro and In Vivo Qi-therapy on

Neutrophil Superoxide Generation in Healthy
Male Subjects

Myeong Soo Lee,*,† Seong Min Jeong,* Hye-Sook Jang,‡ Hoon Ryu*,¶ and Sun-Rock Moon*,§

*Centerfor Integrative Medicine, Institute of Medical Science and

†Professional Graduate School of Oriental Medicine
Wonkwang University, Iksan 570-749, Republic of Korea
‡Department of Nursing, Wonkwang Health Science College, Iksan 570-750, Republic of Korea
§Department of Radiation Oncology, Wonkwang University School of Medicine

Iksan 570-711, Republic of Korea

¶Present address: Department of Neurology, University of Massachusetts Medical School

Worcester, MA 01655, USA

Abstract: The present study investigated the effects of in vitro and in vivo Korean ChunSoo
Qi-Energy Healing on neutrophil superoxide generation. Neutrophil superoxide generation
was measured by a chemiluminescence assay. Superoxide generation was significantly increased
in vitro by emitted Qi-therapy (QT) of 60-second duration and 150-second duration compared
to control (1.59-fold for 60 seconds, p < 0.05; 1.50-fold for 150 seconds, p < 0.05). Neutrophil
superoxide generation increased significantly immediately after 5 minutes of QT in vivo (1.42-
fold, p < 0.05). These results show that QT in vivo and in vitro has an acute stimulatory effect
on neutrophil superoxide generation. This study provides direct scientific support that Qi as
such may positively affect human innate immunity.

Keywords: Qigong; Qi-therapy; Neutrophil; Superoxide.

Introduction

External Qi-therapy (QT) is a process by which vital energy (Qi) is transmitted from a Qi
master to another person for the purpose of preventing and curing disease, as well as protecting
and improving health through regulation of mind and body. This may be a very useful
intervention. Research studies have shown that QT is effective for relief of pain, relaxation
of stress states and increasing immunity (Lee et al., 2001a–c).

Correspondence to: Dr. Sun-Rock Moon, Department of Radiation Oncology, Wonkwang University
School of Medicine, Iksan 570-711, Republic of Korea. Tel: (+82) 63-850-1527, Fax: (+82) 63-850-1528,
E-mail: sunrmoon@wonkwang.ac.kr

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 624 M.S. LEE et al.

In recent years, several studies attempted to reveal a specific in vitro and in vivo effect of
external Qi by modern biochemical and immunological methods (Chien et al., 1991;
Fukushima et al., 2001; Lee et al., 2001a and b; Shah et al., 1999). Chien et al. (1991)
reported that facilitating Qi from a Qigong masters increased the rate of cell growth and
DNA synthesis. Lee et al. (2001a and b) reported psychoneuroimmunological effects of
in vivo QT on humans and stimulatory effect on natural killer (NK) cell activity in vitro by
emitted Qi.
Neutrophils play an important role in the immune system, forming the first defense against
invading microorganisms by phagocytosis and by using toxins such as reactive oxygen species
and lysosomal enzymes. Neutrophil phagocytosis has been found to increase in vitro after
Qigong energy treatment of the phosphate-buffered saline (PBS) into which the cells were
placed (Fukushima et al., 2001). However, no studies have investigated directly the effects
of QT on neutrophil oxidative burst activity which indicates innate immune function.
The purpose of this study was to investigate neutrophil superoxide generation after direct
QT in vitro. In addition, the effect of in vivo QT was examined by treatment of seven male
subjects.

Materials and Methods

Subjects

The experiment was conducted on 14 male volunteers (seven for in vivo and seven for in
vitro experiment) from Iksan, Korea. Their mean physical characteristics were: age 28 ± 5
years, height 167 ± 2 cm, and weight 62 ± 4 kg. The study protocol was approved by the
ethics committee of the Human Subjects Review Board at Wonkwang University Hospital
and School of Medicine, and all the subjects gave their informed consent. They abstained
from smoking and liquids with caffeine or alcohol for at least 6 hours prior to testing. All
subjects were in good health without a history (remote or recent) of chronic disease,
malnutrition, malignancy or renal disease. None was taking any medications such as steroid
hormones that might affect physical activity.

In Vitro Qi-therapy

Separated neutrophils in Cuvettes (4 cm high, Abbott Laboratories, IL) capped with parafilm
were placed 5 cm in front of the palm of the ChunDoSunBup (CDSB) (Ryu et al., 1995) Qi
master (male, aged 25 years, trained in CDSB Qi-training for about 5 years) at room
temperature (total distance from palm to sample was about 9 cm). Six tubes were used in
each experiment. The CDSB Qi master did not know any methods, materials or procedures
of the experiment and only emitted Qi (ChunSoo Energy Healing) with positive thinking
(ChunJon Foundation, 1997; Lee et al., 2001b). Each blood sample was divided into two
control samples and four experimental samples (we measured duplicate samples and averaged)
and all were set to receive QT simultaneously, except two controls were randomly removed
at the start of the experiment and thus had no time to be exposed to Qi at 60 seconds and

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 QI-THERAPY ON NEUTROPHIL SUPEROXIDE GENERATION 625

again at 150 seconds. Two of the four experimental samples were randomly removed and
marked for treatment time. The experiment was repeated seven times, each time with blood
from a different subject.

