Food Chemistry

Advances in characterization and analysis methods of fructoborates- A review

--Manuscript Draft--

Manuscript Number: FOODCHEM-D-23-04791

Article Type: Review Article (max 10,000 words)

Keywords: calcium fructoborate, analytical technique, nutraceutical product, fructoborate analysis

Corresponding Author: Alexandra Bounegru, Ph.D.

Dunarea de Jos University of Galati Department of Chemistry Physics and
Environment
ROMANIA

First Author: Alexandra Bounegru, Ph.D.

Order of Authors: Alexandra Bounegru, Ph.D.

Simona Butan

Veronica Filimon

Abstract: Fructoborates are challenge compounds for scientific world due to their nutritional
importance and potential impact on human health.This review article explores various
aspects of fructoborates, including their chemical composition, bioavailability, and
mode of action in the body, as well as methods for analyzing and identifying their
structure. The significance of calcium fructoborate in human nutrition is highlighted,
given its high content of boron, calcium, and fructose. Calcium fructoborate is
extensively studied and has beneficial effects on bone health, cardiovascular function,
metabolic syndrome, and inflammation. Analytical methods, such as spectroscopy,
chromatography, and thermal analysis, are used to detect and quantify fructoborates in
dietary supplements and other matrices. Future research directions include
investigating their bioavailability and absorption, understanding their molecular
mechanisms of action, and conducting clinical trials to assess long-term efficacy and
safety. Developing improved assay methods and understanding their mechanisms of
action are crucial for effective therapies and health interventions.

Suggested Reviewers: Boris Nemzer

VDF FutureCeuticals Inc
bnemzer@futureceuticals.com

Saad A. Hussain
University of Baghdad
saad_alzaidi@yahoo.com

Petre Rotaru
University of Craiova
protaru@central.ucv.ro

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1 Advances in characterization and analysis methods of

2 fructoborates- A review
3
4 Butan Simona1, Filimon Veronica2, Bounegru Alexandra Virginia1*
5
6 1 “Dunărea de Jos” University of Galaţi, Department of Chemistry, Physics and Environment, Faculty of Sciences and
7 Environment, 47 Domnească Street, 800008 Galaţi, Romania
8 2“Dunarea de Jos” University of Galati, Cross-Border Faculty, 47 Domneasca Street, 800008, Galati, Romania

9
10 Corresponding author: alexandra.meresescu@ugal.ro
11 *Author to whom correspondence should be addressed.
12
13 Highlights
14  Comprehensive synthesis of existing research on the structural and biological
15 characteristics of fructoborates.
16  Detailed presentation of current analytical methods used for the detection and
17 quantification of fructoborates.
18  Identification of the advantages and limitations of each method, providing a solid
19 foundation for decision-making in research and industry.
20  Exploration of new perspectives and directions in the development of fructoborate
21 analysis techniques.
22  Contribution to a deeper understanding of the importance of fructoborates in the medical
23 and dietary supplement fields.
24
25 Abstract
26 Fructoborates are challenging compounds for the scientific world due to their nutritional
27 importance and potential impact on human health. This review article explores various aspects
28 of fructoborates, including their chemical composition, bioavailability, and mode of action in the
29 body, as well as methods for analyzing and identifying their structure. The significance of
30 calcium fructoborate (CaFB) in human nutrition is highlighted, given its high boron, calcium, and
31 fructose content. CaFB is extensively studied and has beneficial effects on bone health,
32 cardiovascular function, metabolic syndrome, and inflammation. Analytical methods, such as
33 spectroscopy, chromatography, and thermal analysis, are used to detect or quantify
34 fructoborates in dietary supplements and other matrices. Future research directions include
35 investigating their bioavailability and absorption, understanding their molecular mechanisms of
36 action, and conducting clinical trials to assess long-term efficacy and safety. Developing
37 improved assay methods and understanding their mechanisms of action are crucial for effective
38 therapies and health interventions.
39
40 Keywords: calcium fructoborate, analytical technique, nutraceutical product, fructoborate
41 analysis
42
43 1. Introduction
44 Fructoborates represent an important class of nutritional compounds that contain
45 calcium, fructose, and boron (B). These compounds have attracted the attention of the scientific
46 world and food industry due to their potential health benefits and impact on human
47 nutrition(EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) et al., 2021; Nielsen,
48 2020; Nielsen & Eckhert, 2020). Calcium Fructoborate (CaFB) is the most studied and well-
49 known fructoborate, obtained through the reaction between calcium, fructose, and B(“Plant-
50 Based Calcium Fructoborate as Boron-Carrying Nanoparticles for Neutron Cancer Therapy,”

1
 51 2019). This compound stands out for its ability to provide both calcium, an essential mineral for
52 bone and dental health, and B, an important micronutrient with various biological
53 roles(Rondanelli et al., 2020).
54 Fructoborates, given their unique properties, are investigated for benefits such as joint
55 health, immune system fortification, bone health, and estrogen level maintenance(Belhan et al.,
56 2021; Hunter et al., 2019; Tkacheva & Eliseeva, 2022). Their antioxidant and anti-inflammatory
57 actions aid cellular protection against oxidative stress and inflammation(Aysal et al., 2023;
58 Kisacam et al., 2020, 2021; Mitruţ et al., 2021). To leverage their nutritional benefits, the
59 implementation of precise analytical methods is pivotal. Such methods facilitate the
60 determination of B, calcium, and fructose content in a variety of fluoroborate-containing foods,
61 dietary supplements, and similar products. Their analysis and identification are crucial to
62 comprehend these nutritionally valuable compounds. Notably, CaFB has garnered scientific
63 interest due to its health-promoting potential. Several analytical methods have been applied
64 over time for quantifying fructoborates in food or pharmaceutical products, as presented in
65 Table 1.
66 Although fructoborates are a topic of interest in the field of nutrition and health, the
67 current literature does not provide sufficient up-to-date research on this subject. Therefore, this
68 review aims to fill this gap and provide an updated and comprehensive perspective on the
69 analysis of fructoborates, thus contributing to the development and improvement of knowledge
70 in this important field.
71 The purpose of this review is to aggregate and interpret available data on the biological
72 effects of fructoborates in humans and the analytical methodologies employed for their
73 identification and quantification in various matrices. The employed methods in fructoborate
74 studies span spectroscopic, chromatographic, and thermal techniques, offering a
75 comprehensive understanding of the B, calcium, and fructose content, molecular makeup, and
76 bioavailability of fructoborates. The critical evaluation of these methodologies aims to identify
77 the most precise and efficient techniques for detecting and quantifying fructoborates in food
78 products, dietary supplements, and similar commodities. Ensuring the accuracy of this
79 information is pivotal for the quality, safety, and appropriate labeling of fructoborate-containing
80 products.
81 Furthermore, this review emphasizes the nowadays challenges and limitations of current
82 analysis methods of fructoborates, providing insights into future directions for research and
83 improvement of these techniques. The development of more sensitive, selective, and non-
84 invasive analysis methods can contribute to obtaining more precise results and a deeper
85 understanding of fructoborates and their impact on human health.
86 2. The nutritional importance and action mechanism of fructoborates
87 Fructoborates are organic compounds of B containing two essential molecules, borate,
88 and fructose. These compounds are naturally found in certain foods such as apples, pears,
89 plums, grapes, and nuts, especially those rich in sorbitol or fructose(Brown & Shelp, 1997) as
90 well as in some medicinal plants like ginseng roots.
91 Researchers have found that calcium binding contributes to the stability of organoborate
92 complexes. Woods(Woods, 1996) found that by introducing a positively charged substituent
93 group (Ca2+), electrostatic stabilization will be obtained. Similarly, Kobayashi et al.(Kobayashi et
94 al., 1996) observed that the presence of calcium ion (Ca2+) significantly improves the stability of
95 the borate-rhamnogalacturonan-II (RG-II) dimeric complex, an insoluble molecule in water
96 present in the cell walls of higher plants. The fructoborate anion consists of two fructose
97 molecules bound to a B atom, with a molecular weight of 367.13 g mol-1, and it can adopt three
98 isomeric forms depending on the structure of the fructose ring (Söderholm et al., 1999). These
99 forms include two isomers with 5-membered rings (α- and β-fructofuranose borate) and one
100 isomer with a 6-membered ring (α-fructopyranose borate), which dynamically interconvert, with
101 the β-fructofuranose borate form, the most predominant structure(R. I. Scorei & Rotaru, 2011).

