Fe(III)-Rhamnoxylan—A Novel High Spin Fe(III) Octahedral Complex Having Versatile Physical and Biological Properties

An iron (III) complex with rhamnoxylan, a hemicellulose from Salvia plebeia seeds, was synthesized and characterized by elemental analysis, spectroscopic and magnetic susceptibility measurements, thermal analysis and scanning electron microscopy. The rhamnoxylan was found to be a branched hemicellulose consisting of β-1,4-linked xylose main chain and rhamnose attached to the chain at β-1,3 positions. The complex was found to contain 18.8% w/w iron. A high-spin octahedral geometry of Fe3+ was indicated by the electronic absorption spectrum of the complex. In other experiments, the complex exhibited good electrical and magnetic properties. In vivo efficacy, as hematinic, of the complex in induced anemia was demonstrated equivalent to that of iron protein succinylate (taken as standard) as evidenced by raised red blood cell count, hemoglobin, hematocrit and total iron in rabbit. The complex was found to be non-toxic with LD50 > 5000 mg kg−1 body weight in rabbit. Thus, iron(III)-rhamnoxylan hold the potential for application as hematinic for treatment of iron deficiency anemia.


Introduction
The iron deficiency anemia is of concern in developed countries and a serious problem in underdeveloped countries. It is caused by low iron diet in developed countries and scarcity of nutritional diet in underdeveloped countries [1]. Iron deficiency causes a drop in hemoglobin level in blood. Typical hemoglobin levels in humans are >13 g dL −1 (males) and >12 g dL −1 (females); these levels may drop to 3 g dL −1 or lower in case of extensive blood loss due to hemorrhage, menstrual flow and bleeding ulcer [2]. In order to treat such conditions iron compounds are administered as therapeutics or included in dietary supplements. The iron salts currently used as therapeutics include ferrous sulphate, ferric chloride, ferric ammonium citrate, ferrous fumarate and complex compounds like ferrous gluconate, iron dextran, iron sorbitex, ferric carboxymaltose, ferric hydroxide polymaltose complex and iron protein succinylate (IPS) [3]. Among these, polymeric iron complexes are considered more useful as simple iron salts, when administered orally, interact with food and other medications resulting in reduced bioavailability [4]. On the other hand, the polymeric complexes are relatively stable and release iron in a sustained manner through ligand exchange processes in the body. Therefore, the complexes are considered beneficial for oral administration. Large molecule polysaccharide metal complexes exhibit higher safety as compared with traditional small molecule metal complexes. Therefore, polysaccharide metal complexes from single polysaccharides can be used as promising novel drugs.
Fe 3+ forms both low-spin and high-spin octahedral complexes with different ligands. High-spin complexes are formed with weak-field ligands such Cl − or OH − ions. Low-spin and high-spin complexes play important role in spin-cross-over reactions in the biological

Synthesis of Fe(III)-Rhamnoxylan
A dark brown complex was obtained by the experimental procedure as discussed in the methods section. The role of NaOH is to facilitate dissolution of rhamnoxylan during complex formation. In order to remove Fe(OH) 3 , that may have formed due to a reaction of FeCl 3 with NaOH, the complex was thoroughly washed with dilute HCl. Further washings with methanol and ether afforded a significantly dry product, which was further dried at 60 • C in an oven to a constant weight and ground to obtain a free-flowing powder in good yield (~90%). The complex was sparingly soluble (approx. 1 g in 100 mL) in dimethyl sulfoxide (DMSO) and dimethylformamide (DMF), and insoluble in other organic solvents and water.

Elemental Analysis
Elemental analysis of the rhamnoxylan sample and the synthesized Fe(III)-rhamnoxylan complex (Table 1) shows the presence of C and H. The percentages in the complex of these elements (C: 31.1%; H: 5.15%) were lower than those of rhamnoxylan (C: 46.73%; H: 6.45%) as expected due to the presence of iron (18.40%) in the complex. However, the C:H molar ratio was similar in the two materials, which confirmed that iron is bonded to the rhamnoxylan. The AgNO 3 gave negative test for Cl − ions. The water content in the complex (11.0%) was higher than that in the rhamnoxylan used (5.98%) indicating the hygroscopic nature of the complex. The monosaccharide analysis in Table 1 clearly indicates the presence of Xylp and Rhap in the complex; their percentages are lower than those in pure rhamnoxylan due to the presence of iron as part of the complex. However, the Xylp:Rhap ratio in the complex is similar to that in the rhamnoxylan. This effect is also noticeable in the relatively higher molar mass of the complex (2.52 × 10 6 D) compared to that of the rhamnoxylan (2.47 × 10 6 D) as determined by GPC (Table 1).

