pharmacopuncture is a new acupuncture therapy based on a combination of meridian theory and herbal pharmacology . in pharmacopuncture , by injecting the extracts of herbs at acupuncture points , doctors gain a synergism of positive effects , i.e. , a physical stimulation due acupuncture and a chemical effect due to the herbal medicine .
recently , in korean medicine , pharmacopuncture has been widely used to treat patients with such diverse diseases as herniated intervertebral discs , arthropathia , skin diseases , and especially obesity . for obesity care , many reports have been published concerning related acupuncture points , such as jusanli ( st36 ) and zhongwan ( cv12 ) , and pharmacopuncture extracts , such as atractylodes lancea , panax ginseng , sweet bee venom , etc .
in addition , mahwang ( ephedra sinica)- cheonoh ( aconitum carmichaeli ) extract is known to be able to inhibit the generation of fat cells , as well as the activation of collateral obese metabolic diseases . as a perennial plant belonging to the family ephedraceae , mahwang , in korean medicine , can perspire , drive out cold , generate diuresis , and reduce edema .
mahwang also contains many alkaloids , such as ephedrine , pseudo - ephedrine , tannin and flavonoids , which can pharmacologically activate sympathetic nerves to reduce appetite , inhibit cholesterol absorption , and accelerate the disintegration of body fat [ 5 , 6 ] .
because of these effects , it has frequently been used as a herb prescription to treat obese patients   and as an ingredient in food for short - term weight loss or for elevating exercise performance . as a perennial plant belonging to the ranunculaceae family
, cheonoh is expected to be able to promote the blood - circulation system and the meridian system on the surface of the body and to stimulate metabolism just as mahwang can ; thus , cheonoh has frequently been used with mahwang as a herb prescription for the treatment of obese patients .
reports have been published indicating that mahwangcheonoh pharmacopuncture is effective in promoting the disintegration of fat cells [ 4 , 9 ] . however , to the best of the authors knowledge , no reports on its toxicity have yet to be published .
accordingly , the authors performed such toxicity tests and obtained significant results demonstrating that mahwangcheonoh pharmacopuncture is a safe , credible pharmacopuncture therapy for treating obese patients .
mahwangcheonoh pharmacopuncture extracts were prepared at the korean pharmacopuncture institute ( seoul , korea ) in a clean room , were stored in a refrigerator at 2.1 - 5.3c , and remained stable for 6 months following production ( lot no . kpi-2013 - 03 ) .
the animals used in this study were 6-week - old specific - pathogen - free ( spf ) sprague - dawley ( sd ) male ( 184.7 - 204.0 g ) and female ( 156.1 - 179.5 g ) rats purchased from orientbio inc .
this study was approved by the institutional animal care and use committees of biotoxtech ( no . 130364 ) . when the animals were received , a visual inspection was done , and weights were recorded using electronic scales ( cp3202s , sartorius , gttingen , germany ) .
the general symptoms and changes were checked once daily during the seven days of acclimatization , after which all animals were checked and found to be normal .
all animals were bred in stainless - steel wire breeding boxes ( 260 mm ( w )  350 mm ( d )  210 mm ( h ) ) kept at a temperature from 22.1 to 24.1c , a humidity from 47.7% to 70.8% , and an illuminance from 150 to 300 lux , with a 12-hour light ( 7:00 am to 7:00 pm)/dark cycle .
each breeding group was divided by three animals during acclimatization periods and was divided by one animal during observation periods .
feed ( teklad certified irradiated global 18% protein rodent diet 2918c , harlan laboratories inc . , usa ) and ultraviolet ( uv ) sterilizer running water were provided freely .
twenty male and twenty female rats with weights closest to the mean weight were selected .
the selected animals were distributed into four groups ( five rats of each sex per group ) .
