Structural, chemical and physiological investigations of bilirubin found in seed arils of strelitzia nicolai.

This study describes new protocols to reveal new insights into the presence of the only animal pigment found in S. An ultrastructural study of the seeds was initiated to integrate additional knowledge.

Introduction

Effect of disturbances in bilirubin metabolism
Strelitziaceae: Bird of Paradise family
Rationale of study
Aims and Objectives of this study
Outline of research presented in this thesis

This, together with the discovery of bilirubin in a plant, was the basis of the first rationale for this study. The ultrastructure and the localization of the bilirubin found in the arils were also not reported before this investigation.

Therapeutic Uses of Animal Bile in Traditional Chinese Medicine: An Ethnopharmacological, Biophysical Chemical and Medicinal Review. Heritage and Innovation of Traditional Chinese Medicine (TCM) Taste Theory and TCM Taste Standardization Principle Theory in Compendium of Materia Medica.

Journal article for publication 1

New insights into the presence of bilirubin in a plant species Strelitzia nicolai (Strelitziaceae)

16 Conclusion: This study reveals new insights into the presence of the only animal pigment found in Strelitzia nicolia arils, the potential benefits of bilirubin in the plant, and indications of its therapeutic value.

Introduction

Heme degradation is initiated by heme oxygenase-catalyzed oxidation of the a-bridge carbon of heme that is converted to CO, leading to the opening of the tetrapyrrole ring which releases the iron molecule.

Metabolism of Bilirubin

In the liver, bilirubin is conjugated, excreted in the bile, modified in the gastrointestinal tract, and lost mainly through the feces.

Bilirubin toxicity

This rare disorder is an inherited form of non-haemolytic jaundice caused by mutations in the UGT1A gene, which codes for bilirubin glucuronosyltransferase. This is associated with a defect in the ability of hepatocytes to excrete conjugated bilirubin into the bile (Paulusma, et al., 1997).

Potential beneficial effects of bilirubin

The Gilbert syndrome is a harmless hereditary variation associated with persistently elevated plasma levels of unconjugated bilirubin (Ritter et al., 1992). This produces increased levels of unconjugated bilirubin which frequently causes brain damage in infants (Ritter et al., 1992).

Bilirubin as an antioxidant and cytoprotectant

By the early 1950s, bilirubin was claimed to protect against oxidation of lipids, for example linoleic acid and vitamin A (Bernhard et al., 1954). In diabetes mellitus, Han et al., (2010) found that a high intensity of serum bilirubin is associated with a poorer risk of the disease.

Bilirubin as an anti-cancer agent

Biliverdin reductase is an evolutionarily conserved enzyme that converts biliverdin to bilirubin, the powerful physiological antioxidant. Bilirubin defends cells in contrast to high levels of hydrogen peroxide (H2O2), which oxidize bilirubin to biliverdin, which is then recycled to bilirubin on the other side by biliverdin reductase (Baranano et al., 2002).

Bilirubin as a protectant in diabetes

22 Overproduction of oxidative particles causes progressive loss of pancreatic β-cells, reducing insulin levels (Yamagishi et al., 2011). Korean scientists reported significant resistance of hyperbilirubinemic Gunn rats to induced diabetes after intraperitoneal exposure to streptozotocin compared with their nobilirubinemic littermates (Fu et al., 2010).

Bilirubin as a protectant in cardiovascular disease

Enhanced expression of haem oxygenase-1, the enzyme responsible for changing hemoglobin to bilirubin, is associated with better insulin sensitivity and glucose metabolism. In addition, serum bilirubin is inversely associated with insulin resistance, as it enhances the expression of glucose transporter-1 and the speed of glucose uptake.

Bilirubin as a protector against neurological diseases

Iwasaki et al., (2005) found that a decreased serum bilirubin concentration is associated with an improved risk of neurodegenerative diseases. In addition, a low serum bilirubin concentration is associated with an increased risk of amyotrophic lateral sclerosis and psychological disorders such as winter depression and schizophrenia.

The breakthrough of the only animal pigment (bilirubin) found in a plant

The initial step in tetrapyrrole biosynthesis is the production of 5-aminolaevulinic acid ALA via two likely pathways: (a) condensation of succinyl CoA and glycine (C4 pathway) by ALA synthase or (b) decarboxylation of glutamate (C5 -pathway) via three different enzymes, glutamyl-tRNA synthetase to charge a tRNA with glutamate, glutamyl-tRNA reductase to reduce glutamyl-tRNA to glutamate-1-semialdehyde (GSA) and GSA aminotransferase to catalyze a transamination reaction for the production of ALA (Grimm, 2003). To make heme and chlorophyll, uroporphyrinogen III requires to be decarboxylated to coproporphyrinogen III by the action of uroporphyrinogen III decarboxylase (as illustrated in Figure 3).

Differences and similarities of the tetrapyrrole metabolic synthesis in animals and plants

Could it be due to the bilirubin found in the sperm cells which is part of their primary diet. If so, this could be the cytoprotective effect suspected to be the underlying feature of bilirubin.

Conclusion

Hyperbilirubinemia reduces streptozotocin-induced pancreatic injury through attenuation of oxidative stress in the Gunn rat. A mutation in the human multispecific organic anion channel transporter gene causes Dubin-Johnson syndrome.

