Chapter 3

Biomarkers

Oldenlandia corymbosa and Oldenlandia umbellata are well-known medicinal plants in various traditional systems of medicine and used to treat bronchitis, asthma, tuberculosis, constipation, and leprosy. We evaluated the phytochemical content, antioxidant potential, enzyme inhibitory action, anti-inflammatory, and antibacterial activities of the whole plant extracts of O. corymbosa and O. umbellata due to their therapeutic importance.

The phytochemical analysis includes qualitative analysis and quantification of total phenolics, flavonoids, tannins, flavonols, proanthocyanidins, and vitamin E. Enzyme inhibitory assays were studied using α-amylase and α-glucosidase enzymes and anti-inflammatory activity was determined by egg albumin denaturation assay. Antioxidant potential was estimated using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethyl benzothiozoline-6-sulfonic acid) diammonium salt (ABTS), ferric reducing antioxidant power (FRAP), hydroxyl radical, superoxide, nitric oxide, metal chelating, and phosphomolybdenum assays. Minimum inhibitory concentrations of extracts were studied against ten bacterial strains by the microdilution method.

The aqueous extract of O.umbellata showed a higher extract yield (5.0%). The highest amount of total phenolics and flavonoids were observed in the ethanolic extract of O.umbellata (77.23±0.46 mg GAE/g) and O.corymbosa (19.01±0.26 mg GAE/g). Ethanolic extract of O.umbellata (IC₅₀ of 31.71±0.32 µg/mL) and aqueous extract of O.corymbosa (30.53±1.83 µg/mL) showed notable α-amylase inhibitory effect and petroleum ether extract of both plants observed good inhibition against α-glucosidase enzyme. Likewise, petroleum ether extract of O.umbellata (IC₅₀ of 57.16±1.88 µg/mL) showed a moderate anti-inflammatory effect. In all extracts, considerable antioxidant potential was recorded specially with aqueous extract in the nitric oxide scavenging assay with an IC₅₀ of 40.72±1.25 µg/mL. Ethanolic extract of O.umbellata and petroleum ether extract of O.corymbosa (MIC of 56.25 µg/mL) showed good antibiotic potency in lower concentrations which proves these two valuable herb's potential in herbal preparations.

The results of the study provide a basis for further studies aiming to evaluate the possibilities of using the O. umbellata and O. corymbosa as natural sources of antioxidants, enzyme inhibitory, and antibacterial agents. Further studies could be directed to exploit these plants in experimental animal models as potential pharmaceutical agents.

Biological oxidation processes are in the core of life energetics, play an important role in cellular biophysics, physiological cell signaling or cellular pathophysiology. Understanding of biooxidation processes is also crucial for biotechnological applications.

Therefore, a plethora of methods has been developed for monitoring oxidation so far, each with distinct advantages and disadvantages. We review here the available methods for monitoring oxidation and their basic characteristics and capabilities. Then we focus on a unique method — the only one that does not require input of additional external energy or chemicals — which employs detection of biological autoluminescence (BAL). We highlight the pros and cons of this method and provide an overview of how BAL can be used to report on various aspects of cellular oxidation processes starting from oxygen consumption to the generation of oxidation products such as carbonyls. This review highlights the application potential of this completely non-invasive and label-free biophotonic diagnostic method.

The consumption of antioxidants from food products can aid in preventing oxidative damage in the human body. Oxidative damage is known to be contributing to the onset of various diseases including cardiovascular disease and certain types of cancer. A significant body of evidence is available on the antioxidant potential of various well-known nutrients such as vitamin C and vitamin E. Scientific studies are furthermore conducted into the potential of other nutrients to function as an antioxidant. Examples of such nutrients include green tea polyphenols, of which epigallocatechin gallate is the most well-known, and curcumin. In order to communicate on the relationship between a food product and the antioxidant potential, scientific evidence must be provided which will be assessed by European Food Safety Authority (EFSA). The requirements of this risk assessment, are discussed in detail. It can be concluded that EFSA provides detailed information on the methodology, inlcuding the biomarkers, for scientific studies researching the antioxidant effects. This information should be taken into consideration when setting up studies for health claim application.

Free radicals are very reactive because of the availability of unpaired electrons in their outer shell. They are generated inside the cell either as by-products of metabolism or due to the mitochondrial dysfunction. Reactive oxygen species (ROS) are nonreactive; however, they have the potential to produce free radicals. They are also the by-product of metabolism generated mainly from the electron leakage during the process of cellular respiration. Oxidative stress (OS), is a disorder situation, where the generation of ROS inside the system surpasses the antioxidant defense system of the body. This disparity may be a result of altered antioxidant capacity because of compromised production, distribution of antioxidant molecule, or the overproduction of ROS from endogenous sources or external stresses. Excess ROS has a detrimental effect on cellular macromolecules such as lipid, proteins, or DNA, which results in improper signal transduction pathways. Therefore the OS has been occupied a place in the growing list of human diseases such as cancer, hepatotoxicity, neurodegeneration, cardiovascular as well as in aging symptoms.

Immunoassays have been extensively applied in the medical diagnostic field. Enzyme-Linked Immunosorbent Assay (ELISA) and Lateral Flow Immunochemical Assay (LFIA) are methods that have been well established to analysis of clinical substances such as protein, hormones, drugs, identification of antibodies and in the quantification of antigen. Over the past years, the application of these methods has been extended to assess the clinical oxidative stress condition based on monitoring of the 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) biomarker levels. The present manuscript provides an overview of the current immunoassays based on ELISA and LFIA technologies applied for a quantitative analysis of the 8-oxodG. The discussion focuses on the principles of development, improvement and analytical performance of these assays. The relationship of the molecule 8-oxodG as a clinical biomarker of the assessment of the oxidative stress condition is also discussed. Commercially available products to 8-oxodG analysis are also presented.

The study aim was to investigate the potential of salivary biochemical assessments to determine smoking cessation in patients under smoking cessation programs. The study validated smoking behavior and degree of nicotine dependence using Fagerström test for nicotine dependence in smokers and non-smokers. Protein components were examined using Raman spectroscopy. Salivary matrix metalloproteinases-8 (MMP-8) levels were analyzed using Quantikine human total MMP-8 immunoassay-kits. Thiocyanate (SCN⁻) concentrations, peroxidase and glutathione-peroxidase activities were determined using spectrophotometric methods. Human mononuclear cell line was examined for M1 polarization. The data were analyzed using Fisher’s exact test and two-tailed paired t-test at α = 0.05. Amide I peak in smoker group was significantly reduced as compared to non-smokers (p < 0.05). Sulfhydryl bond, dialkyl disulfides SS-stretch and corresponding disulphide bond at 500−545 cm^-1 were significantly increased in smokers (p < 0.05). The CH₂ wag showed higher intensity amongst smoker group representing a recoil of carbon atoms. Salivary MMP-8 levels of participants after smoking cessation was significantly lower than smoking salivary levels (p < 0.05). The SCN⁻ and peroxidase concentrations were significantly higher in smoking group whereas an M1 polarization was seen in non-smokers. Salivary biochemical analysis can aid clinicians to verify smoking cessation in patients under smoking cessation programs.