Ultrasound Assisted Extraction of Micro and Macroelements in Fruit Peels Powder Mineral Supplement for Osteoporosis Patients and their Determination by Flame Atomic Absorption Spectrometry


 Osteoporosis is a worldwide disease depicted by the reduced bone mass, an adequate supply of minerals are needed to support bone remodelingand their deficiency causes bone-related diseases, osteoporosis in particular, and have osteo-protective effects. The aim of recent researchwas to quantify the micro (Mn, Fe, Cu, and Zn) and macroelements (Mg, K, and Ca) in the peels powder of some common fruits (pomegranate, orange, lemon, mango, and grapefruit) by flame atomic absorption spectrometer (FAAS). The extraction of micro and macroelements in peels powder was done by using dilute acids in ultrasonic bath. Apple leaves were used as standard reference material (SRM, NIST 1515) to optimize the ultrasound assisted extraction (UAE) method at varied operating parameters. Maximum response was obtained for extracting of minerals in 500 mg SRM at 60 °C temperature, setting a vortexing time of 5 min.while using 5.0 mL extracting agent HNO3 (0.5 M)-H2O2 (10 %) at90 % sonication amplitude of ultrasound bath for 6 min.While analyzing the SRM, the percentage recovery wasobtained in ranged between 96.8-102.7 % to assure the accuracy whereasrepeatability (n = 10) study in terms of % RSDyielding≤ 2.29well support the precision of proposed method and limits of quantitation (µg/g) were 0.034, 0.061, 0.065, 0.057, 0.017, 0.175 and 0.053 for Mn, Fe, Cu, Zn, Mg, K and Ca respectively. The proposed UAE method was reliable,efficient, and advantageous over the conventionally employed acid digestion method with regards to less consumption of reagents and short analysis time for the determination of micro and macroelements in fruit peels powder.


Introduction
Osteoporosis is a worldwide disease depicted by the reduced bone mass followed by the microarchitectural decay of bone tissues which results in the elevated risk of bone fracture. Annually more than 200 million cases of osteoporosis are reported as active case worldwide, with approximately 2 million case are of hip fractures 1,2 . Younger women with estrogen de ciency while older ones undergone the menopause are primarily caught by osteoporosis whereas secondary osteoporosis occurs even occurs in younger individuals (both male and female) by chronic use of steroids like corticosteroids 3 . Its prevalence in Pakistan is also high, according to an estimation 5.6-17.8 % pre-menopausal women and 20-49.3 % post-menopausal women become a victim of it 4 . To prevent the osteoporosis, maintenance and development of bone mass, nutrition particularly mineralization play an important role.
Osteoclasts are specialized, multinucleated cells which are involved in bone development and regeneration, also ensure the bone mineralization. So an adequate supply of minerals (macroelements: Mg, K, Ca and microelements: Mn, Fe, Cu, Zn) and vitamins are needed to support bone remodeling 2,5 . The importance of these minerals other than Ca for good bone health can hardly be overemphasized.
Several studies have been conducted to link these minerals with bone health and their de ciency causes bone-related diseases, osteoporosis in particular, and have osteo-protective effects [5][6][7] .
Most of the Ca (about 99 %) found in body is associated with bones that plays a major role in bone strength and health throughout life and bone loss associated with aging is managed by adequate Ca intake. Marginal Ca intake or its de ciency is an important factor underlying osteoporosis and increased bone fracture risk 3,8 . Hydroxyapatite production is supported by Mg 9 , about 60 % of its total in the body is stored in the skeleton and this constitutes about 1 % of the total bone mineral content. Mg de ciency via hypocalcemia elevates osteoclast activity while the osteoblastic number and activity decline, so it has a major role in Ca metabolism 10,11 . Ca hemostasis in uenced by potassium particularly excretion and conservation of urinary Ca and bone density is closely related to K intake and its higher intake associated with less bone loss 12 . Mn is a constituent of some enzymes and is distributed in tissues throughout including bone. It plays an important role in bone formation, the thickness of the trabecular bone area, and its mineralization. Utilization of Mn in combination with Cu, Zn, and Ca is more effective in preventing spinal bone loss than alone using Ca 2,13 . Fe has a bone protective effect, it plays an enzymatic co-factor to stimulate the synthesis of bone matrix and mineralization via activation of lysyl hydroxylase and 25-hydroxycholecalciferol respectively 14,15 . Skeletal abnormalities associated with Cu de ciency, in osteoporosis malabsorption of Cu has also its role. Cu removes bone-free radicals that cause osteoclast activation and also inhibit osteoclastic bone resorption directly 2,16 . A large portion of the total body burden of Zn exists in skeletal and Zn related proteins regulate cellular function in osteoblasts and osteoclasts. Zn increases osteoblastic activity and inhibits osteoclastic bone resorption 17,18 .
Fruits are consumed across the world as a rich source of nutrients like minerals, vitamins, ber, and other bioactive compounds that are required for a healthy human life. As a result of increased consumption of the fruits that leads to the wastage of the fruits especially their peels and seed. But the recent studies show that the peels and seeds are a rich source of various valuable chemicals as compare to pulp 19 . So the peels of these fruits should be focused on their use in the manufacturing of various cereals used as breakfast, dietary supplements, and in the formulation of a nutraceutical for the treatment of bone-related problems especially in aged women 20 . Nutritive values of edible parts of the fruits are focused more and the peels are usually considered as agro wastes but the study has revealed that some minerals are even more concentrated in fruit peels than in the edible parts of the fruits 21 . Hundreds of tons of fruit peels are produced each year and the wastage of peel means the wastage of minerals.
Mixture of concentrated acids alone or in combination with hydrogen peroxide are widely used as extracting agents for the extraction of metals in different food matrices by employing microwaveassisted digestion or conventional acid digestion methods. High pressure and temperature along with large volume of reagents (acids and hydrogen peroxide) are required for getting adequate results by using the these digestion methods [22][23][24] . Ultrasound-assisted extraction (UAE) is promisingly alternative method to microwave-assisted digestion or conventional acid digestion methods to get adequate results and enough potential for extracting of metals in different food matrices, sediments 25 , animal and plant tissues 26,27 where ultrasonic energy is used to make easier the process of extraction in the presence of matrix analyte. Also keeping in view the principles of green chemistry, UAE method in combination with dilute acids to extract the metals is generating less waste which is harmful to environment, also this method has advantages over the conventional digestion/extractions methods related to operating costs, safety, simplicity, and reduced extraction time 28,29 . The main target of the current research is to investigate the micro (Mn, Fe, Cu, and Zn) and macroelements (Mg, K, and Ca) composition in the peel's powder of some common fruits (pomegranate, orange, lemon, mango, and grapefruit). UAE method was optimized prior to determination of their concentration by ame atomic absorption spectrometer (FAAS). Consequently suggest these fruit peels powder a non-chemical/organic source of micro and macroelements having nutritional bene ts for the patients of osteoporosis in terms to overcome skeletal abnormalities and improve bone health.

Reagents and solutions
Hydrochloric acid (37 % m/m), Nitric acid, (65 % m/m), and hydrogen peroxide (30 % v/v) of reagent grade (Sigma Aldrich, USA) for digestion of fruit peels powder samples and preparation of extracting agents were purchased from Falcon Chemicals, Lahore-Pakistan. Standard solutions of each metal to construct respective calibration curve were prepared by diluting the certi ed reference standard, 1000 mg/L of each metals (Merck, Germany) with HNO 3 (2 % v/v) whereas glassware employed in this procedure was soaked in HNO 3 (20 % v/v) to avoid any contamination. 18 MΩ.cm resistivity quality water was prepared in our own lab by GenPure water system (Thermo Scienti c, USA) for diluting the solutions. Apple leaves were used as standard reference standard (NIST 1515) for FAAS method validation.

Collection of samples
Fresh fruits samples including pomegranate, orange, lemon, mango, and grapefruit were collected from one supplier located in the provincial capital of Punjab province of Pakistan viz. Lahore. Three packets were collected randomly, from each pack of fruit sample, three samples were collected for each fruit, and in total, 45 fruit samples (3 packets x 3 fruits x 5 fruit species) were used in this study. The plant material was authenticated by plant taxonomist from Institute of Agriculture Sciences, University of the Punjab, Lahore-Pakistan. A voucher specimen (DCUET-020219) was deposited at the Institute's herbarium. After collection, the fruit samples were washed with ultrapure water, then peeled off. The fruit peels were dried for 72 h at 60°C after washing with ultrapure water, the dried peels were grinded by using knife mill and temperature of ultrasonic bath was varied from 2-20 min. and 30-90°C respectively. 5.0 mL of extracting agent and 500 mg nely grinded SRM was transferred to round bottom ask, and vortex for 5, 10, and 15 min. then it was placed in an ultrasonic bath at 30, 60, and 90°C temperature for different intervals (2,4,6,8,10,12,14,16,18, and 20 min.). The resulting suspension was centrifuged (Sorvall™ ST 8, Thermo Scienti c™, UK) at 3000 rpm for 3 min. then supernatant was diluted with ultra-pure water and made up to volume in 25 mL of volumetric ask. The nal solution was stored at 4°C till any further analysis. The optimized UAE method was applied for the determination of micro and macroelements in the fruits peels powder under investigation.

Conventional acid digestion
The nely grinded powder of fruit peels was transferred to glass digester containing concentrated HNO 3 (15 mL), initially heated the sample at 100°C for 15 min. then before adding the H 2 O 2 (2 mL) it was kept at 120°C for 2 h and after the addition of H 2 O 2 , further heated for 20 min. at the same temperature. This conventional acid digestion method also applied to SRM for the comparison of UAE with it.
The results were presented in mg 100 g − 1 of dry powder mass while the analysis were carried out in triplicate whereas stock solutions of real samples and SRM were further diluted as per requirement of analysis.

Instrumentation
Air circulation oven (Bio-Science, China) and knife mill (GRINDOMIX GM 300, Thomas Scienti c, USA) were used for drying and grinding of fruit peels respectively. Ultrasonic bath (Model 1800, Thomas Scienti c, USA) and glass digester (Hach DRB 200 Dry Thermostat Reactor, Thomas Scienti c, USA) were employed for UAE and CAD respectively. The composition of Mn, Fe, Cu, Zn, Mg, K, and Ca present in SRM and fruit peels powder were determined by using FAAS equipped with an auto-sampler (Model: PG-990, PG-instruments, UK). AA Win Lab® software was used to interpret the data whereas experimental design and data for optimization of AAS are explained in (Table 2) 30,31 . Operational parameters of AAS in present study are presented in Table 1.  Wavelength accuracy and its reproducibility were checked to optimize the AAS by using hallow cathode lamp of mercury (HCL, Hg) while searching the peaks at 253.7, 546.1, and 871.6 nm whereas bandwidth was xed at 0.2 nm. The wavelength accuracy was measured by difference between standard value and calculated value. For resolution studies of peaks obtained for particular element, the minima and maxima was calculated by using manganese (Mn) HCL, details parameters are provided in Table 2.

Validation of proposed method
For the validation study of proposed UAE method, percentage recovery was calculated to assess the accuracy of method after analyzing the SRM whereas paired t-test at 95 % con dence of interval was performed to check the e ciency of UAE as compared to CAD. Precision was evaluated by the repetition of extraction procedure in replicates of ten (n = 10) under the same conditions and results of repeatability are expressed are in % RSD (100 x σ/µ, where σ is SD and µ is mean value). Limit of detection (LOD = 3 σ/S) and limit of quantitation (LOQ = 10 σ/S) were also estimated where standard deviation (SD) of analytical blank measurement (n = 18) was presented by σ while S is the slope of the calibration curve (y = mx + b) 32,33 .

Optimization of AAS
The peaks were scanned in triplicate at three different wavelengths (253.7, 546.1, and 871.6 nm) by using HCL of Hg the results of obtained wavelength are presented in Table 3, the difference between set and obtained wavelength represents the wavelength accuracy whereas maximum/minimum values were reported at the interval of 6 h to check the wavelength reproducibility which are in compliance with user requirement limit of ± 0.25 nm and ± 0.10 nm respectively.  SRM (NIST 1515) was used to optimize (n = 6, see supplementary data, Table 1S-8S) the UAE method by using three different extracting agents whereas operating parameters including pre-sonication, sonication and temperature were also varied ( Table 5). The proposed UAE method was evaluated on the basis of the recovery studies of seven elements present in the SRM (microelements: Mn, Fe, Cu, and Zn and macroelements: Mg, K, and Ca) and compare with certi ed value.  The results of Mn, Fe, Cu, Zn, Mg, K, and Ca present in SRM obtained by UAE were compared with CAD method, and signi cance difference at 95 % con dence of interval (p = 0.05) was assessed by comparing the t critical (cut off point on the t distribution) and t experimental (experimentally compare the means of two groups) for both the methods (CAD and UAE). The value of t critical (2.57) was more than t experimental at ve degrees of freedom (n-1 = 5) which indicates no signi cant difference in obtained values of Mn, Fe, Cu, Zn, Mg, K, and Ca present in SRM by using both the methods as shown in Table 6. Table 6. Extraction and digestion method validation by using SRM (n = 6)

