GARCÍA VIEYRA, María, Isabel (Privada Constituyentes no 309, Col. Juana de Medin, Moroleón Guanajuato, 38800, MX)
LÓPEZ PÉREZ, Mercedes, Guadalupe (Vialidad Interior no 319, Col. Residencial BosquesC.P, Irapuato Guanajuato, 36670, MX)
GARCÍA VIEYRA, María, Isabel (Privada Constituyentes no 309, Col. Juana de Medin, Moroleón Guanajuato, 38800, MX)
1. A pharmaceutical composition that comprises Agave fructans for stimulating the absorption of minerals and their fixation in bones, and to stimulate the production of osteocalcin in mammals.
2. A pharmaceutical composition according to claim 1 where the agave fructans are composed of a mixing of branched fructans with links β(2-1) and β(2-6), with a high-degree polymerization, of the inulin, Fn, neofructans, and Levan series.
3. A pharmaceutical composition for stimulating the absorption of minerals according to claim 1, 2, or 3, wherein the mineral may be calcium, magnesium, or phosphorus.
4. A composition according to claim 1, 2, or 3, where in addition has a concomitant decrease in body weight in mammals.
5. A composition according to claim 1, 2, or 3, where in addition has a concomitant effect on decreasing blood glucose levels in mammals.
6. The use of the composition of claim 1, 2, or 3 as a supplement to stimulate the absorption of minerals and fixation in bones, and to stimulate the production of osteocalcin in mammals.
7. The use of the composition of claim 1, 2, or 3 as a supplement to stimulate the absorption of minerals and their fixation in bones, and to stimulate the production of osteocalcin in mammals, for osteoporosis treatment or osteoporosis prevention.
8. The use of the composition of claim 1, 2, or 3 as a supplement for weight control in mammals.
9. The use of the composition of claim 1, 2, or 3 as a supplement to lower blood glucose levels in mammals.
10. A method to stimulate the absorption of mineral salts in mammals and their fixation in bone, which comprises administering the composition of claim 1 or 2 to a mammal.
11. The method of claim 10, wherein the stimulation is done in an osteoporosis condition or the susceptibility to osteoporosis.
12. The method of claim 10, where it is also to control body weight in mammals.
13. The method of claim 10, where it is also to lower glucose levels in mammals.
14. The pharmaceutical composition of claim 1, 2, or 3, wherein it stimulates the production of osteocalcin in mammals.
15. The use of the composition of claim 14 for treating or preventing osteoporosis.
Field of the invention.
The present invention relates to novel compositions of Agave fructans species useful to promote the absorption of minerals in bone tissue and their storage in bone, and to stimulate the production of osteocalcin; the invention particularly relates to Agave fructans compositions, and more particularly to the treatment and prevention of osteoporosis.
Background of the invention.
Osteoporosis is recognized as one of the major health problems in the world. About one in three postmenopausal women and a substantial number of men suffer from an osteoporosis-related fracture at some point in their life. If no preventive measures are taken or without and adequate treatment in humans, osteoporosis progresses silently and painlessly until causing a bone fracture, which most often occurs in the hip, the spine and the wrists. Albeit there is a high- mortality rate resulting from this type of fracture, the most important consequence of osteoporosis is a limited mobility of the individual, losing personal independence and reducing the quality of life. Nutritional factors influence skeletal growth during the individual's development and in the bone maintenance during adulthood 1 . Therefore, the nutritional components play a key role in the pathopysiological process of developing osteoporosis 2 . It is suggested that, additionally, a minimized bone resorption in old age and to maximize peak bone mass during adolescence may be the key to postpone or even prevent bone fractures resulting from osteoporosis 3 .
Considering the pathophysiology of osteoporosis, especially in post-menopausal stage, the most relevant fact is the estrogen deficiency, since the ovaries produce less estrogen causing an increase in bone loss. The effects of estrogen deficiency are complex, though we can highlight those that occur at the level of bone remodeling because of the augment in the activation frequency of new remodeling sites in the cortical and cancellous bone; these hormones play an important role in maintaining bone density; its lack makes the loss to become excessive. We can generally say that once the bone has been lost it is difficult to be replaced, and consequently becomes more fragile and susceptible to breakage, which evidences the importance of prevention.
Preventive measures for osteoporosis include regular exercise, taking small doses of fluoride and low doses of vitamin D, which induces the absorption of calcium, along with monthly monitoring of calcemia and calciuria, as both may cause hypercalcemia or hypercalciuria that in turn can lead to renal calculus; the preferred calcium sources are calcium citrate and salmon calcitonin; the use of estrogens is useful during the first five years of establishing menopause. Studies have found that women who get most of their daily calcium requirement from food have healthier bones than women who get their calcium mainly from tablets 4 .
5 The present invention relates to nutraceutical compositions administration containing a functional component of plant fructans, namely, Agavaceae species. The functional components in the alimentary system have increased in the last decade, one of them being the fructans that is, polymers of fructose with glycosyl β(2-1) links and/or β(2-6) that may display a terminal glucose molecule in their structure, and their structure may be linear or branched. The major vegetable0 sources of fructans are chicory roots, Jerusalem artichoke, onions and, nowadays, Agaves.
Fructans are considered functional food ingredients that affect the physiological and biochemical processes in humans, resulting in better health and reduced risks for many diseases 5 .
Inulin is the fructan most widely studied and consequently, the best known of fructans reported. Many experimental studies have demonstrated its use as a bifidogenic, stimulating the body's5 immune system, decreasing the levels of pathogenic bacteria in the gut, alleviating constipation and reducing the risk for osteoporosis by increasing minerals absorption, especially calcium 6 . Fermentation of fructans in the colon and cecum produces short-chain fatty acids (SCFA) contributing to augment the solubility of cations, and this by reducing the pH 7 . This effect may facilitate, e.g., the dissociation of divalent calcium-phytate complexes 8 ' 9 . Additionally, many0 reports have shown that this not only increases the absorption of calcium by the intestine, but also the calcium content in bones; its effects on glucose metabolism and some hormones have also been reported 1 ' 8 ' 9 ' 10 ' 11 ' 12 . However, has been reported the effect that linear-fructans consumption generates on the accumulation of calcium in the bone, such as inulin; nevertheless, the results are inconsistent and its consumption may even be negative for the accumulation of5e magnesium m ■ bones 1,8,9,10
The patent US7812004 relates to inulin products containing a mixture of inulin (linear or branched), both fermentable and those with difficult ferment capacity, used both in fixed proportions, showing an increased ability to promote absorption of calcium and magnesium in the human body, the ameliorated lipid metabolism as measured in the liver and a reduced risk for0 colon cancer is exemplified in animal models. This patent only describes the possible origins of inulin and only works experimentally with inulin from chicory (Chichorium intybus); the work does not address nor suggests what is delineated here as the object of the invention. The patent application WO2005056023 relates to chewable products containing a combination of inulin and fructose in low concentrations, mixed with other nutrients to provide a prebiotic effect without side effects.
Agave fructans do not consist of a mixture of fructans from different origins as these are procured from a single vegetal and natural source. Fructans are soluble sugar polymers that have a complex and highly branched structure with glycosil links both β(2-1) and β(2-6); they also contain external glucose molecules (graminanes) and internal glucose (neofructans); the latter type of fructans has been called agavinas 13 ' 14 . In vitro studies, the Agave fructans stimulate the growth of at least 6 different strains of Bifidobacteria and four different strains of Lactobacilli more effective than inulin-type fructans 15 ' 16 .
The genus Agave is composed of succulent plants belonging to a large family of plants of the same name: Agavaceae. They are known by the common name of agave, pita, maguey, cabuya, fique and mezcal. Agave is endemic to the American Continent; the family Agavaceae sensu stricto consists of 9 genera (293 species) such as Manfreda, Polianthes, Prochnyanthes, Agave, Furcrae, Beschorneria, Yucca, Hesperoyucca and Hesperale. Of these, Mexico has more than 55% of the species of each of them 17 .
Fructans are the most abundant carbohydrates in 15% of the plants blooming. The Agave fructans are composed of fructose monomers (95%>) and glucose (5%). Fructans amount to approximately 21% of the plant's dry weight in the Agave tequilana species and are found in the plant in an average amount of 640 mg/g dry weight; the stems of the plants (known as cones) contain the largest amount of fructans in mature stage (achieved in 6 or 8 years), representing over 60%) of the total fructans in the plant; fructans are used to produce tequila, flour, honey and fructooligosaccharides (FOS) that are used in food 14 ' 18 .
There are currently Agave products of different qualities that are marketed as honey and syrups for consumption 19 . However, the beneficial health effects claimed by the manufacturers and merchants of these products (e.g. the prevention of colon cancer and osteoporosis, among other diseases) are not supported by scientific evidence. These traders know the presence of fructans in Agave 13 , and they likewise suggest that linear fructans procured from chicory may have analogous effects.
There are many industrial processes variety to obtain Agave fructans; however, the most widely used involves the extraction with hot water, where the liquid extract is clarified by centrifugation and dried by spraying. One of the optimized processes consists of the extraction at 77.8°C for 1.4 hours in a relation water/solid of 5.5 (mL/g); after removing the crude extract of clarified fructans, it is vacuum evaporated and lyophilized for dehydration, achieving an extraction rate of 82% fructans; the dried extract has a fructans content of 97.5% w/w 20 .
Therefore, it is necessary to procure new compositions based on Agave fructans to use them in the prevention and/or stimulation of bone mineralization.
Brief description of the invention.
While developing the present invention, a target was set to determine the effect of Agave fructans on bone mineralization. This approach and its results, as delineated below, exceed the reported properties of inulin-type fructans regarding the bioavailability of minerals and their effects on bone mass. These, as fructans are generally extracted from sources other than Agave and the effects are greater than those reported in the state of the art.
In addition, we also assessed the effect of two different commercial Agave fructans on ovariectomized mice as a model of osteoporosis 21 ; the mice were fed with diets supplemented with standard or Agave fructans at 10% or chicory inulin-type fructans (Raftline®) at 10% (positive control) with the detailed results described below.
One of the objectives of the invention is to provide compositions based on Agave fructans to stimulate the absorption of mineral salts in mammals and promote their attachment to bone and stimulate the production of osteocalcin.
The pharmaceutical compositions used as dietary supplements to prevent osteoporosis in susceptible individuals or in the general population are embodiments of the invention; the Practical Guide to Diagnosis, Prevention and Treatment of Osteoporosis disclosed by GEOSUR (Uruguay, 2007) describes the conditions of susceptibility to osteoporosis:
Susceptible individuals that may be given the compositions of the invention are those who have any of the following conditions: history of previous minor trauma fractures; background of minor trauma fractures in immediate relatives (younger patients whose only risk factor is the history of fracture of first grade in family members are not included); corticosteroid therapy (treatment with systemic corticosteroids at doses equal to or greater than 5 mg/day of prednisone or it's equivalent for more than three months is a substantial risk for an osteoporotic fracture); premature ovarian failure; premature menopause (<40 years); hyperthyroidism; hyperparathyroidism; hypogonadism primary or secondary (amenorrhea resulting from nervous anorexy or other chronic diseases); malabsorption syndrome; low body mass index (<20); rheumatoid arthritis; immobilization longer than three months. It is important to contemplate the following factors: inadequate intake of calcium throughout life (this factor can cause a low peak of bone mass in childhood and adolescence, and prevent its maintenance in adulthood), as some studies show that around 60% of the adult population and 45% of children and adolescents have a lower milk intake than recommended; inactivity is a significant risk factor as well as smoking, excessive alcohol intake, excessive caffeine intake (more than three cups of coffee per day), and excessive sodium salt intake.
Other diseases that cause a decreased bone mineral density and therefore, individuals who suffer them are candidates to benefit from the administration of the compositions of the invention, are: osteomalacia, hyperparathyroidism, chronic renal failure, hypercalcemia, inflammatory bowel disease, celiac disease, other inflammatory arthritis, chronic liver disease, chronic blood diseases, or neoplasic conditions.
The people who are receiving drugs that cause diminution of the bone mineral density are also candidates to benefit from the administration of the compositions of the invention; between these drugs they are: corticoids, thyroid hormone at suppressive doses of TSH, gnr analogs, antiandrogens, anticonvulsivants, anticoagulants, and furosemide.
They are also embodiments of the present invention the Agave compositions characterized because they are constituted of a mixture of branched fructans with β(2-1) and β(2-6) links, with high degree of polymerization, of the inulin series, Fn, neofructanes and levanes; the representative chemical structure of the used Agave fructans in the development of the invention is exemplified in figure 1.
One of the objectives of the invention is to provide compositions based on Agave fructans to reduce the pH in the large intestine, favoring the absorption of minerals such as calcium and magnesium.
It is another objective of the invention to provide compositions based on Agave fructans to stimulate the absorption of minerals in the bone marrow of mammals; the mineral salts may be calcium and magnesium.
Another major objective of the invention is to provide compositions of Agave fructans to increase the production of the osteocalcin hormone.
Another embodiment of the invention is to provide methods to stimulate the absorption of minerals and to stimulate the production of osteocalcin in mammals by the administration of compositions based on Agave fructans.
Another embodiment of the invention is to provide methods to concurrently contribute to body- weight control and to decrease glucose levels in blood by administering compositions based on Agave fructans. Brief description of the figures.
Figure 1. Shows the determined structure of Agave branched fructans.
Figure 2. Shows the contents of calcium and magnesium in the plasma of mice fed with standard diet (STD and SHAM), or a diet supplemented with inulin-type fructans (R E), or Agave fructans (FCA1 and FCA2). Mean ± SEM. Mean values with different letters were significantly different (P≤0.05). For details of diets and procedures, see example 1 below.
Figure 3. Shows the calcium (A) and magnesium (B) content in the femur of mice fed with standard diet (STD and SHAM), or with a diet supplemented with inulin-type fructans (RNE), or Agave fructans (FCA1 and FCA2). The mean values with different letters were significantly different (P≤0.05).
Figure 4. Shows the calcium (A) and magnesium (B) content in spine column. Mean ± SEM.
The mean values with different letters were significantly different (P≤0.05). For details of diets and procedures, see example 1 below.
Figure 5. Shows the plasma osteocalcin levels. Mean ± SEM. The mean values with different letters were significantly different (P≤0.05). For details of diets and procedures, see example 1 below.
Figure 6. Shows images by scanning electron microscopy (SEM) of the femur of ovariectomized female mice. A) Control; B) inulin-type fructans; C) FCA1 - type 1 Agave fructans; D) FCA2 - type 2 Agave fructans.
Detailed description of the invention.
The present invention provides compositions based on Agave fructans that stimulate mineral absorption in mammals, mainly at the skeletal system, which allows them to be used in nutritional processes to prevent the occurrence of pathologies associated with the depleting of bone structure, e.g. osteoporosis, or to be administered to stimulate mineral deposition in mammals, e.g. calcium in the bone marrow.
It has often been reported that plant products rich in carbohydrates can affect non-digestible dietary mineral bioavailability. The impairment of calcium and magnesium absorption (Ca and Mg) for dietary fiber is traditionally attributed to phytic acid in food 22 . However, the ingestion of fructans has shown an increase in the absorption of Ca and Mg.
The indigestibility of fructans, namely, the inability to be digested by the upper gastrointestinal tract and their fermentability as well as their effect on mineral absorption has mostly been examined in inulin-type fructans whose characteristic is to have a linear structure. However, little research has been made on branched fructans such as those reported in the Agave species and the present invention.
In the present invention, we evaluate the effect of two different commercial fructans obtained from Agave plants. It is known that this class of fructans is highly complex and branched. We investigate the effect of these fructans in the apparent absorption of minerals like Ca and Mg in the large intestine and in the mineral retention in bone using animal models. Also we compared our results with studies already very known with inulin-type fructans (linear fructans).
The evaluation was made on the murine model of osteoporosis already mentioned; ovariectomized mice were fed with the compositions of the invention containing for example 10% w/w Agave fructans, despite type (linear or branched). The mice groups 1 and 2 (only ovariectomized, (STD) and sham-operated controls (SHAM)) were fed with pellet diet without fructans; group 3 (R E) was fed with a diet containing 10% inulin-type fructans (positive control); and groups 4 (FCA1) and 5 (FCA2) were fed with a diet containing 10% commercial Agave fructans (see example 1).
Agave fructans resulted in a decrease in body- weight gain compared to the STD and SHAM groups.
Some positive effects analogous to those delineated above for inulin-type fructans, such as a decrease in energy consumption and body weight, were also demonstrated along with decreased levels of glucose in the blood; the body-weight loss of mice fed diets supplemented with Agave fructans (FCA1 and FCA2) decreased 17% and 15% in rodents fed with RNE fructans (inulin- type) compared with the standard group.
The results shown here demonstrate that fructans from Agave can stimulate the production of incretins at higher levels than those procured with chicory inulin-type fructans. The incretins are a group of gastrointestinal hormones that augment the quantity of insulin released by the pancreas, which can greatly benefit patients with diabetes or individuals with high-glucose levels in diabetes risk. The incretins augment the feeling of satiety what may inhibit food intake; therefore, the administration of Agave fructans compositions may lead to a body-weight reduction in mammals 23 .
12 23 24
These results were consistent with another research ' ' where glycemia is likely to correlate with weight gain, suggesting that obesity parameters are more related to energy and fat-mass development. However, the effects and mechanisms by which these carbohydrates promote different systemic effects are unknown. The underlying mechanism of increased absorption of minerals by the presence of SCFA is not yet known 25 . As already mentioned, the fermentation of fructans produces short-chain fatty acids (SCFA), which have been reported as directly or indirectly responsible for the effect, and these are credited with the absorption of minerals, especially calcium and magnesium. In vitro experiments with Ussing-type chambers showed that the addition of SCFA or the decrease of the intestinal mucosa pH caused a significant decrease in tissue conductance. These changes in the tissue conductance correlate well with the calcium
25 26 27 "
flow, and therefore, resembled the in vivo luminal composition ' ' .
Previous studies have repeatedly shown that consumption of various inulin-type fructans may ameliorate intestinal mineral absorption 23 ' 27 ' 28 ' 29 in vivo (in humans and animals). Moreover, there is a correlation between the production of SCFA and cecal fermentation parameters. According to these reports, we found that the intake of all fructans used in this study augmented the total production of SCFA in the cecum and colon. However, our results indicate a significantly greater augment in mice that consumed Agave fructans FCA1 and FCA2, where the FCA1 group showed the most blatant increase: 23% in the cecum and 25% in the colon (see table 2).
Notwithstanding the foregoing, references in the state of the art concerning the effect of fructans on the production of SCFA and of these on mineral absorption in the intestine do not elucidate the findings that indicate a more pronounced effect with the administration of Agave fructans. Among the most relevant results are those concerning the effect of diets containing Agave fructans on mineral absorption and bone retention.
As delineated in the examples, the results on the calcium and magnesium content show a more pronounced increase in both groups of mice fed Agave fructans; these differences were significant on the RNE and STD groups. The FCA1 mice group showed the best effect by augmenting 38% the calcium absorption and 20% the bone retention of this mineral; the other groups also showed an increase: FCA2, 36%> and 18%; and RNE, 34% and 18% respectively; magnesium augmented only in the bone content. The calcium and magnesium content excreted in feces tended to be lower in all groups fed fructans regardless the vegetal origin, but the differences were not statistically significant (see table 3, figures 2 and 3).
However, other parameters evaluated in the present invention (in bone calcium content and the preservation of trabecular bone structure) were indeed statistically different showing a better effect when using Agave fructans instead of inulin-type fructans.
Furthermore, the plasma calcium levels showed slight changes during experimental progress, and in the sixth week (final week) fructans-fed groups succeeded in reversing the imbalance in calcium homeostasis caused by ovariectomy.
Regarding the plasma magnesium levels, they remained steady throughout the experiment (see figure 1). The data produced by the present invention correlate well with previous work, despite the type of fructans (linear or branched) supplemented to the diets administered to the mice; the fructans diets prevent imbalance in the calcium homeostasis 1 ' 3 ' 8 . However, as aforementioned, the best results were procured with the FCAl Agave fructans type 1. It is eminently probable that this is owed to the Agave-fructans structure, which should facilitate the access of hydro lytic enzymes in the large intestine of mice. As shown in figure 1, the Agave-fructans structure is branched and the average degree of polymerization (DP) of fructans FCAl is 22, while for fructans FCA2 is approximately half (13). This, because the presence of a large number of branches from the Agave fructans has a greater number of sites for the access of hydro lyzing enzyme; i.e., an enzyme could access each branch to allow the fermentation of Agave fructans, what makes us infer that the fermentation of Agave fructans may occur more efficiently and faster than that of inulin-type fructans whose linear structure would only allow access to a single enzyme. Another aspect concerning the structure of Agave fructans is its polymerization degree, as the fructans presenting the best result were those with higher DP of 22 (FCAl). This may indicate that having a larger size, the fructans produce a better protective effect throughout the intestinal tract as opposed to small fructans (DP 4-7), which would rapidly ferment in the cecum and the proximal intestine leaving the rest of the colon with no benefit. The FCA2 are also branching fructans, but these branches are lower than those of FCAl as they have a lower DP than the latter.
The areas and mechanisms of the Ca intestinal absorption are different from those of the intestinal absorption of Mg 30 . This could elucidate the differential effects that fructans may have over the total or segmental intestinal absorption of these two main minerals in bones. Intestinal absorption of Ca involves two distinct mechanisms. The main route of Ca absorption is a saturable transcellular process, regulated by vitamin D and Ca-binding proteins 31 . This is an eminently regulated process that occurs throughout the colon. Although the mechanisms involved in the intestinal absorption of Mg are a saturable process including a facilitated diffusion and a passive diffusion; thus, the intestinal absorption of Mg by passive diffusion of the distal small intestine and the first tract of the colon is very important 32 .
Regarding the osteocalcin levels observed in the present invention, we found a significant augment (up to 50%) in all groups fed the compositions of the invention containing Agave fructans compared with the standard group. This shows the potential of Agave fructans and inulin fructans from chicory (Cichorium sp) in preventing bone loss (here induced ovariectomy) and to stimulate the formation of bone. Osteocalcin is the most abundant non-collagen protein in bones; it is a marker of osteoblast activity (bone formation marker). This protein is deposited in the bone matrix, and a portion is released into the bloodstream. This protein is specifically expressed in osteoblasts 33 and was identified 30 years ago, albeit initially it was only recognized as a constituent of the extracellular matrix of the bone. Subsequent studies produced the cognizance of its structure, which has three glutamic acid residues that are carboxylated by what is also known as bone Gla protein 34 . This carboxylation confers a high affinity for minerals, so it has been associated with bone mineralization. Its role in bone formation 35 was not demonstrated, despite various efforts to prove this assertion.
In 1996, Ducy and colleagues 36 demonstrated through osteocalcin deficient mice that this protein had a role in bone formation, but failed to establish the mechanisms of action.
It has recently been suggested that this is not a simple protein, but a hormone because of its features, since studies show that this protein is a cell-specific gene, which is found in the bloodstream, generated as a pre-molecule that, after certain modifications, is secreted into the bloodstream and finally, its secretion shows a circadian pattern typical of most hormones. However, to date is has not been possible to find any receiver for it. Besides, the possible role of osteocalcin as a hormone, its role in regulating glucose metabolism and energy has been suggested which augments its medical importance not only in its previous role in preventing osteoporosis, but also diabetes and obesity, as it has been proved that it may regulate insulin secretion 37 and some hormones involved in energy metabolism. So the contribution of the present invention is of great interest as the results show that Agave fructans may affect the production of said protein-hormone.
However and regarding its role in bones, an increase in osteocalcin concentration indicates a high osteoblastic activity and increased bone formation, which can be measured by radioimmunoassay or ELISA (see figure 4).
Osteocalcin correlates well with states of increased bone remodeling (hyperparathyroidism and hyperthyroidism, Paget's disease, acromegaly), or its decrease (hypoparathyroidism, hypothyroidism and hiperglucocorticoidism) as delineated; the administration of inulin-type fructans and Agave fructans has shown then to have a potential effect to augment the production levels of osteocalcin, and potentially to achieve the normalization of its levels in the body. This effect contributes in the treatment of osteoporosis. The state of the art does not have a history on this effect, so that its therapeutic use is part of the main objectives of the invention.
The significant differences observed in the present invention regarding the absorption of both Ca and Mg (see table 4) because of the ingestion of Agave fructans may be related to three major structural features: 1) the lack of linearity; 2) the polymerization degree; and 3) the presence of links β(2-6). Agave fructans are branched neo series fructans and have links to both β(2-1) and β(2-6), while inulin-type fructans (Raftilinie, obtained from chicory) consist of a linear chain of fructose molecules linked only by β(2-1).
Agave fructants are best represented by a high-degree of polymerization (the quantity of fructose containing these molecules), indicating that their molecules are high DP (Degree of Polymerization) fructans. The chromatographic profile of these fructans was procured by anion- exchange chromatography (HPAEC-PAD) and, according to the retention time of the eluted peaks and by comparison with standards, it showed the presence of inulin series and Fn series, and other peaks probably corresponding to certain neofructans or Levan series. It was also possible to identify by gas chromatography (GC-FID) the different residues that form these carbohydrates by derivatization to partially methylated alditol acetates (PMAA), proving the structural diversity of Agave fructans. The determined structure of Agave fructans is shown in formula 1, figure 1. This structure generally represents Agave fructans, as it contains all their characteristic elements such as the structure complexity, the presence of multiple branches and the presence of neofructans series, and finally the presence of links β(2-1) and β(2-6). However, we must explicate that this represents an average structure of Agave fructans FCA1, and that the main difference with the fructans of samples FCA2 is that the polymerization degree in FCA1 is 22 while in FCA2 the polymerization is 13. This difference in the DP may be the reason for FCA1 to show greater effects than those produced when using FCA2.
Based on the information above, the compositions of the present invention provide evident advantages for bone formation and preservation even in unfavorable conditions (menopause, diabetes, etc.) above compositions containing linear fructans.
For example, Bosscher and colleagues 1 describe the effects of inulin-type fructans on bone health, only highlighting the effects on bone calcium accumulation when consuming linear fructans, whereas in the present invention the use of Agave fructans, which has a more complex and branch structure, causes a greater absorption of calcium and consequently its accumulation in the bone, preventing the loss of bone structure. Moreover, unlike that delineated by Bosscher, the present invention provides conclusive evidence of this positive effect of Agave fructans in the accumulation of calcium in bone through microscopic analysis by which it is possible to consistently confirm this effect; likewise, the results procured by the analysis of osteocalcin made herein lead to the conclusion that in the groups of mice receiving Agave fructans there is a greater production of this protein and thus, increased bone formation.
However, Lobo and colleagues 10 delineated essays with male mice fed a restrictive diet of both calcium and iron to evaluate the effect of linear-fructans supplementation on bone mineralization. The results from this study indicate that the administration of linear fructans to calcium-deficient mice worsens the magnesium content in bone, whereas in the present invention the results were completely different and very promising, because magnesium levels remained stable during the experimental period. On the other hand, Lobo and colleagues 10 found no difference in bone calcium content, whereas the present invention showed a greater absorption and retention of calcium and magnesium on bone in mice receiving Agave fructans. Also, the experimental period delineated by Lobo was only 4 weeks, time considered as insufficient the animals used to present any imbalance caused by the restriction of calcium and iron imposed on them. In contrast, herein we induced an imbalance by performing an ovariectomy on the mice, which allowed us to conclusively assess the effect of branched Agave fructans on the animals. All the same, Greger 8 delineates the effect of some carbohydrates such as gums, pectin, resistant starch, lactulose and inulin in ameliorating the bioavailability and mineral absorption. However, this study shows little evidence of the effect of inulin on the absorption of calcium. The present invention provides in contrast a more detailed analysis on these effects of both inulin-type fructans and branched Agave fructans emphasizing the structural distinctions between them, thus providing new cognition on the activation of regulation mechanisms of homeostasis in calcium and the activation of proteins promoting bone formation and osteocalcin.
Finally, Lopez and colleagues 9 delineate studies on the effect of linear fructans to counteract the presence of phytic acid in the diet, a compound that is known to negatively act on the absorption of calcium and other minerals, because it forms complexes with these minerals preventing its absorption. This study indicates that fructooligosaccharides (linear fructans of low-degree of polymerization between four to seven fructose units) may counteract the presence of phytic acid ameliorating thereby the calcium absorption in mice supplemented with this type of fructans. However, this study presents several inconsistencies because the model used was male rats, a model that is known is not the best for studying problems analogous to osteoporosis and calcium absorption; the experimental design was short and only emphasized the comparison of the presence of an inhibitor of calcium absorption with an enhancer of the same, only providing data such as ameliorated digestibility and increased calcium in bone.
Generally, the aforementioned works focus on estimating the effect of calcium absorption by inulin-type fructans (linear) and of chicory with experimental methods that only determine the content of calcium or plasma in bone without inquiring whether there are other effects in this regard. Albeit the report by Lobo and colleagues 10 integrates ovariectomized rats and evaluates the bone microstructure, the results are inconclusive regarding the protective effect on the loss of the trabecular bone network with these treatments. The present invention uses in this sense a new source of fructans such as Agave fructans that as already adverted to have large structural differences compared to fructans procured from chicory; likewise, our results are conclusive on the protective effect of the Agave fructans examined in most of the variables analyzed in ovariectomized-rats, a model that has been established as the best for the study of this type of pathology. Analogously, the present invention includes new valuation data such as the analysis of osteocalcin, a protein involved in the formation of new bone.
To illustrate the present invention, below are the following examples, which are not intended to limit the scope thereof. Example 1. Treatment and animal diets.
Forty-eight female mice of the strain C57BL/6J of 12 weeks of age were housed in a room with controlled temperature and humidity with a cycle of 12h light/dark. Forty were ovariectomized (OVX), while eight were used as sham-operated controls (SHAM). At 14 weeks of age the mice were divided into five groups, four groups of ten ovariectomized mice (STD, RNE, FCAl and FCA2), and a group of eight sham-ovariectomized mice that were subjected to sham surgeries in which the ovaries were not removed (SHAM). All mice were then fed for six weeks as follows: groups one and two (STD and SHAM; subjected to surgery removing the ovaries and without removing them) were fed a standard diet; group three (RNE) fed a diet containing 10% of inulin- type fructans (positive control), and groups four (FCAl) and five (FCA2) fed a diet containing 10% commercial Agave fructans.
After an acclimation period of 14 days anterior to the experiment, mice in the control groups (OVX and SHAM) were fed standard pellet diet (STD) 5053 (Lab-Diet, USA), while mice with FCAl, RNE and FCA2 diet were fed a diet by Lab-Diet USA, mixing 90g of 5053 standard diet with lOg of the correspondent fructan (commercial Agave fructans 1 and 2 and Raftline®, respectively). The standard diet contained the following (g/100 g dry diet): 23.5 protein (consistent of soybean protein and fish), 64.5 total carbohydrates procured from corn, wheat and oats (including 32 starch; 3.0 sucrose, 6.0 cellulose) and 12.0 lipids. The food consumed by each animal was weighed daily. After the sixth week of treatment, mice were killed and samples collected (blood, bone, intestinal and cecal contents, and colon and cecum).
The commercial Agave fructans were obtained from Agave tequilana plants of two different sources, while the inulin-type fructans from chicory used for the positive controls (Raftline®) were from Orafti (Belgium).
The food intake was evaluated daily in the morning. Weight gain and faecal excretion were evaluated once a week. Blood samples were taken weekly from the tails of the mice being tested. Blood samples were collected in tubes with heparin; after centrifugation, plasma was stored at -80°C until used to determine the calcium, glucose and osteocalcin levels. Blood glucose was measured using a Prestige Glucometer kit (Home Diagnostics, Inc., Ft Lauderdale, FL., USA). Plasma osteocalcin levels were determined using an Osteocalcin ELISA kit for mice (Immuno-Biological Laboratories Inc., USA).
After killing the mice (six weeks), the cecal content was transferred to vials and frozen at -80°C until the analysis of short-chain fatty acids SCFA; for this we used gas chromatography with flame ionization detection (GC-FID) 22 . In short, the blind samples and feces were weighed and the solutions were acidified with H 2 SO 4 , adding a solution of internal standard (2 -methyl valeric acid). The SCFA were extracted by stirring the sample solutions with diethyl ether and then centrifuged (10 min at 2000 rpm). Two mL of the ether phase was injected directly into a FFAP capillary column of the GC-FID.
The amounts of calcium and magnesium were measured in feces, blood, spine and femur, and in the diets used; in all cases they were determined with an atomic absorption spectrophotometer.
The samples were dried in an oven to constant weigh at 60°C, first the diets, feces and femurs.
The dried samples were weighed and added a mixture of perchloric nitric acid at a 3 : 1 ratio to digest the samples. Once digestion was completed, the samples were then quantitatively transferred and diluted with 10 mL of distilled water. A 1% solution of potassium iodide was added to calcium. The results were expressed in a dry- weight basis. Reagent blanks were prepared using the same digestion procedures. The blood was diluted with distilled water and directly subjected to atomization.
The absorption of calcium and magnesium was calculated using the following formula 23 with modifications:
Absorption (%) = (intake - fecal excretion) / (intake) x 100 (1)
The results were expressed as mean values with standard deviation. The statistical differences between groups were evaluated using a one-way ANOVA followed by Turkey using the software STATGRAPHICS Plus 5.1. The differences were considered significant from P >0.05. Example 2. Effects of administering the compositions of the invention in a murine model.
During the six-week experimental period, food intake, and initial body weight were equal in all experimental groups. However, at the end of the sixth week, the final body weight increased in the five mice groups. But, the increase was greater in the STD group (21.35 ± 0.87 to 26.01 ± 0.14). However, weight gain was higher in the STD and SHAM group, 3.66 ± 0.37 and 3.38 ± 0.30 (see table 1), while the final levels of glucose decreased with a significant glucose decrease in mice fed with all types of fructans with the progression of the experiment.
Food intake, body weight, excretion of feces and blood glucose levels of mice fed a standard diet (STD and SHAM) or diet supplemented with RNE fructans (inulin-type fructans) or
FCA1 and FCA2 (commercial Agave fructans).
STD SHAM RNE FCA1 FCA2
Intake, mg/day 2.62 ± 0.34 a 2.88 ± 0.06 a 2.52 ± 0.3 Γ 2.5 ± 0.24 a
Initial weight, g 21.35 ± 0.87 a 20.51 ± 0.82 a 21.41 ± 0.8 a 21.57± 0.56 a 20.95 ± 0.58 a
Final weight, g 26.01 ± 0.14 a 23.89 ± 0.52 b 24.09 ±0.28 b 23.12± 0.25 b 23.48 ± 0.35 b
Weight gain, g 3.66 ± 0.37 a 3.38 ± 0.30 a 2.67 ± 0.22 b 1.56 ± 0.31 c 2.53 ±0.23 b
Initial fecal excretion, g/day 6.7 ± 0.29 a 7.65 ± 0.36 a 6.34 ± 0.16 a 5.21 ± 0.2Γ 6.65 ±0.12 a
Final fecal excretion, g/day 7.34 ± 0.23 b 8.34 ± 0.12 a 8.84 ± 0.11 a 9.31 ± 0.12 a 10.35 ± 0.15 a
Initial glucose levels, mmol/L 7.06 ± 0.12 a 6.09 ± 0.9 b 6.99 ± 0.10 a 6.87 ± 0.9 a 6.93 ± 0.8 a
Final glucose levels, mmol/L 7.26 ± 0.23 a 6.49 ± 0.9 a 6.14 ± 0.08 b 6.03 ± 0.1 l b 6.07 ± 0.13 b
Values are mean ± DE, n= 10 for STD, RNE, FCA1 and FCA2; n=8 for SHAM.
Means sharing the same letter do not differ significantly (P< 0.05).
Generally, the supplementation containing fructans significantly augmented fecal excretion by comparison with the STD (FCA1, 9.31 ± 0.12, FCA2, 10.35 ± 0.15, RNE, 8.84 ± 0.11, STD, 7.34 ± 0.23, and SHAM, 8.34 ± 0.12) (see table 1). For the concentration of SCFA, in general the groups fed fructans significantly augmented their production compared to the STD group. However, the FCA1 group showed the highest SCFA total concentration (see table 2).
For the individual concentration on the content of acetate and butyrate in the cecum, the group with the highest content was again FCA1 (34.72 ± 1.01 y 18.67 ± 2.13). With regard to propionate, it was the FCA2 (25.64±1.03). On the other hand, the concentration of SCFA shows the same trend observed for the cecum, where the FCA1 group showed a greater increase in total SCFA concentration (table 2) and the individual concentration of acetate and butyrate (44.03 ± 1.91 y 16.21 ± 2.00).
Generally, the supplementation with the compositions of the invention containing any type of fructans increased the concentrations of short chain fatty acids (SCFA), nevertheless, this increase was greater for group FCA1.
The plasma calcium content (mg/L) was similar in all the groups of mice at the beginning of the experiment (figure 2); however, in the third and sixth week it fell in group STD (67.8 to 63.5 and 53.1, respectively), whereas the groups fed with the compositions of the invention containing fructans and group SHAM presented slight diminutions, although these were not significant. In the case of magnesium, some difference was not observed throughout all the treatment (figure 2).
Concentration of short-chain fatty acids (SCFA's) in cecum and colon (mmol/Kg).
STD SHAM RNE FCAl FCA2
Acetate 27.92 ± 1.23 b 28.26 ± 0.98 b 34.10 ± 3.05 a 34.72 ± 1.01 a 32.64 ± 1.72 a
Propionate 17.82 ± 0.40 b 18.64 ± 1.28 ab 23.50 ± 1.04 a 21.75 ± 1.18 a 25.64 ± 1.03 a
Butyrate 12.54 ± 1.19 b 11.32 ± 1.08 b 17.28 ± 0.64 a 18.67 ± 2.13 a 16.15 ± 0.76 a
Total SCFA's 58.28 58.22 74.88 75.14 74.43
Acetate 33.39 ± 1.51 b 35.21 ± 1.27 ab 41.34 ± 1.73 a 44.03 ± 1.91 a 41.25 ± 1.43 a
Propionate 12.18 ± 0.59 b 13.62 ± 1.89 b 16.85 ± 2.55 a 18.60 ± 1.71 a 20.97 ± 2.91 a
Butyrate 10.63 ± 0.30 b 11.39 ± 1.91 ab 13.06 ± 1.76 a 16.21 ± 2.00 a 15.42 ± 2.4Γ
Total SCFA 's 56.20 60.22 71.25 78.84 77.64 Values are the mean ± SD, n= 10 for STD, RNE, FCAl and FCA2; n=8 for SHAM
Means sharing the same letter do not differ significantly (P< 0.05).
Figure 3 shows the results concerning the content of calcium and magnesium in bone. As shown, the intake of the compositions of the invention containing Agave fructans was indeed differential showing a statistically significant increase (P≤0.05) in Ca concentration in femur, when compared with Ca content in bone of the STD group. In addition, there were differences in the Mg concentration in femur between the experimental groups. FCA2 group showed the highest level of Mg with 4.41±0.63, FCAl; 3.91±0.19, RNE; 3.66±0.49, SHAM; 4.29±0.46; and STD group (ovariectomy) was the lowest with 3.01±0.14.
In the present invention, the calcium content in the spine (figure 4) was also determined; the two groups fed with the compositions of the invention containing Agave fructans (FCAl and FCA2) showed higher calcium content (0.464±0.007 and 0.442±0.015) than the group of inulin-type fructans; the content of the RNE group is quite analogous to the SHAM group (0.455±0.013) (mice with ovaries). The RNE group significantly augmented the calcium content (>20%) only regarding STD group, but as mentioned before, not with respect to the mice that consumed FCAl and FCA2. About the content of magnesium, it augmented significantly in the three fructans groups compared with the STD group. Interestingly, plasma osteocalcin concentrations (ng/mL) augmented significantly in the third and sixth weeks in all mice fed fructans compared to the STD group (figure 5). While the differences were not significant, the FCAl group showed a better response in this marker of bone formation respecting the STD group (51.72 ± 6.0 wk 1, 54.6 ± 6.9 wk 3 and 45.11 ± 3.7 wk 6 vs. 48.27 ± 5.9 wk 1, 31.60 ± 3.2 wk 3 and 27.25 ± 3.8 wk 6, respectively).
Analogously, there was a higher content of Ca in the feces of the control group compared with the groups of fructans (table 3). However, these differences were not significant. The magnesium content in feces showed no differences between groups. So, the FCA1 group showed a higher apparent mean in the absorption of Ca in comparison with the STD group throughout the experimental period, and this difference (P≤0.05) increased by approximately 42%. Regarding Mg, the average fecal content was slightly lower in the fructans group than in the STD group, but no significant differences (FCA1; 1.29±0.02, FCA2; 1.27±0.03, RNE; 1.26±0.04 and STD; 1.37±0.05) were found, and apparent intestinal absorption did not show differences either (table
Calcium and Magnesium absorption in mice.
STD SHAM RNE FCA1 FCA2
Intake (mg) 1 1.50±0.70 12.64±0.93 12.57±0.38 12.24±0.79 12.23±0.72
Excretion (mg) 8.40±0.52 8.87±0.39 8.23±0.47 7.54±0.35 7.77±0.41
% Absorption 26.93 b 29.87 b 34.55 a 38.42 a 36.46 a
Intake (mg) 2.23±0.04 2.45±0.05 2.12±0.03 2.13±0.03 2.13±0.05
Excretion (mg) 1.37±0.05 1.48±0.02 1.26±0.04 1.29±0.02 1.27±0.03
% Absorption 38.66 ab 39.46 a 40.29 a 39.63 a 40.42 a
Values are the mean ± SD, n= 10 for STD, RNE, FCA1 and FCA2; n=8 for SHAM
Means sharing the same letter do not differ significantly (P< 0.05).
For purposes of the invention, by using this model of osteoporosis in ovariectomized mice, the positive effect of the compositions of the invention containing Agave fructans on intestinal calcium absorption and therefore, a higher content of Ca in bone and an increased bone formation by determination of osteocalcin, were shown. Structural differences in the type of fructans did not alter cecal and colonic fermentation or the production of end products of bacterial metabolism (SCFA) nor affected the stimulation of Ca and Mg digestive absorption and its accumulation in bone. Additionally, to our knowledge, this is the first report on the effects of Agave fructans in the absorption of minerals. Example 3. Effect of the administration of the compositions of the invention in the bone mineral absorption at murine model.
We proceeded to analyze the microstructure of the bone: after killing the animals, the animals' femurs were collected (in this case the right femur); cuts were made on the head of the femur and then a cleaning protocol for removal of tissue was performed; the sample was placed in a tube with proteinase K 1 mg/mL and SDS at 10 mg/rnL for 12 h; the sample was washed then three times with distilled water, adding acetone for 12 h; the sample was washed again and dried to constant weight. We proceeded to mount the clean samples on a slide where they were subsequently coated with gold and analyzed by scanning-electron microscopy (SEM), aside from procuring chemical analysis of the samples by spectra of X-ray scattering (SEM/EDS) for semiquantitative analysis (table 4).
Chemical analysis SEM/EDS
Ca(%) (g/lOOg) P(%) (g/lOOg)
STD 13.38 27.15±1.57 d 7.38 15.79 ±1.21 b
SHAM 27.1 1 53.09 ±2.69 a 11.59 31.58 ±1.01 a
RNE 19.98 39.16 ±1.79 c 10.25 30.10 ±1.33 a
FCA1 24.89 48.79 ±1.05 b 12.27 32.39 ±1.1 l a
FCA2 23.91 47.69 ±1.89 b 11.51 31.64 ±1.91 a
Values are the mean ± SD, n= 3.
Means sharing the same letter do not differ significantly (P< 0.05).
The different experimental groups were subjected to an analysis by scanning electron microscopy to assess changes in bone microstructure. The images obtained from this analysis clearly show that there is a preventive effect on bone loss resulting from the consumption of Agave fructans regarding the control group, where it is clear that the loss of trabecular network in the control group is large, and in the groups of Agave fructans it is maintained throughout the experimental period. Figure 5 shows the image by scanning electron microscopy of the femur of ovariectomized mice. As seen, the control sample (figure 5 A) clearly shows the loss of trabecular bone networks, while the samples of inulin-type fructans (figure 5B), Agave fructans 1 (figure 5C) and Agave fructans 2 (figure 5D) show the presence of such networks in femurs of ovariectomized mice that consumed fructans 6 weeks before being killed. The control (A) did NOT consume fructans.
But, the analysis of X-ray scattering (SEM/EDS) showed a difference in mineral composition between the STD group and the groups of fructans (see table 4), where the STD group drastically decreased its mineral content with respect to the groups treated with Agave fructans, which is consistent with the result obtained by atomic absorption spectrometry. However, in this analysis a difference was observed regarding the source of fructans, as for calcium content the highest content was for the groups fed Agave fructans.
These results suggest that the loss of bone structure was higher in the STD group compared to the groups consuming fructans; the chemical analysis shows that coupled with the loss of bone structure there is also a bone demineralization induced by ovariectomy in mice, and that this demineralization is reduced in the groups treated with Agave fructans, which reconfirms its preventive effect on osteoporosis.
Agave fructans have shown a positive effect, an effect similar to that reported for inulin-type fructans of chicory (Ratline®); however, significant differences were seen for medical practice as delineated in the present invention since in most cases the effect of Agave fructans was significantly higher, most particularly when the compositions contained FCA1, so outweighing the beneficial properties for linear fructans described in the state of the art. Therefore, in the present application claims the compositions containing Agave fructans and the therapeutic and preventive properties delineated herein. The use of these Agave-fructan prebiotics in the diet can reduce or prevent osteoporosis by maintaining healthy bone tissue; the value of these compositions increases as they act as an auxiliary in controlling obesity by producing a reduction of body weight, and an auxiliary in the reduction of hyperglycemia; they are also a source of fiber that ameliorates or relieves constipation; they also favor the growth of beneficial bacteria such as Lactobacilli and Bifidobacteria, which are beneficial bacteria to the body; and they are also useful in the control of obesity, as already mentioned, diabetes mellitus, immune system and preventing colon cancer, to name a few. References.
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