thirty two consecutive patients attending the internal medicine out patient department ( opd ) of ma podar ayurvedic hospital , mumbai , india , for treatment of obesity who were prescribed basti treatment as per ayurvedic criteria16 , were invited to participate in this study . patients in the age groups of 18 to 60 yr with a bmi  30 kg / m2 and waist - hip ratio  1.0 in men and  0.9 in women were invited to participate in the study after obtaining their written informed consent . acutely ill patients with severe cardiac , respiratory , hepatic and renal dysfunction and pregnant or lactating women were excluded from the study . the study was carried out during the period from august 2007 to december 2010 . as this was a hypothesis generating proof of concept study where we wished to study whether any changes occurred in metabolic and immune parameters in patients who were receiving basti as therapy for obesity , a sample size of at least completed 30 patients was considered adequate for analysis . following written informed consent to participate , a detailed clinical history was taken from each individual and relevant physical examination including recording of weight and measurement of anthropometric data like waist - hip ratio , upper arm circumference and abdominal circumference was carried out . blood sample ( 45 ml ) was collected aseptically from the ante - cubital vein and processed for measurement of various metabolic and immunological parameters . the study protocol was approved by the institutional ethics committee of ma podar ayurvedic hospital , mumbai . after collection of blood sample at baseline ( s1 ) , the prescribed therapeutic course of 16 bastis in a regular pattern as described in charak samhita ( known as kala basti15 ) was initiated . on day 1 , anuvasan basti ( oil enema ) with sesame oil of 240 ml was administered through the anal canal with the help of syringe and a rubber catheter . the next day asthapana basti ( enema with decoction ) consisting of til tail 120 ml , triphala kwatha ( decoction of terminalia belerica , terminalia chebula & phyllanthus emblica)-700 ml , cow 's urine -150 ml , honey 10 g and black salt 10 g was administered . this course of alternate anuvasana and asthapana basti was administered six times and then anuvasan basti four times on consecutive days . during the course , each participant was observed for symptoms of proper , inadequate or over effects of anuvasana basti and asthapana basti daily . after completion of this course of 16 days , the participants were advised a diet of food items that are easy to digest like khichadi , moong dal , roti and requested to avoid fried and carbohydrate rich foods , cold drinks , etc . during the course of basti . they were also asked to alter their lifestyle regimen ( e.g. to avoid sitting and standing for the prolonged period , excessive / loud speaking , strenuous travelling / walking for long distance , day time sleep , suppression of natural urges , sex , overexposure to breeze , sunlight , smoke , dust , use of pillow of unsuitable thickness , etc . ) for a period of 32 days as per the recommendations of ayurveda15 . at the end of this period the course of basti was considered complete . clinical examination and blood investigations were repeated ( s2 ) at this point as in the baseline visit . the final blood sample ( s3 ) was collected 90 days after s2 along with clinical examination to assess whether the effect of basti was maintained . during this period , thus , the participant was under observation for a total of 138 days after being recruited in the study . the day sample 1 ( s1 ) was collected was designated as day 0 , s2 was collected on day 48 ( 16 basti days and 32 days of lifestyle and diet restrictions ) and s3 on day 138 . anthropometric parameters such as weight , bmi , waist- hip ratio , upper arm and abdomen circumference were measured at the time of s1 , s2 and s3 collections . isolation of peripheral blood lymphocytes ( pbls ) was done from the heparinized peripheral venous blood by ficoll - hypaque ( sigma - aldrich , usa ) centrifugation method17 . immunophenotyping of peripheral blood lymphocytes ( pbls ) : pbls were incubated with phycoerythrin ( pe ) labelled mouse anti - human monoclonal antibodies ( mab ) targeted against cd3 , cd4 , cd8 , cd14 , cd19 , cd56 , cd80 , cd86 , cd209 , cd11c , -t cell receptor ( tcr ) and -tcr ( bd pharmingen , usa ) for surface staining as per manufacturer 's instructions . pbls were stained with fluorochrome conjugated monoclonal antibodies against activation markers such as fluorescein 5-isothiocyanate ( fitc ) conjugated cd25 , cd69 , cd71 and pe - conjugated hla - dr ( bd pharmingen , usa ) and further incubated