traditional healers play an important role in health care delivery system , particularly for the primary health care , in rural parts of india . many of these traditional herbal health practices are popular among the community and claimed to be effective . however , most of these formulations are not scientifically studied for their safety and efficacy . hence , there is a need to evaluate the safety and therapeutic potentials of these formulations . this formulation is being used for the treatment of spasmodic abdominal pain in rural communities of northern karnataka , especially in gokak and hukkeri talukas in belgaum district of karnataka in india . local traditional practitioners claim that , the formulation is safe and effective in relieving abdominal pain . after detailed discussion with the practitioners , regarding the signs and symptoms of the pain , it was speculated that the formulation might be possessing anticholinergic / antihistaminic activity . several reports are available on chemical components and biological activities of the ingredients in the formulation . ( acanthaceae ) , locally known as nelabevu and is mentioned in various ayurvedic formulations for the treatments of fever , expectorant , diarrhea and inflammatory diseases . its various extracts have been studied for antipyretic , anti - ulcerogenic , analgesic , antioxidant and anti - inflammatory activities . the plant is also reported for hepatoprotective , anti - hyperglycemic and renal protective activities . cassia fistula l. ( caesalpiniaceae ) is the other plant in the formulation , which is known as kakke . in ayurveda , aragvadhadi kvatha churna is an important formulation mentioned in ayurveda , in which c. fistula is a major ingredient . the hepatoprotective , hypoglycemic , antitussive and anti - inflammatory activities are reported for the plant . the third plant , foeniculum vulgare mill . , ( apiaceae ) , commonly known as badesop , contains trans - anethole as its major component . indian pharmacopoeia of ayurveda suggest its uses for various ailments such as shoola , agnimandya , kasa , pravahika and raktadosha . the aqueous extract of f. vulgare has been reported for its anti - ulcerogenic activity . analgesic , anti - inflammatory and antioxidant activities are also reported for various extracts of the plant . the fourth ingredient of the formulation is cuminum cyminum l. ( apiaceae ) , known in local language as jeerige . c. cyminum l. mentioned in ayurveda for agnimandya , atisara , and krimiroga . hingvadi curna , jirakadyarista , jirakadimodaka , hinguvacadi curna are the popular formulations comprising this plant . the reported major chemical components from the plant were a - pinene , 1 , 8-cineole , cuminol , cuminic alcohol , -terpinene , safranal , p - cymene and -pinene . the oil from c. cyminum has been reported for analgesic effect , while aqueous extract of the plant is reported for relaxant activity in guinea pig tracheal chain model . however , no reports are available on antispasmodic activity of the formulation as a whole , which is being used in the community for several generations . the present study was , therefore , undertaken to evaluate the safety and efficacy of the formulation . the selected traditional practitioners were frequently visited and interviewed to get the details on disease , diagnosis , treatment , formulation and proportions of the ingredients , method of preparation , mode of administration etc . the plants incorporated in the formulation , that is , a. paniculata nees . c. fistula l. , f. vulgare mill . and c. cyminum l. were authenticated . their voucher specimens were deposited in the herbaria at regional medical research centre ( icmr ) , belgaum . these specimens are numbered as rmrc-0007 , 432 , 501 and rmrc  500 respectively . the parts of medicinal value from the plants were collected with the help of traditional practitioners . the method of preparation was standardized in consultation with the traditional practitioners . in the practice , 2 tsf of this mixture is given orally , early in the morning for 1-week . the coarse powders of a. paniculata stem ( 1.57 g ) , c. fistula stem bark ( 3.16 g ) , f. vulgare seeds ( 2.3 g ) were mixed with c. cyminum seeds ( 2.4 g ) . as there are no models available to evaluate the spasmolytic activity of the powdered material directly , the water extract of this formulation was prepared using cold maceration . the powder prepared is mixed thoroughly with 250 ml of distilled water for 7 days at room temperature with daily muddling up . the supernatant water was carefully decanted on 8 day and was used for the experiments . histamine and acetylcholine were procured from sigma - aldrich chemical co. calcium chloride ( cacl2 ) , magnesium sulfate , sodium chloride ( nacl ) , dextrose , sodium hydrogen carbonate ( nahco3 ) , potassium chloride ( kcl ) and sodium dihydrogen phosphate ( nah2po4 ) were procured from local supplier . guinea pigs of either sex weighing between 300 and 500 g were procured from shri venkteshwara traders , bengaluru , india . they were housed in galvanized cages under standard conditions ( temperature 25c  3c , relative humidity 