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102 Figure 1 depicts the chemical structures of calcium α-D-fructofuranose borate and calcium β-D-
103 fructofuranose borate.
104 Both isomers have been separated by chromatography, but the mixture contains 50
105 times more calcium β-D-fructofuranose borate than calcium α-D-fructofuranose borate(Kim et
106 al., 2008). Figure 2 illustrates the chemical reactions by which CaFB can be obtained from
107 fructose, boric acid, and calcium carbonate in an aqueous solution.
108 Boron binds to fructose at a cellular pH of 7.4, forming fructoborate esters and free boric
109 acid, but not borate anions(I. D. Scorei & Scorei, 2013). From a microbiological standpoint,
110 research suggests that B has an extensive function in the binding to glycoproteins in cell
111 membranes and the mechanism of action of fructoborates may occur through chemical binding
112 to specific cytokine glycoprotein receptors on the cell membrane surface, respectively.
113 Fructoborate has a better ability to interact with glycoproteins than boric acid and may have
114 physiological activity both inside and outside the cell. Boric acid generated by the hydrolysis of
115 the fructoborate complex crystallizes in the hexagonal system, which can influence the toxicity
116 and biological activity of free boric acid. Fructoborate is considered a non-toxic "reservoir" of
117 B(MogoÅŸanu et al., 2016; Yokota & Konishi, 1990). Furthermore, CaFB regulates antioxidant
118 metabolism and even has a protective effect against oxidative DNA damage. A very recent
119 study demonstrated the antioxidant and histopathological effect of CaFB on liver and kidney
120 tissues in rats in the case of CCl4-induced toxicity(Aysal et al., 2023).
121 CaFB disintegrates in the gastrointestinal tract under the influence of low pH and
122 digestive enzymes, releasing fructose and boric acid. Studies have shown that complete
123 dissociation of CaFB occurs in an environment with a pH of 4(EFSA Panel on Nutrition, Novel
124 Foods and Food Allergens (NDA) et al., 2021). In vivo, studies have demonstrated that B from
125 CaFB is available in the circulatory system, with increased levels in the bloodstream after a
126 single dose. Although information regarding absorption and excretion is limited, it is presumed
127 that both calcium and fructose from this compound are systemically available, considering its
128 composition. Thus, each component of CaFB has the potential to exert biological and nutritional
129 effects on the body. Analytical methods such as 11B-NMR spectrometry can be used to study
130 the dissociation and absorption of NF in the gastrointestinal system and provide specific
131 information on its ADME (absorption, distribution, metabolism, and elimination)(EFSA Panel on
132 Nutrition, Novel Foods and Food Allergens (NDA) et al., 2021).
133 From a nutritional standpoint, fructoborates are important due to their B content. Boron is
134 an essential trace element required for strong and healthy bones, as well as for the proper
135 functioning of the nervous system and sex hormones. Although B is needed in small amounts,
136 some research suggests that B deficiency may be linked to certain conditions such as
137 osteoporosis.
138 Research indicates that fructoborates are more readily absorbed by the body compared
139 to other B forms, such as sodium borate. In addition, fructoborates exhibit no toxicity or
140 accumulation risk, positioning them as a safe and beneficial option for dietary supplementation.
141 Their consumption could be a proactive strategy for chronic disease prevention(Donoiu et al.,
142 2018; R. Scorei & Popa, 2013). The potential consequences of a fructoborate-deficient diet are
143 nonspecific, including joint inflammation, reduced bone density, osteoporosis, and weakened
144 immunity(MogoÅŸanu et al., 2016; I. D. Scorei & Scorei, 2013). Borate supplementation has
145 been acknowledged in some countries as beneficial for the prevention of osteoporosis,
146 osteoarthritis, certain cancers (including breast and prostate), and cardiovascular diseases(Li et
147 al., 2021; Ri et al., 2022; Tepedelen et al., 2017; Yıldırım et al., 2021). Studies reveal that
148 fructoborates are predominantly situated in the mineralized portion of bones rather than being
149 collagen-bound, thereby influencing bone structure and strength(Jugdaohsingh et al., 2015).
150 These B compounds play a role in the homeostatic regulation of estrogenic and testosterone
151 hormones, thereby impacting bone mineralization(Bello et al., 2018; Rondanelli et al., 2020).
152 Additionally, individual health status and age influence the total B concentration in blood and

