Pharmacopuncture therapy is a new acupuncture therapy to treat  diseases based on herbal medicine, acupuncture & moxibustion  medicine, and meridian theories [  1  ]. Pharmacopuncture fluid is extracted from herbs and injected into  acupoints or sore spots [  2  ].Through a single procedure, it can achieve both the effects of  acupuncture and herbal medicine [  3  ].

The  Samgihwalryeok  pharmacopuncture consists of  Panax ginseng, Cervus elaphus sibericus, Angelica gigas  Nakai, Liriope platyphylla  , and  Schisandra chinensis Baillon  and can be used to treat lethargy and chronic fatigue from qi  deficiency, blood deficiency or both qi and blood deficiency [  4  ]. This study was performed to examine the single-dose toxicity of  Samgihwalryeok  pharmacopuncture. The testing methods were visual observation of  general symptoms, body weight changes, hematological tests,  biochemical analyses, necropsy and histopathological observations  with 6-week-old Sprague-Dawley (SD) rats. All experiments were  conducted at Biotoxtech (Chungwon, Korea), an authorized institution  for non-clinical studies, under the regulations of Good Laboratory  Practice (GLP) of Korea Food & Drug Administration (KFDA)  Notification No. 2012-86 (Test guidelines for non-clinical studies,  Aug 24, 2012) [  5  ].

Samgihwalryeok  pharmacopuncture solution was prepared in a sterile room at the  Korean pharmacopuncture institute (KGMP). After the mixing process  with pure water, the pH was controlled to between 7.0 and 7.5. NaCl  was added to the pharmacopuncture solution to make a 0.9%  isotonic solution. The completed extract was stored in a  refrigerator (2.1 - 6.6°C).

The animals used in this study were 6-week-old SD rats. The mean  weights were 185.4 - 209.0 g and 153.0 - 174.5 g for the male and  the female rats, respectively, at the time of injection of the  pharmacopuncture. Visual inspections were conducted for all  animals; all animals were weighed using a CP3202S system  (Sartorius, Germany). During 7 days of acclimatization, the general  symptoms of the rats were observed at the end of the day. The  weights of the rats were recorded on the last day of  acclimatization. No abnormalities were observed. The temperature of  the lab was 21.0 - 23.2°C, and the humidity was 40.9% -  59.4%. Enough food (Teklad Certified Irradiated Global 18%  Protein Rodent Diet 2918C) and UV-filtered water were provided.

Group separations were done after 7 days of acclimatization. The  animals were randomly distributed into 4 groups of 5 male and 5  female rats per group (Table  1  ): the control, low-dose, mid-dose and high-dose groups.

In a pilot test (Biotoxtech Study No.: B12876P), 1.0 mL/animal,  referring to a 1.0 mL dose for each clinical application, was  administered by intramuscular (i.m.) injection (left thigh) to one  male and one female rat and resulted in no deaths. From this result,  the doses for  Samgihwalryeok  pharmacopuncture were set up as follows: animals in group 1 (G1, the  control group) were injected with 0 mL/animal of pharmacopuncture  and 1.0 mL/animal of normal saline solution (ChoongwaePharma Corp.,  Korea), animals in group 2 (G2, the low-dose group) were injected  with 0.1 mL/animal of pharmacopuncture, animals in group 3 (G3, the  mid-dose group) were injected with 0.5 mL/animal of  pharmacopuncture, and animals in group 4 (G4, the high-dose group)  were injected with 1.0 mL/animal of pharmacopuncture. Using a  disposable syringe (1 mL, 26 G), we administered the  Samgihwalryeok  pharmacopuncture solution by i.m. injection in the animals of the  low-dose and the mid-dose groups once on the left thigh; for the  high-dose and the control groups, we administered one 0.5-mL  injection of pharmacopuncture and of normal saline, respectively, in  each thigh. All experiments were conducted at Biotoxtech (Chungwon,  Korea), an authorized institution for non-clinical studies, under  the regulations of GLP of KFDA Notification No. 2012-86 (Test  guidelines for non-clinical studies, Aug 24, 2012) [  5  ].

The general symptoms (side effects, revealing times recovery time  etc.), as well as the mortalities, were examined for 10 seconds at  30 minutes and at 1, 2, 3, and 4 hours after injection on the day of  dosing (day 0).

From the 1  ^st  day to the 14  ^th  day of treatment, the general symptoms were examined once a day. The  body weights were measured immediately before treatment and at 3, 7  and 14 days after injections.

All animals were fasted for more than 18 hours before autopsy. The  rats were anesthetized by using isoflurane, and blood samples were  collected from the abdominal aorta on the day of autopsy (15 days  after injection). An automatic hematology analyzer (ADVIA120,  SIEMEMS, Germany) was used to analyze blood for the hematological  examinations. For the blood coagulation test (3,000 rpm, 10  minutes), 2-mL samples of blood were placed in a tube with 3.23%  sodium citrate to collect blood plasma. The RBC, HGB, HCT, MCV, MCH,  MCHC, PLT, etc. were measured for the hematological examinations and  the prothrombin time (PT) and the activated partial thromboplastin  time (APTT) were determined for the coagulation tests. The results  were obtained using an Automated Coagulation Analyzer (Coapresta  2000, SEKISUI, Japan). For the biochemical tests, the blood  remaining after carrying  out the hematological tests was centrifuged at 3,000  rpm for 10 minutes, and the serum was collected. Measurements  were done using a biochemistry analyzer (7180,  HITACHI, Japan) and an electrolyte analyzer (AVL9181,  Roche, Germany).

After the observations, organs and tissues from the entire  bodies of all animals were visually inspected and examined  under an optical microscope. Tissue samples of all  the animals were fixed in 10% neutral buffered formalin.  Routine histological methods, such as trimming, dehydration,  and paraffin embedding, were conducted on the  fixed organs and tissues. Fixed samples were then sliced  using a microtome and stained with hematoxylin & eosin  (H&E).

All the results from the experiments were analyzed by using  SAS (version 9.2, 9.3, statistical analysis system [SAS]  Institute Inc., USA). The Bartlett test was conducted to  evaluate the homogeneity of the variance and the significance.  If the sample variances were equal, the significant  result was obtained using a one-way analysis of variance  (ANOVA) test. If the sample variances were not equal, a  Kruskal-Wallis test was conducted post-hoc. Statistical  significance was associated with  P  ≤ 0.05.

During the observation, no mortality or clinical signs  were observed in the all of experimental (0.1, 0.5, and  1.0 mL/animal) or control groups (Table  2  and  3  ).

There was no significant change in body weight shown  by comparison of the experimental (0.1, 0.5, and 1.0  mL/animal) and the control group (Fig  1  ).

There was no significant change in the hematological  test results observed from comparison of the experimental  (0.1, 0.5, and 1.0 mL/animal) and the control  group (Table  4  ).

There was no significant change in the biochemical  test results from experimental and control group (Table  5).

No abnormalities were observed when the visual inspection  was conducted on all of the animals in experimental  and control group (Table  6  ).

No abnormalities were observed in the local tolerance  test on the injection sites (Table  7  ).

Pharmacopuncture is a type of new acupuncture technique  that combines acupuncture and drug therapies. It  can be seen as a unique treatment technique of Korean  Oriental medicine that provides the effects of meridian  theory in acupuncture therapy and flavor theory in drug  therapy [  6  ]. The  Samgihwalryeok  pharmacopuncture consists  of  Panax ginseng, Cervus elaphus sibericus, Angelica  gigas Nakai, Liriope platyphylla,  and  Schisandra chinensis  Baillon  [  4  ] and is prepared in a sterile room at the Korean  pharmacopuncture institute (KGMP, pH 7.0% - 7.5%, 0.9%  NaCl).

Recent reports have suggested that  Panax ginseng  pharmacopuncture  has the effects of increasing body weight  [  7  ], heart rate variability [  8  ] and immune response [  9  ],and of decreasing NOS expression induced by noise stress  [  10  ] and anti-cancer effects [  11  ].  Cervus elaphus sibericus  pharmacopuncture has the effects of increasing heart rate  variability [  8  ] and body-weight [  7  ] and decreasing the osteoporosis  induced by an ovariectomy [  12  ]. Decreased  cerebral infarction or ischemia damage [  13  ] and improvement  in hypothyroidism induced by thiourea [  14  ] were  reported in  Angelica gigas  pharmacopuncture studies.  Liriope  platyphylla  was reported to have anti-inflammation  [  15  ] and anti-cancer effects [  16  ], and  Schisandra chinensis  Baillon  was reported to have anti-inflammation effects  [  17  ].  Samgihwalryeok  pharmacopuncture made with all of  these was reported to affect chronic fatigue and insomnia  [  4  ].

This study was performed to determine the safety of using  Samgihwalryeok  pharmacopuncture in SD rats. Animal  testing is the most fundamental and basic way to perform  safety assessments [  18  ]. The  Samgihwalryeok  pharmacopuncture  used in study was prepared in a sterile room at the  Korean pharmacopuncture institute (KGMP). The animals  were randomly distributed into 4 groups, 5 male and five  female rats per group: control (0 mL/animal and 1.0 mL  of saline), low-dose (0.1 mL/animal), mid-dose (0.5 mL/  animal), and high-dose (1.0 mL/animal) groups. After  intramuscular injection of  Samgihwalryeok  pharmacopuncture,  general symptoms, body-weights, hematological  factors, blood-biochemical factors, necropsy features and  histopathological features were observed. These observations  produced no significant findings. All conditions of  this study followed The Korea Food & Drug Administration’s  testing protocol guidelines for the study of toxicity,  and all experiments were conducted following the GLP  regulations [  19  ]. According to the all of the above results,  Samgihwalryeok  pharmacopuncture can be used as a  safe treatment, but further studies should be conducted  to yield more concrete evidence to support this safety and  prove its efficacy.

This study was designed to investigate the safety of  Samgihwalryeok  pharmacopuncture for single-dose intramuscular  injection (0.1 - 1.0 mL/animal, 5 rats per group). The  following results were found: No mortalities or abnormal  clinical signs, no changes in body weights, and no differences  in hematological and biochemical analyses, necropsy  findings and histopathological findings were observed  in this study. Therefore, the approximate lethal dose of  Samgihwalryeok  pharmacopuncture must be considered  to be more than 1.0 mL/animal in both male and female  rats.