Curcumin

Objective: To study the neuroprotective effect of curcumin against tartrazine-induced neurobiochemical and histopathological changes in the gray matter of motor cortex of rat brain. Study Design: Lab-based experimental study. Place and Duration of Study: Department of Anatomy, Islamic International Medical College, from Sep 2019 to Sep 2020. Methodology: Forty five adult male albino rats (250-300 gm) were divided randomly into three groups (n=15). Group A, rats were given standard rat diet. Group B, rats were given tartrazine orally by dissolving in tap water in the dose of 7.5 mg/ kg body weight. Group C, rats were given 200 mg/kg of curcumin along with tartrazine orally by dissolving in tap water, daily for 28 consecutive days. At the end, all animals were dissected and their brain were removed. The neurobiochemical parameters including lipid peroxidation marker level along with microscopic changes widening of Virchow’s robin space and neuropil vacuolation in neurovascular unit were observed. Results were analyzed using SPSS version 21. Results: The curcumin administration significantly improved the MDA levels (p-value <0.001 on intergroup comparison) and widening of Virchow’s robin space (group A=3.57 ± 0.78 μm, group B=19.39 ± 0.68 μm, group C12.1 ± 0.83 μm; p-value <0.001). Neuropil vacuolation was reduced to negligible in 60% rats of curcumin treated group. Conclusion: Curcumin displayed a neuroprotective role in improving the tartrazine induced neurobiochemical and histopathological changes in the motor cortex of rat brain.


INTRODUCTION
Tartrazine, a synthetic azo dye that is certified by FDA as a colorant (E102, FD&C Yellow 5) is among the most commonly used food color to impart lemon yellow color in various food products 1 . The World Health Organization (WHO) has approved the acceptable daily intake for tartrazine to be 7.5mg/kg/day or less 2 . Azo-fission of orally ingested tartrazine in intestine and liver lead to formation of aromatic amines, sulfanilic acid, which can cross the blood brain barrier and generate reactive oxygen species (ROS) in brain 3 . Various neurotoxin may lead to damage to neurovascular unit (NVU) that includes cellular and extracellular components (neurons, perivascular astrocytes, microglia, pericytes, endo-thelial cells, and basement membrane) that are involved in regulating cerebral blood flow and blood-brain barrier function 4 . Tartrazine induces marked neuroinflammatory changes in the neurovascular unit of brain by promoting the marked rise in malondialdehyde (MDA), lipid peroxidation metabolites and ROS in the brain cortex even at prescribed accepted daily intake (ADI) levels. Histopatho-logical changes in the brain of tartrazine-treated rats included significant widening of Virchow robins space (perivascular space) and vacuolation in neuropil 5 . The former is associated with tartrazine induced oxidative stress and inflammation which stimulate the release of pro-inflammatory cytokines. These cytokines in turn augment neutrophil aggregation and IL-6 synthesis by vascular endothelium, leading to increased blood flow in blood vessels 6 . Tartrazine by enhancing lipid peroxidation alters the vascular membrane permeability leading to accumulation of cellular infiltrates in the perivascular space surrounding the blood capillaries in neuropil 7 . Vacuolization is an initial histological indicator of interference in fluid balance between the neuronal cell membrane and surrounding interstitium. This may be attributed to the exhaustion of the antioxidants in combating ROS that were generated by tartrazine administration. Vacuolation in astrocytes (glial cells) results in neuropil vacuolation 8 . As these cells constitute the blood brain barrier, this may alter the permeability across their foot processes (aquaporin 4 gated channels) and lead to abnormal fluid diffusion and aggravate the neuroinflammation 9 .
Curcumin, a natural food color, the main bioactive component in the rhizome of the turmeric plant is a safe and highly pleiotropic molecule with multiple biological targets. Being a potent antioxidant it exerts its effects primarily by up regulating the brain antioxidants enzymes and neuroprotective markers. It enters brain tissue in biologically relevant concentrations, reduces acute and transient microglia activation, proinflammatory mediator production, and the behavioral symptoms of sickness. By hindering the activation of the transcription factor, nuclear factor kappa B (NF-KB) the curcumin play a vital role in the cell survival, cytokine production, and other cellular functions 10 . Considering the beneficial effects of curcumin a current study was undertaken to investigate the neuroprotective potential of curcumin against tartrazine induced neurobiochemical and histological alteration in the (neurovascular unit) gray matter of motor cortex of adult male rats.