with fitc or pe conjugated mouse anti - human monoclonal antibody targeted against cd3 . estimation of intracellular calcium release : pbls ( 1x10/ml 0.01 m pbs ) were loaded with 5 m fluo-3-am for 30 min at 37c . one g of anti - cd3 mab was added as stimulant and fluorescence intensity was measured immediately on the facscalibur flow cytometer for the determination intracellular calcium release at different time points as previously described18 . measurement of intracellular reactive oxygen species in lymphocytes : reactive oxygen species ( ros ) level in pbl was measured using 2 , 7 dichlorofluorescein diacetate ( dcfh - da ) dye ( sigma , usa ) . anti - cd3 mab ( 1 g ) was added as stimulant and increase in mean fluorescence intensity was measured from 0 to 45 min , as described earlier19 . analysis was performed using cell quest software and results were expressed as mean fluorescence intensity ( mfi ) . lymphocyte turnover by annexin fitc staining : pbls were incubated with pe labelled mouse anti - human monoclonal antibodies targeted against cd3 and cd19 ( bd pharmingen , usa ) for surface staining . lymphocyte turnover ( apoptosis ) was further monitored by annexin v fitc ( bd pharmingen , usa ) staining method as per manufacturer 's guidelines and the data analyzed using the cell quest software . lymphocyte - proliferation assay : proliferative responses in pbls were analyzed using tritiated thymidine incorporation assay , according to standard protocol20 . tritiated thymidine incorporation was measured in a liquid beta scintillation counter ( model 1900 , packard , usa ) as counts per minute ( cpm ) . stimulation index ( s.i . ) was calculated as the ratio of mean cpm of cultures stimulated with anti - cd3 mab and unstimulated cultures . cytokine and immunoglobulin elisa : pbls ( 1.5x10/200 l crpmi / well ) were stimulated with 1 g anti - cd3 mab for 48 h at 37c . cell - free supernatants were collected and analyzed for secreted cytokines interferon gamma ( ifn- ) , interleukin ( il)-10 , il-4 , il-8 , tnf- , il-6 and il-1 using opt - eia sandwich elisa kit ( bd , biosciences , usa ) as per the manufacturer 's instructions . immunoglobulins , igg and igm levels in serum samples from obese individuals were measured using the igg and igm detection kit ( bethyl laboratories inc . , pbls ( effectors ) were co - cultured with cr - labelled k562 targets ( 5000 cells per well ) in effector to target e : t cell ratios 40:1 , for 4 h at 37c , according to standard protocol21 . the percentage of cytotoxic activity was calculated using the following formula : % specific lysis = ( sample cpm - spontaneous cpm)/ ( maximal cpm - spontaneous cpm )  100 . complete blood count ( cbc ) was done on haematology analyzer sysmex kx-21 ( sysmex corporation , kobe , japan ) and erythrocyte sedimentation rate ( esr ) was done by the westergren method22 . fasting blood sugar , liver function tests [ serum bilirubin , serum alanine transaminase ( alt ) , aspartate transaminase ( ast ) alkaline phosphatase ( alp ) , -glutamyl transferase ggt ] , renal function tests ( blood urea nitrogen , serum creatinine , protein , albumin and uric acid ) , minerals ( serum calcium , phosphorus , iron and copper ) , lipid profile ( serum cholesterol , triglycerides , hdl - cholesterol and ldl - cholesterol ) were analyzed using standard biochemical kits ( amp diagnostics , austria ) on a fully automated biochemical analyzer ( biochemical systems international srl , italy ) . fasting insulin , vitamin b12 and endocrine markers ( t3 , t4 , tsh , cortisol ) were measured using standard radioimmunoassay kits ( shinjin medics inc . homeostatic model assessment - insulin resistance ( homa - ir ) index was calculated using the formula [ sugar ( mmol / l ) x insulin]/22.5 . glycosylated haemoglobin and high sensitive ( hs)-crp were measured using nycocard reader ( axis shield , norway ) . vitamin a levels were measured using standard hplc kit ( recipe chemicals , + instruments , gmbh germany ) . ferritin levels were measured using standard elisa kits ( calabasas , ca , usa ) . for assurance of quality , both internal and external quality control ( obtained from christian medical college , vellore , india and bio rad , usa ) samples were processed . for maintaining the integrity of metabolic data all the kits procured for analyzing the parameters immunologic variables - the results were analyzed using spss software ( version 15.0 , spss inc . , usa ) or graphpad prism software ( version 5.01 ) , ( graphpad software , inc . overall significance was assessed by using one - way anova or friedman test ; wilcoxon signed rank test was applied for multiple comparisons at p<0.02 ( after applying the bonferroni