Vortexing and sonication time in uence on extraction recoveries
The required quantity of SRM and each fruit peels powder along with extracting agent was added to vortexing tube separately and subject to pre-sonicated (vortexing) for different time intervals (5-15 min.). After the vortexing the tube was placed in ultrasonic bath, 5 min. vortexing was noted as the optimum time for extracting both the micro and microelements from SRM and all samples (pomegranate, orange, lemon, mango, and grapefruit) understudy to achieve excellent recoveries. Increase in vortexing time (10 or 15 min.) did not show any increment in recoveries (supplementary data, Table 9S-11S). UAE e ciency increased while increasing the sonication time (Fig. 1a) but the maximum recoveries of micro and microelements from SRM was obtained at 6 min. sonication time by using HNO 3 -H 2 O 2 as extracting agents. It was observed that the sonication more than 6 min. did not show any increase in recovery of micro and microelements from SRM. So the optimum recoveries were achieved by using dilute acid with hydrogen peroxide as extracting which offers practical advantages over CAD methods where a extended time is required to digest the sample 34 .

Extracting agents in uence on extraction recoveries
The  3] were also studied over the extraction of micro and macroelements from SRM and fruit peels powder. A vortexing and sonication time was xed at 5 and 6 min. respectively at temperature of 60°C while using the extracting agents and higher recoveries were obtained by using EA-3, which is an acid-oxidant mixture (Fig. 1b). The extraction recoveries of micro and macroelements from SRM by using rest of two extracting (single acid, EA-1 and combination of two acids, EA-2) agents were comparative lower (supplementary data,

Temperature in uence on extraction recoveries
Variation in temperature (30, 60, 90°C) of ultrasonic bath was demonstrated for extraction of micro and macroelements in fruit peels powder to assess the effect of temperature on extraction performance. Increasing the temperature of extracting agents along with increment in sonication time resulted in formation of free radical which accelerate the reaction involved in the digestion of samples. Extraction recoveries were also assess by increasing time of vortexing along with varied condition of temperature (30, 60 and 90°C). The data showed that ( Fig. 2 and

Validation of proposed method
The errors in the analytical methods determine the accuracy of results on the basis of difference between the actual values and determined values of SRM which enable us to make the decision about the method adopted for analysis. The magnitude of sample matrix, purity of reference standard, environmental condition of laboratory as well as stability of instrument play a vital role to get accurate results, so to ensure accuracy during the analysis of micro and macroelements in real samples, SRM was analyzed and results are presented in terms of percentage recovery studies (Table 6) Table 7. The lower values of detection limits (LOD and LOQ) indicate that the method provided adequate sensitivity as shown in Table 7. The repeatability (n = 10) were performed to check the precision of method and results were presented in % RSD which were obtained in ranged of 0.43 to 2.29 (Table 7) during the analysis of SRM which is good enough and in compliance with the FDA manual (% RSD < 7).

Analysis of fruit peels powder samples
The optimized UAE procedure was applied to fruit peels for extraction of the micro (Mn, Fe, Cu, and Zn) and macroelements (Mg, K, and Ca) and subsequently, these were determined by FAAS, results are presented in Table 8. The highest Mn content was found in mango peel ranged between 0.49-0.60 mg/100 g (Table 8S, g), and red grapefruit (0.10 mg/100 g) from Turkey 20 is less than that the results are found in our study (Table 8), while 0.21-0.34 mg/100 g Mn was found in orange cultivar (lima and pera orange) from Brazil 37 which is comparable with concentration found in our samples under investigations.
Our study showed that mango peel contained the highest Fe concentration (6.5 mg/100 g) that is 80.2 % of the estimated average requirement (EAR, 5.0-8.1 mg/day) and 36.1 % of recommended dietary allowance (RDA, 8-18 mg/day), so both parameters cover the DRI as 5.0-18 mg/day for both males and females. Grapefruit also contained an appreciable amount of Fe (3.53 mg/100 g) while lemon and pomegranate have almost the same concentration (Table 8). The concentration of Fe reported for the peels of orange (0.51 mg/100 g), lemon (0.34 mg/100 g) and red grapefruit (0.23 mg/100 g) from Turkey 20 is less than that of the results are found in our study (Table 8), while 1.01 mg/100 g Fe was found in orange cultivar (lima orange) from Brazil 37 .
The Cu content in all the fruit peels was found in the range between 0.06-0.29 mg/100 g (Table 8S, supplementary data) while the highest concentration was found in mango peel ranged between 0.27-0.31 mg/100 g which contribute 32.2 % of DRI (0.7-0.9 mg/day) including EAR, 0.70 mg/day and RDA, 0.90 mg/day for both males and females with aged group ranged between 18-70 years. The Cu content was found in lima and pera orange was 0.06 mg/100 g and 0.09 mg/100 g originate to Brazil 37 while the concentration of Cu reported for the peels of orange (0.15 mg/100 g), lemon (0.04 mg/100 g) and red grapefruit (0.08 mg/100 g) from Turkey 20 .
The Zn content in all the fruit peels was found in ranged between 0.78-1.10 mg/100 g (Table 8S, supplementary data) while the highest concentration was found in mango peel (1.03 mg/100 g) which contribute 9.4 % of DRI (6.8-11.0 mg/day) including EAR, ranged 6.8-9.4 mg/day and RDA, ranged 8-11 mg/day for both males and females with aged group ranged between 18-51 years. The Zn content was found in lima and pera orange was 0.35 mg/100 g and 0.21 mg/100 g respectively originate to Brazil 37 while the concentration of Zn reported for the peels of orange (0.25 mg/100 g), lemon (0.28 mg/100 g) and red grapefruit (0.33 mg/100 g) from Turkey 20 .
The highest content of Mg was found in mango peel ranged between 133-141 mg/100 g while the least concentration was found in pomegranate (45-51 mg/100 g) and orange (47-51 mg/100 g) which are comparable (Table 8S, supplementary data). The DRI contribution by mango peel is 33.6 % including EAR, ranged 265-350 mg/day and RDA, ranged 320-420 mg/day for both males and females with aged group ranged between 18-70 years. The Mg content was found in lima and pera orange was 23.8 mg/100 g and 27.8 mg/100 g respectively originate to Brazil 37 while the concentration of Mg reported for the peels of orange (13.2 mg/100 g), lemon (11.5 mg/100 g) and red grapefruit (10.0 mg/100 g) from Turkey 20 .
The present study indicated that the mango peel is rich in K (1501.67 mg/100 g) content while the least amount was found in orange peel (680 mg/100 g). The K content was found in lima and pera orange was 258.7 mg/100 g and 266 mg/100 g respectively originate to Brazil 37 while the concentration of K reported for the peels of orange (154 mg/100 g), lemon (127 mg/100 g) and red grapefruit (132 mg/100 g) from Turkey 20 . The DRI contribution of K by mango peel is 31.9 % as AI (adequate intake, 4700 mg/day), for both the genders with aged group ranged between 18-70 years.
Among all the fruit peels, the highest Ca content was found in grapefruit peel (801 mg/100 g) while the least concentration was found in pomegranate (277. 33  In conclusion, mango peel comprised of the highest concentration of Mn, Fe, Cu, Zn, Mg, and K, while the Ca content was found less than orange, lemon and pomegranate peels. Special attention should be paid to such a nutritionally rich peel powder of mango for its potential use as a component of functional food. Different food cereals can be forti ed with these fruit peel powders especially mango peel powder to meet the body's ongoing demand for individual minerals. Since mineralization (Fig. 3) is an important modi able factor in the development and maintenance of bone mass and the prevention of osteoporosis.
Concerning nutrition and health, this research showed that fruit peels of pomegranate, orange, lemon, mango, and grapefruit contain an appreciable amount of micro and macroelements that are good for bone health and could have osteo-protective effects.

Conclusions
In this study, an ultrasound-assisted extraction method was optimized by varying the parameters of extraction and using different extracting agents for the micro (Mn, Fe, Cu, and Zn) and macroelements (Mg, K, and Ca) composition in the peels powder of some common fruits including pomegranate, orange, lemon, mango and grapefruit.The composition of mentioned elements was consequently determined by optimized and validated FAAS method. SRM was also analyzed by CAD method to check the performance and comparison of UAE to determine the signi cant difference between both methods. The signi cance difference at 95 % con dence of interval (p = 0.05) was assessed by comparing the t critical (cut off point on the t distribution) and t experimental (experimentally compare the means of two groups) for both the methods (CAD and UAE). The value of t critical (2.57) was more than t experimental at ve degrees of freedom (n-1 =5) which indicates no signi cant difference in obtained values of Mn, Fe, Cu, Zn, Mg, K, and Ca present in SRM by using both the methods. Concerning nutrition and health, fruit peels powder is a very valuable source of micro and macroelements for the patients of osteoporosis in terms to overcome skeletal abnormalities and improve bone health. On the basis of literature, hypothesis was established for the management of osteoporosis by minerals, and fruit peels are rich source of minerals and is a potential organic source of minerals rather to use their salts as source of minerals. This research is just to Vortexing time and temperature in uence on micro and macroelements recovery from SRM SupplementarymaterialSciReports.docx