FT-IR and Raman Analysis
The FT-IR spectra of rhamnoxylan and the Fe(III)-rhamnoxylan complex are shown in Figure 1. All the absorption bands of the sugar moieties in rhamnoxylan were present in the spectrum of the iron complex. The characteristic bands due to ν(OH) at 3337-3371 cm −1 , ν(C-C) in rhamnonosyl side chain at~1059 cm −1 and β-glycosidic C-H bending at~910 cm −1 were observed in both the samples. A broad absorption at 3371 cm −1 in the spectrum of the complex appears to be shifted to a higher energy from 3337 cm −1 due to ν(O-H) [11] in the pure rhamnoxylan [12,13]. This clearly indicates formation of a coordinate covalent bond between the hydroxyl groups and iron. Slight shift of glycosidic linkage, C-O-C was observed at 1034 cm −1 [13][14][15][16]. A Fe-O absorptions below 450 cm −1 that falls in the far IR region is generally indicative of formation of new Fe-O bond; this could not be recorded due to instrumental limitation. For that Raman spectrum of the complex was compared with that of the rhamnoxylan shown in Figure 1b and ferric chloride reported in literature [17]. Prominent Raman shifts in the region 300-420 cm −1 were observed in the spectrum of the complex that confirmed the existence of Fe-O bond in the complex and the absorption at 255-258 cm −1 assigned to an A 1g (Fe-Cl) mode [17] was absent in our spectrum indicating that the complex was free from FeCl 3 . The AgNO 3 test also confirmed the absence of Cl − ion in the complex. groscopic nature of the complex.

Monosaccharide Analysis
The monosaccharide analysis in Table 1 clearly indicates the presence of Xylp and Rhap in the complex; their percentages are lower than those in pure rhamnoxylan due to the presence of iron as part of the complex. However, the Xylp:Rhap ratio in the complex is similar to that in the rhamnoxylan. This effect is also noticeable in the relatively higher molar mass of the complex (2.52 × 10 6 D) compared to that of the rhamnoxylan (2.47 × 10 6 D) as determined by GPC (Table 1).

FT-IR and Raman Analysis
The FT-IR spectra of rhamnoxylan and the Fe(III)-rhamnoxylan complex are shown in Figure 1. All the absorption bands of the sugar moieties in rhamnoxylan were present in the spectrum of the iron complex. The characteristic bands due to ν(OH) at 3337-3371 cm −1 , ν(C-C) in rhamnonosyl side chain at ~1059 cm −1 and β-glycosidic C-H bending at ~910 cm −1 were observed in both the samples. A broad absorption at 3371 cm −1 in the spectrum of the complex appears to be shifted to a higher energy from 3337 cm −1 due to ν(O-H) [11] in the pure rhamnoxylan [12,13]. This clearly indicates formation of a coordinate covalent bond between the hydroxyl groups and iron. Slight shift of glycosidic linkage, C-O-C was observed at 1034 cm −1 [13][14][15][16]. A Fe-O absorptions below 450 cm −1 that falls in the far IR region is generally indicative of formation of new Fe-O bond; this could not be recorded due to instrumental limitation. For that Raman spectrum of the complex was compared with that of the rhamnoxylan shown in Figure 1b and ferric chloride reported in literature [17]. Prominent Raman shifts in the region 300-420 cm −1 were observed in the spectrum of the complex that confirmed the existence of Fe-O bond in the complex and the absorption at 255-258 cm −1 assigned to an A1g(Fe-Cl) mode [17] was absent in our spectrum indicating that the complex was free from FeCl3. The AgNO3 test also confirmed the absence of Clˉ ion in the complex.

