Patent Application: US-201214234622-A

Abstract:
the present invention provides methods of producing dosage forms for formulas of elemental compositions encompassing acetate salts of calcium , magnesium and zinc along with vitamin d 3 . the acetate salts could be extracted from natural sources such as pearls , coral , and oyster or compounded using synthetic materials . the dosage and ratio of calcium to magnesium was estimated using in vitro and in vivo estimations . the dosage for promoting bone health and alleviation of osteoporosis is about a quarter to a third of the conventional dose .

Description:
in general , soluble calcium salts have a lower percentage of calcium . for example , calcium ascorbate has only 9 % of calcium . the content is several folds lower than that of the insoluble calcium carbonate ( 40 % calcium ). among soluble calcium , calcium acetate has the highest calcium content ( 25 % calcium ). this makes calcium acetate a suitable candidate for making a solid dosage . the addition of elements and vitamins to a formula lowers the percentage of calcium . this poses a severe challenge to prepare a dosage form that has an acceptable size to consumers . the present invention describes methodologies for preparing dosage forms with acceptable sizes . the present invention also provides a method of preparing tablets comprising calcium acetate , magnesium acetate , zinc acetate and vitamin d 3 , comprising the steps of : ( i ) blending a calcium composition comprising calcium acetate , magnesium acetate , and zinc acetate with a composition comprising vitamin d 3 ; and ( ii ) blending the composition obtained from ( i ) with a calcium composition comprising calcium acetate , magnesium acetate , and zinc acetate , thereby obtaining tablets comprising calcium acetate , magnesium acetate , zinc acetate and vitamin d 3 . in one embodiment , the calcium composition comprises at least 10 percent by weight of calcium acetate , at least 5 percent by weight of magnesium acetate , and at least 0 . 2 percent by weight of zinc acetate . the present invention also provides a tablet produced by the method described above . the present invention also provides a method of preparing soft gel capsules comprising calcium acetate , magnesium acetate , zinc acetate and vitamin d 3 , comprising the steps of : ( i ) dissolving vitamin d 3 in fish oil , flaxseed oil , or other oils containing either omega 3 or omega 3 - 6 - 9 ; ( ii ) mixing the composition obtained from ( i ) with a calcium composition comprising calcium acetate , magnesium acetate , and zinc acetate , thereby obtaining soft gel capsules comprising calcium acetate , magnesium acetate , zinc acetate . in one embodiment , the calcium composition comprises at least 10 percent by weight of calcium acetate , at least 5 percent by weight of magnesium acetate , and at least 0 . 2 percent by weight of zinc acetate . the invention will be better understood by reference to the experimental details which follow , but those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative , and are not meant to limit the invention as described herein , which is defined by the claims which follow thereafter . throughout this application , various references or publications are cited . disclosures of these references or publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains . it is to be noted that the transitional term “ comprising ”, which is synonymous with “ including ”, “ containing ” or “ characterized by ”, is inclusive or open - ended and does not exclude additional , un - recited elements or method steps . a pearl extract was prepared by adapting the patented method reported by li and li ( 31 ). briefly , pearls are pulverized to a size between 80 to 120 mesh . the powder is soaked in a mixture of saturated sodium chloride solution with titrated amount of acetic acid . electrical current is applied to the mixture for several days . after dilution with water and magnetization , the mixture was filtered and precipitated . the precipitate , rich in calcium acetate , is dried and ready for consumption as a dietary supplement . a detailed list of elements present in the extract is presented on table 1 : this extract , a1 , is fortified with acetate salts of magnesium to provide ca / mg ratios of 0 . 5 / 1 ( a6 ), 1 / 1 ( a4 ) and 2 / 1 ( a5 ). the major elemental content of the pearl extract and its fortified mixtures are listed on table 2 : besides pearl , the method described in this example can also be used to extract multiple acetate salts of calcium , magnesium and zinc from natural sources such as corals , oysters , mineral mines , etc . the composition of formulas a1 , a4 through a6 could also be achieved by mixing appropriate amounts of acetates salts of calcium , magnesium and zinc . experimental data on elemental solubility . the gastrointestinal tract is a complex organ . there are a number of factors which could alter the solubility of elements including calcium , magnesium and zinc ; subsequently , their rate of absorption and bioavailability . examples 2 - 5 highlight some of the physiological factors which have been postulated to have a significant impact on the solubility of elements . the solubility of calcium in the four formulas in an artificial gastric ( ph = 1 ) and intestinal fluid ( ph = 7 ) was tested using a method developed for icp - oes ( inductively coupled plasma optical emission spectrometer ) ( perkinelmer optima 4300dv ). two commercial samples , caltrate ™ and calcium acetate , were also tested in parallel for comparison . the results are shown in table 3 . compared to caltrate ™, the solubility of calcium acetate is approximately 45 times higher in the artificial gastric juice and 26 , 000 times higher in the artificial intestinal juice . the solubility of the pearl extract , a1 , comprising mostly calcium acetate , is similar to that of calcium acetate in the artificial gastric juice and intestinal juice ( p & gt ; 0 . 