Methods for Preclinical Evaluation of Bioactive Natural Products

Antioxidants are groups of substances able to prevent and delay the oxidation of easily oxidizable molecules and avoid free radicals’ formation. In living organisms, the main free radicals are reactive oxygen species and reactive nitrogen species. At low levels, they are involved in the regulation of diverse physiological processes, but an imbalance between free radicals and the ability of the body to eliminate them results in a pathological condition called oxidative/nitrosative stress. Oxidative/nitrosative stress causes damage to cellular structures such as lipids, nucleic acid, and proteins, compromising cellular health and viability and inducing the development of several diseases. Physiological systems are able to contrast the free radical excess, through the endogenous enzymatic materials (e.g., uric acid, glutathione etc.), and via transcription factor activation. The uptake of natural antioxidants can contribute to prevent cellular damage and exert beneficial effects. Natural antioxidants are generally derived from plant sources and they play an important role by directly scavenging free radicals or increasing antioxidant defences. Natural antioxidants have gained remarkable interest and several methods have been developed for identifying their antioxidant capacity. This chapter reviews the major in vitro and in vivo assay procedures for the antioxidant activity estimation describing materials, extract types, extracts/pure compounds' concentrations, step by step processes and calculations for each assay. Advantages and limitations, as well as the molecular mechanisms of each method have been reported.

Diabetes mellitus (DM) is a metabolic disease characterized by the destruction of pancreatic β cells or reduced insulin secretion and action, and is one of the most common health problems worldwide. Its incidence is increasing at a high rate, resulting in enormous social costs. Various drugs show their effectiveness by improving insulin sensitivity, and reducing glucose production in the liver or other tissues. Several preclinical studies on diabetes-induced in animals using surgical, pharmacological or genetic methods demonstrated the effectiveness of these drugs. The anti-diabetic activity of plants has been attributed to the occurrence of primary and secondary metabolites characterized by many beneficial effects with advantages over chemical treatments. A number of studies have demonstrated the potential health benefits of phytocomponents in treating DM by acting on multiple molecular targets. Therefore, it is important to test in vitro assays. This review includes methods for the evaluation of preclinical anti-diabetic activities and summarizes the potential of natural resources to prevent and/or treat diabetes. In addition, the database contains information including the plant name, useful plant parts, active compounds, and their mechanisms of action, in which in vitro and in vivo methods were studied.

The inflammatory process can be unleashed by a wide variety of biological, chemical, and physical factors, and arises to counteract these dangerous enemies. In case of failure by the organism to defeat these exogenous stimuli, a chronic inflammatory status occurs, hence potentially leading to several diseases. Therefore, anti-inflammatory drugs, from both synthetic and natural sources, represent valuable allies to fight the phlogistic process. The search for novel candidate drugs is never ceasing, also from the plant kingdom, known to provide products with generally lesser or more tolerable side effects than synthetic drugs. Researchers can take advantage of a wide plethora of in vitro and in vivo methods in order to investigate the anti-inflammatory potential of unknown natural products. Cell cultures (i.e., stimulated macrophages) offer a fast and highly reproducible first-line screening, while animal models, thanks to their complexity, grant to achieve a broader and multifactorial view of the inflammatory process. The underlying mechanism of action of candidate drugs can also be explored by several cell-free assays, which are crucial to assess the activity of key enzymes involved in the inflammatory cascade. Here, we report the most widely employed models for the assessment of the anti-inflammatory potential of natural products, discussing the overall procedure, and also providing examples of plant drug screening. Together, these approaches represent the basis for a thorough and proper investigation of the anti-inflammatory activity of novel candidates. 

Plant extracts are widely utilized for their antimicrobial effect, alone or in combination with traditional drugs. However, the current global lack of harmonization on the assays used to investigate their biological effect makes the result comparison very challenging. In the present chapter, we highlight the variables used for the antimicrobial testing, which may affect the end result. The in vitro antimicrobial methods, including diffusion methods, dilution methods and thin-layer chromatography-bioautography are reported, together with a range of assays aimed at evaluating the mechanisms of action of the extract. Furthermore, in vivo test procedures are evaluated, with a focus on the impact of the differences in the testing animal and the procedure used.

Natural products have formed the basis of traditional medicine systems throughout human history. Today, drug discovery studies from natural origins continue rapidly and efficiently with modern methods. Among the many activities, cytotoxic activity is related to the behaviour of test material on cell viability and cellular growth. Cytotoxicity methods, used as a screening test or initial test for guiding other activities, provide useful information for biocompatibility studies for medical devices or materials, drug discovery and development processes, toxicity evaluation of cosmetics, research of disease mechanisms and treatments, and determination of chemopreventive agents. In vitro cytotoxicity analyses have emerged as an alternative to in vivo studies and have become preferable due to their ease of application, standardization, rapid, low cost, and compatibility with data from in vivo studies. With cell-based cytotoxicity studies, basic information about the cytostatic and cytotoxic effects of the tested substance is obtained. In studies dealing with natural products, the most appropriate cytotoxic method should be selected according to the properties and chemical structures of natural compounds, the ultimate goal of the study, cell types, etc. Although there are many cytotoxicity methods, this chapter is an introductory overview of the most commonly used assay methods to estimate the cytotoxic activity in natural products. 