3
153 bones(Donoiu et al., 2018). Notably, vitamin D deficiency might lead to a decrease in blood B
154 levels, although the exact mechanism remains to be elucidated(Simaz et al., 2021). Boron
155 intake from the diet has been found to influence serum calcitonin and Ca2+ concentrations in
156 humans(Devirian & Volpe, 2003; Hunter et al., 2019).
157 A diet low in fructoborates is also associated with increased serum triglyceride
158 levels(Kuru et al., 2019), and it is presumed to contribute to an elevation in blood glucose
159 concentration(López-Cabrera et al., 2018). Fructoborates cannot be synthesized in the human
160 body from fructose and boric acid; their intake must be supplemented through the diet and they
161 are absorbed in the small intestine(EFSA Panel on Nutrition, Novel Foods and Food Allergens
162 (NDA) et al., 2021). The action of fructoborates is enhanced when administered concurrently
163 with calcium, magnesium, zinc, iron, and copper. On the other hand, the intake of iodine,
164 fluoride and silicon may diminish or even nullify the effect of fructoborates due to their
165 antagonistic effects(Popova et al., 2017; Singh et al., 2021). One way to increase fructoborate
166 intake through the diet is by using dietary supplements that contribute to enhancing immunity
167 and promoting a healthy gut flora(Nandwana et al., 2022).
168 3. The Effects of Fructoborates on Health
169 3.1. Fructoborates in the inflammatory process
170 Inflammation can disrupt the normal process of bone remodeling and contribute to the
171 loss of bone mineral density. Levels of C-reactive Protein are inversely correlated and
172 independently associated with total bone mineral density. CaFB, a patented compound
173 containing calcium, fructose, and B, has been shown in clinical studies to reduce serum levels
174 of CRP in humans. Research suggests that CaFB may be beneficial for bone health by
175 controlling inflammation associated with the loss of bone mineral density(I. D. Scorei & Scorei,
176 2013).
177 Among the available supplements containing B and borate, CaFB is the most studied
178 and offers the highest potential for human health(Rondanelli et al., 2020). CaFB has a
179 stimulating effect on humoral and cellular immune response (increased plasma concentrations
180 of immunoglobulins IgG and IgM, and increased percentages of CD4+ and CD8+ lymphocyte
181 subgroups)(Taranu et al., 2011). Moreover, CaFB regulates the production of inflammatory
182 mediators by macrophages and suppresses cytokine production.
183 A recent study reported a novel chemisorption deposition method to form a coating with
184 CaFB on a titanium and hydroxyapatite (HApTiCaFb) implant, followed by evaluating
185 histological characteristics related to bone healing at the interface of a bioceramic material in an
186 animal model. The results showed that the release of CaFB from HApTiCaFb occurred rapidly in
187 the first three days after immersion in a phosphate-buffered solution, followed by a linear
188 release up to 14 days. SEM analysis revealed similar particle morphology and size diameter for
189 both implants. No fibrosis or inflammation was observed around the porous HApTiCaFb implant,
190 suggesting that coatings with CaFB promote the osseointegration of the HApTi implant in rabbit
191 femurs and significantly reduce inflammation(Department of Research Methodology, University
192 of Medicine and Pharmacy of Craiova, Romania & Văruţ, 2021).
193 Both animal and human studies have demonstrated that CaFB has a potent anti-
194 inflammatory action without generating side effects(Rogoveanu et al., 2015; I. D. Scorei &
195 Scorei, 2013; R. I. Scorei & Rotaru, 2011). The mechanism of action of CaFB involves
196 modulating serine proteases derived from inflammation-activated leukocytes, synthesizing
197 modulated leukotrienes, and reducing reactive oxygen species generated during neutrophil
198 bursts. CaFB can also modulate the synthesis of eicosanoids (pro-inflammatory prostaglandins)
199 derived from arachidonic acid , capable of scavenging superoxide ions and exhibiting anti-
200 inflammatory activity(R. I. Scorei et al., 2010). A study showed that treatment with CaFB in
201 lipopolysaccharide (LPS)-stimulated macrophage cells led to the inhibition of interleukin (IL)-1β,
202 IL-6 and nitric oxide release in cell culture media and an enhancement in the generation of
203 tumor necrosis factor-alpha (TNF-α), respectively. Therefore, no effect on the expression of

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204 LPS-induced cyclooxygenase-2 (COX-2) protein has been identified(R. I. Scorei et al., 2010; R.
205 I. Scorei & Rotaru, 2011).
206 Although we can assert that CaFB influences macrophage production of inflammatory
207 mediators, further studies are needed to establish whether CaFB treatment can suppress the
208 production of pro-inflammatory cytokines and prevent the exacerbation of endotoxin-associated
209 diseases. The complete mechanism of CaFB's effectiveness as an anti-inflammatory agent
210 compared to anti-inflammatory drugs is currently unknown.
211 3.2. Fructoborates in osteoporosis
212 Osteoporosis can affect individuals regardless of gender or age, requiring long-term
213 treatment with high social costs. The disease can lead to complications that immobilize patients
214 and cause multiple deficiencies(Miljkovic et al., 2009). Nutritional B has beneficial effects in
215 reducing the negative consequences of vitamin D deficiency in rodents. Studies suggest that B
216 can increase the level of 25-hydroxyvitamin D by inhibiting the enzyme 24-hydroxylase.
217 Additionally, increased regulation of 17β-estradiol levels in women has been observed through
218 B consumption, including in postmenopausal women. Nutritional B can inhibit microsomal
219 enzymes that catabolize estradiol and 25-hydroxyvitamin D through vicinal
220 hydroxylations(Miljkovic et al., 2004).
221 Over time, it has been found that CaFB can also influence bone density in animals with
222 vitamin D deficiency(Miljkovic et al., 2009). Furthermore, CaFB can help protect bone health in
223 humans by controlling inflammation associated with loss of bone mineral density. Clinical
224 studies have shown that CaFB significantly reduces levels of CRP, a marker of inflammation, in
225 humans(I. D. Scorei & Scorei, 2013).
226 A pilot study investigated the effect of CaFB supplementation on inflammatory and lipid
227 markers in individuals with primary osteoarthritis. Participants were divided into four groups and
228 received treatment or a placebo for 2 weeks. The results showed that CaFB had a beneficial
229 effect on inflammatory markers and induced slight changes in lipid metabolism. The study
230 suggests that short-term supplementation with CaFB may be useful to reduce the inflammation
231 of individuals with osteoarthritis(R. Scorei et al., 2011).
232 A comprehensive clinical research program was conducted to explore the effects of
233 CaFB on osteoporosis, utilizing Fortex Margarine as the delivery vehicle for this compound. This
234 study included 100 osteoporosis patients, and the preliminary results indicated a substantial
235 alleviation of bone pain and joint discomfort, enhanced mobility, and elevated blood calcium
236 levels(Manda et al., 2009). This outcome could be attributed to enhanced calcium absorption or
237 more efficient regulation of calcium deposits within the body. The increased blood calcium could
238 then trigger a feedback mechanism leading to calcium incorporation into deposits (i.e., bones),
239 which is likely a crucial factor in pain reduction. Blood tests have demonstrated that CaFB
240 administration elevates blood calcium levels, potentially contributing to the alleviation of bone
241 pain. Furthermore, this compound may aid in the reconstruction of joint cartilage, resulting in
242 decreased discomfort and improved joint movements. Laboratory tests showed an increase in
243 bone density in more than two-thirds of the participants, along with relatively positive
244 symptomatology(Ghivercea et al., 2004).
245 A pivotal study was designed to assess the efficacy of CaFB supplementation on
246 osteoblast mineralization in a controlled lab environment. This study utilized mesenchymal
247 stromal cells from bone marrow and trabecular bone osteoblasts from pigs. CaFB was tested
248 both in combination with dexamethasone and independently. The results indicated that
249 dexamethasone promoted alkaline phosphatase activity in bone marrow cultures but inhibited it
250 in trabecular osteoblasts. CaFB showed a minimal impact on alkaline phosphatase activity in
251 trabecular osteoblasts but marginally increased it in differentiated osteoblasts. Furthermore, the
252 joint treatment of dexamethasone and CaFB resulted in pronounced mineralization during the
253 initial stages of differentiation. Thus, CaFB effectively stimulates osteoblast differentiation and
254 acts in synergy with dexamethasone to bolster mineralization(Manda et al., 2009).