Structural Analysis of Rhamnoxylan by MALDI-ToF Mass Spectroscopy
The MALDI-ToF mass spectrum of the water soluble hemicellulose is shown in Figure 2. The assignments of fragment ions were done by mass interval analysis. The masses of resid- were calculated as (132) n + 23, (132) n + 23 + 18 Da, (146) n + 23 Da, (146) n + 23 + 18 Da and (146) n + (132) n + 23 Da, where n is the number of monosaccharide units and 18 Da the mass of reducing end consisting of two hydrogen and one oxygen atoms. The mass difference between the daughter and the parent ions was used to identify the glycoside position in the monosaccharides. Such fragmentation patterns have been categorized into various types by Domon and Costello [18,19].   High intensity peaks were observed in the 600-900 m/z range. The intense peak at m/z 703 was assigned to a [5 Xyl + Na] + adduct, where Xyl is the xylose unit. The neighboring peaks at 643 and 626 m/z with a mass difference of 60 and 78 Da indicate β-1,4 linkages of xylosyl residues in the xylan backbone due to loss of C 2 H 4 O 2 and C 2 H 4 O 2 + 18 (crossring A/X-type cleavage). A peak at m/z~850 represents a [Xyl-Xyl-Xyl-Xyl-Xyl-Rh + Na] + residue, where Rh is the rhamnose unit. Two low intensity signals at 834 and 730 m/z having mass differences of 16 and 120 Da, respectively, suggest that rhamnose is also linked to xylose through β-1,4-linkage. Relatively higher intensities of these signals indicate that these groups are present as branches at the xylan chain. This is because bulky groups are more vulnerable to fragmentation. A low intensity peak at m/z 661 was assigned to [Xyl-Xyl-Xyl-Xyl-Xyl + H] + xylosyl residues. The lower intensity signal of the fragment is understandable because the carbohydrates are not easily protonated [20]. Very low intensity peaks were observed in the 900-3500 m/z range. There is a series of weak signals spaced by 132 Da; around m/z 1028, 1157, 1290, 1554 (dimer) and 1818.237 (dimer). Above m/z 1800, the rhamnosyl residues produced peaks frequently.
Some lower intensity peaks at m/z 1906, m/z 1894 and m/z 1866, having mass difference of 78, 90 and 118 Da from m/z 1984, due to cross-ring fragmentation (A/X-type) suggested that xylosyl and rhamnosyl residues are also linked through β-1,3 positions along with β-1,4. The lower intensities may be indicative of a rigid main chain difficult to undergo fragmentation. In the m/z 8000-13,000 range bunches of peaks were observed. These bunches most probably consist of a series of signals separated by 132 and 146 Da corresponding to xylosyl and rhamnosyl residues, respectively. Several signals with a difference of 16 and 17 Da were observed due to loss of O and OH, which are abundantly present in the polysaccharide structure. There was no evidence of the presence of uronic acids because of the absence of peaks having a mass difference of 178 Da.
This analysis shows that the main chain of polysaccharide consists of β-1, 4-D-xylose linkages branched with β-1, 3-linked rhamnose units. Thus, a block of the polymeric structure of rhamnoxylan can be proposed as shown in Figure 3. According to this, a mole of rhamnoxylan (2.47 × 10 6 Da) would consist of around 850 such blocks and 61,000 monosaccharide units. This analysis shows that the main chain of polysaccharide consists of β-1, 4-D-xylose linkages branched with β-1, 3-linked rhamnose units. Thus, a block of the polymeric structure of rhamnoxylan can be proposed as shown in Figure 3. According to this, a mole of rhamnoxylan (2.47 × 10 6 Da) would consist of around 850 such blocks and 61,000 monosaccharide units.

Electronic Absorption Spectrophotometry
Electronic absorption spectra of iron complexes in the UV-Visible range provide useful information about the oxidation state of the metal ion, chromophores in the ligand, charge transfer transitions and geometry of the complex ion. High-spin Fe 3+ complexes contain five unpaired electrons in the d orbital, so all d-d transitions are spin forbidden, hence, should be weak. Most of the Fe 3+ complexes exhibit weak d-d absorption bands, while some have been reported to show quite intense absorptions. The spectrum of the Fe(III)-rhamnoxylan was characteristic of a complex formation and entirely different from that of FeCl 3 ·6H 2 O shown in Figure 4. The spectrum of FeCl 3 ·6H 2 O consisted of a single absorption at about 300 nm [21] whereas, that of the complex exhibited three absorptions in the visible region centered at 400 (very broad), 570 (broad), 645 and 690 nm (sharp). This pattern resembles those of six-coordinate Fe 3+ complexes. The absence of any absorption in the regions 420-450 nm and 800-900 nm rules out the presence of Fe 2+ ion in the complex [22]. The assignment of the observed absorptions is straightforward as for the octahedral complexes [23][24][25][26] and can be assigned as: ~400-6 A1g  ( 4 A1g, 4 Eg); ~570 nm-6 A1g  4 T2g; ~645 nm (charge transfer, O-Fe LMCT); ~690 nm-6 A1g  4 T1g. The sharpness of low-energy bands in the spectrum of the complex may be attributed to combination vibrational bands of the ligand.

Mössbauer Specroscopy
The Mössbauer spectrum recorded at room temperature is shown in Figure 5. The spectrum was fitted to a single doublet to compare it with a reported iron polysaccharide complex [27]. The ferrous doublet was absent that confirmed the absence of Fe 2+ ion in the sample. The isomeric shift and quadrupole splitting were found to be 0.35 and 0.71 mm s −1 , respectively. These values correspond to ferrihydrite iron core [28].
Based on the analytical (elemental and monosaccharide analyses) and spectroscopic (FT-IR/Raman and electronic absorption) evidence presented so far, the coordination sphere of Fe 3+ ion in the complex appears to have an octahedral geometry, where the macromolecule acts as an anionic ligand. A structural block of the Fe(III)-rhamnoxylan complex can be shown as in Figure 6. Accordingly, a mole of the complex (2.52 × 10 6 Da) would consist of around 578 such blocks. This indicates that the degree of polymerization has been reduced to ~68% on complex formation. The salient features of the proposed The absence of any absorption in the regions 420-450 nm and 800-900 nm rules out the presence of Fe 2+ ion in the complex [22]. The assignment of the observed absorptions is straightforward as for the octahedral complexes [23][24][25][26] and can be assigned as: 400-6 A 1g → ( 4 A 1g , 4 E g );~570 nm-6 A 1g → 4 T 2g ;~645 nm (charge transfer, O-Fe LMCT); 690 nm-6 A 1g → 4 T 1g . The sharpness of low-energy bands in the spectrum of the complex may be attributed to combination vibrational bands of the ligand.