Endometriosis is an estrogen-dependent disease that causes pelvic pain and reduces fertility by developing inflammatory lesions outside the uterine. Although many studies have been performed to investigate the etiopathogenesis of endometriosis, there is still no direct evidence. Endometriosis, a common gynecological disease, often recurs even if treated. In vivo studies are used to investigate its etiopathogenesis and potential treatment methods. Hormonal therapy is generally used for endometriosis cases. This conventional therapy aims to lower estrogen levels in the body, however, it can be inadequate for the treatment and has numerous negative effects. The urgency of finding novel and cheap long-term safety therapies for endometriosis is highlighted by the need to manage it as a chronic disease. Medicinal plants and natural plant-derived compounds are interesting options for this purpose. Indeed, there is an increasing interest in using herbal therapy to treat endometriosis. Several studies have been conducted on natural products to find a drug candidate for the management of the mentioned problem. Many of these drugs have a pleiotropic action profile, meaning they block multiple processes involved in endometriosis pathogenesis, including proliferation, inflammation, reactive oxygen species (ROS) production, and angiogenesis. As a result, including them in multimodal treatment approaches may help to improve therapeutic efficiency and reduce adverse effects in future endometriosis treatments. Several methods have been described for the assessment of the potential effectiveness of the bioactive agents against endometriosis. In the present chapter, we aimed to give general information regarding endometriosis, the use of plant products in the treatment of this disease, and the methods that have been used for the activity investigation.

Polycystic ovary syndrome (PCOS) is one of the most common gynecological disorders among women of reproductive age. The secretion rate and metabolism of estrogens and androgens are disrupted in patients with PCOS. Patients have systemic inflammation, hyperinsulinemia, hyperandrogenism, and polycystic ovaries. Several experimental models have been developed and studied for the investigation of this disease. These studies aim to explore the pathophysiology of PCOS and new treatment approaches. Some plant extracts and phytochemicals obtained from them are shown to be effective against PCOS in literature. In this chapter, general information about PCOS, plant-based treatment approaches, and activity methods are explained in detail.

Hypertension is one of the leading causes of chronic kidney disease (CKD) due to the deleterious effects that increased blood pressure (BP) has on kidney vasculature. Patients with CKD and hypertension often require a combination of antihypertensive medications to target BP. Several classes of antihypertensive/diuretic agents may have a role in the treatment of CKD and hypertension. Besides these conventional therapies, it is extremely useful to identify new natural sources (herbs and botanicals) of diuretics and their targets in the kidney that can be used alone or in combination with traditional synthetic drugs. The use of herbal medicine and natural products to contrast hypertension has increased many-fold over the past decade driven by documented experimental evidence. However, many nephrologists are unaware of these potential therapeutic benefits in treating hypertension, mainly for the lack of knowledge of molecular mechanisms underlying their effects. Indeed, rigorous preclinical assays are necessary to scientifically validate the utilization of medicinal plants for the treatment of hypertension. This chapter provides detailed methods to study the effects of two representative herbal extracts on renal transporters involved in blood pressure regulation by in vitro, in vivo, and ex vivo approaches. Of note, these methods can be applied to any herbal extracts. 

This chapter provides a collection of methods generally used in experimental pharmacology to test compounds with different activities in the cardiovascular system. There are many models in vitro and in vivo and we have highlighted those used in the field of natural compounds research. For the in vitro procedures, the focus was on the inhibition of enzymes, such as an angiotensin-converting enzyme or nitric oxide synthase, which plays a pivotal role in maintaining vascular control and overall blood pressure. Moreover, test procedures on isolated vessels were also considered for compounds whose activity is exerted on specific targets expressed in the vasculature. Regarding the in vivo methods, heart failure and hypertension models were highlighted for their high incidence of overall deaths cause. In particular, we have analyzed models of heart failure in rats and mice, animals generally used in preclinical studies. In addition, it also evaluated useful models of hypertension involving mainly rats; however, in many cases, the procedure could be translated to different animal species.

This chapter provides an overview of the main features that need to be assessed with respect to the general pharmacology (pharmacokinetics, PK; pharmacodynamics, PD) of all molecules, including natural compounds. Here, the attention has been focused on bioavailability, the volume of distribution, and clearance to describe the physiological processes of absorption/distribution and elimination of drugs from the body. It is worth pointing out that PD studies always require an in vitro preliminary approach, not issued here, that needs to be assessed and confirmed in vivo. Indeed, PD studies aim to the individuation of specific molecular targets, responsible for an observed pharmacological effect. Thus, the use of animal models as a complex approach does not allow to dissect the molecular mechanisms of natural compounds but can confirm their efficacy in vivo. Therefore, specific PD approaches to determine a specific action by a compound are addressed in the relevant chapters within this book.