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255 Combining drug treatments with fructoborate-based supplements has become a
256 common and preferred practice for enhancing treatment response. Another clinical study
257 evaluated the effects of CaFB and sodium tetraborate (NTB) as supplements for patients with
258 active rheumatoid arthritis (RA) receiving etanercept treatment. After 60 days of treatment, both
259 forms of B significantly improved symptoms and reduced inflammation levels in patients. CaFB
260 proved to be more effective than (NTB) to improve the clinical status and inflammatory marker
261 levels. The use of B as an adjunct with etanercept may be a promising new strategy for RA
262 treatment and could help avoid issues associated with biological medications(Hussain et al.,
263 2016).
264 3.3. Fructoborates in cardiovascular conditions
265 Studies have shown that fructoborates, including CaFB, can exert a protective effect on
266 cardiovascular health. They have demonstrated a good potential for inflammation reduction,
267 blood lipid levels modulation and endothelial dysfunction improvement, which are factors
268 involved in the development of cardiovascular diseases. Fructoborates have also been studied
269 for the prevention and treatment of cardiovascular conditions such as angina pectoris(Militaru et
270 al., 2013), atherosclerosis and ventricular dysfunction.
271 For example, a double-blind, active-controlled study tested supplementation with CaFB
272 in the case of patients with stable angina pectoris. After 60 days of treatment, a significant
273 reduction of CRP levels and improvements in lipid profile were observed. Likewise, there were
274 recorded significant reductions in angina episodes and changes in inflammation markers within
275 a relatively short time. These results indicate the possibility of using CaFB and other natural
276 adjuncts to improve standard anti-angina therapies(Militaru et al., 2013). Another similar study
277 evaluated the effects of CaFB on inflammatory markers and blood lipids in middle-aged healthy
278 subjects. The results showed that supplementation with CaFB for 30 days at a daily dose of 112
279 mg significantly reduced total cholesterol, LDL cholesterol, triglycerides, CRP and homocysteine
280 levels while increasing HDL cholesterol levels. A significant reduction of pro-inflammatory
281 cytokines IL-1β, IL-6 and MCP-1 was also observed. These findings suggest that CaFB can be
282 used as a dietary supplement to support cardiovascular health and prevent unhealthy cardiac
283 conditions(Rogoveanu et al., 2015).
284 In addition, these B-based compounds have proven to be an optimal solution to improve
285 markers for cardiovascular conditions due to their evident benefits on health and supplementing
286 animal feed with them(Miljkovic et al., 2009). A Romanian study demonstrated that a diet of
287 organic eggs enriched with CaFB has positive effects on lipid metabolism. This led to a
288 significant decrease in total cholesterol, uric acid, as well as CRP and fibrinogen levels. This
289 potential adjunctive treatment was applied in the case of patients with cardiovascular disorders
290 and osteoarticular conditions. The study showed that functional organic eggs enriched with
291 CaFB have no side effects and are well-tolerated by patients. So, these food products can be
292 included in the diet of patients with various conditions and even healthy individuals (Mitrut P,
293 Scorei IR, Badea P, Dumitrescu A. S, n.d.).
294
295 3.4. Fructoborates and Oxidative Stress
296 The compounds of boron, especially fructoborates, have been associated with beneficial
297 effects on the skin and wound healing. Experiments have been conducted on keratinocyte cell
298 cultures, which play an important role in the skin healing process(Turner et al., 1998). It has
299 been observed that calcium fructoborate does not generate oxidative stress and has an
300 antioxidant effect, reducing cellular damage caused by free radicals. This effect is attributed to
301 the ability of calcium fructoborates to scavenge superoxide radicals through organic
302 peroxiborates. Furthermore, CF has demonstrated the ability to inhibit the auto-oxidation of
303 pyrogallol, suggesting that it can act as a scavenger of superoxide radicals. These findings
304 indicate that CF has antioxidant activity and may protect cells from oxidative damage(R. Scorei
305 et al., 2005).

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306
307 3.5. Fructoborates in neoplastic conditions
308 Several studies have shown that fructoborates have potential as antineoplastic agents,
309 which are those substances that can inhibit the growth and development of cancerous tumors.
310 These compounds have demonstrated the ability to inhibit the growth of cancer cells and induce
311 their programmed cell death (apoptosis). Moreover, fructoborates have shown potential to
312 reduce angiogenesis (the formation of new blood vessels necessary for tumor growth) and
313 inhibit the metastasis of cancer cells to other parts of the body.
314 In a study conducted by Scorei et al., 2008, it was found that both boric acid and CaFB
315 inhibit the proliferation of breast cancer cells in a dose-dependent manner. Treatment with
316 CaFB caused increased levels of p53 and bcl-2 proteins, as well as activation of the apoptotic
317 pathway through high expression of pro-caspase-3 proteins, cytosolic cytochrome c levels and
318 caspase-3 activity. These findings suggest the possibility of using B acid and CaFB as
319 chemotherapeutic agents in the treatment of human cancer, although further studies are
320 mandatory to understand the mechanisms involved in this process(R. Scorei et al., 2008).
321 In a more recent study, the effect of CaFB on MDA-MB-231 breast cancer cells was
322 tested. Treatment with CaFB led to reduced cell viability and increased expression of proteins,
323 which are involved in apoptosis and metastasis inhibition. The results indicate that these B-
324 based compounds may have not only therapeutic potential but also preventive benefits in breast
325 cancer(Tepedelen et al., 2017).
326 In 2022, the cytotoxic effect of CaFB on colon cancer cells (Caco-2) was investigated.
327 CaFB showed the highest efficiency at a concentration of 10 mM for 24 hours. The decrease in
328 Bcl-2 levels and the increase in Bax levels at this concentration indicate the apoptotic effects of
329 CaFB. Akt, p70S6K and 4EBP1 levels tended to decrease, while PTEN and TSC2 levels were
330 found to increase after treatment with CaFB. Moreover, CaFB increased Hck expression and
331 decreased Fyn expression. In conclusion, treatment with CaFB at a concentration of 10 mM can
332 prevent the proliferation of colon cancer cells by inducing apoptosis and, possibly, activating
333 autophagy(Kisacam et al., 2022).
334 Mehmet Ali Kisacam et al. have explored the involvement of CaFB in experimentally
335 induced skin cancer in vivo in mice. Various treatments were used, including CaFB in
336 combination with DMBA and TPA. Topical application of DMBA and TPA led to increased
337 protein levels and mRNA expression of factors involved in skin cancer. Additionally, an increase
338 in the number of TUNEL-positive cells was observed. Treatment with CaFB reduced protein
339 levels and mRNA expression of factors involved in skin cancer and decreased the number of
340 TUNEL-positive cells. Therefore, this study indicates that CaFB may have preventive effects on
341 skin cancer(Kisacam et al., 2020).
342
343 4. Analysis methods of fructoborates
344 The analysis of fructoborates is important to ensure the safety and efficacy of dietary
345 supplements containing these compounds. Fructoborates are used as dietary supplements to
346 improve bone and joint health(Reyes-Izquierdo et al., 2012), and their analysis can help to
347 determine their exact content and purity.
348 Besides, the analysis of fructoborates can be useful in monitoring the stability and quality
349 of products during storage and transportation. Various analysis methods can be useful in
350 research and development processes to better understand the molecular structure and
351 properties of fructoborates and optimize the formulation of dietary supplements.
352 Overall, the analysis of fructoborates is crucial to ensure the safety and efficacy of these
353 compounds and the products that contain them, as well as to facilitate the development of new
354 products and applications in the field of dietary supplements and health.
355 In addition, the presence of B isotopes has an impact on environmental samples.
356 Therefore, some analysis methods focus on monitoring environmental quality.