Mössbauer Specroscopy
The Mössbauer spectrum recorded at room temperature is shown in Figure 5. The spectrum was fitted to a single doublet to compare it with a reported iron polysaccharide complex [27]. The ferrous doublet was absent that confirmed the absence of Fe 2+ ion in the sample. The isomeric shift and quadrupole splitting were found to be 0.35 and 0.71 mm s −1 , respectively. These values correspond to ferrihydrite iron core [28].
Polymers 2022, 14, x FOR PEER REVIEW 7 of structure are: (i) the rhamnoxylan acts as an anionic ligand under highly alkaline cond tion, (ii) coordinated water (1-3 molecules per iron atom) is present, (iii) varying numb (1-2) of free OHˉ ions in the alkaline medium are also found in the coordination spher The structure represents a polynuclear ferrihydrite iron core similar to the Enteromorph polysaccharide-iron (III) complex [29]. The Fe(III)-rhamnoxylan complex pre-dominant appeared to be hydrophobic (lipophilic) as shown by the solubility data. . Based on the analytical (elemental and monosaccharide analyses) and spectroscopic (FT-IR/Raman and electronic absorption) evidence presented so far, the coordination sphere of Fe 3+ ion in the complex appears to have an octahedral geometry, where the macromolecule acts as an anionic ligand. A structural block of the Fe(III)-rhamnoxylan complex can be shown as in Figure 6. Accordingly, a mole of the complex (2.52 × 10 6 Da) would consist of around 578 such blocks. This indicates that the degree of polymerization has been reduced to~68% on complex formation. The salient features of the proposed structure are: (i) the rhamnoxylan acts as an anionic ligand under highly alkaline condition, (ii) coordinated water (1-3 molecules per iron atom) is present, (iii) varying number (1-2) of free OH ions in the alkaline medium are also found in the coordination sphere. The structure represents a polynuclear ferrihydrite iron core similar to the Enteromorpha polysaccharide-iron (III) complex [29]. The Fe(III)-rhamnoxylan complex pre-dominantly appeared to be hydrophobic (lipophilic) as shown by the solubility data.

Powder X-Ray Diffraction Analysis
The p-XRD spectra of rhamnoxylan and the Fe(III)-rhamnoxylan complex are shown in Figure 7. Rhamnoxylan appeared to be mainly in amorphous state, whereas the complex exhibited four peaks at 2θ values 33°, 45°, 57° and 75° indicating a crystalline nature of the complex [30][31][32][33][34] and favoring octahedral geometry. The sharp peak around 33 °Corresponds to that of iron oxide nanoparticle [31,32,35], whereas at 45° is related to the iron crystal [32]. The appearance of peaks represents different crystal sizes ranging from nanoto micro-meter [23]. The significant difference in the spectra suggests a phase change due to complex formation. These peaks represent different crystal sizes ranging from nano-to micro-meter scale. Attempts to grow a single crystal for further structure elucidation were not successful.

Powder X-ray Diffraction Analysis
The p-XRD spectra of rhamnoxylan and the Fe(III)-rhamnoxylan complex are shown in Figure 7. Rhamnoxylan appeared to be mainly in amorphous state, whereas the complex exhibited four peaks at 2θ values 33 • , 45 • , 57 • and 75 • indicating a crystalline nature of the complex [30][31][32][33][34] and favoring octahedral geometry. The sharp peak around 33 • corresponds to that of iron oxide nanoparticle [31,32,35], whereas at 45 • is related to the iron crystal [32]. The appearance of peaks represents different crystal sizes ranging from nano-to micro-meter [23]. The significant difference in the spectra suggests a phase change due to complex formation. These peaks represent different crystal sizes ranging from nano-to micro-meter scale. Attempts to grow a single crystal for further structure elucidation were not successful.

Magnetic Properties
The magnetic moment was found to be 5.9 B.M. that corresponds to the spin only value of 5.92 B.M for Fe(III) complexes. This result confirms the high spin configuration of Fe(III) ion in the complex. Thus, the complex exhibits paramagnetism [33,35,36]; the property that may be exploited in biomedical applications including cell separation, immunoassay, magnetic resonance imaging and drug delivery.

Scanning Electron Microscopy
The SEM images of rhamnoxylan and the Fe(III)-rhamnoxylan complex are shown in Figure 8. A comparison of the two micrographs clearly shows the formation of a new material with change in the morphology after complexation.

