Formulation and biochemical evaluation of designer diet enriched with botanicals for bone health

Abstract The study designed to evaluate the role of sesame, fenugreek flax, and nuts seeds as best alternatives for therapeutic agents to improve bone mineral status. Effect of these plant seeds on proximate composition, antioxidant potential, mineral, fatty acid, and amino acid profile of supplements was studied. The sesame, fenugreek, and flaxseeds were incorporated with nuts at level of 35% to develop supplement. Improved contents of protein, fat, and NFE were recorded in range of 25.72–32.23, 18.92–23.15, and 34.25%–39.59%, correspondingly. The antioxidant potential of supplements is high (450.56 mg GAE/ 100g) prepared with fenugreek followed by flaxseeds and sesame seeds. Calcium as fundamental bone mineral found greater (525.15%–790.21%) in seed‐based supplement. Enhanced contents of Oleic, linoleic, and linolenic acids ranged 17.32–41.78, 21.72–44.23, and 1.00%–47.12%, respectively. Nutritional enriched supplements may be best alternatives for improving bone health by lowering osteoclast and increasing osteoblast mechanism in bone development.


| INTRODUC TI ON
Our skeleton is a major module of body as it provides a strong defensive fundamental structure. Due to which body is able to cope with daily routine and maintain whole physique. Regeneration and maintenance of bone tissues is a continuous progression. Bone is not merely a mineral reservoir but much of its composition is protein, fatty acid, and polysaccharides which is an organic segment.
And it also comprehended a complex of nerves, blood vessels, and marrow at center. Bones are major reservoir of calcium about 99 percent of whole body (Song, 2017), which is available in form of hydroxyapatite. Other minerals (including Ca, P, Mg, Vitamin K, and Vitamin D) constitute the inorganic part of bone for its development and maintenance.
One of several risk factors for reducing bone strength and several disorder is low bone frame, and these factors may characterized as skeletal and nonskeletal. Most cases of low bone density occur in aged subjects, particularly postmenopausal women indeed, a decline of estrogen levels in females at menopause leads to loss of bone mass and strength and contributes to the development of bone diseases such as osteopenia and osteoporosis.
Skeletal factors involve the alteration in biomechanical strength determining the bone size and shape act without interference of any other related issue (Weaver et al., 2016). Environmental | 2985 YASMEEN Et Al.
factors, especially dietetic behavior and routine diet plan are major contributing aspect of bone mass loss. Alcohol and nicotine taking, low physical activity, dependency on medication are determinants of these factors (Martha et al., 2019). Diet is one of several important parameters for optimum bone density and treating of bone ailments. As the significance of calcium and vitamin D is concerned, it has been much discussed in previous studies in regards to improve bone health (Peters & Martini, 2010). Balanced nutrition enriched diet besides fulfilling daily energy requirement also play an important role in providing essential micronutrients, which ultimately helps to maintain bone mass and bone mineral density (Anastasilakis et al., 2017).
Nutritional therapy has abundant impact on bone health and key role in management of bone disorders like osteopenia, osteoporosis, and arthritis. It is an amendable element to strengthen bone health and inhibit the initiation of bone-related issue.
Furthermore, studies have shown that diets high in fruits and vegetables have positive effects on bone mineral status and that nutrients and vitamins, including vitamin K, vitamin C, phosphorus, potassium, magnesium, protein, and sodium, are important for the maintenance of optimal bone health (Prynne et al., 2006). The findings of many studies have related the intake and/or serum levels of several vitamins, including A, B complex, C, E, and K, as well as the homocysteine (Hcy) level, to bone health in animals and in humans.