05 ). the solubility of calcium acetate is ph dependent ; it is lower in the artificial intestinal fluid when compared to the artificial gastric juice . magnesium has a tendency to lower the solubility of calcium . when the ratio of ca / mg decreases , the solubility of the extract decreases , a5 & gt ; a4 & gt ; a6 . nevertheless , a6 , the least soluble pearl extract formula , is ˜ 12 times more soluble in artificial gastric juice and 8 , 500 times more soluble in artificial intestinal juice than that of caltrate ™ therefore , unlike caltrate ™, solubility of acetate salts should not be an issue in gastrointestinal tract fluids because the acetate salts will still be in solution . the solubility profile of magnesium salts is very similar to that of calcium ( table 4 ). in general , acetate salts of magnesium are highly soluble . they are more soluble in artificial gastric juice than artificial intestinal juice . in contrast to magnesium acetate , the solubility of magnesium carbonate in caltrate ™ is low . the solubility profile of zinc salts is also similar to that of magnesium and calcium , except the magnitude of difference between salt forms under differing ph and environmental conditions is less drastic ( table 5 ). this set of experiments thus leads to the conclusion that acetate salts are preferred salts in the disclosed formulations for their high solubility . in this example , the effects of ph ( ranging from 1 to 9 ) on the solubility of three elements of the four pearl formulas ( a1 , a4 , a5 and a6 ), a commercial product ( caltrate ™) and a synthetic compound ( calcium acetate , ca ace ) were investigated . solution ph was adjusted using appropriate amounts of acetic acid ( acoh ), nitric acid ( hno 3 ) or ammonium hydroxide ( nh 4 oh ). saturated solutions were prepared by dissolving each preparation in a solution with a final ph value ranging from 1 to 9 . the resultant mixtures were incubated in a water bath at 37 ° c . for one hour . each sample was then filtered ( with or without centrifugation ) immediately , and the filtrate was diluted to an appropriate concentration for elemental analysis . the concentration of calcium , magnesium , and zinc was measured using icp - oes . the results are shown in tables 6 - 8 . statistical analysis was performed using one - way anova and the p value was set at 0 . 05 . throughout the ph range tested , both a1 and calcium acetate showed significantly higher calcium content in solution than the other preparations . caltrate ™ had the lowest calcium content ( p & lt ; 0 . 05 ). a1 and calcium acetate have the highest solubility at ph 1 ( table 6 ) magnesium has a negative effect on the content of calcium in solution ; the rank order in terms of solubility is a5 & gt ; a4 & gt ; a6 . except for caltrate ™, calcium acetate and a1 , which are more soluble at ph 1 , ph has no effect on the solubility of magnesium in solution ( table 7 ). similarly , the amount of zinc in solution correlated well with the zinc content in different formulations ( a5 & gt ; a4 & gt ; a1 & gt ; a6 ) ( table 8 ). for all four acetate formulas tested , ph values higher than 5 were associated with higher solubility of zinc than that at ph 2 and 3 . since intestinal ph values are typically higher than 6 , the present formulations present advantages in terms of solubility , when compared with the solubility of calcium carbonate in caltrate ™ under such ph conditions . these results are consistent with those reported in table 3 . the following analyses using anions which are present in abundance in gastro - intestinal tract fluids were performed on the four test formulas ( a1 , a4 , a5 and a6 ), caltrate ™ and calcium acetate in order to assess the solubility and subsequently , their rate of absorption and bioavailability . the following are the standard ranges of common anions and cations in the human gastrointestinal fluids . the following analyses using anions which are present in abundance in gastro - intestinal tract fluids were performed on the four test formulas ( a1 , a4 , a5 and a6 ), caltrate ™ and calcium acetate in order to assess the solubility and subsequently , their rate of absorption and bioavailability . in this example , the effects of bicarbonate and phosphate ( hco 3 − and po 4 3 − ) on the solubility of calcium , magnesium , and zinc were studied at ph 7 . furthermore , the effects of chloride on the absorption of these three elements at ph 1 and ph 7 were also studied . the procedures described in example 3 for ph adjustment and solubility measurements were used . icp - oes was used to quantify calcium , magnesium and zinc . statistical analysis was performed using one - way anova and the level of significance was set at p & lt ; 0 . 05 . tables 10 - 12 are the results of chloride effects at ph 1 . this condition mimics that of the acidic environment in the stomach . chloride has the most intense effect on the solubility of calcium , magnesium and zinc in caltrate ™ at ph 1 ( tables 10 - 12 ). at a cl − concentration of 200 mm , the solubility of calcium was the highest . the maximum magnesium and zinc solubility was reached at cl − concentrations of 50 mm and 120 mm , respectively . the fluctuations of calcium , magnesium and zinc solubility are minimal in all the acetate formulations : calcium acetate , a1 , a4 , a5 and a6 . significant differences are often obtained at the highest cl − concentration ( p & lt ; 0 . 05 ). at ph 7 , the solubility of calcium in caltrate ™ is dramatically lower than that at ph 1 in the presence of chloride ( compare values in tables 10 and 13 ). as chloride concentration increased , the solubility of calcium in caltrate ™ increased . the ph and chloride effects are not pronounced for the acetate formulations . in general , maximum calcium solubility is reached at chloride concentrations between 50 to 100 mm . in the presence of chloride , ph has less of an effect on magnesium solubility ( compare values between tables 11 and 14 ). in general , the solubility of magnesium at ph 7 is slightly lower for all formulas and the chloride effect is not pronounced . in the presence of chloride , the solubility of zinc in caltrate ™ at ph 7 is less than half of that at ph 1 ( compare values between 11 and 14 ). however , this difference is not as pronounced in the acetate formulas . there is a tendency for zinc solubility to increase with the increase of chloride concentration . maximum zinc solubility is reached at 120 mm chloride when