Thermal Analysis
TGA was used to determine thermal stability of the complex and to further verify the presence of organic component in it. Thermograms of rhamnoxylan and the Fe(III)-rhamnoxylan are shown in Figure 9. A three-stage decomposition of the complex was observed. The stage I at ambient to ~120 °C corresponds to a loss of absorbed moisture (~11.3%) in the complex; whereas in case of rhamnoxylan this loss was ~5.8% due to absorbed moisture at ~100 °C. It was noted that decomposition of the complex starts at a higher temperature (~271 °C) compared to that of the rhamnoxylan (~225 °C); this suggests that the complex is thermally more stable than the rhamnoxylan. Two major losses occurred at stages II and III, which account for the decomposition of the polymeric ligand. A relatively lower weight loss (~20%) at stage II probably represents side chain decomposition compared

Magnetic Properties
The magnetic moment was found to be 5.9 B.M. that corresponds to the spin only value of 5.92 B.M for Fe(III) complexes. This result confirms the high spin configuration of Fe(III) ion in the complex. Thus, the complex exhibits paramagnetism [33,35,36]; the property that may be exploited in biomedical applications including cell separation, immunoassay, magnetic resonance imaging and drug delivery.

Scanning Electron Microscopy
The SEM images of rhamnoxylan and the Fe(III)-rhamnoxylan complex are shown in Figure 8. A comparison of the two micrographs clearly shows the formation of a new material with change in the morphology after complexation.

Magnetic Properties
The magnetic moment was found to be 5.9 B.M. that corresponds to the spin onl value of 5.92 B.M for Fe(III) complexes. This result confirms the high spin configuratio of Fe(III) ion in the complex. Thus, the complex exhibits paramagnetism [33,35,36]; th property that may be exploited in biomedical applications including cell separation, im munoassay, magnetic resonance imaging and drug delivery.

Scanning Electron Microscopy
The SEM images of rhamnoxylan and the Fe(III)-rhamnoxylan complex are shown i Figure 8. A comparison of the two micrographs clearly shows the formation of a new ma terial with change in the morphology after complexation.

Thermal Analysis
TGA was used to determine thermal stability of the complex and to further verify th presence of organic component in it. Thermograms of rhamnoxylan and the Fe(III)-rham noxylan are shown in Figure 9. A three-stage decomposition of the complex was observed The stage I at ambient to ~120 °C corresponds to a loss of absorbed moisture (~11.3%) i the complex; whereas in case of rhamnoxylan this loss was ~5.8% due to absorbed mois ture at ~100 °C. It was noted that decomposition of the complex starts at a higher tempe ature (~271 °C) compared to that of the rhamnoxylan (~225 °C); this suggests that the com plex is thermally more stable than the rhamnoxylan. Two major losses occurred at stage II and III, which account for the decomposition of the polymeric ligand. A relatively lowe weight loss (~20%) at stage II probably represents side chain decomposition compare

Thermal Analysis
TGA was used to determine thermal stability of the complex and to further verify the presence of organic component in it. Thermograms of rhamnoxylan and the Fe(III)rhamnoxylan are shown in Figure 9. A three-stage decomposition of the complex was observed. The stage I at ambient to~120 • C corresponds to a loss of absorbed moisture (~11.3%) in the complex; whereas in case of rhamnoxylan this loss was~5.8% due to absorbed moisture at~100 • C. It was noted that decomposition of the complex starts at a higher temperature (~271 • C) compared to that of the rhamnoxylan (~225 • C); this suggests that the complex is thermally more stable than the rhamnoxylan. Two major losses occurred at stages II and III, which account for the decomposition of the polymeric ligand. A relatively lower weight loss (~20%) at stage II probably represents side chain with a major loss (~40%) at stage III due to decomposition of the main polymer chain. The residue of the complex was ~26% that corresponds to Fe2O3.

Electrical Properties
The impedance spectra of the samples are shown in Figure 10. The resistance and ionic conductivity data at different temperatures are presented in Table 2. It can be seen that resistance decreases and conductivity increases with a rise in temperature in accordance with the kinetic theory. These data show that the complex can be used as a solid polymer electrolyte.

Electrical Properties
The impedance spectra of the samples are shown in Figure 10. The resistance and ionic conductivity data at different temperatures are presented in Table 2. It can be seen that resistance decreases and conductivity increases with a rise in temperature in accordance with the kinetic theory. These data show that the complex can be used as a solid polymer electrolyte.
Polymers 2022, 14, x FOR PEER REVIEW 9 of 1 with a major loss (~40%) at stage III due to decomposition of the main polymer chain. Th residue of the complex was ~26% that corresponds to Fe2O3.

Electrical Properties
The impedance spectra of the samples are shown in Figure 10. The resistance and ionic conductivity data at different temperatures are presented in Table 2. It can be seen that resistance decreases and conductivity increases with a rise in temperature in accord ance with the kinetic theory. These data show that the complex can be used as a solid polymer electrolyte.

Stability Study
The results of the six-month accelerated stability study are depicted in Figure 11. It was observed that the Fe 3+ content remained unchanged throughout the study period and there was no Fe 2+ impurity detectable in the product. This is understandable because Fe 2+ , the only expected impurity, may be produced under reducing environment and cannot creep up in the presence of air and humidity. Moreover, no significant change in the FT-IR and electronic absorption spectra was observed during this period. So, based on this study and TGA data, it can be concluded that the product is highly stable.

Stability Study
The results of the six-month accelerated stability study are depicted in Figure 11. was observed that the Fe 3+ content remained unchanged throughout the study period an there was no Fe 2+ impurity detectable in the product. This is understandable because Fe the only expected impurity, may be produced under reducing environment and cann creep up in the presence of air and humidity. Moreover, no significant change in the F IR and electronic absorption spectra was observed during this period. So, based on th study and TGA data, it can be concluded that the product is highly stable. Figure 11. Fe 3+ content in the Fe(III)-rhamnoxylan complex over a period of six months stored at ± 1 °C and 75% relative humidity.

Acute Oral Toxicity
According to OECD guidelines, 24 h after administration of the test dose (5000 m kg −1 body weight) none of the animals was found to be dead and the animals showed n significant change in eye color, hair fall and weight for next 48 h. During that time, the remained active with normal heart and breathing rate and no irritation. The eye reflex were also positive as assessed by a standard method [37]. No redness or ulceration w found in the stomach walls, indicating a good tolerability needed for long term treatmen The isolated stomach, spleen, liver, intestine, heart and kidneys were found to be norm as examined microscopically. The results of this test suggested that the synthesized Fe(III rhamnoxylan complex is non-toxic and may be categorized as 'unclassified' according GHS categorization [38].

In Vivo Absorption and Excretion of Iron
After oral administration of 5000 mg kg −1 body weight of Fe(III)-rhamnoxylan in th acute toxicity test, the stomach material was microscopically found to contain folds of th hemicellulose (Figure 12a) indicating that the complex remains intact in the stomach an reaches intestine slowly releasing iron there, after dissolution of rhamnoxylan at the alk line pH in the duodenum. The complex is predominantly lipophilic and slowly dissolv in alkaline medium. Some amount of iron in feces and significant amount in blood we detected indicating that iron from the complex is released and distributed throughout th body. Stomach was almost clear of the complex after 4 days (Figure 12b). Figure 11. Fe 3+ content in the Fe(III)-rhamnoxylan complex over a period of six months stored at 50 ± 1 • C and 75% relative humidity.

Acute Oral Toxicity
According to OECD guidelines, 24 h after administration of the test dose (5000 mg kg −1 body weight) none of the animals was found to be dead and the animals showed no significant change in eye color, hair fall and weight for next 48 h. During that time, they remained active with normal heart and breathing rate and no irritation. The eye reflexes were also positive as assessed by a standard method [37]. No redness or ulceration was found in the stomach walls, indicating a good tolerability needed for long term treatment. The isolated stomach, spleen, liver, intestine, heart and kidneys were found to be normal as examined microscopically. The results of this test suggested that the synthesized Fe(III)-rhamnoxylan complex is non-toxic and may be categorized as 'unclassified' according to GHS categorization [38].

In Vivo Absorption and Excretion of Iron
After oral administration of 5000 mg kg −1 body weight of Fe(III)-rhamnoxylan in the acute toxicity test, the stomach material was microscopically found to contain folds of the hemicellulose (Figure 12a) indicating that the complex remains intact in the stomach and reaches intestine slowly releasing iron there, after dissolution of rhamnoxylan at the alkaline pH in the duodenum. The complex is predominantly lipophilic and slowly dissolves in alkaline medium. Some amount of iron in feces and significant amount in blood were detected indicating that iron from the complex is released and distributed throughout the body. Stomach was almost clear of the complex after 4 days (Figure 12b). As the storage of iron in the body depends upon several factors including that in stomach, spleen, liver, macrophages, absorption from GIT and simultaneous release through bile [39], the bioavailability of iron cannot be accurately determined from the AUC. So, the bioavailability was assessed as the % increase of iron in blood after 14 days (Table 3) and it was estimated to be ~13.29% that was slightly higher than that of the IPS standard (~9.78%). Table 3. Bioavailability data of Fe(III)-rhamnoxylan and IPS.

Therapeutic Evaluation
The Figure 13a shows the % change in Hb level for 14 days experiment. The minimum levels of Fe, Hb, HCT and RBCs count were observed on the 6th day. However, the minimum levels of all these parameters were better in the treatment groups compared to those for the untreated control (Table 3). A comparison of the Hb levels on the day 14 indicates that recoveries were: 89% (Fe(III)-rhamnoxylan), 87% (IPS) and 70% (negative control/anemic), suggesting that the Fe(III)-rhamnoxylan complex is equally or slightly more effective in raising the Hb level than IPS (the standard drug). Similar trends were observed in RBCs (Figure 13b), HCT ( Figure 13c) and blood iron (Figure 13d). Overall, the in vivo study demonstrates better (p < 0.05) efficacy, bioavailability and toxicity profile of Fe(III)-rhamnoxylan, in the animal model under investigation, compared with a commercially available standard (IPS). Fe(III)-rhamnoxylan can be considered as a potential candidate for clinical trials. As the storage of iron in the body depends upon several factors including that in stomach, spleen, liver, macrophages, absorption from GIT and simultaneous release through bile [39], the bioavailability of iron cannot be accurately determined from the AUC. So, the bioavailability was assessed as the % increase of iron in blood after 14 days (Table 3) and it was estimated to be~13.29% that was slightly higher than that of the IPS standard (~9.78%).

Therapeutic Evaluation
The Figure 13a shows the % change in Hb level for 14 days experiment. The minimum levels of Fe, Hb, HCT and RBCs count were observed on the 6th day. However, the minimum levels of all these parameters were better in the treatment groups compared to those for the untreated control (Table 3). A comparison of the Hb levels on the day 14 indicates that recoveries were: 89% (Fe(III)-rhamnoxylan), 87% (IPS) and 70% (negative control/anemic), suggesting that the Fe(III)-rhamnoxylan complex is equally or slightly more effective in raising the Hb level than IPS (the standard drug). Similar trends were observed in RBCs (Figure 13b), HCT ( Figure 13c) and blood iron (Figure 13d). Overall, the in vivo study demonstrates better (p < 0.05) efficacy, bioavailability and toxicity profile of Fe(III)-rhamnoxylan, in the animal model under investigation, compared with a commercially available standard (IPS). Fe(III)-rhamnoxylan can be considered as a potential candidate for clinical trials.

Synthesis of Fe(III)-Rhamnoxylan Complex
Rhamnoxylan (2 g) was suspended in hot water (at 75-80 °C) and allowed to soak for about an hour under constant stirring by a magnetic stirrer. To the stirred suspension, FeCl3•6H2O (5.35 mL of 75% aqueous solution) was added followed by dropwise addition of NaOH (40% aqueous solution) to adjust the pH to 11.5. The mixture was stirred for further 60 min at 80 °C. The brown colored complex formed as precipitate was isolated by filtration under vacuum. The complex was washed with dilute HCl to remove any ferric hydroxide that may have formed followed by several washings with methanol. The com-

Synthesis of Fe(III)-Rhamnoxylan Complex
Rhamnoxylan (2 g) was suspended in hot water (at 75-80 • C) and allowed to soak for about an hour under constant stirring by a magnetic stirrer. To the stirred suspension, FeCl 3 ·6H 2 O (5.35 mL of 75% aqueous solution) was added followed by dropwise addition of NaOH (40% aqueous solution) to adjust the pH to 11.5. The mixture was stirred for further 60 min at 80 • C. The brown colored complex formed as precipitate was isolated by filtration under vacuum. The complex was washed with dilute HCl to remove any ferric hydroxide that may have formed followed by several washings with methanol. The complex was finally washed with ether and dried at 60 • C in an oven and ground into a powder (yield 1.8 g; 90% of the rhamnoxylan used). The complex formation occurred in the pH range 8-11.5 with better yield at 11.5. Similar pH has been used in the synthesis of an iron-saccharide complex [41]. The purity of the complex was ascertained by the single peak appearing in the gel permeation chromatogram.

Characterization
The elemental (carbon and hydrogen) analysis of the synthesized complex was carried by Leco 4201 elemental analyzer (Leco, St Joseph, MI, USA). Iron was determined by atomic absorption spectrophotometery. For this, the complex (100 mg) was digested in aqua regia (15 mL) followed by dissolution of the iron salt in water to make 100 mL of the solution. The solution was filtered and analyzed by flame atomic absorption spectrometer AAS FS240 (Varian, Palo Alto, CA, USA) using iron hollow cathode lamp set at 248.3 nm and an air-acetylene flame. The standards (prepared from 1.0 mg mL −1 stock solution, lot# D2-FE03121 in 2% HNO 3 v/v, from Inorganic Ventures, Christiansburg, VA, USA Cat# AAFE1-5) and samples were analyzed in triplicate. The R 2 value of the six-point calibration curve was 0.9998. The AgNO 3 test was performed to detect Cl − ions if any. Moisture content of the complex was determined by Karl-Fischer titration using Titrino 701KF (Metrohm, Herisau, Switzerland) auto titrator.
Monosaccharide analysis was carried out after hydrolysis of the rhamnoxylan and the iron complex with sulfuric acid and electro chemical detector according to a reported method [42]. Rhamnose and xylose (Sigma Aldrich, St. Louis, MO, USA) were used as standards. The chromatographic conditions were: isocratic elution using 95% water and 5% 0.2 M NaOH at room temperature (25 ± 1 • C); flow rate 0.5 mL min −1 ; injection volume 50 µL.
The FT-IR spectra were recorded at 2 cm −1 resolution, in transmission mode, using a Carry 630 FTIR spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The powdered sample was placed on the diamond crystal and the spectrum was recorded in the 4000-630 cm −1 range. Raman spectra were recorded by use of a reflex microscope (inVia™ confocal Raman microscope, Renishaw). A near-infrared diode laser was used for excitation at 785 nm. Electronic absorption spectra were recorded in diffuse reflectance mode by UV-1800 spectrophotometer (Shimazdu, Kyoto, Japan) in the 200-800 nm range.
MALDI-ToF mass spectrometric analysis was carried out to determine structural features of the rhamnoxylan used for complex formation. This technique is appropriate as it produces singly charged ions; this has been demonstrated in our recent work [20]. The spectra were recorded in the 500-14,000 m/z range by MALDI-ToF/ToF (Bruker Daltonics Inc, Fremont, CA, USA) spectrometer using single ToF option in the reflectron mode. The desalted sample (1.5 mL) was spotted in triplicate on MTP 384 polished steel BC targets (Bruker Daltonics) and the samples were overlaid with 2,5-DHB (1.5 mL) and dried. The rest of the procedure was same as reported earlier [20].
The Mössbauer spectrum was recorded at room temperature using a 25 mCi 57 Co(Rh) source. The velocity was calibrated by laser interferometry. The powdered sample (~125 mg) was pressed in a brass ring with area of 1.75 cm 2 and sealed with benzenestyrofoam. Aluminum foil was used as the backing. The spectrum was fitted to single doublet assuming a Lorentzian line shape.
Magnetic susceptibility measurements were performed by using MSB MK 1 magnetic susceptibility balance (Sherwood Scientific, Cambridge, UK). The balance was calibrated by using MnCl 2 , standard (QQ153, Sherwood Scientific Ltd., UK). The effective magnetic moment (µ eff ) was calculated by the formula: µ eff (B.M.) = 2.828(χAT) 1/2 , where χA is the atomic susceptibility of the paramagnetic ion (Fe 3+ in this case) corrected for the ligands and T is the absolute temperature.
Surface morphology was studied by scanning electron microscopy (SEM) using JEOL JSM-6480LV microscope after sputter coating with gold (in case of rhamnoxylan) and without sputter coating (in case of Fe(III)-rhamnoxylan as the complex was self-conducting due to the presence of iron in it). For this, samples were fixed on the stub with a doublesided adhesive tape and images were recorded at different magnifications.
Thermal analysis was carried out in TGA mode by SDT Q600 analyzer (TA Company, La Vergne, TN, USA). Samples (approx. 6 mg) were heated in aluminum sample pans from 10 • C to 700 • C at a heating rate of 10 • C min −1 under nitrogen flow (30 mL min −1 ).
To determine electrical properties, sample was cold-pressed into cylindrical pellets (10 mm dia) at 40 MPa. The pellets were painted with silver paste and impedance spectra were recorded in air at 40-200 • C (heating rate: 15 • C) using Gammry Interface 1000 potentiostat. The total resistance was determined by the formula: R t = R g + R gb , where R g and R gb are the resistance of grain interior and grain boundary, respectively. The ionic conductivity (σ) was calculated using the formula: σ = l/RA, where l is the thickness of the sample and A the cross-section area.
A six-month accelerated stability study was performed, according to WHO Q1F Stability Guideline [43] as follows. Three different batches of the complex contained in plastic PP Securitainers ® (VWR International Ltd., Lutterworth, UK) were stored at 50 ± 1 • C and 75% relative humidity and assayed for Fe 3+ according to a validated method [44]. In addition to this the FT-IR and electronic absorption spectra were also recorded before and after six months.

Biological Evaluation
The synthesized complex was subjected to acute toxicity test and therapeutic evaluation in vivo in standard Dutch rabbit. These studies were approved by the institutional review board (vide IRB-124/12-2018) of the Forman Christian College (A chartered University), Lahore, Pakistan. All animal experiments complied with the ARRIVE guidelines in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines (EU Directive 2010/63/EU for animal experiments).

Acute Oral Toxicity Test
This test was performed according to OECD guidelines for determination of oral toxicity [38]. Ten male rabbits (800-1000 g) were fed with fodder and free water adaptively for a week. Five of the animals were orally fed with the synthesized complex at a dose of 5000 mg kg −1 body weight, while the other group of five was kept as control. Both the groups were monitored for 48 h for any physical and behavioral changes including eye color, eye reflexes, hair fall, weight change, heart rate, breathing rate, irritability and activity. Eye reflexes were examined by flashing light into the side of the rabbit's eye. Corneal reflexes were examined by smoothly touching the cornea with a clean cotton swab to check for any induced blink reflex [37].

Therapeutic Evaluation
Twenty male rabbits (6-8 weeks old, weighing 1000 ± 20 g) were purchased from Tollinton Market, Lahore, Pakistan and housed in the animal storage facility at Forman Christian College. They were fed with plain water and a low iron diet, adaptively for seven days. The animals were divided equally into four groups as: A (untreated), B (negative control/anemic), C (positive control/IPS) and D (Fe(III)-rhamnoxylan). Iron-deficiency anemia was induced as reported earlier [45] by oral administration of 11 µg kg −1 per day