Plant-based curative composites have positive effects against various human diseases without any hazardous side effects. These compounds are widely used to cure the disorders along providing essential nutrients and improving performance (Shirwaikar, Khan, Kamariya, Patel, & Gajera, 2010). Plant-based compounds are being used in many traditional systems mostly in rural areas but on increasing urbanization synthetic foods and medicines has been in practice.
Instead, plant-derived medication has been investigated much safer than synthetic pharmacological elements. Out of many plant-based sources sesame, fenugreek and flaxseeds are important and generally ignored food stuffs which can contribute to strengthen the body potential against disorders.
Sesame seed is delicious flavored food containing health benefitting nutrients like essential fats, minerals, antioxidants, and vitamins. Sesame seed is considered to be helpful for its potential to provide recommended daily allowance (RDA) of certain nutrients like calcium, phosphorus, iron, magnesium, and potassium. As a study revealed that sesame seed provides 100% of the recommended daily allowance (RDA) for manganese and potassium, 57%-65% of the RDA of phosphorus and iron, and 13%-35% for zinc, calcium, and copper while its recommended daily intake is 25 to 50 grams (Pathak, Rahman, Bhagawati, & Gogoi, 2017). Lignan glycosides have naturally potential against oxidation as antioxidant which is rich in sesame seed. This antioxidant ability helps to prevent bone inflammation consequently prevent bone resorption and increase bone formation (Kim, Lee, & Lee, 2006).
Fenugreek (Trigonella foenum-graecum Linn) is seasoning ingredient used in foods have much medicinal and therapeutic value. Fenugreek seed is complex of many dietary nutritional components like amino acids, fatty acids, vitamins, and saponins such as disogenin, flavonoids, polysaccharides, and glycosides recommended for therapeutic use (Umesh, Najma, & Baquer, 2014). Fenugreek seed extract have positive effect on lowering the consumption of dietary fat in humans, and it highly modifies the feeding behavior which is responsible for weight management. This investigation is a major factor for lowering bone loss by weight controlling especially in females. Fenugreek has antiosteoporotic ability in presence disogenin and helps to reduce process of osteoclast which contribute to bone mass loss ultimately (Nesma, El-Nasr, El-Denshary, Mahmoud, & Nofal, 2018).
Flaxseed is a oil containing kernel rich in essential fatty acids, dietary fiber, and proteins. As containing high amounts of omega 3 fatty acids and α-linolenic acid, it contributes to stimulation of osteoblast process during which bone mass substance secreted during estrogen deficiency. Linolenic acid is a precursor of eicosapentaenoic acid is higher in flaxseed enriched diet (56%) than fish is effective in reducing bone loss consequently preventing osteoporosis (Boulbaroud, Mesfioui, Arfaoui, Quichou, & El Hessni, 2008).
Phosphorus and magnesium are major mineral elements for bone strength, and composition is in higher amounts in flaxseed (Branski, Fasano, & Troncone, 2006).
All available treatments for bone-related diseases include pharmaceutical pharmacological and synthetic agents which act by reducing rate of bone mineral loss and increasing bone potential to avoid fracture risk. But these therapeutic managements have side effects like gastrointestinal infection, hypercalcemia, dyspepsia. Thus, there is a need to explore natural plant-based sources for enhancing skeleton strengthening before commencing bone losses.

| Procurement of raw materials
Fenugreek, Sesame, and Flaxseeds of autumn season were procured from Ayub Agricultural Research Institute (AARI), Faisalabad, Pakistan. Other ingredients were purchased from local market.
Analytical reagents and HPLC grade standards were obtained from Merck (Darmstadt, Germany) and Sigma-Aldrich.

| Preparation of raw materials
The seeds of sesame, fenugreek, and flaxseed were cleaned by sieving followed by sorting. Afterward, milling of seeds along with other ingredients was carried out using Laboratory (Perten-3100) mill to get straight grade fine powdered sample. The resultant powder was stored in air tight containers prior to analysis.

| Product development
Three different supplement formulations were developed from the selected raw materials. These supplements comprise similar composition except one major ingredient. As first, second, and third supplement contains a major ingredient like sesame seed, fenugreek, and flaxseed respectively as shown in Table 1. All ingredients were mixed homogenously. Grapes, papaya, and mud apple concentrate were prepared after peeling, slicing, and grinding one by one. The concentrate was then freeze dried to get powder to use it as flavor. Finally this powder was mixed in their respective formulation.

| Proximate composition
Supplement samples were analyzed for moisture, crude protein, crude fat, crude fiber, ash, and nitrogen-free extract by using triplicate samples (AOAC, 2016).

| Mineral profile
The samples were subject to mineral assay through wet digestion considering the protocols of AOAC (2016). For the estimation of sodium and potassium, Flame Photometer-410 will be used while calcium, iron, magnesium, Phosphorus measured through Atomic Absorption Spectrophotometer.

| Fatty acid profile
Fatty acid methyl esters (FAMEs) were prepared from the oil samples according to protocol described by (Lutterodt, Slavin, Whent, Turner, & Yu, 2011). Briefly, 1 mg of oil was reacted with 0.1 mol/L NaOH-MeOH for 5 min, followed by reacting with 1.1 mol/L HCl-MeOH for 5 min at ambient temperature. After adding water to stop the reaction, FAMEs were extracted with iso octane. GC analysis was conducted with a Shimadzu GC-2010 equipped with a FID and a Shimadzu AOC-20i auto sampler.

| Total phenolic contents
Total phenolic content determination was performed for prepared supplement by using procedures described by Singleton, Orthofer, and Lamuela-Raventos (1999). Decant two mL of aqueous extract in a 10 ml volumetric flask, then added 5 ml of distilled water followed by 0.3 ml of 5% NaNO 2 and after 5 min -0.6 ml of 10% AlCl 3 was added. After another 5 min stay, 2 ml of 1 M NaOH was added and volume was made up to 10 ml with distilled water. Take one mL from this solution after mixing this solution, add 9 ml distilled water and measured absorbance by using spectrophotometer at 765 nm. Total phenolic content was calculated and expressed as Gallic Acid Equivalent (mg Gallic Acid/100g).

| Total favonoids
Total flavonoids contents of supplements were evaluated using the procedure described by Zhishen, Mengcheng, and Jianming (1999).
Took 2ml of aqueous extract of sample in a 10 ml volumetric flask, and added 0.3 ml of 5% NaNO 2 then 5 ml distilled water and after 5 min -0.6 ml of 10% AlCl3 was added. A 2 ml of 1 M NaOH was added after 5 min stay, and volume was made up to 10 ml with distilled water. Take one mL from this solution with 9 ml distilled water and measured absorbance by using spectrophotometer at 510 nm.
Total flavonoids were expressed as Catechin equivalents per dry matter.

| Ferric reducing antioxidant power (FRAP)
Ferric reducing antioxidant power was measured by using the method of Muller et al. (2011). An aliquot of prepared supplement samples extract (50 μl each) was taken with 3 ml of FRAP reagent following incubation at 37°C for 30 min. The increase in absorbance was noted at 593 nm using Spectrophotometer. The results were compared with the calibration curve, prepared by using various concentrations of trolox as standard.

| Statistical analysis
The results for chemical assays were expressed as mean ± SD of three independent analyses. One-way analysis of variance (ANOVA) was used to verify the difference between the sample groups.
Statistical significance of the difference was tested while using LSD test at p ≤ .05. The data obtained from all the determinations were statistically analyzed by using the statistical software Statistix 8.1 (Informer Technologies).

| Proximate composition
As balanced nutrition diet is essential for better human health. A food compound of various ingredients delivers minerals, vitamins, and macro nutrients required for body well-being, which play crucial role against diseases caused by malnourishment consequently reducing risk of numerous ailments. The results depicted that moisture, ash, fat, protein, fiber, and NFE contents significantly different with respect to different treatments and is given in Table 2, which showed the maximum protein contents in flaxseed-based supplement i-e 32.23 following by fenugreek and sesame seed-based supplement 28.56 and 25.72, respectively. Flaxseed fortified product showed increased amount of protein as studied by Patil, Sheety, Todkar, and Bodhankar (2013); Masoodi, Aeri, and Bashir (2012).
Protein is most important nutrient to enhance body immunity and resistance against initiation of diseases.
High protein diets are key to increase the anabolic insulin like growth factors which is resultantly play a crucial role in bone structure development. These sufficient proportioned protein diets are accompanying with proper calcium absorption. On contrary, lower protein content diet consumption reduce the calcium absorption results rise in parathyroid hormone rise, which aid the bone mass loss (Heaney & Layman, 2008). On the basis of its nutrition, botanical constituted diet have ability to change acid-base status making alkaline which is favorable for bone health (James et al., 2016). Essential fatty acid containing fat contents of current study ranged between at maximum of 23.15% in sesame seed-based supplement and at minimum of 18.92% in fenugreek-based supplement. Similar results found by (Kasaye & Kumar, 2015) in which the crude fat content of samples

| Mineral profile
Minerals are important constituent of human bone composition. Any reduction of particular mineral quantity in human serum affects the bone strength quality. Absorption of these minerals depends upon the whole composition of diet taken. The results showed that mineral profile of prepared supplements has a significant difference among each other and is given in Potassium is one of the important minerals in any health improvement supplement playing crucial role in cell membrane potential and ion transport through membrane. Potassium and magnesium of flaxseed powder product were reported 200.97 and 125.86 respectively by Kaur and Das (2015), which is relatively is lower than that of present study due to the addition of comparable higher amount of flaxseed powder along in presence of nuts. Calcium, potassium, and magnesium of walnut in a study reported as 1,062, 2,771, and 1,426 respectively on whole weight basis (Cindric, Zeiner, & Hlebec, 2018) which is major mineral enrichment ingredient in present study as a minor amount ingredient.
Fenugreek having high nutritious value can be used as value addition ingredient in many products like bakery, snacks and other cereal-based fortified flours. To obtain multiple benefits, it is a mineral enriched daily diet constituent which will help in body metabolism.

Bread and biscuits prepared by adding various percentages of fenu-
greek powder showed a range of minerals like calcium, magnesium, iron, and zinc from 5.71 to 78.04 (Kasaye & Kumar, 2015) But in present study 35% fenugreek seed-based supplement with inclusion of nuts the range of mineral profile is much greater like 616.7% potassium to least 12.73% iron.

| Amino acid composition
The amino acid composition has a significant difference with respect to treatments and is given in Table 4. Glutamic acid ranked the highest concentration in sesame (19.23g/100g), fenugreek seed (17.86g/100g), and flaxseed (16.87g/100g)-based supplement. This observation is similar to that of reflected by (Fasuan, Saka, Gbadamosi, & Omobuwajo, 2018). Amino acid profile results showed least contents amino acids are methionine, cysteine, and tryptophan. Among the essential amino acids, lysine is estimated to be highest in all three supplements 8.05 g/100g in T1, 8.90g/100g I T2, and 6.12g/100g in T3 than other. These findings are opposite to observed by Fleddermann et al. (2013) who concluded the higher essential amino acid is leucine. Valine, tryptophan, and isoleucine contents of fenugreek are similar to those evaluated by Hegazy (2011)  Note: All data are the mean ± SD of three replicates. Means followed by different letters (a, b, and c) within the same row are significantly different (p < .05) from each other using a one-way analysis of variance (ANOVA) and LSD test. 18:3 (Linolenic) 1.00 ± 0.02 c 20.62 ± 0.11 b 47.12 ± 0.34 a Note: All data are the mean ± SD of three replicates. Means followed by different letters (a, b, and c) within the same row are significantly different (p < .05) from each other using a one-way analysis of variance (ANOVA) and LSD test. 16:0, palmitic acid; 16:1, palmitoleic acid; 18:0, stearic acid; 18:1, oleic acid; 18:2, linoleic acid; 18:3, linolenic Acid.

| Fatty acid profile
Fatty acids have important role in bone metabolism and potential to resist against bone loss preventing osteoporosis. The fatty acid profile has significant difference with respect to treatments and is given in Table 5. Supplement prepared in current study showed a positive trend of fatty acids profile for bone health improvement. Omega 3 fatty acids are one of the classes of fatty acids having potential of improving bone turnover. Plants and nuts are major sources of omega 3 fatty acids (Ricard, Mònica, & Salas-Salvadó, 2016). Fatty acid composition of sesame seed-based supplement exhibited best of linoleic 42.32, Oleic 41.78, and palmitic acid 9.68 values, which are slightly higher than estimated by Elleuch et al. (2007). Reported values of sesame seed along with nuts supplement are also comparable with sesame seeds fatty acid composition evaluated by Biglar et al. (2012).
As Table 3  20.62 respectively compared with report by Sulieman et al. (2008) with improvement in fatty acid profile in the presence of nuts and fruit sources.

| Antioxidant potential
The results regarding the total phenolic content (TPC), free radical scavenging activity (DPPH), total flavonoids, and FRAP showed significant difference among different treatments and is given in Table 6.
The results indicated the highest value 450.00 mg GAE/100g in T2 (fenugreek-based supplement) followed by 156.00 mg GAE/100g in T3 and 123.23 mg GAE/100g in T1. The principal mechanism of antioxidant is scavenging free radicals by transferring hydrogen atom.
Results of antioxidant analysis revealed that amount of total phenolic contents not proportional with DPPH activity of scavenging free radicals. This finding is similar to that of Kaur and Das (2015).  (Apak, Ozyurek, Guçlu, & Çapanoglu, 2017). In a study reported by Kenny, Smyth, Hewage, and Brunton (2013), frap evaluation of fenugreek seeds powder distinctly showed value of 0.135 to 77.352 TE mg/g, which is comparable to current evaluation of fen- ugreek with various nuts. FRAP value of flaxseed powder extract estimated to be 0.58 to 1.08 mg TE/g by Deng et al. (2017), which is lower than that of supplement prepared in current study by the addition of nuts as 3.72.
Flavonoids are bioactive polyphenol and one of the important nutrient found abundantly in fruits, vegetables, nuts, and seeds. Sesame seed being adequately nutritious has been esteemed as a food additive capable of having potential against many diseases. Table 5 presents the total antioxidant potential including Total flavonoids, which indicated the lower flavonoids value of 1.85 ± 1.56 in sesame seed-based supplement than other treatments. This record is comparable and lower than 30% sesame-supplemented diet calculated by William, Faulet, Koné, Gnanwa, and Kouamé (2018). Flaxseed-based supplement (35%) contents calculated in current study are (2.13 ± 1.35 mg CE/g) in the presence of nuts and fruit ingredients, which is more than flaxseed reported flavonoid contents (35-70 mg/ 100 g) (Katare, Saxena, Agrawal, Prasad, & Bisen, 2012). Fenugreek seed is comparatively a good source of bioactive components like alkaloids, flavonoids, and saponins (Kasaye & Kumar, 2015) and attributed to antioxidant potential.
Flavonoid contents of fenugreek-supplemented formulation depict the higher value (3.75 ± 2.10) than other treatments.

| CON CLUS ION
This study focused on the formulation and biochemical evaluation of plant-based nutrients against bone health. The outcomes of current study revealed that Sesame, fenugreek, and flaxseeds are good sources of proteins, fats, NFE. Increased protein contents in prepared supplements observed more in (T3) but differ in mineral profile trend. The results obtained from mineral analysis showed significant improvement in the calcium and phosphorus contents as major bone minerals of (T1) sesame seed supplement. Addition of nuts in prepared diet plan (T2) in the presence of 35% fenugreek amplified the omega fatty acids, linoleic, and linolenic contents that ultimately increased bone turnover potential. Amino acid profile of prepared product showed that substitution of nuts and used botanical seeds significantly improved the essential amino acid value that plays effective role in collagen structure during fracture healing.
Oxidation inhibition activity of (T2) fenugreek-based supplement observed higher reduce the oxidative stress and enhance the product shelf life. Overall, improved nutrition of products has positive effect on availability and absorption of each nutrient.

ACK N OWLED G M ENTS
The authors are highly thankful to Government College University Faisalabad and PCSIR Laboratories, Lahore for providing the facilities in conducting the research work.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
This study has nothing to do with human and animal testing.

D ECL A R ATI O N
The authors did not use the human subjects.