male wistar rats ( 200 - 250 g ) obtained from the animal room of the kerman university of medical sciences .
they were kept in a temperature - controlled environment on a 12:12 h light / dark cycle with free access to food and water .
the procedures were in accordance with the guidelines for the care and use of laboratory animal of kerman university medical science ( iaec no . :
this experimental study was carried out on 40 male wister rats to study therapeutic effects of mumijo extract on acetic acid - induced gastric ulcer .
animals were randomly ( n = 10 in each group ) divided into four groups : ( i ) control received normal saline ( 2 ml / kg ) , ( ii ) sham - operated group received 0.2 ml of acetic acid to induce gastric ulcer , ( iii ) mumijo ( 100 mg / kg / daily ) was given for 4 days postacetic acid administration , ( iv ) ranitidine group received the standard drug , ranitidine ( 20mg / kg ) , dissolved in normal saline .
twenty - four hours before each experiment , animals were deprived of food , but free to drink water .
immediately , it was washed thoroughly with running tap water and cut into small pieces .
then the plant material was shade dried at temperature 21 - 24c and ground mechanically into a coarse powder and stored in an airtight container .
powdered plant material ( 150 g ) was macerated with 400 ml of distilled water at 21 - 24c temperature for 3 days with frequent shaking . after 3 days
, the extracts were filtered and to the marc part 300 ml of the solvent was added and allowed to stand for next 2 days at same temperature for second time maceration ( re - maceration ) and after two days , again filtered similarly . the combined filtrates ( macerates ) were evaporated in vacuo at 40c and the dry extract obtained was stored in a vacuum desiccator for future use .
all the test samples were administered by oral lavage in a volume of 1 ml/100 g body weight once a day to each rat .
the ulcers were induced by the local application of acetic acid to serosal surface of the stomach as described earlier . under anesthesia ,
the midline incision was made , and the stomach was taken out . on the serosal surface of the glandular portion of the stomach , 0.2 ml of 100% acetic acid ( anterior gastric wall ) was injected .
after the treatment , the rats were sacrificed , and stomachs were removed and weighed , fixed in 10% neutral buffered formalin , embedded in the paraffin wax , sectioned at 5 m , stained with hematoxylin and eosin and then examined by the light microscopy .
briefly , 2 ml of 2.5% bovine hemoglobin plus 0.5 ml of 0.3 n hcl and 0.5 ml of gastric effluent were maintained in separate tubes at 37c for 10 min and then mixed . mixtures were incubated for 10 min at 37c , and the reaction was stopped by the addition of 5 ml 0.3 n trichloroacetic acid .
after agitation and filtration , optical density was measured at 280 nm by using a spectrophotometer ( unikon 930 , kontron instrument , italy ) .
the results were compared to a standard curve , which was generated in an identical manner using known amounts of porcine pepsin ( 1 g = 3 peptic units ) , and were expressed as micrograms of pepsin . in order to know whether or not the intragastric ph is contributing to the mucosal lesions , intragastric ph was measured 4 days after administration of mumijo and ranitidine .
the pylorus of the stomach and esophagocardiac junction were immediately ligated , and the stomach was then removed from each rat , and the gastric contents were collected , centrifuged , and the supernatants were used for ph measurement using ph meter (  50 ph meter , beckman , fullerton , ca , usa ) .
this study protocol was approved by the ethics review committee , kerman university of medical sciences , kerman , iran .
the results were expressed as mean  standard error of mean , and statistical significance was evaluated using one - way anova , followed by a tukey 's post - hoc test .
male wistar rats ( 200 - 250 g ) obtained from the animal room of the kerman university of medical sciences .
they were kept in a temperature - controlled environment on a 12:12 h light / dark cycle with free access to food and water .
the procedures were in accordance with the guidelines for the care and use of laboratory animal of kerman university medical science ( iaec no . :
this experimental study was carried out on 40 male wister rats to study therapeutic effects of mumijo extract on acetic acid - induced gastric ulcer .
animals were randomly ( n = 10 in each group ) divided into four groups : ( i ) control received normal saline ( 2 ml / kg ) , ( ii ) sham - operated group received 0.2 ml of acetic acid to induce gastric ulcer , ( iii ) mumijo ( 100 mg / kg / daily ) was given for 4 days postacetic acid administration , ( iv ) ranitidine group received the standard drug , ranitidine ( 20mg / kg ) , dissolved in normal saline .
twenty - four hours before each experiment , animals were deprived of food , but free to drink water .
immediately , it was washed thoroughly with running tap water and cut into small pieces .
then the plant material was shade dried at temperature 21 - 24c and ground mechanically into a coarse powder and stored in an airtight container . powdered plant material ( 150 g )
was macerated with 400 ml of distilled water at 21 - 24c temperature for 3 days with frequent shaking .
after 3 days , the extracts were filtered and to the marc part 300 ml of the solvent was added and allowed to stand for next 2 days at same temperature for second time maceration ( re - maceration ) and after two days , again filtered similarly .