5060% and natural light and dark cycle ) . the animals were fed with a balanced diet formulated in the department ( cauliflower , cabbage , gram and wheat flour ) and water was available ad libitum . the study was approved by institutional animal ethics committee , as per the guidelines of committee for the purpose of control and supervision of experiments on animals . a single dose ( 2000 mg / kg body weight ) of herbal formulation was administered next day , between 10:00 and 11:00 am and the animals were observed as per organization for economic co - operation and development ( oecd ) guideline 4232002 . overnight fasted ( with free access of tap water ) guinea - pigs of both sexes were sacrificed with halothane over anesthesia . the terminal portions , of about 1020 mm in length , were taken after discarding the portion nearest to the ileocecal - junction . the intestinal content was eliminated by washing with tyrode solution ( composition nacl 136.9 ; kcl 2.7 ; cacl2 1.3 ; nahco3 11.9 ; mgcl2 1.1 ; nah2po4 0.4 and glucose 5.6 ) as prescribed by jabeen et al . 1.5 cm ileum tissue was mounted in 15 ml organ bath containing tyrode solution at 37c , continuously bubbled with air . effect of acetylcholine and histamine , in a single dose ( 1 g ) , was recorded individually in the absence of herbal formulation . similarly , effect of herbal formulation ( 0.5 ml ) alone was tested in the absence of both acetylcholine and histamine . the test with a single dose of acetylcholine was repeated after wash period to confirm the tissue response . the tissue response for combination of herbal formulation ( 0.5 ml ) and acetylcholine ( 1 g ) was recorded , followed by the test with a single dose of acetylcholine . tissue response for a single dose of histamine was recorded , and test was conducted with a combination of herbal formulation ( 0.5 ml ) and histamine ( 1 g ) . finally histamine , in a single dose , was tested again to confirm the tissue response . all the tests were carried out twice , and the washing was repeated before each test . the percent of ileum relaxation induced by herbal formulation was calculated by assuming the contraction induced by spasmogens ( acetylcholine and histamine ) as 100% . the ileum relaxation or contraction was expressed as mean  standard error of the mean ( sem ) results were analyzed using one - way anova and bonferroni post - hoc test by considering p  0.05 as significant . the selected traditional practitioners were frequently visited and interviewed to get the details on disease , diagnosis , treatment , formulation and proportions of the ingredients , method of preparation , mode of administration etc . the plants incorporated in the formulation , that is , a. paniculata nees . c. fistula l. , f. vulgare mill . and c. cyminum l. were authenticated . their voucher specimens were deposited in the herbaria at regional medical research centre ( icmr ) , belgaum . these specimens are numbered as rmrc-0007 , 432 , 501 and rmrc  500 respectively . the parts of medicinal value from the plants were collected with the help of traditional practitioners . the method of preparation was standardized in consultation with the traditional practitioners . in the practice , 2 tsf of this mixture is given orally , early in the morning for 1-week . the coarse powders of a. paniculata stem ( 1.57 g ) , c. fistula stem bark ( 3.16 g ) , f. vulgare seeds ( 2.3 g ) were mixed with c. cyminum seeds ( 2.4 g ) . as there are no models available to evaluate the spasmolytic activity of the powdered material directly , the water extract of this formulation was prepared using cold maceration . the powder prepared is mixed thoroughly with 250 ml of distilled water for 7 days at room temperature with daily muddling up . the supernatant water was carefully decanted on 8 day and was used for the experiments . histamine and acetylcholine were procured from sigma - aldrich chemical co. calcium chloride ( cacl2 ) , magnesium sulfate , sodium chloride ( nacl ) , dextrose , sodium hydrogen carbonate ( nahco3 ) , potassium chloride ( kcl ) and sodium dihydrogen phosphate ( nah2po4 ) were procured from local supplier . guinea pigs of either sex weighing between 300 and 500 g were procured from shri venkteshwara traders , bengaluru , india . they were housed in galvanized cages under standard conditions ( temperature 25c  3c , relative humidity 5060% and natural light and dark cycle ) . the animals were fed with a balanced diet formulated in the department ( cauliflower , cabbage , gram and wheat flour ) and water was available ad libitum . the study was approved by institutional animal ethics committee , as per the guidelines of committee for the purpose of control and supervision of experiments on animals . a single dose ( 2000 mg / kg body weight ) of herbal formulation was administered next day , between 10:00 and 11:00 am and the animals were observed as per organization for economic co - operation and development ( oecd ) guideline 4232002 . overnight fasted ( with free access of tap water ) guinea - pigs of both sexes were sacrificed with halothane over anesthesia . the terminal portions , of about 1020 mm in length , were taken after discarding the portion nearest to the ileocecal - junction . the intestinal content was eliminated by washing with tyrode solution ( composition nacl 136.9 ; kcl 2.7 ; cacl2 1.3 ; nahco3 11.9 ; mgcl2 1.1 ; nah2po4 0.4 and glucose 5.6 ) as prescribed by jabeen et al . 1.5 cm ileum tissue was mounted in 15 ml organ bath containing tyrode solution at 37c , continuously bubbled with air . effect of acetylcholine and histamine , in a single dose ( 1 g ) , was recorded individually in the absence of herbal formulation . similarly , effect of herbal formulation ( 0.5 ml ) alone was tested in the absence of both acetylcholine and histamine . the test with a single dose of acetylcholine was repeated after wash period to confirm the tissue response . the tissue response for combination of herbal formulation ( 0.5 ml ) and acetylcholine ( 1 g ) was recorded , followed by the test with a single dose of acetylcholine . tissue response for a single dose of histamine was recorded , and test was conducted with a combination of herbal formulation ( 0.5 ml ) and histamine ( 1 g ) . finally histamine , in a single dose , was tested again to confirm the tissue response . all the tests were carried out twice , and the washing was repeated before each test . the percent of ileum relaxation induced by herbal formulation was calculated by assuming the contraction induced by spasmogens ( acetylcholine and histamine ) as 100% . the ileum relaxation or contraction was expressed as mean  standard error of the mean ( sem ) results were analyzed using one - way anova and bonferroni post - hoc test by considering p  0.05 as significant . there was no mortality over a period of observation for 14 days in animals treated with a single dose of 2000 mg / kg . there were no other signs of toxicity and adverse effects . the results of contraction and relaxation are expressed in mm ( millimeter ) and represented as mean  sem [ table 1 ] . the herbal formulation reduced the ileum contraction induced by histamine and acetylcholine agonists [ figure 1 ] . the significant decrease ( p < 0.01 ) was observed in histamine with herbal formulation ( 26.01  3.53 ) , compared to histamine alone ( 53.38  8.29 ) . similar significant decrease ( p < 0.05 ) was observed also in acetylcholine with herbal formulation ( 20.83  4.24 ) , compared to acetylcholine alone ( 44.38  4.84 ) . the herbal formulation alone did not showed any changes ( contraction / relaxation ) in the tissue . changes in treated and nontreated ileum contraction / relaxation effect of herbal formulation on ileum contraction . * p < 0.05 , * * p < 0.01 . hista : histamin , hista + hf : histamin + herbal formulation , ach : acetylcholine , ach + hf : acetylcholine + herbal formulation there was no mortality over a period of observation for 14 days in animals treated with a single dose of 2000 mg / kg . there were no other signs of toxicity and adverse effects . the results of contraction and relaxation are expressed in mm ( millimeter ) and represented as mean  sem [ table 1 ] . the herbal formulation reduced the ileum contraction induced by histamine and acetylcholine agonists [ figure 1 ] . the significant decrease ( p < 0.01 ) was observed in histamine with herbal formulation ( 26.01  3.53 ) , compared to histamine alone ( 53.38  8.29 ) . similar significant decrease ( p < 0.05 ) was observed also in acetylcholine with herbal formulation ( 20.83  4.24 ) , compared to acetylcholine alone ( 44.38  4.84 ) . the herbal formulation alone did not showed any changes ( contraction / relaxation ) in the tissue . changes in treated and nontreated ileum contraction / relaxation effect of herbal formulation on ileum contraction . * p < 0.05 , * * p < 0.01 . hista : histamin , hista + hf : histamin + herbal formulation , ach : acetylcholine , ach + hf : acetylcholine + herbal formulation the herbal formulation showed significant inhibition in the amplitude of spontaneous contraction of guinea pig ileum . results showed that the herbal formulation is capable of inhibiting acetylcholine and histamine - induced contractions . the current study also demonstrated that the herbal formulation has neither relaxing nor contracting effect on ileum ; it antagonizes the effect of spasmogens such as acetylcholine and histamine . among the ingredients of the present formulation , only c. cyminum has been reported to possess relaxant like activity in guinea pig tracheal chain tissue . however , no such reports were available for the other ingredients . the observed antagonism against the spasmogens suggests that the spasmodic action of the formulation was produced by all ingredients of the used herbal formulation . the plants of herbal formulation are not yet reported for such relaxant activity except c. cyminum . based on the data obtained , it can be concluded that , the traditional formulation is found to be safe and effective in the experimental model of the spasmodic pain . the study supports the claim of the traditional practitioners that this formulation can be used for the spasmodic pain treatment . further work is being carried out to determine the responsible fraction of the plant by employing the suitable model .