Heme-Mimetic Potential of Iron Conjugated Pheophytin-I in Attenuating Oxidative Stress-Induced Cellular and Vascular Toxicity.

Purpose of the Study: Heme is the cardinal porphyrin in systemic physiology, apart from hemoglobin it forms structural skeleton of physiological antioxidants such as catalase and peroxidases. Aim: The current study presents evidence that iron chelated pheophytin (Fe-Ph-I) created in resemblance to heme can exert significant heme-mimetic efficacy in mitigating oxidative stress-induced cellular and vascular damage. Materials and
Methods: Fe-Ph-I was synthesized by incorporating ferrous ion into the porphyrin core of Ph-I moiety. The candidate drugs (Ph-I and Fe-Ph-I) were characterized by spectroscopic analysis and heme-mimetic attribute of Fe-Ph-I was established by comparing the efficacy of Fe-Ph-I with reference to its unmetallated parent Ph-I as well as un-chelated ferrous ions in a host of in vitro, ex vivo, and in vivo bioassays paradigms.
Results: The study confirmed that Fe-Ph-I, Ph-I, and free ferrous ions all exerts significant in vitro anti-radical efficacy, however, while un-chelated ferrous ions intensifies, Ph-I and Fe-Ph-I mitigate ex vivo oxidative stress with Fe-Ph-I exhibiting superior potency. Also from in vivo assessment of oxidative stress-induced hemolytic anemia, it was observed that Fe-Ph-I is significantly superior than Ph-I in alleviating intravascular hemolysis, thereby endorsing that not ferrous ions alone but ferrous ion chelated with porphyrin yielding a heme-mimetic structure is responsible for superior potency of Fe-Ph-I over Ph-I.
Conclusion: In conclusion, Fe-Ph-I is cost-effective and therapeutically safe biological macromolecule of clinical potency against pathologies either mediated by or themselves precipitate oxidative stress-induced cellular or vascular damage. Copyright:

INTRODUCTION

Reactive oxygen species (ROS) are flag bearers of oxidative stress. Within systemic physiology, they create hypoxic tissue microenvironment resulting in intravascular erythrolysis and anemia.[1] The objective of the current study was to evaluate the efficacy of iron-conjugated pheophytin (Fe-Ph-I) in protecting against oxidative stress-induced cellular and vascular damage. Fe-Ph-I is a porphyrin compound created in resemblance to heme [Figure 1]. Heme forms the structural skeleton of physiological antioxidants such as catalase and peroxidases [Figure 1].[2] Moreover, phytol possesses antioxidant capability of its own.[3] Thus, the rationale governing our current study was that having a structural skeleton similar to that of heme along with the added ambience of a phytol chain, Fe-Ph-I will be a potent anti-radical agent capable of addressing oxidative stress and hemolytic anemia. As a proof of concept, we evaluated the comparative anti-radical and cytoprotective efficacy of both Ph-I and Fe-Ph-I in in vitro and ex vivo bioassays. Furthermore, to assess whether ferrous ions or chelated ferrous ions are responsible for the variance in the bioactivity of Fe-Ph-I from Ph-I, we simultaneously evaluated the efficacy of FeCl2 in the aforementioned bioassays. Finally, heme-mimetic efficacy of Fe-Ph-I was evaluated against oxidative stress-induced intravascular hemolysis in rodent animal model. Thus, the aim of the current study was to derive information that will be of use in determining the possible clinical fate of iron conjugated naturally occurring pheophytin (Fe-Ph-I) against inflammatory, oncological or hemopoietic pathologies that either mediated by or themselves precipitate oxidative stress-induced cellular or vascular damage.

Figure 1

Structural similarly between iron chelated pheophytin-I and Heme. The figure also shows examples of hemoproteins responsible for scavenging excess reactive oxygen species in systemic physiology

MATERIALS AND METHODS

Material

All chemicals employed were of laboratory grade. Ferrozine and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were procured from Sisco research laboratories Pvt. Ltd. and ferrous chloride (FeCl2) was purchased from Loba Chemi Pvt. Ltd.

Preparation of pheophytin-I and iron-pheophytin-I

Briefly, the acetonic solution of extracted chlorophyll (Chl-a) was acidified by dropwise addition of 1N hydrochloric acid until the color of the solution changes from green to olive-brown. The resulting mixture was then diluted with petroleum ether and washed with cold distilled water to Ph-I from the petroleum ether [Figure 2].[4] Fe-Ph-I was synthesized by stirring acetonic solution of Ph-I with Iron (II) chloride (1.3M) and sodium acetate (25M) dissolved in glacial acetic acid for 30 min at 80°C to obtain bright green-colored solution from which Fe-Ph-I was extracted by dissolving in petroleum ether and washing with excess distilled water [Figure 2].[5]

Figure 2

Scheme of synthesis of pheophytin-I and iron chelated pheophytin-I

Characterization of iron chelated pheophytin-I

Structural characterization of both Ph-I and Fe-Ph-I were analyzed performed using Orion aqumate 8000; 5 beam UV-Vis spectrophotometer (ThermoFischer), FT-IRSpectrometer SN340 BrukerTensor – 37, and Bruker's AVANCE-III 500 MHz NMR Spectrometer.