the mahwangcheonoh pharmacopuncture extracts were injected into the animals via a muscular route , and the high dose was set at 1.0 ml per animal because no mortalities had occurred in a preliminary test ( biotoxtech study no . :
b13474p ) in which a single dose of 1.0 ml of mahwangcheonoh pharmacopuncture extract had been injected into the muscles of male and female rats .
based on this high dose , we set 0.5 ml per animal as the medium dose and 0.25 ml per animal as the low dose .
for the control group , 1.0 ml of normal saline was injected . in the low - dose group ( g2 ) and the medium - dose group ( g3 ) ,
the mahwangcheonoh pharmacopuncture extracts were injected into the left femoral muscle . for the control group ( g1 ) and the high - dose group ( g4 ) , 0.5 ml of normal saline and 0.5 ml of mahwangcheonoh pharmacopuncture extract , respectively , were injected into the left and the right femoral muscles , for a total of 1.0 ml per animal .
on the day of injection ( day 0 ) , general symptoms ( types of toxic indications , times of toxic expression , recovery times ) and any deaths were recorded at 30 minutes and at 1 , 2 , 4 and 6 hours after injection . from day 1 until day 14 after injection ,
body weights were measured on the day of injection ( before injection ) and on days 3 , 7 and 14 after injection .
all animals were denied access to food and water ( fasted ) for at least 18 hours prior to necropsy . on the day of necropsy ( 15 days after injection ) , the animals were anesthetized with isoflurane , and blood was collected from the abdominal aorta .
one ml of the collected blood was put into a tube containing ethylene diamine tetraacetic - acid ( edta ) , and the hematology parameters , the prothrombin time ( pt ) and the activated partial thromboplastin time ( aptt ) were measured using a hematology analyzer ( advia 2120i , siemens , germany ) .
two ml of the collected blood were put into a tube containing 3.2% sodium citrate ; then , the plasma was collected after centrifugation at 3,000 rpm for 10 minutes .
a coagulation examination was performed using coagulation time analyzers ( coapresta 2000 , sekisui , japan ) to measure the pt and the aptt .
the blood biochemistry was determined by using serum taken from blood that had been collected from the abdominal aorta and had been centrifuged at 3,000 rpm for 10 minutes . a blood chemistry analyzer ( 7180 , hitachi , japan ) and an electrolyte analyzer ( avl 9181 , roche , germany ) were used for the measurements . for the histopathology , the organs and the tissues collected from all animals after necropsy were inspected .
after necropsy , tissues surrounding the injection regions were excised and fixed in 10% neutral buffered formalin solution , after which local resistance tests were performed on those tissues . fixed organs and tissues sections
the weights , as well as the hematological and the blood biochemical results , were analyzed by using sas ( version 9.2 , 9.3 , sas institute inc .
a bartlett test was conducted to evaluate equal variance between the test and the control groups ( p < 0.05 ) .
the one - way analysis of variation ( anova ) test was conducted when equal variance was recognized on the bartlett test ( p < 0.05 ) , and the dunnett s t - test was conducted when equal variance was recognized on the anova test ( both p < 0.05 , p < 0.01 ) . also , the kruskal - wallis test was conducted when equal variance was not recognized on the bartlett test ( p < 0.05 ) , and the steel - test was conducted when equal variance was recognized on the kruskal - wallis test ( both p < 0.05 , p < 0.01 ) .
during the observation period , no abnormal symptoms or deaths were observed in either the male or the female rats in the test and the control groups ( table 1 ) during that period , the weights of the male rats in the control and the test groups changed from 192.6  8.7 g ( g1 ) , 195.6  5.6 g ( g2 ) , 195.3  4.3 g ( g3 ) , and 193.2  7.4 g ( g4 ) to 314.1  11.9 g ( g1 ) , 315.0  18.0 g ( g2 ) , 314.2  16.3 g ( g3 ) , and 318.5  16.2 g ( g4 ) , respectively .