Abstract

However, the body depends on external (exogenous) sources to acquire the rest of the necessary antioxidants (Diplock et al., 1998). A further study in Belgium by Temme et al., (2001) showed an inverse relationship between serum bilirubin levels and cancer mortality.

Materials and methods

Recently, our understanding of cancer has advanced in the realization that apoptosis and the genes that control it have a profound effect on the malignant phenotype. This, together with the discovery of the only animal pigment in a plant (bilirubin), led us to the present study investigating whether the aryl extract containing bilirubin is a potential antioxidant and induces apoptosis in HeLa cell lines. There are fundamental differences between the application of a pure chemical and the same chemical in a plant matrix (Murthy et al., 2002).

The plate was then removed from the chamber, allowed to air dry and the Rf value calculated. 10 mg of the sample was dissolved in 0.5 ml of CDCl3 and the 1H and 13C NMR spectra were recorded and referenced to the deuterio-chloroform signal at  7.24 relative to the internal standard TMS. The scavenging activity (antioxidant capacity) of the plant phytocompounds on the stable radical DPPH was evaluated according to the method of Murthy et al., (2002) with some modifications.

The percentage free radical scavenging activity was calculated according to the following equation: % scavenging activity = Ac-As/Ac x 100. Non-viable normal cells were identified as those with chromatin colored light orange and with an organized structure, while non-viable with apoptotic nuclei were observed to have highly condensed and fragmented chromatin.

Results

The coverslips were then removed and mounted on a slide and viewed with a fluorescence microscope set for fluorescence (495 nm primary filter and a 515 nm secondary filter). Cells were viewed under the 10X objective where viable normal cells stained bright green with intact structured nuclei and viable apoptotic cells stained green with highly condensed or fragmented nuclei. Data were analyzed using the Graphpad Prism 5 for individual comparisons and data were expressed as the mean ± standard deviation.

Discussion

These treatments indicate that the anti-cancer effects are related to two straightforward processes: inhibition of cell proliferation and initiation of apoptosis on HeLa cells in vitro. While this study was an in vitro study, in vivo experiments may provide further insight into the use of this extract in the treatment of cancer. Studies on antioxidant activity of pomegranate (Punica granatum) peel extract using in vivo models, Journal of agriculture and food chemistry.

Effect of Strelitzia nicolai seed arils containing bilirubin on Sprague Dawley rats

In a previous study, Dwarka et al., (in press) identified the presence of bilirubin in the arils of S. The granular layers of the treated animals did not differ much compared to the control. Overall, the brain cells look healthy with no signs of myelin breakdown.

61 Acute toxicity of the pancreas causes the improper secretion and activation of proenzymes within the pancreas. This leads to bleeding, edema, ischemia and necrosis of the pancreatic cells (Berardi and Montgomery, 2005). The experimental groups do not show signs of expansion in the tissue septa and any signs of disruption of the acinar arrangement.

Drug-induced cirrhosis can be excluded as it is associated with reduced platelet counts (Bureau et al., 2011) and is not evident on liver histology. All control groups and most of the experimental group indicate the presence of nitrites in the urine.

Preparation techniques of Strelitzia nicolai aril tissue for scanning electron microscopy

70 Sample preparation is critical, with the critical concern being that the prepared sample accurately represents the natural sample (Boyde and Boyde, 1980). This implies that all biological material prepared for SEM must first be fixed. Therefore, care must be taken in the preparation steps to ensure that the inherent structure of the organism is preserved. One of the more recent methods that can image biological samples without preparation is Variable Pressure Scanning Electron Microscopy (VP-SEM).

This improved method allows for less sample shrinkage than critical point drying (Takao and Hitoshi, 1988). Therefore, the aim of this study was to compare the appearance of aril tissue prepared by different SEM methods. Compared to all other methods tested in this study, this method appears to be the most suitable with the least sample distortion.

This study also shows that the amount of time delicate plant material spends in the freeze dryer also affects specimen shrinkage. Using environmental scanning electron microscopy to image the spore adhesive of the marine alga Enteromorpha in its naturally hydrated state.

A comparative microstructural study of the animal pigment, bilirubin, in seed arils of Strelitzia nicolai

Summary of study

General Discussion
Conclusion
Recommendations for future research

In the nutrient-poor tropical rainforest region, the food source of the Amazon forest dwellers is Phenakospermum (powdered seeds used to make porridge). The in vitro results demonstrated the effectiveness of the aril extract as an antioxidant and anticancer agent. However, a chronic, more comprehensive and in-depth study still needs to be initiated to verify the safety of the aril extract containing bilirubin.

The next section of this study focused on developing an understanding of the structure of the arils. An attempt was made to look at the surface structures of the arils at different stages. Light and electron microscopy, along with modified Hall staining, showed the presence of bilirubin on the nodule surface.

Further research is required to determine the effectiveness of aril extract in the treatment of cancer. However, the result of this study was insufficient to conclude the safety of the extract if administered intravenously.

NMR spectroscopy of bilirubin standard and S. nicolai aril extract

Animal ethics and histology of treated animals

SEM micrographs of stages 2, 3, 4 and 5 using different preparation methodologies