Assessment of ferrous ion chelation by in vitro ferrozine assay

Briefly, test samples were acidified with sulfuric acid and vortexed separately at room temperature (RT) and at 80°C for 30 min. Then, ferrozine solution was added (2 mM, 1 mL), and the reaction mixtures were incubated at RT for 10 min. Absorbance was measured at 562 nm to determine for iron chelation.[6]

2,2-Diphenyl-1-picryl-hydrazyl in vitro radical scavenging assay

Briefly, 0.1 mM DPPH solution was prepared in methanol. To this freshly prepared DPPH solution (1 mL) varying dilutions of the sample (1 mL) were added and incubated in dark for 15 min at RT. A decrease in absorbance was measured at 517 nm to quantified anti-radical potency. Ascorbic acid was used as a positive control (PC) for the study.[7]

Hydrogen peroxide-induced in vitro free radical scavenging assay

Briefly, a solution of 40 mM hydrogen peroxide (H2O2) was prepared in phosphate buffer (pH 7.4) and varying concentrations of the candidate drug solutions (1 mL) were added to H2O2 solution (1 mL) and incubated at 37°C ± 2°C for 15 min to measure absorbance at 230 nm and quantify anti-radical potency. Ascorbic acid was used as PC for the study.[8]

Ex vivo oxidative hemolysis assay

To evaluate the protective potency of candidate drugs on oxidative stress-induced hemolysis, different concentrations of sample solutions were individually incubated for 15 min at RT with H2O2 (1 mL, 40 mM). To this reaction mixture erythrocyte suspension (5%, 1 mL) freshly prepared from whole human blood was added then again incubated at 37°C ± 2°C for 30 min. Postincubation the reaction mixtures were centrifuged and absorbance of the supernatant was measured at 560 nm. Percentage potency (% inhibition) against oxidative stress-induced erythrolysis was quantified.[9]

Oxidative stress-induced hemolytic anemia in vivo bioassay

To evaluate the potency of the candidate drug against oxidative stress-induced vascular toxicity, phenylhydrazine (PHZ) induced hemolytic anemia model was employed. The animal study protocol was dully approved by IAEC (379/Go/ReBI/S/01/CPCSEA 2019/17). Herein, Wistar Albino rats of either sex were randomly assigned to control, negative control (NC), and treatment groups and treated for 6 consecutive days. The animals allocated to control and NC group were treated with physiological saline while the treatment group animals received 50 mg/kg dose of the candidate drugs (Ph-I and Fe-Ph-I) respectively. On day 4 of the study, all animals except the animals of the control group were administered with 40 mg/kg. i.p. PHZ for the next 3 consecutive days. 24 h following the last PHZ challenge, all animals were euthanized to collect blood and spleen for hematological and histopathological assessment (hemoglobin [Hb], hematocrit, mean cell volume, mean cell Hb concentration, mean corpuscular Hb, and red blood cell morphology). Histopathology of spleen was performed using Confocal Laser Scanning Microscope (CLSM) MEA 53100 Nikon Corporation.[10]

In vivo acute toxicology

Acute toxicity studies of the test compounds were formed as per OECD TG 420. Wherein animals were dosed stepwise using fixed-dose levels of 5, 50, 200, 300, 2000 mg/kg b.w. A period of 24 h was allowed between administrations of each subsequent dose levels. All the animals were observed for 14 days for symptoms of morbidity or mortality.[11]

Statistical analysis

Results of both in vitro as well as in vivo studies were analyzed using software GraphPad Prism version 9.1.2 (226) software (GraphPad Software, San Diego, California USA).

RESULTS AND DISCUSSION

Characterization of pheophytin-I and iron chelated pheophytin-I

The hypsochromic shift observed in the UV-visible spectrum of Fe-Ph-I in reference to Ph-I is indicative of the conversion on nonmetalated Ph-I into metallated Fe-Ph-I [Figures 3 and 4]. The distinctive pyrrolic N-H peak present in FTIR-spectrum of only Ph-I at about 3541.25 cm-1 indicates the conversion of Ph-I into Fe-Ph-I [Figures 5, 6 and Table 1]. Lastly in NMR spectra [Figures 7 and 8, Table 2] peaks corresponding to the N-H protons were observed between -1 to -2 ppm for only Ph-I and not in Fe-Ph-I. Thus concluding that N-H proton belonging to porphyrin nucleus were occupied by Fe++ converting Ph-I to Fe-Ph-I.