correction ) . , intracellular ca+ flux and ros ) was done using medcalc software ( version 11.4.4 ) , ( medcalc software bypa , belgium ) . clinical and metabolic variables - all results were analyzed using graphpad instat software ( version 3.5 ) and graphpad prism software ( version 5.01 ) . the normally distributed data were expressed as mean  sd ( 95% confidence interval ) and analysed using paired  t test . the data not normally distributed were expressed as median ( iqr ) and statistical analysis was done by wilcoxon signed rank test . immunologic variables - the results were analyzed using spss software ( version 15.0 , spss inc . , usa ) or graphpad prism software ( version 5.01 ) , ( graphpad software , inc . overall significance was assessed by using one - way anova or friedman test ; wilcoxon signed rank test was applied for multiple comparisons at p<0.02 ( after applying the bonferroni correction ) . , intracellular ca+ flux and ros ) was done using medcalc software ( version 11.4.4 ) , ( medcalc software bypa , belgium ) . clinical and metabolic variables - all results were analyzed using graphpad instat software ( version 3.5 ) and graphpad prism software ( version 5.01 ) . the normally distributed data were expressed as mean  sd ( 95% confidence interval ) and analysed using paired  t test . the data not normally distributed were expressed as median ( iqr ) and statistical analysis was done by wilcoxon signed rank test . immunologic variables - the results were analyzed using spss software ( version 15.0 , spss inc . , usa ) or graphpad prism software ( version 5.01 ) , ( graphpad software , inc . overall significance was assessed by using one - way anova or friedman test ; wilcoxon signed rank test was applied for multiple comparisons at p<0.02 ( after applying the bonferroni correction ) . , intracellular ca+ flux and ros ) was done using medcalc software ( version 11.4.4 ) , ( medcalc software bypa , belgium ) . clinical and metabolic variables - all results were analyzed using graphpad instat software ( version 3.5 ) and graphpad prism software ( version 5.01 ) . the normally distributed data were expressed as mean  sd ( 95% confidence interval ) and analysed using paired  t test . the data not normally distributed were expressed as median ( iqr ) and statistical analysis was done by wilcoxon signed rank test . of the 74 participants who were prescribed basti treatment during the study period , 48 were included . of these , 15 participants did not come for s2 and s3 collections and were excluded from the analysis . the mean age (  sd , range ) of the participants was 42.5 (  8.44 , 22.00 - 58.00 ) yr . a significant ( p<0.001 ) decrease occurred in weight , bmi , upper arm and abdominal circumference immediately after the course of basti ( at s2 ) and this was maintained at s3 . 1 , a marked decrease in levels of ifn- ( 3.771  4.63 to 1.54  2.23 pg / ml ; p<0.02 ) and il-6 ( 111.2152.05 to 52.85  111.24 pg / ml ; p<0.02 ) was observed at s2 as compared to s1 . il-6 levels remained significantly low in serum even at s3 compared to s1 ( 27.79  79.29 pg / ml ; p<0.002 ) . a reduction in il-1  levels was observed at s3 and a gradual decrease in il-8 levels was noted , although not significant . * p<0.02 , p<0.002 . during the course of basti therapy ( s1 , s2 , s3 ) , no significant alterations were observed in the percentages of t cell subsets ( cd3 , cd4 , cd8 , -tcr and -tcr ) , natural killer ( nk , cd56 ) , b cells ( cd 19 ) , macrophages ( cd14 ) , dendritic cells ( cd209 ) , and regulatory t cells ( cd4 cd25 ) . the activation status of t lymphocytes as measured by the expression of early ( cd3 cd69 ) and late ( cd3 cd25 ) activation markers was also not altered ( table ii ) . expression of immunophenotypic markers and activation status of lymphocytes a marked increase in production of ifn- ( p < 0.02 ) was observed in culture supernatants of pbls stimulated with anti - cd3 mab at s2 , which further increased ( p<0.002 ) at s3 as compared to that noted at s1 . in contrast , a gradual reduction in il-8 production was noted in pbl - culture supernatants upon anti - cd3 mab stimulation ( p<0.02 ) ( fig . 2a ) . there was a positive correlation between il-6 and il-8 levels at s1 ( r= 0.87 ) , s2 ( r= 0.57 ) and s3 ( r= 0.87 ) . no significant changes in levels of cytokines il-6 , il-1  , tnf-  , il-4 and il-10 were observed in pbl - culture supernatants before and after stimulation with anti - cd3 mab . measurement of cytokine production in cell - free culture supernatants of unstimulated peripheral blood lymphocytes ( pbls ) and after stimulation with anti - cd3 mab ( 1 g ) [ ifn- and il-8 ] . figures represent mean values in pg / ml  sd ( n=32 ) ( * p<0.02 , # p<0.002 ) . a significant increase in generation of ros was observed over a time - period of 45 min at s2 compared to that at s1 . when ros - generation was measured at s3 also , a rapid increase in calcium flux was noted at 20 seconds after stimulation with anti - cd3 mab in pbls at s2 , while peak in calcium release was noted at 30 seconds after addition of stimulus ( anti - cd3 mab ) at s3 . no significant modulations in nk - cytotoxicity were observed in obese individuals undergoing basti therapy ( i.e. at s2 and s3 ) . following basti therapy , pbls exhibited higher proliferative response to tcr - agonist at s2 compared to s1 but the difference was not significant ( fig . there was a gradual increase in turnover of t - cells with slight reduction in b - cells at s2 ( fig . 2c ) . generation of ros , intracellular ca release , nk - cytotoxicity and proliferative response of pbls upon stimulation with anti - cd3 mab at different time points . figure represents mean of per cent positive cells  sd ( n=32 ) . as seen in fig . 3 , the levels of igm were markedly elevated from 1.06  0.68 mg / ml at s1 to 3.76  4.08 mg / ml at s2 ( p<0.02 ) with a significant reduction at s3 ( 1.671.10 mg / ml ; p<0.02 ) as compared with s1 . a marginal decrease in levels of igg was at s2 with significant drop ( p<0.02 ) at s3 . a positive correlation between igg and il-6 levels was observed at s2 ( r=0.39 ) and s3 ( 0.66 ) . figures represent mean values of igm and igg in mg / ml  sd ( n=32 ) ( * p<0.02 ) . no significant change was observed on the metabolic parameters like fasting blood sugar , hba1 c , homa - ir , liver function , renal function , serum phosphorus , iron , sodium , potassium , copper , vitamin b12 , t3 , t4 , thyroid stimulating hormone ( tsh ) and any of the variables of lipid metabolism after the course of basti . a decrease was observed in serum insulin at s2 compared to s1 and this reduction was maintained at s3 . a significant decrease was observed in serum cortisol levels at s2 and s3 as compared to s1 ( table iii ) . this was a systematic study carried out to assess certain immunological or metabolic changes in obese individuals after a therapeutic course of basti . a significant decrease was observed in weight , bmi , upper arm and abdominal circumference in obese individuals and this decrease was maintained over a period of three months . the mean weight loss of 3.09 kg at the end of basti treatment ( day 48 from baseline ) was comparable to the various pharmacological interventions like sibutramine , orlistat , phentermine , diethylpropion or bupropion and fluoxetine in obesity23 . further , no side effects were observed during our study over a period of three months indicating the safety of the procedure . the basti treatment lasted 16 days followed by a 32 period of diet and lifestyle change which was common to all patients . it appeared that the whole package of basti and diet in the prescribed package led to a sustained weight loss associated with changes in upper arm and abdominal circumference . according to ayurveda , the ingredients of basti reach up to the small intestine and get absorbed through the gut wall , are distributed in the body and thus exert systemic effects24 . our study confirmed that the therapeutic procedure brought about a weight loss that was sustainable . however , one can not comment on its mechanism of action at this point of time . perhaps as an indicator toward the mechanism of action , was the observation that the elevated serum levels of il-6 and ifn- of these obese patients reduced significantly after basti . the low grade chronic inflammation in obesity is characterized by accumulation of macrophages and other activated immune cells ( t cells , b cells , eosinophils , neutrophils , mast cells and natural killer cells ) in the adipose tissue . m1 macrophages are characterized by enhanced pro - inflammatory cytokine production ( tnf- , il-6 and il-1 ) . on the other hand , m2 macrophages are characterized by the production of anti - inflammatory cytokines such as il10 . adipokines / cytokines released from activated immune cells contribute to insulin resistance that is associated with obesity25 . in obese individuals , increased production of adipokines influences multiple functions such as appetite and energy balance , immunity , insulin sensitivity , angiogenesis , blood pressure and lipid metabolism including production of high amount of tnf- and il-626 . enhanced activity of tnf and il-6 are involved in the development of obesity - related insulin resistance27 . macrophages and dendritic cells infiltrate into expanding adipose tissue28 , where they produce ifn-. basti therapy downregulated levels of il-6 and ifn- at s2 . il-8 has been shown to be produced and released from human adipose tissue and/or adipocytes suggesting il-8 involvement in obesity29 . thus , the results demonstrate that basti treatment ameliorates th1 type inflammatory cytokine response in obese individuals that may attenuate adipose tissue inflammation and insulin resistance without disrupting other immune functions . it was interesting to note that functional properties of t - cells such as proliferative response , intracellular calcium flux and generation of ros were modulated in obese individuals after undergoing basti therapy . fat accumulation is known to increase systemic oxidative stress independent of hyperglycaemia and may relate to the dysregulated production of adipocytokines30 . moreover , regulation of intracellular ca ( [ ca]i ) plays a key role in obesity , insulin resistance and hypertension . our results demonstrated increased generation of ros and higher levels of intracellular calcium in pbls of obese individuals at s2 , which returned to baseline at s3 . a significant increase in serum calcium was seen after the course of basti . though serum calcium decreased after 90 days , it was still higher than baseline level . there have been several isolated reports of an inverse relationship between dietary and serum calcium and indices of obesity31 . no clinically significant changes were observed in haematological parameters , as well as liver and renal function tests over the three month study period in any of the patients indicating safety of the intervention . serum ferritin reduced significantly after the basti treatment and this decrease was maintained up to 90 days . serum ferritin is generally considered as reflective of iron storage , but it is also increased in response to inflammation and is considered as a measure of chronic inflammation32 . there is increasing evidence indicating that higher ferritin levels are observed in obesity ( which is associated with increase in multiple markers of low grade inflammation)33 which further contribute to insulin resistance . the reduction seen in ferritin levels , therefore , indicates the beneficial effects of basti in obesity . serum cortisol levels decreased after the 48 days treatment period , which further decreased after 90 days . the primary reason for endocrine malfunction in human obesity is hypersensitivity and/or hyper - responsiveness of the hypothalamo - pituitary - adrenal ( hpa ) axis . the decrease in cortisol levels observed in our study suggests normalizing of the hpa axis . the basti treatment , however , did not demonstrate significant change in levels of any other hormone . in conclusion , our findings showed that in obese individuals , the ayurvedic basti ( enema ) treatment modulated immune responses by regulating pro - inflammatory cytokines , immunoglobulins and functional properties of t - cells . these changes were associated with a reduction in the body weight which was maintained even after three months .
background & objectives : basti ( medicated enema ) is a popular ayurvedic intervention recommended for obesity . 
 however , there are no data to show whether any physiological or biochemical changes occur following this treatment . 
 this study was conducted to identify the immunological and metabolic changes in obese individuals after a therapeutic course of basti.methods:thirty two obese individuals ( 18 and 60 yr ) with a body mass index ( bmi ) 30 kg / m2 who received a therapeutic course of 16 enemas ( basti ) followed by a specific diet and lifestyle regimen for a period of 32 days as their treatment for obesity , were enrolled in the study . clinical examination , 
 measurement of immune and metabolic markers were done before ( s1 ) , immediately after ( s2 ) and 90 days after the completion of therapy ( s3).results : a significant reduction ( p<0.001 ) in weight , bmi , upper arm and abdominal circumference was seen at s3 , along with a decrease in serum interferon ( ifn)- ( p<0.02 ) , interleukin ( il)-6 ( p<0.02 ) and ferritin ( p<0.05 ) and increase in igm levels ( p<0.02 ) . peripheral blood lymphocytes ( pbls ) stimulated with anti - cd3 monoclonal antibodies showed significant increase in reactive oxygen species ( ros ) generation and calcium flux after basti . 
 all organ function tests revealed no changes.interpretation & conclusions : our study documents that a therapeutic course of basti modulates immune responses by regulating pro - inflammatory cytokines , immunoglobulins and functional properties of t - cells . 
 these changes are associated with a reduction in the body weight which is maintained even after three months of treatment . 
 the study also documents the safety of basti procedure .