In Vivo Qi-therapy

In this experiment, in vivo QT was performed by same master who did the in vitro experiment.
He did not know any methods, materials and procedures of the experiment and only emitted
Qi with positive thinking.
After subjects had rested for 5 minutes, while supine on beds in a quiet room under
constant conditions, the subjects received QT with no touch on the body for 5 minutes
following methods described previously (Lee et al., 2001a).
The peripheral blood was obtained by venipuncture using heparinized (10 U/ml) syringes
from the median cubital vein. Blood was drawn 10 minutes before QT (Pre), immediately
after QT (Post I) and 1 hour after QT (Post II). The samples were immediately treated for
separating neutrophils.

Materials

Hank’s balanced salt solution (HBSS) and phosphate-buffered saline (PBS) were prepared
by conventional methods and sterilized. HBSS contained 5 mM D-glucose. A single batch
of opsonized zymosan (used for all experiments) was prepared by incubating 10 mg of
Zymosan-A particles (highly glycosylated fragments of yeast cell walls) (Sigma, St. Louis,
MO) with 1 ml of fresh human plasma for 30 minutes at 37°C. The suspension was centrifuged
at 400 × g for two minutes and the pellet washed twice in PBS before resuspension in PBS
at a final concentration of 10 mg/ml. Aliquots (l ml) were stored frozen and used only once
after thawing. Counting by hemocytometer revealed that this preparation contained
approximately 106 zymosan particles/microliter.

PMN Isolation

Human venous blood (10 ml) was collected from volunteers into heparinized tubes. To obtain
neutrophils from peripheral blood, the heparinized blood was centrifuged for 10 minutes at
1000 rpm to remove platelet-rich plasma. After 2% dextran sedimentation of erythrocytes
for 30 minutes, neutrophils were isolated under sterile conditions by density gradient
centrifugation on Ficoll-Paque cushions in conical tubes. The tubes were centrifuged at 2500
rpm for 30 minutes in swing-out buckets at room temperature. Contaminating erythrocytes
were lysed with hypotonic solution containing NH4Cl-EDTA and then washed twice. The
cells were gently resuspended in magnesium-free HBSS containing 1.6 mM CaCl2; then the
cell number and viability were determined. The entire procedure was conducted in sterile
conditions at room temperature. The final cell preparation comprised at least 97% neutrophils
and < 0.2% monocytes, as assessed by Wright-Giemsa differential staining. The viability of
neutrophils was > 98% as determined by trypan blue exclusion.

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 626 M.S. LEE et al.

Measurement of Superoxide Anion Production

Superoxide anion (O2−) was measured by a chemiluminescence assays. Ten microliters of

lucigenin (10, 10'-dimethyl-9,9'-acridinum) (Sigma) (0.3 mM final concentration), as a
chemiluminescence source, was added to each tube containing 1 × 106 cells in a 300 µl
volume of Veronal buffer (containing BSA-Bovine serum albumin, glucose, Ca2+ and Mg2+).
The tubes were immediately placed in a lightproof and thermostatic chamber of a six-channel
Biolumant LB9505 (Berthold, Bad Wildbad, Germany) and stabilized for 20 minutes. For
the enhancement of O2− production, the triggering agent, opsonized zymosan (50 particles/
cell) (Sigma Co., St. Louis, MO) was added to the tubes. The number of resultant photons
from lucigenin-dependent chemiluminescence was recorded in counts per minute (cpm) for
60 minutes.

Statistical Analysis

Two-tailed paired t-tests were used to test for significant differences between control and
experimental groups.

Results

Figure 1 demonstrated the in vitro superoxide generation of neutrophil according to QT

time. The values of O2− were significantly increased by 60 seconds (1.59-fold, p < 0.05) and
150 seconds (1.50-fold, p < 0.05) QT compared to control.
In vivo neutrophil superoxide generation was significantly increased immediately after
QT compared to before QT (1.42-fold, p < 0.05) and went to baseline at one hour after QT
(Fig. 2).

5
Superoxide generation

4
× 108 cpm)

3
(×

0
Control 60 sec 150 sec

Figure 1. Effect of in vitro QT on superoxide (O2−) generation by zymozan-stimulated neutrophils. *p < 0.05
compared to control. Duration of QT: 60 seconds and 150 seconds. Error bars indicate standard deviations.

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 QI-THERAPY ON NEUTROPHIL SUPEROXIDE GENERATION 627

6
Superoxide generation
5
× 108 cpm)
4
(×

0
Pre Post I Post II

Figure 2. Effect of in vivo QT on superoxide (O2−) generation by zymozan-stimulated neutrophils. *p < 0.05
compared to before. Pre: Before QT; Post I: after QT; Post II: 1 hour after QT. Error bars indicate standard
deviations.

Discussion

The present study investigated the in vitro and in vivo effects of Korean QT on neutrophil
superoxide generation. Superoxide generation was significantly increased by in vitro treatment
for 60 seconds and 150 seconds. Neutrophil superoxide generation also increased significantly
after 5 minutes of QT in vivo. These results show that emitted Qi has similar acute stimulatory
effects on neutrophil superoxide generation both in vivo and in vitro.
The in vivo result is consistent with our recent report that demonstrated 10 minutes of
QT increased neutrophil oxidative burst activity compared to placebo control (Lee et al.,
2001b). To date, there have been no studies that have examined the changes in neutrophil
oxidative burst activity in vitro with QT. But Fukushima et al. (2001) reported that neutrophil
phagocytic activity was stimulated by PBS treated with external Qigong. Because the oxidative
burst process is very closely connected with phagocytic activity, both neutrophil functions
might be influenced by QT. Therefore, it is possible to speculate that the increase of oxidative
burst activity reflected direct effects of QT on neutrophil immune capacity both in vivo and
in vitro.

Acknowledgments

This work was supported by a grant-in-aid (2001) from Wonkwang University, Korea.

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