Figure 3

Absorption spectrum for pheophytin-I

Figure 4

Absorption spectrum for iron chelated pheophytin-I

Figure 5

FTIR spectrum for pheophytin-I

Figure 6

FTIR spectrum for iron chelated pheophytin-I

Table 1

Assignment of infrared frequencies

Origin	Frequency (cm−1)		Assignment	Origin	Frequency (cm−1)		Assignment
	Ph-I	Fe-Ph-I			Ph-I	Fe-Ph-I
C-H	3003.2	3007.44	Vinyl stretch (phytol chain)	C=N	1383.74	1311.17	Aromatic ring stretch
	2936.01	2921.91	Methyl and methylene stretch (phytol chain)	C-H	1239.21	1221.21	In plane aromatic C-H bending
C=O	1747.21	1731.39	Ester linkage (porphyrin macrocycle)	C-O	1048.46	1195.29	Ester linkage ( porphyrin macrocycle)
C=O	1696.22	1616.63	Keto group attached to ring V	C-H	915.46	984.22	Vinyl C-H out of plane bending
C=C	1579.79	1529.11	Aromatic ring stretch	C-C/C-N/C-N-C	>800		Pyrrole ring bending vibrations
CH3	1469.88	1445.95	CH3 anti-symmetric bending	N-H	3541.25	-	Pyrrolic N-H

Fe-Ph-I: Iron chelated pheophytin-I

Figure 7

NMR spectrum for pheophytin-I

Figure 8

NMR spectrum for iron chelated pheophytin-I

Table 2

Assignment of[1] HNMR chemical shifts δ (ppm)

Position	R	Ph-I		Fe-Ph-I		Position	R	Ph-I		Fe-Ph-I
		δ(ppm)	J (Hz)	δ(ppm)	J (Hz)			δ(ppm)	J (Hz)	δ(ppm)	J (Hz)
2a	CH3	3.130 (s)	-	3.328 (s)	-	18	H	4.156 (t)	6.5	4.091 (q)	6.5
3a	CH	7.762 (t)	6.0	7.653(q)	4.0	18a	CH3	1.321 (d)	6.0	1.225 (d)	9.0
3b1	CH	6.194 (d)	5.5	6.296(d)	5.5	20	H	8.577 (s)	-	8.511 (s)	-
3b2	CH	5.915 (d)	5.5	5.807(d)	6.0	P1	CH2	4.606 (d)	4.5	4.311 (d)	5.5
5	H	9.373 (s)	-	9.318 (s)	-	P2	CH	5.183 (t)	7.5	5.260 (d)	6.0
7a	CH3	3.111 (s)	-	3.293 (s)	-	P3’	CH3	1.752 (s)	5.5	1.655 (s)	7.5
8a	CH2	3.497 (t)	6.5	3.507 (q)	7.0	17	H	4.347 (q)	6.0	4.792 (q)	7.0
8b	CH3	1.452 (t)	3.0	1.318 (d)	7.0	17a	CH2	2.275 (d)	-	2.312 (d)	7.0
10	H	9.651 (s)	-	9.599(s)	-	17b	CH3	2.232 (s)	6.5	2.184 (d)	6.5
12a	CH3	3.202 (s)	-	3.342 (s)	-	17b	CH3	2.232 (s)	-	2.184 (d)	7.0
13b	CH	6.223 (s)	-	6.518 (s)	-	18	H	4.156 (t)	6.5	4.091 (q)	6.5
13c	O-CH3	3.366 (s)	-	3.659 (s)	-	18	H	4.156 (t)	6.5	4.091 (q)	6.5
17	H	4.347 (q)	5.5	4.792 (q)	7.5	Phytol chain protons		1.211-0.876	-	1.156-0.635	-
17a	CH2	2.275 (d)	6.0	2.312 (d)	7.0	N1	H	−1.401	-	-	-
17b	CH3	2.232 (s)	-	2.184 (d)	7.0	N2	H	−1.583	-	-	-

Fe-Ph-I: Iron chelated pheophytin-I, HMNR: Proton NMR

Assessment of iron chelation by ferrozine assay

In the present study [Figure 9], it was observed that the absorbance of reaction mixture composed of FeCl2 and Fe-Ph-I was significantly more (P < 0.0001) while that of Ph-I was significantly less (P < 0.005) than the absorbance of nascent ferrozine solution. This is because Fe-Ph-I yields free Fe++ into the reaction mixture while Ph-I does not, therefore indicating the conversion of Ph-I into Fe-Ph-I.