METHODOLOGY
It was a laboratory based experiment and nonprobability consecutive sampling technique was used to draw the sample. This study was carried out in the department of Anatomy, Islamic International Medical College Rawalpindi in collaboration with the National Institute of Health (NIH), after seeking approval from the Ethics Review Committee [Riphah/IRC/19/0373], Islamic International Medical College, from September 2019 to September 2020.
The study was performed on 45 Albino Sprague Dawley adult male rats as a mammalian model. Two months old adult male rat weighing 300gm were included and rats <300gm and female rats were excluded from the study. The animal care and handling was done according to the guidelines set by the ethics review committee, Islamic International Medical College, Rawalpindi.
Tartrazine and curcumin in powdered form was weighed first on electronic weighing scale according to a dose of 7.5mg/kg/day and 200mg/kg/day respectively. Then dissolved in tap water to make 20ml solution for each rat which was given daily to 30 rats of experimental group B & C via oral route 2 . For neurobiochemical assays, MDA calorimetric assay kits and ELISA test kits were purchased from Multi LinkX Enterprises. Phosphate buffer saline PBS was purchased from local chemical shop.
Rats were kept in cages under the supervision of Animal house of NIH, Chak Shehzad Islamabad. Forty five rats weighing approximately 300gm were kept under standard temperature at 22 ± 0.5 o C in air conditioned room and were shifted into clean stainless steel cages under 12 hour light and dark cycle with 50% humidity. They were given food and water ad libitum for 7-days to acclimatize. Rat pellets and water were used as food during the whole experiment. Each group comprised of 15 male rats. Group A (control group) was kept on standard diet orally throughout the experiment. Experimental group B was given tartrazine at dose 0.031 gm/day and those of group C same dose of tartrazine along with 0.9 gm/day curcumin by dissolving in tap water via oral route for four weeks. Following 24 hours of last dose administration, the rat was anaesthetized with chloroform soaked cotton balls till they lost consciousness (euthanasia). Brains were taken out, rinsed in cold saline.
A 2mm section of each rat brain in region of motor (frontal) cortex was removed and shifted to phosphate buffer saline (PH 7.5) containers for biochemical assays. While the rest of brain tissues were fixed in 10% formaldehyde solution which were processed to obtain paraffin blocks, from which 5μm thick coronal sections were prepared and conserved for histopathological analysis.
Tissue was stained by using H&E staining. For the assessment of qualitative parameter of neuropil vacuolation all the slides were observed under 10X magnification by using image J software to assess the presence and degree of neuropil vacuolation. Vacuolation was identified in neuropil as single or clustered, oval to round, colorless spaces ( fig-1(a-c)). Scoring was done according to criteria given by International Harmonization of Toxicological Research (fig-1) 11 . For the assessment of quantitative parameter of Virchow Robin space measurement, the widening of maximally dilated vascular zone (a-b) was measured in the transverse plane of blood vessel in the four zones under 10X magnification and then mean of four reading was taken (table-I). Four images of each slides were taken by using eye piece camera YW-100 2.0 MP and then all images were taken transferred to image J software.
Lipid peroxidation metabolite MDA level was estimated by calorimetric assay kits through TBA method. A 10% tissue homogenate of motor cortex was prepared in PBS (pH 7.4) and was used for the determination of MDA. The brain tissue (motor cortex) was minced into small pieces, then weighed and homogenized in normal saline on ice, the volume of normal saline: weight of tissue (gm)=9:1. The tissue homogenate is centrifuged for 10min at 10000g. The supernatant was collected was used to estimate the level of MDA with the help of spectrophotometer.
Data was entered and analyzed in SPSS-21 and results were expressed as mean ± SD. One way analysis of variance (ANOVA) was applied for the mean comparison of quantitative variables between control and experimental groups. Post hoc tukey's test was applied for the intergroup comparisons among groups. Qualitative variable (vacuolation) was expressed by frequency and percentage. Chi square test was used for intergroup comparison among this variable. A p-value of ≤0.05 was considered significant.

RESULTS
An examination of hematoxylin/eosin-stained brain tissue sections by microscopy revealed that coadministration of curcumin significantly lessened the widening of the Virchow robin's space (perivascular space) (fig-1, table-I) and vacuolation in neuropil (fig-2, table-II) as compared to tartrazine intoxicated rats. A significant improvement was also observed in neurobiochemical assay in curcumin administered group.
The mean measurements of Virchow's Robins Space was found to be 3.57 ± 0.78µm, 19.39 ± 0.68µm and 12.1 ± 0.83 µm respectively. On comparing the mean measurements of Virchow's Robins Space between all the three groups ANOVA showed significant results (p-value <0.001). On intergroup comparison   -1b). While the comparison of group C with the groups B and A were also found to be statistically significant (p-values of <0.001) (fig-1, table-I).
Regarding the neurobiochemical assays the mean MDA level in group A was found to be 156.6 U/ml ± 0.757 while a significant elevation was observed in mean MDA levels of group B with the value of 172.8 U/ml ± 0.687 (p-value <0.001). The group C presented a significant decrease 159.4 U/ml ± 1.24 (p<0.001) in MDA concentration as compared to tartrazine exposed rat highlighting the neuroprotective role of curcumin. The p-value for intergroup comparison was <0.001, which was statistically significant (table-III).

DISCUSSION
Among the food colorants, tartrazine is considered as toxic to the human beings if consumed in excess amount and even within ADI limit 12,13 . In this study the ADI dose of tartrazine, 7.5 mg/kg body weight was used 2 .
Addition of the natural antioxidant, curcumin to diet improved cellular metabolism by increasing capacities of cellular antioxidants. Several studies supported the anti-inflammatory, antioxidant, and neuroprotective effects of curcumin as an active constituent of turmeric. Because of its biological properties, curcumin is an appropriate candidate for improving injuries after any toxic insult to brain tissue [14][15][16] . Curcumin has a broad range of biological actions. Because of its lipophilic property, orally administered curcumin seem to cross the blood brain barrier (BBB) and inhibit lipid peroxidation (as a chain-breaking antioxidant) and to enhance endogenous antioxidant mechanisms 10,16 . Curcumin possesses antioxidant property by virtue of its phenolic hydroxyl group in its structure that donates electrons to quench free radicals 17 .
Among the brain sub-regions the frontal motor cortex of cerebrum was found to be more prone to oxidative stress induced by tartrazine as evident in the current findings of neurobiochemical test in experimental rat frontal motor cortex 2 . The curcumin treated group showed significant decline of MDA level in the frontal motor cortex of brain tissue in our study as compared to the tartrazine intoxicated group.
Guo et al carried out a study to see the possible protective effect of curcumin in conservation of morphological and biochemical features of cerebral cortex after exposure to acrylamide and concluded that curcumin display anti-apoptotic, antioxidant and anti-inflammatory effects 18 . Dolatabadi et al conducted a study to ruled out that curcumin could improve memory and neurological deficits and restore irregular neuronal distribution following the cerebral ischemia 14 . Curcumin response toward the amendment of widening of perivascular space and neuropil vacuolation were not observed previously. Our study witnessed the neuroprotective potential of curcumin in these parameters.
Curcumin administration to tartrazine treated rats were found to significantly re-equilibrate antioxidant parameters back to normal values 19 . Curcumin administration improved the structural and serological alterations of the frontal motor cortex with significant reduction in tissue MDA levels 18 . Results of Bhat et al showed a significant protective effect of curcumin in neurodegenerative conditions, which may be due to amending lipid peroxidation and augmenting antioxidant defense system 20 . The limitation of study was the lack of immunohisto-chemical marker GFAP to rule out the astrocytes injury.

ACKNOWLEDGMENT
We are highly obliged to Riphah International University, Rawalpindi for providing us with immense help and opportunities for carrying out our research. We are also thankful to Dr. Hussain Ali, Scientific Officer, National Institute of Health, Islamabad for his immense help in animal handling and housing of animals. We thank Mr. Ijaz Lab technician in NIH, provision and establishing protocols of MDA assay kits.

CONCLUSION
The curcumin administration markedly halted the tartrazine induced oxidative stress and neuroinflammation which was replicated in the form of restoration of neurovascular unit of gray matter of motor cortex. Therefore, it was concluded that curcumin administration improves the tartrazine induced neurobio-chemical and histopathological changes in the gray matter of motor cortex of rat cerebrum.