the combined filtrates ( macerates ) were evaporated in vacuo at 40c and the dry extract obtained was stored in a vacuum desiccator for future use .
all the test samples were administered by oral lavage in a volume of 1 ml/100 g body weight once a day to each rat .
the ulcers were induced by the local application of acetic acid to serosal surface of the stomach as described earlier . under anesthesia , the midline incision was made , and the stomach was taken out . on the serosal surface of the glandular portion of the stomach , 0.2 ml of 100% acetic acid ( anterior gastric wall ) was injected .
after the treatment , the rats were sacrificed , and stomachs were removed and weighed , fixed in 10% neutral buffered formalin , embedded in the paraffin wax , sectioned at 5 m , stained with hematoxylin and eosin and then examined by the light microscopy .
briefly , 2 ml of 2.5% bovine hemoglobin plus 0.5 ml of 0.3 n hcl and 0.5 ml of gastric effluent were maintained in separate tubes at 37c for 10 min and then mixed . mixtures were incubated for 10 min at 37c , and the reaction was stopped by the addition of 5 ml 0.3 n trichloroacetic acid .
after agitation and filtration , optical density was measured at 280 nm by using a spectrophotometer ( unikon 930 , kontron instrument , italy ) .
the results were compared to a standard curve , which was generated in an identical manner using known amounts of porcine pepsin ( 1 g = 3 peptic units ) , and were expressed as micrograms of pepsin .
in order to know whether or not the intragastric ph is contributing to the mucosal lesions , intragastric ph was measured 4 days after administration of mumijo and ranitidine . the ph measurement was performed as previously reported .
the pylorus of the stomach and esophagocardiac junction were immediately ligated , and the stomach was then removed from each rat , and the gastric contents were collected , centrifuged , and the supernatants were used for ph measurement using ph meter (  50 ph meter , beckman , fullerton , ca , usa ) .
this study protocol was approved by the ethics review committee , kerman university of medical sciences , kerman , iran .
the results were expressed as mean  standard error of mean , and statistical significance was evaluated using one - way anova , followed by a tukey 's post - hoc test .
ph data are shown in figure 1 . the ph was 3.43  0.32 and 3.93  0.54 , respectively , in sham and ranitidine - treated animals and this difference was not significant .
however , when the extract was administered to the acid acetic - treated group , there was a significant increase in ph of gastric contents to 4.54  0.78 ( p < 0.01 ) .
data are presented as mean  standard error of the mean . * * p < 0.01 , mumijo versus sham group gastric pepsin levels in mumijo group were significantly lower than ranitidine group ( p < 0.05 ) . however , there was no difference between ranitidine or sham groups compared to control [ figure 2 ] .
* p < 0.05 , mumijo versus ranitidine group acid acetic caused histopathological lesions including ulcer with transmural necrosis and degeneration of the gastric tissue .
treatment with mumijo extract offered significant protection against all damages to mucosa by acid acetic [ figure 3 ] .
( a ) the normal gastric morphology of the control animals ( h and e , original magnification , 20 ) .
( b ) the histopathological changes in the gastric tissue of the rats in the sham group .
these changes include gastric ulcer with transmural necrosis ( h and e , original magnification , 10 ) .
( c ) the histopathology changes in the ranitidine group including moderate inflammation with small ulcer ( h and e , original magnification , 10 ) .
( d ) the histology changes in mumijo group including extensive repair tissue ( h and e , original magnification , 10 )
ph data are shown in figure 1 . the ph was 3.43  0.32 and 3.93  0.54 , respectively , in sham and ranitidine - treated animals and this difference was not significant .
however , when the extract was administered to the acid acetic - treated group , there was a significant increase in ph of gastric contents to 4.54  0.78 ( p < 0.01 ) .
data are presented as mean  standard error of the mean . * * p < 0.01 , mumijo versus sham group
gastric pepsin levels in mumijo group were significantly lower than ranitidine group ( p < 0.05 ) . however , there was no difference between ranitidine or sham groups compared to control [ figure 2 ] .
acid acetic caused histopathological lesions including ulcer with transmural necrosis and degeneration of the gastric tissue .
treatment with mumijo extract offered significant protection against all damages to mucosa by acid acetic [ figure 3 ] .
( a ) the normal gastric morphology of the control animals ( h and e , original magnification , 20 ) .
( b ) the histopathological changes in the gastric tissue of the rats in the sham group .
these changes include gastric ulcer with transmural necrosis ( h and e , original magnification , 10 ) .
( c ) the histopathology changes in the ranitidine group including moderate inflammation with small ulcer ( h and e , original magnification , 10 ) .