those of the female rats changed from 166.3  6.0 g ( g1 ) , 168.3  6.9 g ( g2 ) , 169.2  9.1 g ( g3 ) , and 167.4  7.4 g ( g4 ) to 227.5  10.1 g ( g1 ) , 235.4  9.9 g ( g2 ) , 230.5  15.3 g ( g3 ) , and 223.4  9.2 g ( g4 ) , respectively .
however , no statistically significant changes in weights associated with injection were recognized ( figs .
no significant differences were observed in the hematology between the test groups and the control group for either the male or the female rats ( tables 2,3 ) .
the change observed in the lowdose female group ( g2 ) was a minor variation because it was observed sporadically and exhibited no a dose dependency .
that change was determined to be an accidental change with no statistical significance ( tables 4,5 ) .
the results on the local resistance tests were not affected by the injections . however , a weak inflammation was observed in the region of injection in a rat in the control group .
because the inflammation occurred in the control group , it was determined to have no toxicological significance , but rather to be a spontaneous or accidental change without injection relevance .
according to national health statistics , the obesity rate in korean adults , based on the body mass index ( bmi ) , has reached 31.5% , being 37.7% in males and 25.3% in females .
also , the teenage obesity rate in korea has reached 11.5% . because of this , the people s interest in obesity has also increased . according to a survey ,
85.4% of the people were aware of being overweight , and 60% indicated that they were actively trying to lose weight .
moreover , during 2008 , the social and economic cost associated with the treatment of obese patients in the country was estimated to be approximately 1.6 billion us dollars .
because of this , many medical workers can not but be interested in research on treatments for obesity . in korean medicine , much research has been done on the treatment of obese patients by using acupuncture , electro- acupuncture , moxibustion , herbs , subcutaneous acupuncture , and exercise therapy . among these possible treatments , pharmacopuncture injection of one - tenth the volume of a medicine dose directly into fatty areas or acupuncture points
moreover , many pharmacopuncture solutions for obesity treatment have already undergone many safety and reliability tests in both experimental and clinical studies .
pharmacopuncture extracts known to be effective in the treatment of obese patients are ephedra , atractylodes lancea , coix lacryma - jobi , astragalus propinquus , angelica , ginseng [ 14 , 15 ] , and the like .
all of these have been found through laboratory research to be able to reduce effectively blood lipids or fat cells , and body weight , to have no toxicity , and to result in no abnormalities in the blood levels .
mahwangcheonoh pharmacopuncture has also been proven to be effective in the treatment of obese patients [ 4 , 9 ] , but its toxicity and safety have not been adequately addressed .
thus , this research was conducted to establish the toxicity , safety , and stability of this pharmacopuncture so that it could be added as another reliable pharmacopuncture for the treatment of obese patients .
this study observed the general symptoms , weight changes , and hematological and biochemical blood changes caused by single - dose intramuscular injection of mahwangcheonoh pharmacopuncture in sd 6-week - old male and female rats .
the rats were also subjected to necropsy to identify any changes in tissues or organs due to the injections .
no deaths or abnormalities occurred any of the rats , and no significant differences in body weights were observed during the 14-day observational period following injection of the pharmacopuncture .
finally , no abnormal changes due to the pharmacopuncture were observed on the histopathological examinations after the necropsy .
based on the above results , the lethal single dose of mahwangcheonoh pharmacopuncture for rats is over 1.0 ml / animal .
in addition , the mahwangcheonoh pharmacopuncture at that dose is stable and safe to use and has no toxic effects on viscera , muscles or functions .
although this study has shown that mahwangcheonoh pharmacopuncture is safe to use at doses of 1.0 ml or less , no research on its side effects has yet been officially reported .
in addition , because this research involved investigating the effect of a single - dose injection over a 14-day observation period , research on its long - term effects and on the effects of multi - dose injections is needed if mahwangcheonoh pharmacopuncture is to be firmly established as an effective treatment for obese patients .
the objective of this study was to analyze the safety and the toxicity of mahwangcheonoh pharmacopuncture .