caltrate ™ was evaluated . for the acetate formulas , maximum zinc solubility occurred when chloride concentration reached 200 mm . the solubility of calcium in caltrate ™ increased with the increase of bicarbonate concentration ( table 16 ). however , the opposite is true for calcium acetate . the solubility was reduced at least 40 %. the reduction for all the pearl extract formulas was less , approximately 20 to 25 %. the solubility of magnesium in caltrate ™ increased with bicarbonate concentration ( table 17 ). bicarbonate effect was minimal for the acetate formulas . the solubility of zinc in caltrate ™ increased in the presence of bicarbonate ( table 18 ). maximum zinc solubility was reached at 70 mm . for calcium acetate , the trend is similar to that of caltrate ™. bicarbonate has very little effect on the pearl extract formulas . phosphates have insignificant effects on the solubility of calcium in caltrate ™ ( table 19 ). as phosphate concentrations increased the solubility of calcium decreased in all acetate formulations . maximum reduction ( up to 40 %) of the solubility of calcium was observed in formulas containing higher percentage of magnesium ( a4 , a5 and a6 ). considering the range of phosphate concentration tested , 10 , 000 - fold , the change of calcium solubility is not significant . magnesium solubility decreased as phosphate concentration increased ( table 20 ). the reduction ( 80 %) is most significant for the magnesium in caltrate ™. for the other formulas , the maximum reduction was approximately 50 %. again , the effect of phosphates was not that significant considering the range of concentration tested . among the three elements , phosphates have the most intense effect on the solubility of zinc ( table 21 ). all formulas were affected to the same extent and the maximum reduction was approximately 70 %. considering the range of phosphate concentration tested , again , the effects of phosphates were not that significant . the following analyses using cations which are present in abundance in gastro - intestinal tract fluids were performed on the four test formulas ( a1 , a4 , a5 and a6 ), caltrate ™ and calcium acetate in order to assess the solubility and subsequently , their rate of absorption and bioavailability . the effects of na + concentration on the solubility of the three elements in the four formulations ( a1 , a4 , a5 , and a6 ), caltrate ™ and caace were investigated at gastric ph ( ph = 1 ) and intestinal ph ( ph = 7 ), respectively . tables 22 and 23 show the results tested at ph 1 . no significant effects of na + concentration on calcium and magnesium solubility of all formulations were observed . solubility of zinc in caltrate ™ and calcium acetate , which contained trace amounts of zn , increased significantly with an increase in sodium concentrations ; however , no significant differences were obtained for all the acetate formulations ( table 24 ). tables 25 - 27 show the effects of sodium ion at ph 7 . na + has no significant effects on calcium , magnesium and zinc solubility in general . it is interesting to note that all three elements in caltrate ™ could be not detected in the presence of na + at ph 7 . there is a tendency for the solubility of calcium to increase with an increase in potassium ion concentration ( table 28 ). however , most of the differences are not statistically different ( p & lt ; 0 . 05 ). in a5 , the calcium solubility increased by more than 50 %; this difference is significant ( p & lt ; 0 . 05 ). magnesium solubility profiles for the acetate formulas show a similar trend ( table 29 ) to that of calcium . there is a three - fold increase in the magnesium solubility in caltrate ™, ( p & lt ; 0 . 05 ). however , the magnitude of increase in inconsequential when compared to that of a4 , a5 and a6 . an increase in potassium is associated with an increase in zinc solubility for caltrate ™ and caace ( table 30 ). potassium has insignificant effect on the solubility of zinc in the four formulas ( p & gt ; 0 . 05 ). again , the magnitude of increase in zinc solubility is inconsequential when compared to a4 , a5 and a6 . there was a tendency for the solubility of calcium to increase with an increase in potassium concentration , however , the difference is not significant , p & gt ; 0 . 05 ( table 31 ). no calcium could be detected in preparations using caltrate ™. similar observations to that of calcium were obtained for the solubility of magnesium and zinc ( p & gt ; 0 . 05 ) in all formulas containing acetate salts ( tables 32 - 33 ). no measurable magnesium and zinc was reported for preparations using caltrate ™. two diets , one with normal calcium and the other is calcium free , were used for the studies . the nutrient composition of the diets is listed on table 34 : male sprague - dawley rats ( about 6 - 7 weeks ), with an initial weight between 220 g to 250 g , were randomly divided into different treatment groups . all the rats were housed in individual metabolic cages in a temperature - controlled room . each rat received free access to the normal diet ( table 34 ) before the experiment . both normal and calcium free diets ( table 34 ) were used in this set of studies . de - ionized water was provided ad libitum . all the rats were weighed before treatment . two sets of studies were performed : a normal diet and calcium free diet . in each study , there were seven treatment groups . thirty five animals were randomly assigned to one of the treatment groups in which one of the following were administered : caltrate ™, calcium acetate ( ca ace ), a1 , a4 , a5 , a4 plus vitamin d 3 and a5 plus vitamin d 3 ( n = 5 per group ). rats participating in the normal diet study received normal diet ad libitum throughout . rats participating in the group of calcium free diet received the calcium free food ad libitum starting five days before and throughout treatment . in both study groups , animals received one dose a day for five days . amounts of calcium , magnesium and zinc in individual formulation and in each diet were determined using icp - oes . values of dosage and dietary intake were measured for the calculation of elemental balance . for rats that were fed the normal diet , average daily elemental intake of calcium , magnesium and zinc was 625 , 155 and 10 mg / kg / day , respectively . daily elemental dosages , similar to that of human &# 39 ; s , are 53 . 14 mg / kg for calcium , 0 . 38 to 55 mg / kg / day for magnesium and 0 . 017 to 2 . 5 mg / kg / day for zinc . vitamin d 3 , 1 . 06 μg / kg / day ( 42 . 512 iu / kg / day ; 1 iu = 0 . 025 μg ), was added to each dosage preparation prior to administration . the vehicle for preparing each dose was de - ionized water . the concentration of calcium in all dosage preparations was 15 . 94 mg / ml . one ml of each preparation was administered by gavage . body weight , elemental dosage and diet consumption were recorded daily . animals were housed individually in a metabolic cage five days before the study . food consumption was evaluated daily . urine and feces were collected daily for four days and the content of calcium , magnesium and zinc was determined . on day 5 , each animal received its treatment . these treatments were administered once a day for four days . after the last treatment , each animal was anesthetized shortly before peak elemental blood concentration was achieved . blood was collected using a heparinized syringe via cardiac puncture . immediately after blood collection , the animal was then sacrificed with an overdose of isoflourane . each blood sample was centrifuged at 1900 rpm at room temperature ; plasma was harvested and stored at − 20 ° c . until analysis . urine was measured daily ; it was diluted with de - ionized water , filtered and an aliquot was stored at − 20 ° c . until analysis . daily fecal output was collected and lyophilized . each sample was weighed and digested using a mixture of three volume of nitric acid and one volume of perchloric acid . for every gram of dried feces , 10 ml of acid mixture was added . each sample was digested for three days . the volume of the digested sample was measured and an aliquot of the digest was stored at − 20 ° c . until analysis . the content of calcium , magnesium and zinc in plasma , feces and urine were determined using icp - oes . cumulated calcium balance and % cumulated net calcium balance were calculated using equations ( 1 ) and ( 2 ), except , the sum of daily intake and excretion was used for calculation . the balance for magnesium and zinc was also calculated using the concept of equations ( 1 ) and ( 2 ). cumulated elemental balance and % cumulated net elemental balance were calculated in a similar fashion as described above . in general , urinary excretion accounted for less than 5 % of fecal excretion . therefore , fecal excretion practically determines the quantity of elemental balance . all results were analyzed using two - way anova . p & lt ; 0 . 05 was considered to be significantly different . the data are presented as mean ± s . d . and mean ± s . e . m . in tables and figures , respectively . table 35 shows the body weight of rats during the study . stools from study animals were soft and this observation could be related to low elemental intake . insufficient elements from the diet and dosage may have also caused the lack of weight gain for this set of animals . there is a statistical difference ( p & lt ; 0 . 05 ) among the starting body weights of the study animals ( table 35 ). there is also a slight in decline in body weight during the treatment period ; is not the difference significantly different . the addition of magnesium and zinc to a formula promotes the retention of calcium . a1 , a composition with miniscule amounts of magnesium and zinc , has a lower calcium retention ( 17 %, table 36 ); whereas the retention of calcium is significantly higher when the ratio of ca / mg was increased to 2 / 1 ( a5 ), the calcium retention is 49 % ( table 36 ). a higher proportion of magnesium , such as that present in a4 , does not produce more changes in calcium retention ( 49 %, table 36 ). with respect to the minimum amount of magnesium required to provide the highest calcium retention , it appears a 2 / 1 ca / mg ratio is optimal . the addition of vitamin d 3 increases calcium retention significantly ( fig2 and table 36 ). calcium retention increased to 62 % when vitamin d 3 was added to a5 ( table 36 ). this value is more than five times higher than that of the caltrate ™ and caace groups . magnesium appears to be required in order to maintain magnesium balance ( i . e . to avoid magnesium depletion ) ( table 37 ). formulas ( caltrate ™, caace and a1 ) that have miniscule amounts of magnesium caused a net loss of magnesium ( fig3 and table 37 ). the addition of vitamin d 3 has no significant effect on the retention of magnesium . the cumulative net percentage of magnesium did not change significantly after vitamin d 3 was added to a4 and a5 ( fig3 and table 37 ). the retention of zinc is highly variable ; it is particularly true with formulas such as caltrate ™, calcium acetate and a1 that contain minute amounts of zinc ( table 38 ). the results also show that zinc balance became negative when the amount of zinc is low . the addition of zinc to formulas such as a4 and a5 did not significantly improve zinc balance ( table 38 ). the addition of magnesium to the formulas may have caused zinc balance to stay negative ( fig4 ). however , the addition of vitamin d 3 to a4 and a5 made zinc balance positive ( fig4 and table 38 ). the importance of vitamin d 3 on zinc is clearly demonstrated in this set of studies . fig5 shows plasma elemental profiles after each treatment . there were no significant differences observed after elemental treatments . the pattern of calcium retention appears to be similar to that obtained from rats that received calcium free diet ( compare tables 36 and 40 ); suggesting calcium balance is dependent upon elemental treatments , despite the fact that the amount of calcium administered was approximately 10 % of the animal &# 39 ; s daily dietary intake (˜ 130 to 140 mg of calcium per day ). this observation strongly suggests that dietary calcium , present in the least absorbable carbonate form , was enhanced by elemental treatments . the treatment with caltrate ™ has minimal effect . it is not surprising because caltrate ™ contains only calcium carbonate . the treatment with a5 has the most pronounced effect ( fig6 and table 40 ). average dietary intake of magnesium by the study animals was approximately 35 mg . magnesium balance for all study groups was positive ( fig7 and table 41 ). this observation is consistent with the observation obtained from animals receiving the calcium free diet , in that magnesium intake is required to maintain a positive balance ( tables 37 and 41 ). interestingly , the day to day trend showed that animals treated with acetate formulas ( caace , a1 , a4 and a5 vs . caltrate ™) had consistently higher percentages of magnesium balance . there were no statistical differences among elemental treatments in terms of zinc balance ( fig8 and table 42 ). the quantity of zinc administered via elemental formulas was no more than 30 % of the daily dietary intake . it was noted that the addition of a high quantity of magnesium tended to lower zinc balance , a trend observed with a4 treatment ( fig8 and table 42 ). this observation is similar to that observed in the calcium free diet study ( table 38 ). contrary to the calcium free diet study ( table 38 ), zinc balance was positive in this study ( table 42 ). this was achieved without vitamin d 3 ( fig4 and 8 , tables 38 and 42 ). this apparent discrepancy may be due to the quantity of total zinc intake and / or the rate at which zinc was consumed . elemental consumption , along with other nutrients , occurred throughout the feeding period which may last up to 12 hours ; whereas elemental treatments were given as a bolus . concentration and ratio of nutrients presented to the intestinal wall may have a huge difference between bolus administration and dietary consumption . these differences could account for the difference in zinc balance . the results from the calcium free and normal diet studies clearly suggest that adequate dietary intake of elements is key to elemental balance . elemental and vitamin d 3 supplementation are necessary if the diet in deficient in these nutrients . fig9 shows plasma concentration of calcium , magnesium and zinc after individual elemental treatments . there were no statistical differences in the concentration of these elements in plasma after elemental treatments ( p & gt ; 0 . 05 ). the objective of this study was to evaluate elemental balance when the daily intake of calcium , magnesium and zinc was replaced with elemental treatments . animals , received de - ionized water ad libitum ( di water group ), were fed normal calcium diet . animals , substituting their daily calcium intake by a1 or a5 , were fed calcium free diet . it is apparent that the gavage procedure did not have an effect on the body weight of the animals ( table 43 ). elemental treatments , however , induced a significant reduction in body weight . contrary to the results obtained from the normal and calcium free diet studies , magnesium has a minor effect in enhancing calcium retention ( fig1 and table 44 ). the administration of a soluble form of calcium , calcium acetate , significantly enhanced calcium balance ( fig1 and table 44 ). despite a higher amount of zinc administered with a5 , zinc balance was significantly lower than that of the di water group , providing further support that high calcium and magnesium concentration in the intestine could have diminished zinc absorption . ( fig1 and table 46 ). the amounts of zinc administered between the di water and a1 groups were similar . however , similar to that of a5 , zinc balance was significantly lower than that of di water ( fig1 and table 46 ); suggesting high solution concentration of calcium in the intestine may interfere with zinc absorption . this set of results suggest that elemental dietary intake of elements does not produce the same effects when compared to that of an equivalent bolus dose . taking all the study results into consideration , a5 produces the most consistent calcium balance under different experimental / dietary conditions ( compare results on tables 36 , 40 and 44 ). the addition of vitamin d 3 enhances calcium retention of a5 when the subject is deficient in dietary elements ( table 36 ). fig1 shows plasma concentrations of calcium , magnesium and zinc after each elemental treatment . no statistical differences were found in these profiles ( p & gt ; 0 . 05 ). the objectives of this study were to evaluate the effects of salt , mineral composition and vitamins on the rate of bone loss in an ovariectomized rat model . one hundred 4 . 5 - month - old female sprague - dawley rats were used and housed at the laboratory animal services center at the chinese university of hong kong with 12 - h light - night cycle . free cage movement was allowed with access to the normal calcium pellets and tap water . daily consumption of calcium was approximately 140 mg , similar to that recorded in animals who participated in the balance studies . ovariectomy ( ovx ), the removal of ovaries from the female rats , was performed on all rats at 6 - month of age with the exception of the sham control . three weeks after ovx , all the rats recovered from the trauma of the surgery . the rats were randomly divided into different treatment groups or control groups and each group contained six rats . four calcium formulas ( a1 , a4 , a5 and a6 ) and caltrate ™ were investigated in the present study . the caltrate ™ group served as an elemental treatment control . all formulas were dissolved in distilled water , while caltrate ™ was in suspension in distilled water . the solution or suspension was given to the rats daily for 8 weeks by gavages . the dose of all formulas was calculated based on a calcium dose of 53 . 14 mg / kg / day . dose of vitamin d 3 and vitamin k 2 was 12 . 75 iu / kg / day ( equivalent to 800 iu / 70 kg man / day ) and 1 . 71 μg / kg / day ( equivalent to 120 μg / 70 kg man / day ), respectively . all the treated rats were weighed daily and the mass data were recorded . the rats in two control groups ( sham control and normal control ) were given the equivalent volume of distilled water in parallel . for the groups with the treatment of bisphosphonate , alendronate ( 14 μg / kg / 2 - week ) was injected subcutaneously on the back of the rats once every two weeks . at the end of 8 weeks , the rats were anesthetized using isoflourane . blood sample was then taken via heart puncture . the rats were then euthanized under anesthesia by neck dislocation , and right hip , right femur and right tibia of each rat were collected for analysis . plasma was collected from blood samples centrifuged at 1500 g for 15 min . plasma concentrations of calcium , magnesium , and zinc were measured using icp - oes . results show that plasma calcium levels were not statistically different from that of the sham control ( p & gt ; 0 . 05 ) and the values are all within normal levels ( 90 - 110 mg / l ). all plasma concentrations of mg were within the normal range ( 18 - 36 mg / l ). no significant difference in magnesium plasma concentrations was observed except normal control ( without surgery ) has a mean value higher than that of a4 + vit d + vit k ( p & lt ; 0 . 05 ). similarly , plasma concentrations of zn in all rats reached the rat normal concentration at about 1 . 26 mg / l . zn plasma concentrations of rats in the normal control was significantly higher than that of sham control rats and also the rats treated with a5 + vitamin d and a4 + vitamin d + vitamin k ( p & lt ; 0 . 05 ). body weight changes for different treatment groups are shown in fig1 . as expected , weight gains in the ovx rats were significantly greater than the normal rats ( p & lt ; 0 . 05 ). the effects of test substances on bone mineral density ( bmd ) are shown on fig1 and 16 . trabecular bmd of distal femur bmd values of groups a1 , a5 + vit d , bis + a1 + vit d , bis + a4 + vit d , bis + a5 + vit d and bis + caltrate + vit d are significantly higher than that of the ovx control ( fig1 ), suggesting these treatments significantly slow down the rate of loss of bone mass . the addition of vitamin k did not have any significant effect on reducing the rate of bone loss . similar observations were obtained for the average values of trabecular bmd of proximal tibia , except the value of caltrate ™ was high enough to become statistically different ( p & lt ; 0 . 05 , fig1 ). again , vitamin k did not have any significant contribution . the treatment with a5 + vit d provided consistently higher bmd at distal femur and proximal tibia , suggesting this formula may have an advantage over the other elemental formulas . although , the addition of bisphosphonate provides consistently better results , the difference , when compared to a5 + vit d and other elemental formula , such as a1 , was not significant ( fig1 and 16 ). the bmd results of a1 are similar to that of a5 + vit d . this is not surprising because a1 animals were fed normal calcium diet which contains a significant amount of magnesium . the ovx rat model used in this study did not permit evaluation of maximum bending force and failure energy after each treatment because the values obtained from the ovx control and that of the sham were insignificantly different from each other ( p & gt ; 0 . 05 ). fruit juices contain a number of acids such as malic acid , citric acid , etc . which may alter the solubility and hence the recovery of the three key elements in the formulae , hence changing the absorbability of these elements when administered in juice format . the objectives of this study were to evaluate the effects of temperature and storage on the recovery of calcium , magnesium and zinc in a5 after mixing with filtered and unfiltered orange , grape and carrot juice . a 2 . 6 g or 500 mg amount of a5 was weighed accurately and mixed with 330 ml of water or either filtered or unfiltered grape , orange or carrot juice . the specimens were prepared at either 4 or 21 ° c . the elemental content was measured using icp - oes . small quantities of calcium , magnesium and zinc were found in orange , grape and carrot juice ( tables 47 , 50 and 53 ). temperature and filtration had no effects on the recovery of calcium , magnesium and zinc of a5 when 2 . 6 g of a5 was used for the study ( tables 48 , 51 and 54 ). storage at 4 ° c . for a week did not change the recovery of calcium , magnesium and zinc when 2 . 6 g of a5 was dissolved in 330 ml of filtered and unfiltered orange and grape juice ( tables 48 and 51 ). however , when 500 mg of a5 was used instead , the recovery of calcium and magnesium was significantly lowered from the unfiltered orange juice ( table 49 ). the lower recovery of calcium from unfiltered orange juice suggests that the pulp in orange juice may bind ca and mg in a5 . carrot juice did not have this problem ( table 54 ). this set of studies suggests that a5 can be used to fortify a number of juices and water . the 2 . 6 g of a5 provides a daily requirement of the three key elements for the prevention of osteoporosis : 300 mg of calcium , 150 mg of magnesium and 5 . 6 mg of zinc . 500 mg of a5 is intended to provide a serving of these elements in the functional food format . the relatively low calcium content in a5 has posed a challenge in creating a solid form with a size that is acceptable to end - users . the following formulation was created in tablet form ( table 55 ): assay value : 100 , 000 iu vitamin d 3 / g (= 2500 microgram cholecalciferol / g ). the target weight of vitamin d 3 per tablet is 2 . 5 mg . 30 % extra vitamin d 3 has been added per tablet as overage . the manufacturer assay value is 100000 iu / g i . e . 100 iu / mg . since 2 . 5 mg ( 3 . 25 mg with 30 % overage ) has been used each tablet has ˜ 250 iu of vitamin d 3 . step 1 : calcium acetate blend provided was sieved through 40 mesh screen and 100 / 120 mesh screen . the fraction that passed through the 40 mesh screen and was retained on 100 / 120 mesh screen was used for formulation . the fraction of calcium acetate above 40 mesh and below 100 mesh was not used for formulation . this fraction was chosen to keep the particle size similar to other ingredients — vitamin d 3 and kollidon va 64 . step 2 : blending 01 : 6 . 5 g of dispensed dry vitamin d 3 100 gfp / hp and 65 g of kollidon va 64 were blended for 5 minutes at a speed of 25 rpm using a small tumble blender to produce blend 01 . step 3 : blending 02 : 250 g of dispensed calcium acetate blend ( blend 01 * 3 . 49 ) prepared in step 1 was mixed with blend 01 prepared in step 2 for 5 minutes to produce blend 02 ( using tumble blender at 25 - 30 rpm ). step 4 : blending 03 : 250 g of dispensed calcium acetate blend prepared in step 1 was mixed with blend 02 prepared in step 3 for 5 minutes to produce blend 03 ( using double cone blender at 25 - 30 rpm ). step 5 : blending 04 : 600 g of dispensed calcium acetate blend prepared in step 1 was mixed with blend 03 prepared in step 4 for 9 minutes to produce blend 04 ( using double cone blender at 25 - 30 rpm ). step 6 : blending 05 : 5 . 86 g of dispensed magnesium stearate was mixed with blend 04 prepared in step 5 , for 2 minutes . step 7 : the final blend prepared above was dispensed using a rotary table press with target tablet weight of 588 . 7 g . the calcium acetate blend in the above table comprises 14 % calcium acetate , 7 % magnesium acetate and 0 . 7 % zinc acetate . magnesium stearate was used as a lubricant . the dry vitamin d 3 100 gfp / hp composition ( as mentioned in the certificate of analysis provided by basf ) is as presented above . assay value : 100 , 000 iu vitamin d 3 / g (= 2500 microgram cholecalciferol / g ). the target weight of vitamin d 3 per table is 5 mg . 30 % extra vitamin d3 has been added per tablet to account for loss due to degradation . the manufacturer assay value is 100000 iu / g i . e . 100 iu / mg . since 5 mg ( 6 . 5 mg with 30 % overage ) has been used each tablet has ˜ 500 iu of vitamin d 3 . step 1 : calcium acetate blend provided was sieved through 40 mesh screen and 100 / 120 mesh screen . the fraction that passed through the 40 mesh screen and was retained on 100 / 120 mesh screen was used for formulation . the fraction of calcium acetate above 40 mesh and below 100 mesh was not used for formulation . this fraction was chosen to keep the particle size similar to other ingredients — vitamin d 3 and kollidon va 64 . step 2 : blending 01 : 13 g of dispensed dry vitamin d 3 100 gfp / hp and 130 g of kollidon va 64 were blended for 5 minutes at a speed of 25 rpm using a small tumble blender to produce blend 01 . step 3 : blending 02 : 500 g of dispensed calcium acetate blend ( blend 01 * 3 . 49 ) prepared in step 1 was mixed with blend 01 prepared in step 2 for 5 minutes to produce blend 02 ( using double cone blender at 25 - 30 rpm ). step 4 : blending 03 : 500 g of dispensed calcium acetate blend prepared in step 1 was mixed with blend 02 prepared in step 3 for 5 minutes to produce blend 03 ( using double cone blender at 25 - 30 rpm ). step 5 : blending 04 : 1200 g of dispensed calcium acetate blend prepared in step 1 was mixed with blend 03 prepared in step 4 for 9 minutes to produce blend 04 ( using double cone blender at 25 - 30 rpm ). step 6 : blending 05 : 11 . 72 g of dispensed magnesium stearate was mixed with blend 04 prepared in step 5 , for 2 minutes . step 7 : the final blend prepared above was dispensed using a rotary table press with target tablet weight of 1 . 17 g . the size of these two formulations has proven to be acceptable to a test population . a gel cap formula for the calcium acetate blend was created to enhance end user acceptance , increased solubility of vitamin d 3 and increased efficacy on bone mineral density . vitamin d 3 is an oil soluble vitamin . it can be dissolved using lipophilic substances . fish oil containing omega 3 - 6 - 9 fatty acids is known to have beneficial effects on bone health ( 32 ). this oil also has the advantage of dissolving vitamin d 3 , obviating the granulation process of calcium acetate blend as described in example 9 . fish oil has been found to have the ability to increase the bulk density of calcium acetate blend by displacing air from the powder . examples of oil to calcium acetate blend ratios include , but are not limited to , about 1 : 1 , 1 . 5 : 1 and 2 : 1 . examples of oil to calcium ratios include , but are not limited to , about 1 : 0 . 14 , 1 . 5 : 1 and 2 : 1 . examples of oil to magnesium ratios include , but are not limited to , about 1 : 0 . 07 , 1 . 5 : 0 . 07 and 2 : 0 . 07 . examples of oil to zinc ratios include , but are not limited to , about 1 : 0 . 007 , 1 . 5 : 0 . 007 and 2 : 0 . 007 . the dosage of vitamin d 3 ranges from 30 to 300 iu . soft gel capsules can be manufactured using conventional methods ( 33 ). gel capsules made with this blend in dose sizes amounting to two to four capsules a day will be acceptable . the size of a gel capsules will be equivalent to or smaller than that described in example 9 . the objective of this example is to design an elemental formula which would provide an optimal mix of vitamin d 3 and acetate salts of calcium , magnesium and zinc for supporting bone health . it is a general belief that the bioavailability of calcium is independent of the solubility of calcium salts ( heaney , 1999 ). low levels of magnesium and zinc are associated osteoporosis ( mutlu et al ., ( 11 ). vitamin d 3 enhances calcium absorption ( christakos et . al ., 2011 ) and therefore , is an important component of an ideal elemental formula . results presented in this invention clearly show that the bioavailability calcium is dependent on the solubility of a calcium salt in the gastrointestinal fluids . an optimal ratio of calcium to magnesium is required to enhance calcium absorption . vitamin d 3 is responsible for increasing calcium absorption and preventing zinc depletion . a formula containing calcium , magnesium , zinc and vitamin d 3 may not work because the form of the elements and the amount of vitamin d 3 , are not necessarily formulated in the right ratios in terms of absorbable fractions . the lack of clinical effect of a blend of calcium , magnesium , zinc and vitamin d 3 is a good example ( braam et . al ., 2003 ). the confusion in the literature relating to calcium absorption and the equivocal clinical trial results on bone mineral density by calcium supplementation has created problems for experts skilled in the art in designing an optimal formula of a calcium blend . using the acetate salts of calcium , magnesium and zinc with the appropriate addition of vitamin d 3 , an optimum calcium supplement is designed . the ratio of calcium to magnesium is generally 2 : 1 , the ratio of magnesium to zinc is 10 : 1 and the daily dosage of vitamin d 3 ranges from 500 to 1000 iu . the bioavailability of calcium described in this invention is appropriately 2 to 3 times higher than that of caltrate . the dosage of calcium should be half to one third of that of caltrate ™. the recommended intake of calcium from all sources is 1000 mg . the average intake of calcium from dietary sources is 400 mg . it is recommended that 600 mg of calcium should be provided as a supplement ; usually this implies that the source of calcium is from calcium carbonate . the recommended dose of calcium from this invention is 200 to 300 mg . this will provide 100 to 150 mg of magnesium and 5 to 7 . 5 mg of zinc . in addition to dietary intake , the supplementation of magnesium and zinc will also provide an adequate daily requirement of the elements . 1 . ilich j z , brownbill r a , & amp ; tamborini l ( 2003 ) bone and nutrition in elderly women : protein , energy , and calcium as main determinants of bone mineral density . european journal of clinical nutrition 57 ( 4 ): 554 - 565 . 2 . ilich j z & amp ; kerstetter j e ( 2000 ) nutrition in bone health revisited : a story beyond calcium . journal of the american college of nutrition 19 ( 6 ): 715 - 737 . 3 . seelig m s , altura b m , & amp ; altura b t ( 2004 ) benefits and risks of sex hormone replacement in postmenopausal women . journal of the american college of nutrition 23 ( 5 ): 482s - 496s . 4 . heaney r p ( 1993 ) thinking straight about calcium . the new england journal of medicine 328 ( 7 ): 503 - 505 . 5 . heaney r p ( 1993 ) nutritional factors in osteoporosis . annual review of nutrition 13 : 287 - 316 . 6 . riis b , thomsen k , & amp ; christiansen c ( 1987 ) does calcium supplementation prevent postmenopausal bone loss ? a double - blind , controlled clinical study . the new england journal of medicine 316 ( 4 ): 173 - 177 . 7 . hunt c d & amp ; johnson l k ( 2007 ) calcium requirements : new estimations for men and women by cross - sectional statistical analyses of calcium balance data from metabolic studies . am j clin nutr 86 ( 4 ): 1054 - 1063 . 8 . bass m , ford m a , brown b , mauromoustakos a , & amp ; keathley r s ( 2006 ) variables for the prediction of femoral bone mineral status in american women . southern medical journal 99 ( 2 ): 115 - 122 . 9 . kanders b , dempster d w , & amp ; lindsay r ( 1988 ) interaction of calcium nutrition and physical activity on bone mass in young women . j bone miner res 3 ( 2 ): 145 - 149 . 10 . celotti f & amp ; bignamini a ( 1999 ) dietary calcium and mineral / vitamin supplementation : a controversial problem . the journal of international medical research 27 ( 1 ): 1 - 14 . 11 . mutlu m , argun m , kilic e , saraymen r , & amp ; yazar s ( 2007 ) magnesium , zinc and copper status in osteoporotic , osteopenic and normal post - menopausal women . the journal of international medical research 35 ( 5 ): 692 - 695 . 12 . abrams s a & amp ; atkinson s a ( 2003 ) calcium , magnesium , phosphorus and vitamin d fortification of complementary foods . j nutr 133 ( 9 ): 2994s - 2999s . 13 . lowe n m , lowe n m , fraser w d , & amp ; jackson m j ( 2002 ) is there a potential therapeutic value of copper and zinc for osteoporosis ? the proceedings of the nutrition society 61 ( 2 ): 181 - 185 . 14 . saltman p d & amp ; strause l g ( 1993 ) the role of trace minerals in osteoporosis . journal of the american college of nutrition 12 ( 4 ): 384 - 389 . 15 . angus r m , sambrook p n , pocock n a , & amp ; eisman j a ( 1988 ) dietary intake and bone mineral density . bone and mineral 4 ( 3 ): 265 - 277 . 16 . angus r m , pocock n a , & amp ; eisman j a ( 1988 ) nutritional intake of pre - and postmenopausal australian women with special reference to calcium . european journal of clinical nutrition 42 ( 7 ): 617 - 625 . 17 . abraham g e & amp ; grewal h ( 1990 ) a total dietary program emphasizing magnesium instead of calcium . effect on the mineral density of calcaneous bone in postmenopausal women on hormonal therapy . the journal of reproductive medicine 35 ( 5 ): 503 - 507 . 18 . bo - linn g w , et al . ( 1984 ) an evaluation of the importance of gastric acid secretion in the absorption of dietary calcium . the journal of clinical investigation 73 ( 3 ): 640 - 647 . 19 . tsugawa n , et al . ( 1995 ) bioavailability of calcium from calcium carbonate , d l - calcium lactate , l - calcium lactate and powdered oyster shell calcium in vitamin d - deficient or - replete rats . biological & amp ; pharmaceutical bulletin 18 ( 5 ): 677 - 682 . 20 . heaney r p , dowell m s , & amp ; barger - lux m j ( 1999 ) absorption of calcium as the carbonate and citrate salts , with some observations on method . osteoporos int 9 ( 1 ): 19 - 23 . 21 . heaney r p , dowell m s , bierman j , hale c a , & amp ; bendich a ( 2001 ) absorbability and cost effectiveness in calcium supplementation . journal of the american college of nutrition 20 ( 3 ): 239 - 246 . 22 . tsugawa n , et al . 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( 2005 ) study of magnesium bioavailability from ten organic and inorganic mg salts in mg - depleted rats using a stable isotope approach . magnes res 18 ( 4 ): 215 - 223 . 25 . lee h h , prasad a s , brewer g j , & amp ; owyang c ( 1989 ) zinc absorption in human small intestine . the american journal of physiology 256 ( 1 pt 1 ): g87 - 91 . 26 . abrams s a , griffin i j , & amp ; herman s ( 2002 ) using stable isotopes to assess the bioavailability of minerals in food fortification programs . food and nutrition bulletin 23 ( 3 suppl ): 158 - 165 . 27 . smith j c , jr ., morris e r , & amp ; ellis r ( 1983 ) zinc : requirements , bioavailabilities and recommended dietary allowances . prog clin biol res 129 : 147 - 169 . 29 . walsdorf n b , alexandrides g , & amp ; pak c y c ( 1991 ) office uspat . 30 . jackson s d & amp ; blumberg j b ( 1997 ) office uspat . 32 . weiss l a , barrett - connor e , & amp ; von muhlen d ( 2005 ) ratio of n - 6 to n - 3 fatty acids and bone mineral density in older adults : the rancho bernardo study . the american journal of clinical nutrition 81 ( 4 ): 934 - 938 . 33 . anderson j t , et al . ( 1975 ) remington &# 39 ; s pharmaceutical sciences ( mack publishing company , easton ) 15th ed .