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357 There are several analysis methods of fructoborates, such as infrared spectroscopy
358 (e.g., FTIR)(Dumitru et al., 2010), liquid chromatography-mass spectrometry(Xia et al., 2017),
359 nuclear magnetic resonance spectroscopy(Edwards et al., 2014), thermal analysis(Rotaru et al.,
360 2010), and elemental analysis such as inductively coupled plasma optical emission
361 spectrometry (ICP-OES)(Pedrinelli et al., 2019).
362
363 4.1 Spectroscopic Techniques
364 FTIR instruments utilize the interference between two IR beams to produce a signal,
365 called an interferogram, which is a function of the path length difference between the two
366 beams(Dutta, 2017). This is typically achieved using a Michelson interferometer. The signal
367 generated from a Michelson interferometer can then be decomposed into frequency
368 components to form a spectrum using Fourier transform algorithms (Valand et al., 2020).
369 Fourier-transform infrared spectroscopy (FTIR) is a method used to identify and characterize
370 fructoborates based on the absorption of infrared light by the compound molecules. The FTIR
371 spectrum can be used to determine the molecular structure and functionality of the compounds.
372 The research conducted by C.C. Wagner and his team focused on the characterization
373 of CaFB samples with the chemical formula Ca(fruBor)2·3.5H2O. These samples were analyzed
374 chemically and spectroscopically using methods such as infrared and Raman spectroscopy, as
375 well as thermogravimetric and differential thermal analysis. Additionally, theoretical studies were
376 conducted using density functional theory for seven different structural models of the
377 fructoborate fragment and the most stable structure was derived from these calculations. The
378 study allowed a better understanding of the vibrational-spectroscopic behavior of the compound
379 and the detailed analysis of the infrared and Raman spectra. All these data experimental can be
380 used to assign the vibrational modes of the B–based compound. Regarding specific aspects of
381 vibrational-spectroscopic behavior, it was found that in the high-frequency region, the FTIR
382 spectrum exhibits a very strong and broad band centered at approximately 3380cm −1, similar to
383 those typically found in metal carbohydrate complexes. Furthermore, a significant broadening of
384 the band was observed in the spectral range between 900-1200cm−1. In the case of the Raman
385 spectrum, a general broadening of most bands accompanied by significant intensity decreases
386 was observed for fructoborate compared to D-fructose. Moreover, the study highlighted the
387 existence of specific stretching and deformation motions of groups and bonds in the compound,
388 which can be identified in the IR and Raman spectra. For example, the stretching motion of the
389 terminal B-O bond in fructoborate can be identified as a relatively strong IR band at 1658cm−1
390 but is absent in the Raman spectrum. On the other hand, the very strong IR band at 1342cm −1
391 corresponding to δ(OH) + τ[C(1)H2OH] experiences a drastic intensity decrease after the
392 formation of fructoborate and is even absent in the Raman spectrum of this compound(Wagner
393 et al., 2008).
394 In a more recent study, M. Dumitru thoroughly examined the molecular composition of
395 CaFB in a dietary supplement through FTIR and Raman spectroscopic analysis. Experimental
396 measurements identified the correct molecular formula of CaFB, which contains twice as much
397 B as reported in other studies. FTIR analysis determined the chemical bonds and functional
398 groups of the organic substances. The spectra of CaFB, fructose, boric acid and calcium
399 carbonate were recorded in the wavelength range of 4000-650cm−1. In the transmission IR
400 spectrum of CaFB, the vibrational bands of fructoborate were observed due to common
401 functional groups, but the intensity of these bands was diminished due to new interactions in the
402 CaFB molecule (Dumitru et al., 2010).
403 In 2015, the possibility to obtain new bionanocomposites using CaFB-doped
404 hydroxyapatite nanopowders through the sol-gel method was investigated. The resulting
405 bionanocomposites were analyzed using scanning electron microscopy, energy-dispersive X-
406 ray spectroscopy, FTIR and biological tests. According to the results, the active substance,
407 CaFB, was well dispersed among the hydroxyapatite crystals and the composites exhibited

8
408 good cellular compatibility with skeletal myoblasts, offering the potential to use as biomaterials
409 in various medical applications such as bone cement, dental filling materials and resorbable
410 materials for osteosynthesis. Moreover, FTIR spectra showed that the composite contained
411 phases of hydroxyapatite and CaFB and structural studies revealed that the bionanocomposites
412 exhibited lamellar aggregates with intergranular micropores. The results of the biological
413 analysis suggested that the nano-hydroxyapatite-CaFB biocomposites are promising materials
414 that can prevent further bone loss and aid in the growth or restoration of bone mass, making
415 them suitable for medical implants. This alternative method to obtain bioactive materials with
416 anti-inflammatory properties can be useful in the development of biomedical devices(Barna et
417 al., 2015).
418
419 4.2 Chromatographic Techniques
420 Chromatography is a technique used to separate and identify fructoborates from a
421 complex mixture of compounds. By combining liquid chromatography separation technology
422 with precise mass spectrometry detection, the compounds can be accurately identified and
423 quantified.
424 Fruitex-B® CaFB, a patented product in the USA (5,962,049, Miljkovic, 1999), has been
425 analyzed in numerous studies using different techniques.
426 In 2017, a group of researchers reported the identification of the fructoborate complex in
427 food products using liquid chromatography coupled with quadrupole Orbitrap mass
428 spectrometry. The method was established by optimizing the mobile phases and directly
429 infusing the FruiteX-B CaFB solution into the Q Exactive Orbitrap mass spectrometer. The
430 results were verified and compared using solid-state and multinuclear liquid-state spectroscopy.
431 The method proved to be useful to identify and quantify the fructoborate complex at low
432 concentrations in samples of fruits, vegetables, nuts, seeds and even biological samples(Xia et
433 al., 2017). Figure A. 3 shows the MS/MS mass spectrum of the target compound from the
434 apricot extract and the fructoborate from CaFB.
435 In the same year, Andrei Biță's team presented a new, precise and selective
436 chromatographic method used for the simultaneous identification and quantitative determination
437 of boric acid and CaFB in dietary supplements, both in bulk and in accurately dosed forms such
438 as tablets/capsules. The stationary phase consisted of glass plates pre-coated with silica gel C
439 60 F254 and the mobile phase was a mixture of 2-propanol-water in a ratio of 8:2 (v/v). Using
440 this method, the two B-based compounds were successfully separated, with Rf values of 0.83 ±
441 0.01 for boric acid and 0.59 ± 0.01 for CaFB. Therefore, monitoring and quality control in the
442 dietary supplement industry can easily be achieved by applying this method(BITa et al., 2017).
443 The detection and quantification of fructoborates through HPLC require a rigorous
444 scientific approach and in-depth studies. These studies should focus on the HPLC parameters
445 optimization such as the choice of mobile phase and stationary phase to ensure proper
446 separation of fructoborates and other compounds from a complex matrix. Factors such as
447 sensitivity and specificity of detection, as well as method validation according to industrial
448 standards and safety regulations, should also be taken into consideration.
449 4.3. Inductively Coupled Plasma Mass Spectrometry
450 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is a powerful analytical tool
451 employed to trace elements in a diverse array of samples. Over the past decade, this method
452 has become more prevalent, replacing older techniques like atomic absorption or atomic
453 emission spectroscopy. Researchers need to understand both the analytical facets and
454 limitations of ICP-MS, including potential spectroscopic and non-spectroscopic interferences,
455 along with strategies for their minimization or elimination(Wilschefski & Baxter, 2019). ICP-MS
456 operates by using a robust ionization source to break down proteins into elemental or molecular
457 ions, which are subsequently filtered and analyzed based on their mass-to-charge ratio. Its
458 strength lies in quantitative analyses owing to its high sensitivity, expansive linear dynamic