the results after a single intramuscular dose and observations of the general symptoms over a 14-day period showed no abnormal symptoms or mortalities in either the test groups or the control group .
in addition , no significant changes in weight , hematology , blood biochemistry , and histopathology between the test groups and the control group were found .
based on these results , we consider the lethal dose of mahwangcheonoh pharmacopuncture to be far beyond 1.0 ml / animal ; thus , mahwangcheonoh pharmacopuncture may be considered a safe therapeutic agent for the treatment of patients with obesity .
s.d . , standard deviation ; rbc , red blood cell ; hgb , hemoglobin ; hct , hematocrit ; mch ,
mean corpuscular hemoglobin ; mcv , mean corpuscular cell volume ; mchc , mean corpuscular cell hemoglobin concentration ; plt , platelet ; reti , reticulocytes ; wbc , white blood cell ; neu , neutrophils ; lym , lymphocytes ; mono , monocytes ; eos , eosinophils ; baso , basophils ; pt , prothrombin time ; aptt , active partial thromboplastin time .
s.d . , standard deviation ; rbc , red blood cell ; hgb , hemoglobin ;
hct , hematocrit ; mch , mean corpuscular hemoglobin ; mcv , mean corpuscular cell volume ; mchc , mean corpuscular cell hemoglobin concentration ; plt , platelet ; reti , reticulocytes ; wbc , white blood cell ; neu , neutrophils ; lym , lymphocytes ; mono , monocytes ; eos , eosinophils ; baso , basophils ; pt , prothrombin time ; aptt , active partial thromboplastin time .
s.d . , standard deviation ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; alp , alkaline phosphatase ; ggt , gamma glutamyltranspeptidase ; glu , glucose ; bun , blood urea nitrogen ; crea , creatinine ; t - bili , total bilirubin ; t - chol , total cholesterol ; tg , triglycerides ; tp , total protein ; alb , albumin ; a / g ratio , albumin / globulin ratio ; p , phosphorus ; ca , calcium ; na , sodium ; k , potassium ; cl , chlorine .
s.d . , standard deviation ; alt , alanine aminotransferase ; ast , aspartate aminotransferase ; alp , alkaline phosphatase ; ggt , gamma glutamyltranspeptidase ; glu , glucose ; bun , blood urea nitrogen ; crea , creatinine ; t - bili , total bilirubin ; t - chol , total cholesterol ; tg , triglycerides ; tp , total protein ; alb , albumin ; a / g ratio , albumin / globulin ratio ; p , phosphorus ; ca , calcium ; na , sodium ; k , potassium ; cl , chlorine .
p < 0.05 , which implies the existence of a statistically significant difference from the control group based on the steel test
 objectives : this study was conducted to analyze the single - dose toxicity and the safety of mahwangcheonoh pharmacopuncture extracts.methods:six-week-old sprague - dawley rats were used for this study . 
 doses of mahwangcheonoh pharmacopuncture extracts were set at 0.25 ml ( low - dose ) , 0.5 ml ( medium - dose ) and 1.0 ml ( high - dose ) for the test groups . 
 a dose of 1.0 ml of normal saline solution was set for the control group . during 14 days , general symptoms , mortalities , and changes in hematology , 
 blood biochemistry and histopathology of all rats were observed.results:no death was observed in all test groups . 
 any abnormal symptom was not observed in all of the groups . 
 no significant changes in weight between the control group and the test groups were observed . 
 in addition , no significant differences in the hematology signs , the blood biochemistry levels and the histopathological signs related to the mahwangcheonoh pharmacopuncture extracts injection were observed.conclusion:the findings of this study indicate that mahwangcheonoh pharmacopuncture at doses of 1.0 ml or less may be consider safe and non - toxic . 
 so , it can be used for therapy of obesity sufficiently . 
 but further studies on this subject must be performed to confirm and verify this conclusion . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion