Effect of natural sources rich in calcium on treated rats induced osteoporosis

The present study aimed to treat female rats’ osteoporosis using natural sources rich in calcium (permeate with kiwi or fig). Thirty-two female rats weighting 150 ± 10 g were used in this experiment. After adaptation period (7 days), rats were divided to 8 groups (four in each group); first group worked as a control negative and other groups were injected with 1 mg Dex. /kg/bw glucocorticoid (dexamethasone) for 7 days to induce osteoporosis. Second group was considered as a positive group and other groups were treated with beverage (kiwi with permeate or fig with permeate) at different concentrations, i.e., 20, 30 and 40%. The DPPH test was recorded 69.19, 87.01, 68.95, and 44.88% for fresh kiwi, refrigerator kiwi beverage, fresh fig, and refrigerator fig beverage respectively. Meanwhile, using ABTS recorded of 32.6, 39.07, 36.66 and 41.99 Trolox/100 g for above mentioned treatments. In general, total phenol and total flavonoid values of fig treatments were high compared with kiwi treatments. The antimicrobial examination showed an increase in lactic acid bacteria due to refrigeration in both beverages and less total count. Mold, yeast, and coliform not detected. The biological assay resulted in decrease weight gain and feed intake while feed efficiency ratio slightly affected due to beverages. Ca and P content of osteoporotic femur have shown an increase more than the potassium content. Also, serum Ca, P, PTH, Vit. D and protein increased significantly as the result of beverage treatments. The X- ray showed an improved of bone in all treatments compared with (+) control. It could be concluded that permeate mixed with kiwi or fig can be used as a therapeutic diet for subjects, who suffered from osteoporosis.


Introduction
Osteoporosis is the most common health condition among metabolic bone disease, which is related to a decrease in bone minerals density. And microstructural deterioration of bone tissue. A good relation between osteoporosis and increasing constituents a major public health problem, i. e., hyper thyrodism, hyperparathyroidism, hypogonadism, bone destruction which led to tumor metastasis, rheumatoid arthritis and chronic obstructive pulmonary disease [1]. The diagnosis of osteoporosis should be established by the measurements of the bone minerals density [2]. Which recommended by WHO [3]. Due to the demographic changes, more than of 40 million American citizens over 50 years of age are risk of osteoporosis fracture, this number will at least double until year 2040 [4]. Also mentioned that the increase in body mass density (BMD) is leading to an increase in bone fragility and susceptibility fractures of some confectioned antiosteoporotics are effective in the treatments and prophylaxis of osteoporosis [5]. Calcium is vital for life muscle function, nerve activity and bone mineralization depend on an accurate balance between the extracellular and intracellular calcium [6]. Vitamin D directly raise the serum calcium by protein synthesis in the intestinal wall which promotes intestinal calcium transport and absorptions. Vitamin D increases the reabsorptions of calcium and phosphorus from renal tubular [7]. Parathyroid hormone is very sensitive and response to any change in extracellular calcium which acts in conjunction with active vitamin D to regulate calcium. Many studies were carried out to investigate the different mechanisms of parathyroid hormones regulation and restricted that in three distinct mechanisms: (1) it increase reabsorptions of calcium from the renal tubular, (2) increase bone turnover to release calcium and phosphorus from the bone, (3) enhance the conversion of hydroxyl vitamin D to an active form [6]. As far as food health goods are concerned, milk permeate is one that hasn't been widely used. More than 85% of the milk used to make cheese is wasted in this way, although it is still considered a waste product of the ultrafiltration (UF) processes [8]. When milk is ultra filtered, a substantial amount of permeate is produced as a byproduct. Getting rid of the rest amount of liquid was produced by the dairy sector needs a considerable outlay of resources [9]. Adding milk permeate to meal increase its nutritional value since it includes electrolytes (sodium, potassium, magnesium, zinc and calcium) that are present in alternative products [10]. The use of permeate in the manufacturing of several beneficial products has received great attention [8]. On the other hand, employing milk permeate was able to generate an excellent grade produce fruit drinks [11]. However, employing the permeate has economic relevance since it is regarded useful alternative to skim milk as partial or entire replacement [12]. Fig and kiwi are rich sources of sugar, organic acid, mineral, vitamins, polyphenols, flavonoids and fiber [13,14]. They found that fruity milk residue is rich source of important electrolytes.
The present investigation aimed to produce fruity milk residue and study its effects as natural source rich in calcium on osteoporosis female rats.

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were subjected to sensory evaluation by test panelists of FTRI and reported that the ratio of sweetened permeate to the fruits of 1:1 (w/v of fruit and permeate) was the best ratio to produce the beverage which was in parallel with the finding of Atallah [16].

Chemical composition analysis
As stated by the American Association of Analytical Chemistry (AOAC), titratable acidity and pH values were obtained using the AOAC [17].
Mineral composition of the raw material i.e. kiwi, fig and permeate and the produced beverage were determined according to procedures archived in A.O.A.C [17].

Determination of DPPH and ABTS radical savaging activity
DPPH was determined according to the method described by Liu et al. [21] while ABTS was determined according to the method of Re et al. [22].

Determination of total phenols
Total phenols were determined of the produces beverages according to the method described by Chang et al. [23].

Determination of total flavonoids
The method of Liu et al. [21] was used to determined total flavonoids.

Fractionation of sugar components by HPLC
The chromatographic system Agilent (series 1200) coupled to the refractive index detector was equipped with a quaternary pump, degasser and auto injector. The chromatographic data were acquired using the Agilent software. The samples obtained as described above were analyzed using an Aminex-carbohydrate HPX-87 column under isocratic condition with deionizes water. The flow rate was 0.5 ml/min. The column temperature was maintained at 85 °C and the detector at 50 °C. Sample detection was performed by comparing retention time's standards [24].

Biological assay
Thirty-two female albino rats of Sprague Dawley strain, weight 150 ± 10 gm (3 months as age) were raised in the animal house of Food Technology Research, Agriculture research center. Giza, Egypt.). The animals were allowed to free access to water and basal diet for 7 days as an adaptation period. The basal diet was formulated according to AIN [25]. All animal experiments were conducted according to guidelines of the ethical principles established by national institutes of health guide for the care and use of laboratory of animal (NIH Publication 2011). The rats were divided into 8 groups (G1-G8 n = 4 for each group) according to the following scheme ( Fig. 1), functional beverage were added to Basel diet at different concentration (20, 30,  Biological experimental design32 female rats fed basal diet for 7 days as an adaptation period. At the end of the experimental period, (45 days), the rats were fasted overnight. The blood samples were obtained from orbital plexus Venus by means of fine capillary glass tubes [26]. The blood samples were placed in dray and clean centrifuge tubes and allowed to clot for 1 h at room temperature. Serum was removed using a Pasteur pipette and centrifuged for 20 min at 3000 rpm. The clean supernatant sera were then kept frozen until analysis.

Collection of the feed intake
The food intake was calculated daily and the body weight gain was recorded weekly.

Feed efficiency ratio
Feed Efficiency Ratio (FER) for all treatments were given in 1 ml volume from stock solution by stomach tube all over the period of the study was assigned for 3 months. The feed intake was calculated daily and the body weight gain was recorded weekly [27].

Serum analysis
Serum total calcium and PTH parathyroid hormone were estimated using ELISA technique according to the manufacturer's instructions (BioSource host-osteocacin, INC., Camarillo, California, USA. Total Vit. D [28]. While total calcium and phosphorus were determined according to the method of Weatherburn et al. [29].

Radiography of bones by X-ray
Both femurs were separated and cleaned from the surrounding soft tissue. Radiography was performed using X-ray (Fischer Imaging) at 40 Kv and 10 Mas [30]. The length of each femur was measured using a Varnier caliper [31].

Determination of bone mineral content
After incinerated femur bone (550 °C until ashing) using muffle furnace, the ash content was calculated and solubilized using HCL 6N and quantitatively transferred into volumetric flask and complete to 100 ml using HCL (6N). Atomic spectrophotometer (parker Elmer) was used for determination of calcium [32]. Meanwhile phosphorus content in bone was determined colormetrically using uv-visible spectrophotometer [29].

Statically analysis
The obtained data were statistically analyzed using computerized SPSS version 16 (Statistical Package for the Social Sciences). Effects of different treatments were analyzed by one-way ANOVA (Analysis of variance) test using Duncan's multiple range test and P ≤ 0.05 was used to indicate significance between different groups [33].

The chemical composition of raw materials
Data in Table 1 displayed the chemical composition of raw materials used in manufacture of the functional beverages. Data presentenced are similar to Nasir et al. [34] for fig fruit, and with Ragab et al., [35] for kiwi fruit, as for permeate the results is in agreement with Rizk [36].

Chemical composition of functional beverages
The chemical composition of the prepared functional beverages (the total solids, ash, fat and protein contents) increased slightly in all treatments during the storage periods. Generally, there were significant differences (P ≤ 0.05). The slight increase of total solids, acidity and ash during storage may be attributed to the loss of some moisture content and fermentation during the cold storage. And these findings are in agreement with other authors [16,37,38] that the acidity values of fermented beverages prepared with deferent LAB strains for fermentation gradually increased in the course of fermentation, as a result of the LAB metabolism of carbohydrates presented there in milk permeate.

Mineral content of the raw material and produced beverage
Mineral content of raw materials i.e. kiwi, fig as fruits and permeate were reported in Table 2. The data showed that Ca content was the highest mineral in all raw material being 52 ± 0.32, 58 ± 0.45 and 45 ± 0.12 mg/100 g respectively. For permeate, fig and kiwi. Kiwi fruit was characterized by the highest amount of phosphorus (40 ± 3.45 mg/100 g) which was about 3 times as that of fig (14 ± 2.75 mg/100 g) while permeate was found to be poorest material. On the other hand, permeate showed to superior in Mg content (140 ± 5.7 mg/100 g) which was about 8.2 and 4.7 fold as that of fig and kiwi. The data reveled that potassium found to be the predominate mineral in kiwi (332 ± 8.3 mg/100 g) followed by fig (232 ± 5.7 mg/100 g) and permeate (12.6 ± 2.4 mg/100 g). the other minerals showed a small amounts ranged between 0.01 and 3.0 ± 1.63 mg/100 g. the same minerals were determined after producing the beverage and refrigerated stored for 15 days (10 °C). The results showed that the mineral for kiwi beverage were ranked in  Table 3 observed sugar fraction in functional beverage when fresh or during cold storage. In the beginning the results concluded that the lactic acid bacterial used (Str. thermophiles, Lb. bulgaricus, Lb. acidophilus, bifibacteria bifidus  [39] stated that during the fermentation of milk permeate the LAB starter cultures showed post-acidification, thus with the concomitant reduction of pH and increase of TTA. Atallah [16] reported that the total carbohydrate of the prepared functional beverages significantly decreased (P ≤ 0.05) during storage at ~ 4 ºC up to 30 days. Also, Zokaityte et al. [38] added that as a matter of fact, the main sugar in milk permeate was lactose, which was fermented by LAB. All LAB grew well in MP and LAB strain exhibited a significant (P ≤ 0.05) influence on galactobiose and galactotriose synthesis in the fermentable MP substrate.

Total sugar fraction in functional beverage (mg/100 ml)
Sugar fraction in T1 included: sucrose˃ ribose˃ arabinose˃ fructose ˃ galactose at the end of storage period it becomes sorbitol˃ arabinose ˃sucrose ˃ribose ˃ galactose. This results indicated that there is degradation in poly saccharide during cold storage and the activity of probiotic bacteria to utilization of polysaccharide for her growth and fermentation. Sugar fraction in T2 mannose˃ glucuronic˃ rhmanose˃ arabinose at the end of storage period mannitol ˃ arabinose˃ sorbitol˃ inulin. The predominant sugars of kiwi polysaccharides reported are Ara, Gal and Glc, although the molar ratios of the simple sugar composition [40].

Antioxidant activity of the produced beverage
Many fruits contained bioactive component which have many beneficial effect on bone health namely flavonoids and phenols. As mentioned in methods section, antioxidants were determined by two methods, i.e. DPPH and ABTS. The obtained resulted in Table 4 reveled that DPPH resulted in 69.19 ± 1.56%, 87.01 ± 1.98% for T1, T2. After storage period it was 68.95 ± 1.52% and 44.88 ± 1.04% for T3 and T4 respectively. Meanwhile, using ABTS method, resulted in 32.6 ± 1.10, 39.07 ± 0.98, 36.666 ± 0.14 and 41.99 ± 0.95% for T1, T2, T3 and T4 respectively. It worth to mention that DPPH and ABTS resulted showed increase in antioxidant activity of kiwi beverage due to refrigeration while it decreases for fig beverage. It was shown that fig fermented beverage contains total phenols and flavonoid more than kiwi beverage with significant differences in fresh samples was 17.32, 16.89 mg/100 g as Gallic acid and 459.5, 156.5 mg/100 g as catachin. During refrigerated storage, a gradual decrease in all treatments was found which was 16.48, 15.72 mg/100 g as Gallic acid, 265.75, 132.12 mg/100 g as catachin for fig and kiwi respectively. In this respect, Atallah [16] and Rizk [36] whom reported that the biologically active components and antioxidant activity were significantly decreased (P ≤ 0.05) during refrigeration due to their low stability. The antioxidant reacts with free radical which produced by aerial oxygen and depleted. So the concentration of phenols, flavonoids and ascorbic acid decreased during storage, even though stored at refrigerator, which was in parallel with the present results.

Weight gain, feed intake and feed efficiency ratio
The mean values of body weight gain (BEG), feed intake (FI), and feed efficiency ratio (FER) of the osteoporosis rats fed on the replacement of basal diet with 20, 30, and 40% of either kiwi or fig beverage was reported in Table 6. All treated groups resulted in decrease in weight gain compared with the negative control. Similar trend was found concerning feed consumption which affected the decrease in weight gain. On the other hand, feed efficiency rates ranged between 0.128 and 0.415. The major feed efficiency ratio was found in the positive control (0.415). The results can be attributed to the satiating effect of fruits fiber content or due to the presence of permeate in highly amounts which leads to decrease feed intake and parallel weight gain as mentioned by Smith [41]. It is worth mentioning that treatments with beverage increased weight gain, feed intake and feed efficiency ratio compared with positive control because the functional beverage contains a lot of nutrients like carbohydrate, protein, mineral and vitamin beside probiotic bacteria that stimulate the gut function and digestion.

Effect of different level of produced functional beverages on the calcium and phosphorus contents of osteoporotic femur bone
From the results in Table 7, it could be found that the ash content ranged from 0.55 to 0.78 g/100 g bone. The major amount was resulted in negative control (0.78 g/100 g) while the minimum amount found in positive group. Regarding calcium and phosphorus in negative control resulted in maximum amounts being 11.3 ± 2.2 and 7.3 ± 1.33 mg/g ash, respectively. The results showed that Ca and P in all treatments are near to G-and higher than positive control. The treatment with 30% fig beverage, 20, 30 and 40% kiwi beverage were the best treatments. The obtained results confirmed by the mineral content of fig and kiwi and the produced beverage using permeate (Table 1). In this respectively, Lobo-Alexander et al. [42] and Contreras-Padilla et al. [43] reported that fig dietary fiber composition can influences short chain fatty acids formation through fiber fermentation cased colonic bacterial in large intestine. The short chain fatty acids decrease the intestinal pH which intern dissolve insoluble mineral salts particularly Ca, Mg and which increasing their absorption. Kiwi exceeds most other fruits in its content of key micronutrients including K, P, Mg, Fe and folate. It is also contained exceedingly high level of ascorbic acid which increase the bioavailability of nonheme Fe and Ca [44].

Effect of different levels of produced functional beverage on the organs weight
From the results in Table 8, it could be noticed that all treatments affected the organ weight which showed an increase that of negative control. The increase in organs weight may be due to fruit and permeate in produced functional beverages which contained minerals in organic structure which help the maintenance of bone mineral density (BMD) and prevent internal organs from damage [43,44]. Table 9 exhibits serum Ca, P, Protein, Vit. D and parathyroid hormone content of the osteoporotic rats treated with produced beverage. Serum Ca content ranged between 9.3 ± 0.02 mg/dl for the negative control to 14.56 ± 0.23 mg/dl for the positive one significantly. P increased also and ranged from 3.5 ± 0.94 to 4.5 mg/dl with nonsignificat differences between treatments. On the contrary, PTH, Protein and Vit. D resulted in significant decrease than these of negative control. Worthy, treatments cased on slight increase concerning all treatments compared with positive control. The most effective formulas were G3 and G6 for Ca, G4 and G7 for PTH, G3 and G6 for P, G4 and G7 for protein and G4 and G7 for Vit. D., respectively. Consumption of the phytoestrogen and is flavones precursors found in kiwi, metabolic conversion occurs in the gastrointestinal tract resulting in the formation of heterocyclic phenols that are similar to estrogen structure. As steroidal estrogen is use in preventing osteoporosis [45]. Ficus carica was chosen for dietary source of Ca. fig has also been reported to modulate bone remodeling. The mechanism of bone remodeling is composed of balance between bone resorption phase regulated by osteoclast and bone formation phase regulated by osteoblast [46].

Radiography of bones by X-ray
The positive control group recorded Subchondral sclerosis and the emergence of common space (Narrow joint space) compared to negative control group with significant differences. Where the lowest incidence was recorded with 2 followed by 4. The bone composition was significantly altered by drugs: the mineralization degree decreased as period increased. Histomorphometry of the growth plates of lead-exposed rats shows defective remodeling, altered growth plate thickness due to the loss of proliferating cells, and disorganization of the growth plate architecture. Our study was concluded in Fig. 2 and found that histomorphometric measures of bone density (i.e., bone volume, trabecular number, and trabecular thickness) were lower in positive control rats than treatment groups. Positive control rats had lower DEXA-based BMD than normal control group. Osteoporosis defined by decreasing bone density can be appreciated by decreased loss of bony trabeculae in the early stages in radiography. Bones like the vertebra, long bones (proximal femur), calcaneus and tubular bones are usually looked at for evidence of osteoporosis. Figs are a good source of both calcium and potassium. These minerals can work together to improve bone density, which can, in turn, prevent conditions like osteoporosis [46,47]. They suggested that a potassium-rich diet, in particular, can improve bone health and reduce bone turnover. Kiwi contained vitamin C which improve the absorption of calcium and vitamin K can not only increase bone mineral density in osteoporotic people but also actually reduce fracture rates [45]. The effect of vitamin K on bone collagen content may be important for bone quality. The material properties of bone, degree of mineralization, and micro damage accumulation are all influenced by collagen cross-link formation. In particular, the Type-I collagen fibers wired with crosslinks fibers form the framework that binds matrix proteins and mineral crystals. If the arrangement of collagen fibers is altered and the mineral crystal remains immature, these changes in material properties can cause an impairment of bone elasticity [48].

Conclusion
From the obtained data, fruits such as kiwi and fig conserved a rich source of mineral such as ca, P and Mg. when these fruits mixed with fermented permeate to make a fermented beverage, it can be used as a thereby diet for humans suffered from osteoporosis.
Author contributions All authors read and approved the final manuscript. HA: methodology, biology, writing, original draft DF: methodology, prepared beverage, microbiology, original draft, writing IZ: chemical analysis, biology, original draft, writing. All authors review the manuscript.
Funding The research paper supported and funding by agriculture Research Center, Egypt.

Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.
Code availability Not applicable.

Declarations
Ethics approval and consent to participate All experimental animal conduct in this study was approved by the National Institute of the healthy guide for laboratory animal care and used NIH (publication No. 8023 revised 1978 and updated 2011). Animal experiments strictly complied with the legal requirements or guidelines in the country and/or state or province for the care and use of animals including [Arrival guideline. 2.0 updated in July 2020].

Competing interests
The authors declare no competing interests.
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