( d ) the histology changes in mumijo group including extensive repair tissue ( h and e , original magnification , 10 )
gastric ulcers are due to inequality between aggressive ( acid - pepsin secretion , helicobacter pylori , bile , increased free radicals , and decreased antioxidants ) and defensive factors ( mucus , bicarbonate secretion , prostaglandins , blood flow and the process of restitution , and regeneration after cellular injury ) of the gastric mucosa . in the present study , it was recognized that the mumijo had protective effects against the gastric ulcer induced by acetic acid .
protective effects of mumijo on the stomach may be related to its anti - secretary action as our results showed that the mumijo reduced the amount of gastric acidity and pepsin levels in the damaged stomach . according to the some reports , mumijo has significant repairing effects .
it was shown that the presence of some polyphenols compounds such as fulvic acids ( fas ) , 4-methoxy-6-carbomethoxybi - phenyl , tirucallane - type triterpenoids , and benzoic acid in mumijo samples have very important role in decreasing of acid - pepsin secretion , cell shedding , and gastric ulcer index .
it also can increase the mucin secretion and carbohydrate / protein ratio which have very important role as anti - oxidant and anti - inflammatory effects as reported by other researchers , so they stated its use in gastric ulcer and wound healing .
in general , mumijo has an expected broad biochemical and pharmacological activities due to its contents of fa and other antioxidant materials .
fas have been taken orally as a therapy for gastritis , stomach ulcers , and colitis . in our study
, it was also recognized that mumijo has regenerative and repairing effects as shown in the histopathological findings .
it was reported that mumijo pretreatment at the dose of 100mg / kg orally reduced ulcer index in immobilization and aspirin induced gastric ulcers . in duodenal ulcers also , mumijo pretreatment significantly reduced the incidence of ulcers induced by cysteamine in rats and histamine in guinea pigs .
it may be partly related to its role in decreasing of acid - pepsin secretion as mentioned above but the other functions of this plant is also important . in some studies ,
it was shown , that alterations in the antioxidant status following ulceration , implying that free radicals may be associated with gastric mucosal damage in rats .
the reactive oxygen species generated by the metabolism of arachidonic acid , platelets , macrophages , and smooth muscle cells may contribute to gastric mucosal damage .
therefore , by scavenging of free radicals , it may be useful for protecting the gastric mucosa from oxidative damage .
mumijo as antioxidant , inhibit lipid peroxidation and other free radical - mediated process , and therefore , they protect the human body from several diseases attributed to the reactions of radicals . additionally , mumijo has significant anti - inflammatory effects in chronic inflammation .
there are some evidence showing the effects of mumijo on increasing superoxide dismutase , catalase , and glutathione peroxidase activities in rats .
, it has also been reported that mumijo has anti - allergic effects on histamine release and causes mast cells degranulation .
antioxidant properties of mumijo extract can be attributed to the presence of dibenzo - pyrones and fa . on the other hand ,
anti - anxiety activity and anti - stress effects of mumijo have roles in healing of gastric ulcer as stated by frawley and lad , who indicated that mumijo had significant anxiolytic and anti - stress activity .
furthermore , due to bacteriostatic and anti - inflammatory action , mumijo extract facilitates the process of wound cleaning from necrotic tissues , granulation , and epithelization and decreases the period of wound healing .
overall , the results of the present study showed that mumijo administration has gastroprotective effects in acetic acid - induced ulcers through decreasing gastric acid and pepsin .
furthermore , the antioxidant and anti - inflammatory effects of this extract can be mentioned as probable mechanisms to improve gastric tissues .
 objective : gastric ulcer is an important clinical problem , chiefly due to extensive use of some drugs . the aim was to assess the activity of mumijo extract ( which is used in traditional medicine ) against acetic acid induced gastric ulcer in rats.materials and methods : the aqueous extract of mumijo was prepared . 
 animals were randomly ( n = 10 ) divided into four groups : control , sham - operated group ( received 0.2 ml of acetic acid to induce gastric ulcer ) , mumijo ( 100 mg / kg / daily ) were given for 4 days postacetic acid administration , and ranitidine group ( 20 mg / kg ) . 
 the assessed parameters were ph and pepsin levels ( by anson method ) of gastric contents and gastric histopathology . 
 ranitidine was used as reference anti - ulcer drug.results:the extract ( 100 mg / kg / daily , p.o . ) inhibited acid acetic - induced gastric ulceration by elevating its ph versus sham group ( p < 0.01 ) and decreasing the pepsin levels compared to standard drug , ranitidine ( p < 0.05 ) . 
 the histopathology data showed that the treatment with mumijo extract had a significant protection against all mucosal damages.conclusion:mumijo extract has potent antiulcer activity . 
 its anti - ulcer property probably acts via a reduction in gastric acid secretion and pepsin levels . 
 the obtained results support the use of this herbal material in folk medicine . 
Materials and Methods
Animals
Study design
Plant material and extraction
Acetic acid-induced ulcer model
Evaluation of pepsin
Measurement of intragastric pH
Ethical considerations
Statistical analysis
Results
pH of gastric contents
Pepsin levels of gastric contents
Histopathological findings of gastric tissue
Discussion