9
459 range, low detection limits, and applicability across various sample matrices. Additionally, ICP-
460 MS enables the measurement of absolute quantities of biomolecules in intricate samples
461 without requiring specifically targeted standards. Its ability to use stable isotopes significantly
462 enhances the precision of biomolecule quantification, including proteins(Feng, 2021).
463 This precise and reproducible method was applied by Catherine Guerrot and her team
464 (Guerrot et al., 2011) for measuring B isotope ratios in natural waters using a MC-ICP-MS
465 (Neptune) after wet chemical sample purification. The sample matrix was removed as it can
466 drastically affect the isotopic ratio by altering the mass. The study proposed an adapted method
467 for water samples that allowed for the purification of 100 ng of B with the direct recovery of B in
468 2 mL of 3% v/v HNO3. Boron from reference materials, including seawater (IAEA-B1) as well as
469 two groundwater reference materials (IAEA-B2 and IAEAB3), was chemically purified. The
470 reproducibility of the entire procedure was ± 0.4‰ (2s), and accuracy was verified by
471 comparison with positive TIMS values and recommended values. The results showed that the
472 three IAEA natural waters can be used as reference samples for B isotopes, allowing for a
473 better understanding of their isotopic ratios and improving the quality of B isotope ratio
474 measurements for all laboratories(Guerrot et al., 2011).
475 In 2015, a rapid method was reported for the extraction and precise measurement of B
476 concentration and isotopic ratio in various vegetation samples, including bark, wood and tree
477 leaves, using ID-ICP-MS and MC-ICP-MS. The procedure involved microwave digestion, cation
478 exchange chromatography and micro-sublimation. The entire procedure could be completed in
479 a few hours. The B recovery rate was complete, meanwhile the measurement repeatability for
480 the isotope ratios was up to 0.36‰ (± 2s). The main source of uncertainty came from the cation
481 exchange step, but it was not influenced by the nature of the vegetation sample. Regarding the
482 determination of the isotopic ratio δ11B by MC-ICP-MS, it is important to avoid excessive
483 dissolution loads that can lead to chemical impurities and to maintain a reasonable amount of
484 total salts or dissolved organic carbon (Roux et al., 2015).
485 In a very recent study, a new method for the analysis of B in silicate samples was
486 developed using complex wet acid digestion with B-mannitol and inductively coupled plasma
487 mass spectrometry (ICP-MS) to achieve precise results. To enhance the decomposition of
488 silicate samples, the mixture of hydrofluoric acid and nitric acid was improved by adding
489 mannitol. After fluoride removal, the samples were treated with nitric acid and left to mature
490 overnight. The subsequent dilution was carried out with nitric acid solution to directly measure
491 the B content by ICP-MS. It was found that the digestion temperature had no effect on the B
492 content and the accuracy of the measurements decreased at B contents greater than 7250 ng.
493 The digestion efficiency was evaluated and recoveries between 49.5-98.0% were obtained, with
494 a significant decrease in recoveries observed when the pressure reduction step was omitted.
495 The improved method allowed a precise determination of B content in silicate samples and can
496 be used for subsequent analyses of B isotopes and associated geochemical studies. This
497 method can be applied at a wide range of digestion temperatures and illustrates the utility of B
498 as a geological tracer(Tan et al., 2023).
499 As observed, the ICP-MS method offers significant advantages for the determination of
500 B as borates, such as the detection of very low levels of B in various types of samples. In
501 addition, this method is rapid and precise, making it suitable for routine laboratory analysis.
502 Furthermore, ICP-MS is a reproducible method, providing an opportunity to reuse samples for
503 further analyses. On the other hand, there are limitations to this method in terms of B analysis.
504 For example, certain interferences may occur in the ICP-MS analysis of B, such as
505 interferences with silver and chloride ions. So, this method is sensitive to the conditions of
506 sample preparation and proper sample digestion is important before analysis.
507 4.4. Thermal Analysis

10
508 Thermal analysis (TGA, DSC) is a method used to characterize the thermal stability of
509 fructoborates and other organic compounds. By measuring the changes in mass or heat during
510 heating or cooling, thermal properties can be evaluated and compared to other compounds.
511 For example, in Claudia Cecilia Wagner's study, the thermal behavior of CaFB samples
512 with the formula Ca(C6H10O6BO)2·3.5H2O was investigated. Thermogravimetry revealed that
513 CaB2O4 was generated as a residue during pyrolysis. Based on these observations, the authors
514 proposed a new and straightforward synthetic procedure for preparing high-purity samples of
515 this calcium borate. The compound was characterized using powder X-ray diffraction and IR
516 spectroscopy, respectively(Wagner & Baran, 2008).
517 A comparative thermal analysis of CaFB (FruiteX B®), fructose, boric acid and calcium
518 carbonate was also conducted by P. Rotaru. Through thermal analysis, it was found that none
519 of the precursor substances (fructose, boric acid or calcium carbonate) were retained in the final
520 product, indicating that CaFB is a distinct chemical entity (Figure A.4).
521 The results showed that fructose has a melting point between 115 and 155°C and
522 undergoes oxidative degradation in multiple stages starting at 155°C. CaFB exhibits similar
523 decomposition processes in the temperature range of 155 to 580°C.
524 Thermogravimetric and thermal analysis of CaFB revealed six decomposition stages
525 with specific temperatures. The suggested general formula for CaFB is Ca[(C6H10O6)2B]2·4H2O.
526 A comparative study of CaFB (FruiteX B®) and a laboratory-prepared sample of CaFB
527 confirmed that both have the same molecular composition, Ca[(C6H10O6)2B]2·4H2O. The
528 thermanalytical results demonstrated that the thermal decomposition processes are similar
529 between the two samples and the mass losses and residues obtained agree with the theoretical
530 values. Comparative analysis of the thermanalytical curves supports the conclusion that FruiteX
531 B® and the laboratory-prepared CaFB sample are compositionally identical(Rotaru et al., 2010).
532 4.5. Nuclear Magnetic Resonance (NMR)
533 NMR is a powerful analytical method that can be applied to a wide range of liquid and
534 solid matrices without altering the sample or producing toxic waste. Although the sensitivity and
535 detection limits of NMR still need improvement, it offers certain advantages over other common
536 analytical tools such as high-pressure liquid chromatography (HPLC), gas chromatography (GC)
537 and mass spectrometry (MS)(Hatzakis, 2019; Marcone et al., 2013). NMR can be used to
538 analyze the molecular structure and dynamics of fructoborates, determining atom connectivity
539 and molecular orientation.
540 John C. Edward focused on the investigation of the plant mineral complex FruiteX-B® -
541 CaFB (FrxB) using NMR spectroscopy in the study. The 13C and 11B NMR methods allow
542 detailed observation of chemical structure information, such as the ratio of di- and mono-esters,
543 the distribution of free B and chelated B in the FrxB complex. The study showed that no
544 differences in the chemical or physical properties of NMR spectra were observed between the
545 liquid and solid states of FrxB samples subjected to thermal stability tests. These findings
546 indicate that the samples were stable within the temperature range of 35 to 70 ºC and during
547 heat exposure ranging from 2 to 18 hours. By utilizing NMR techniques, reliable quantitative
548 spectral correlations can be obtained between FrxB in different matrices of adulterants (Figure
549 A.5) or mixtures with excipients or other functional ingredients(Edwards et al., 2014).
550 Therefore, NMR technique is a powerful method for the identification, quantitative
551 analysis and stability studies of CaFB. Likewise, this technique can be effectively used in borate
552 chemistry research, product standardization and industrial quality control, respectively. NMR
553 can also be used for the detection and possible measurement of CaFB in biological systems
554 and fluids, following further optimization of the technique.
555 Another study to evaluate the therapeutic benefits of the FruiteX-B product for patients
556 with knee osteoarthritis has been reported. NMR characterization of the preparation and the
557 11B NMR spectra obtained in solid and liquid states were compared to those of boric acid and
558 fructose to confirm their quality(Reyes-Izquierdo et al., 2012).

11
559 NMR techniques can be very useful in the analysis of fructoborates, providing detailed
560 information about their composition and B distribution. Also, it can be used to determine the
561 amount of FrxB in ingredient mixtures and can be helpful to ensure consistent dosage levels
562 and compliance with regulated product labeling requirements. Additionally, NMR can be utilized
563 for stability studies and product standardization. However, NMR is still relatively underutilized in
564 the food industry due to its high cost and relatively low sensitivity. In the future, NMR may be
565 employed for the detection and measurement of fructoborates in biological systems and fluids.
566 Therefore, the development of new technologies and methods could lead to broader usage and
567 improved analysis efficiency.
568 4.6. Associated analysis techniques.
569 It is important to employ multiple analysis techniques for the characterization of
570 fructoborates because each technique provides unique and complementary information
571 regarding the properties and structure of the compounds. Furthermore, utilizing multiple analysis
572 techniques can help confirm and validate data obtained through other methods.
573 For example, thermal analysis can provide information about the thermal stability of the
574 compound, while Fourier Transform Infrared Spectroscopy (FTIR) can be used to identify
575 functional groups in the compound. Raman spectroscopy can offer insights into the molecular
576 structure of the compound, and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) can
577 be utilized to determine the quantities of elements and impurities in the compound. By
578 employing multiple analysis techniques, we can obtain a more detailed and accurate picture of
579 the properties and structure of fructoborates, which can be highly useful in the development and
580 optimization of the utilization of these compounds in various applications, including
581 chemotherapy for diseases.
582 One such detailed study, in which multiple analysis techniques were used, was reported
583 by P. Rotaru(Rotaru et al., 2010) for a commercial dietary supplement containing calcium
584 fructoborate used as an adjuvant in chemotherapy. Analytical techniques such as thermal
585 analysis, X-ray diffraction (XRD), FTIR spectroscopy, Raman spectroscopy, and ICP were
586 applied for the most detailed characterization of the product. The results obtained from
587 measurements in air showed that the commercial sample is indeed a hydrated calcium
588 fructoborate. Thermogravimetric analysis was correlated with elemental analysis, and the
589 correct molecular formula of FruiteX B® was identified as Ca[(C6H10O6)2B]2·4H2O, which
590 contains twice as much boron as reported in other research, and no traces of precursors were
591 found. Starting from 152°C, decomposition occurs in six stages, the first two being weakly
592 endothermic and exothermic, respectively, and the next four being exothermic. The heat
593 released from the last four stages was calculated, and it is attributed to the decomposition of
594 fructose from the complex with a mass loss of 77.8 ± 1% (Rotaru et al., 2010).
595 Vibrational spectroscopy is a method used to analyze metal-organic compounds,
596 particularly through FTIR analysis and Raman spectroscopy. In addition to thermal analysis and
597 XRD analysis, this study conducted an FTIR analysis to compare the laboratory-prepared
598 calcium fructoborate with FruiteX B® and performed a Raman analysis to compare calcium
599 fructoborate with fructose. The FTIR and Raman spectra were compared to those of other
600 similar compounds, and characteristic functional groups of the studied compounds were
601 identified, including B-O bonds and groups such as CH2OH, OH, COH, CH2, C-CH, C-C, and C-
602 O-C, as well as the water of crystallization of metal complexes of carbohydrates. The FTIR
603 spectrum of FruiteX B® and the Raman spectrum of calcium fructoborate are similar to those of
604 other compounds studied within the same frequency range. The XRD result indicated weak
605 crystallinity of calcium fructoborate, and no evidence of fructose, boric acid, or calcium
606 carbonate was found. Thus, FruiteX B® was identified as a naturally occurring calcium
607 fructoborate found in products (Rotaru et al., 2010).
608
609 Conclusions and research perspectives