Figure 9

Effect of treatment groups on absorbance of ferrozine. ***P < 0.0005 and ****P < 0.0001 in reference to control group

As shown in Figure 10, no significant difference was spotted between the % DPPH scavenging potential of PC and Fe-Ph-I treated groups and while anti-radical activity of both Ph-I and Fe-Ph-I increased dose-dependently, the efficacy of Fe-Ph-I was found to be significantly (P < 0.0001) more than that of Ph-I. Last but not the least, the efficacy of FeCl2 was found to be significantly higher than Ph-I (P < 0.0001) thus it can be concluded that incorporation of ferrous ion (Fe-Ph-I) causes significant increase (P < 0.005) in free radical scavenging capacity of Ph-I.

Figure 10

Efficacy of different treatment groups in 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) assay. Comparative dose response analysis of treatment in DPPH assay. All data is expressed as mean ± standard error of the mean. ****P < 0.0001 with reference to FeCl2, ####P < 0.0001 with reference to Fe-Ph-I

As observed in Figure 11, both Ph-I and Fe-Ph-I are dose-dependent scavengers of H2O2. However, the efficacy of Ph-I is significantly less than both Fe Ph-I (P < 0.05) and PC (P < 0.005), thereby indicating that incorporation of ferrous ion is responsible for increasing the H2O2 scavenging potency of Ph-I.

Figure 11

Comparative efficacy of treatment groups in scavenging Hydrogen peroxide (H2O2). All data is expressed as mean ± standard error of the mean. ****P < 0.0001 with reference to FeCl2, ####P < 0.0005 with reference to Fe Ph I

As indicated in Figure 12, both Ph-I and Fe-Ph-I offered dose-dependent protection against oxidative stress-induced hemolysis, however the efficacy of Fe-Ph-I was significantly superior (P < 0.001) than Ph-I, thereby ascertaining that incorporation of Fe++ increases the efficacy of naturally occurring Ph-I. Furthermore, it was observed that no significant difference was registered between the efficacy of Fe-Ph-I and PC-I and PC-II, while efficacy of FeCl2 was found to be significantly less (P < 0.0001) than that of all other treatment groups. Thus while free ferrous ion (FeCl2) scavenges H2O2 by Fenton reaction generating ROS and increasing hemolysis, Fe-Ph-I scavenges H2O2 following the heme-mimetic approach similar to that of heme protein catalase thus explaining the increase observed in protective potency of Ph-I by causing incorporation of ferrous ion into its prophyrin core (Fe-Ph-I).

Figure 12

Comparative efficacy of treatment groups against oxidative stress induced erythrolysis. All data are expressed as mean ± standard error of the mean. ****P < 0.0001 with reference to FeCl2, ####P < 0.0005 with reference to iron chelated pheophytin-I

With reference to NC group, hematological parameters were significantly more (P < 0.0001) in control, Ph-I, and Fe-Ph-I treated groups while no significant difference was registered with FeCl2 treatment [Figure 13]. As compared to the control massive hemolysis and clumping is observed in NC and FeCl2 groups, while mild to no clumping and erythrolysis is observed, with Ph-I and Fe-Ph-I treatment [Figure 14A]. Also in comparison to control group, proper demarcation of red and white pulp is absent in both NC and FeCl2 treated groups [Figure 14B], with prominent presence of hemosiderin-loaded macrophages in both groups which is indicative of hemolytic anemia. With Ph-I and Fe-Ph-I very few hemosiderine loaded macrophages are visible as remnants of PHZ challenge. Thus it can be advocated that anti-anemic potency of Fe-Ph-I is superior than that of both Ph-I and un-chelated free ferrous ion, reaffirming that, heme-mimetic structure is the cause of Fe-Ph-I potency in hemolytic anemia.

Figure 13

Heat map erythrogram of animals from different treatment groups postphenyl hydrazine induced oxidative challenge. The numbers in the heat map represents median values of hematological indices. ####P < 0.0001 in reference to control group, ****P < 0.0001 in reference to NC group

Figure 14

Effect of treatment on red blood cell (RBC) morphology and splenic histopathology postphenyl hydrazine (PHZ) induced oxidative challenge. A. Photomicrograph of RBCs obtained from different treatment groups post PHZ induced oxidative challenge. B. Confocal photomicrograph splenic histoarchitecture obtained from different treatment groups post PHZ induced oxidative challenge

Acute toxicology study

Acute toxicology study indicated no sign of morbidity nor mortality even at 1000 mg/kg b.w. for either Ph-I or Fe-Ph-I. Thereby concluding that both Ph-I and Fe-Ph-I are devoid of any untoward adverse effects.

CONCLUSION

Fe-Ph-I is cost-effective, safe, and clinically useful against therapeutic agent having anti-radical and heme-mimetic efficacy against oxidative stress-induced cellular and vascular damage.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.