12
610 Fructoborates, compounds with nutritional significance, have unique properties and
611 health benefits. CaFB has been extensively studied to understand its biological actions.
612 Research indicates that CaFB has multiple positive effects on bone health, glucose metabolism,
613 the immune system, and cognitive function. It also shows potential in treating conditions like
614 arthritis, osteoporosis, and diabetes.
615 To accurately evaluate fructoborates' content and properties, advanced analysis
616 methods have been developed. These include thermal analysis, spectroscopy, atomic
617 absorption analysis, and other techniques. These methods enable the determination of
618 fructoborate composition, structure, and properties, providing essential information for research
619 and the development of nutraceutical products.
620 Improving fructoborate analysis methods offers several benefits. Firstly, these methods
621 ensure the correct identification and characterization of compounds, maintaining consistency
622 and quality of market products. Secondly, enhanced analysis methods provide information
623 about fructoborate stability under varying conditions, aiding the development of products with an
624 efficient and stable release of bioactive compounds in the body. Furthermore, advanced
625 analysis contributes to understanding fructoborates' absorption, distribution, metabolism, and
626 elimination mechanisms. This knowledge is crucial for evaluating bioavailability, optimizing
627 dosages, and administration regimens.
628 In conclusion, continued research and improved analysis methods are essential in
629 exploring the increasing use and importance of fructoborates in the field of health. These
630 advanced methods ensure the quality, efficacy, and safety of fructoborate-based products while
631 facilitating a deeper understanding of their mechanisms of action.
632 Future research directions in the field are diverse and promising. Detailed investigation
633 of bioavailability and absorption in the body is necessary. Understanding their absorption,
634 metabolism, and distribution in various tissues and organs is crucial. Elucidating the molecular
635 mechanisms through which fructoborates exert their biological effects and health benefits is
636 another important aspect. This involves studying their interactions with cellular signaling
637 pathways and enzymes involved in physiological processes. Rigorous long-term clinical studies
638 are required to validate the effectiveness and safety of fructoborates, assessing their impact on
639 human health, including effects on the skeletal and cardiovascular systems.
640 Developing improved administration methods and formulations is also a significant
641 research perspective. Exploring optimal delivery methods, such as controlled-release systems,
642 and developing stable formulations with increased bioavailability are important goals.
643 Addressing the long-term safety of fructoborate use is a crucial consideration for future
644 studies. Evaluating long-term effects and identifying potential side effects are necessary for
645 ensuring safe and sustainable use of these compounds. Lastly, comparing different types of
646 fructoborates available on the market is an important research perspective. This involves
647 studying their composition, bioavailability, efficacy, and safety.
648 By addressing these research perspectives, we can gain a deeper understanding of
649 fructoborates and their potential in the field of health. This paves the way for the development of
650 more efficient and personalized therapies and interventions.
651
652 Funding
653 This research did not receive any specific grant from funding agencies in the public,
654 commercial, or not-for-profit sectors.
655 Declaration of Competing Interest
656 The authors declare that they have no known competing financial interests or personal
657 relationships that could have appeared to influence the work reported in this paper.
658 Acknowledgments
659 The authors thanks the Dunarea de Jos University for the support provided.
660 Appendix of figures (A)

13
661 Appendix of tables (B)
662
663
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19
Cover Letter

Dear Editor,

I am writing to you regarding a potential publication in the prestigious journal Food Chemistry of
a review article titled "Advances in characterization and analysis methods of fructoborates- A
review." We would like to inform you that this article is designed to provide researchers with new
perspectives and ideas concerning the detection and quantification methods of fructoborates,
especially in pharmaceuticals and dietary supplements.

Before proceeding, we would like to emphasize that research in the field of fructoborates is not
very recent; however, despite this fact, we believe that there is a significant need to consolidate
and, more importantly, expand the existing knowledge. Our goal is to synthesize the information
and provide an overview of the progress made in the structural and biological characterization of
fructoborates, as well as in the development of analytical methods used for their analysis.

The structuring of this review has been inspired by the observation that there is currently no
article that synthesizes the main categories of fructoborate analysis methods and the reported
results, despite their growing importance in the medical and pharmaceutical fields. Through this
article, we aim to make a significant contribution in this field by providing researchers and
healthcare professionals with a valuable source of up-to-date and relevant information.

Additionally, we are aware of the need to evaluate and compare the currently available analysis
methods, as well as to identify their gaps and associated challenges. We would like to
emphasize that our article will also include discussions on the advantages and disadvantages of
each method, as well as prospects for development and innovation in this field.

Taking these into consideration, we strongly believe that our article is of significant interest to
the readers of your journal and will contribute to the advancement of current knowledge in the
field of fructoborates and associated analytical methods. Furthermore, we are open and
prepared to collaborate with colleagues in the field to improve and expand this research in the
future.

Thank you for your time and consideration of this article. We look forward to your feedback and
the possibility of contributing to your prestigious journal.

Sincerely,

The Authors,

Butan Simona1
Filimon Veronica2
Bounegru Alexandra Virginia1*

1 “Dunărea de Jos” University of Galaţi, Department of Chemistry, Physics and Environment, Faculty of
Sciences and Environment, 47 Domnească Street, 800008 Galaţi, Romania
2 “Dunarea de Jos” University of Galati, Cross-Border Faculty, 47 Domneasca Street, 800008, Galati,

Romania
Corresponding author: alexandra.meresescu@ugal.ro
Highlights (for review)

Highlights:

 Comprehensive synthesis of existing research on the structural and biological

characteristics of fructoborates.
 Detailed presentation of current analytical methods used for the detection and
quantification of fructoborates.
 Identification of the advantages and limitations of each method, providing a solid
foundation for decision-making in research and industry.
 Exploration of new perspectives and directions in the development of
fructoborate analysis techniques.
 Contribution to a deeper understanding of the importance of fructoborates in the
medical and dietary supplement fields.
Figure(s) Click here to access/download;Figure(s);Appendix of
Figures.docx

Appendix of Figures

Figure A.1. Chemical structures of calcium α-D-fructofuranose borate (a) and calcium β-D-
fructofuranose borate (b)(Scorei & Rotaru, 2011)

Figure A.2 Chemical reactions to obtain CF from β-D-fructofuranose (Scorei & Rotaru,
2011)
 Figure A. 3. MS/MS Spectrum of calcium fructoborate in raisin extract (top) and 30%
NCE (normalized fragmentation energy) calcium fructoborate solution (bottom) (Xia et al.,
2017).

Figure A. 4 Thermoanalytical curves of FruiteX B®, in air flow (Rotaru et al., 2010).
Figure A. 5. Liquid-state 11B NMR spectra and calibration of maltodextrin mixed with 0, 5, 10,
20, 50, 80, and 100 wt% FrxB (Edwards et al., 2014)
Table (Editable version)

Appendix of Tables

Table B.1 Determination and quantification of fructoborates based on analytical

methods.
Type of Amount
Sample Analitical method Ref.
fructoborates found
16.2±0.6
Liquid Chromatography
Foods (apricots, mg/g 13
Coupled with Q Exactive
raisins) 79.3±8.8
Orbitrap Mass Spectrometry
mg/g
Dietary supplements
14
(tablets, powders, Multinuclear NMR -
CaFB liquids)
Dietary supplements
A tablets 106.5 mg
B capsules 223 mg 15
HPTLC- densimetric analysis
C capsules 110 mg
D capsules 55 mg
Fruitex-B® bulk 96.8 %
Declaration of Interest Statement

Declaration of interests

☒The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: