In-vitro therapeutic bioactivity testing of Medicago sativa L. (Alfalfa/Lucerne)

Abstract

A drug (moriana) is a natural or a synthetic material that affects humans’ physiology and psychology (behaviour and mood) when consumed. Therapeutic drugs are compounds that are used for the purpose of preventing, curing/healing, treating, and diagnosing of diseases. This treatise is about the laboratory-based testing of therapeutic biological activity (bioactivity) of Medicago sativa L leaf extracts. Medicago sativa L. is a flowering perennial legume of medicinal, environmental, and agricultural importance that is known around the world by different names. The name Alfalfa is commonly used in America and some parts of North Africa, while in South Africa, New Zealand, Australia, and the United Kingdom the names Lucerne and Holy hay are commonly used to identify the father of all foods: Medicago sativa. The leaf is safe for human and animal consumption and medicinal uses includes as an aperient, haemostatic, antiscorbutic, oxytocic, tonic, nutritional supplement, anti-diabetic, and many more. The plant’s therapeutic benefits are well known or ethno-pharmaco-botanically established; there exists a lack of empirical evidence in literature to validate the claims. This treatise records the therapeutic bioactivity measuring of Medicago sativa L. leaf extracts towards hyperglycaemia (DM), cancer, pathogenic micro-organisms, oxidative stress, and inflammation relief for the first time. As a response to the existing global burden of diseases, the United Nation’s SGD3 aims to ensure healthy lives and to promote well-being for all people at all ages by the year 2030. The urgent need for developing of therapeutics is motivated by the novel emerging infectious diseases, for example, the COVID-19 pandemic, drug resistance to currently existing clinical drugs, the high cost of medicines, the negative side effects induced by current clinical therapeutics, and the evident unequal access to healthcare and medicines. To satisfy the urgent SDG3, new and novel therapeutics are required for combating the persistent rise in premature morbidity and mortality caused by the top ten global causes of disease. For therapeutic drug development, medicinal plants remain the leading source of ingredients due to their chemical, structural and compound diversity. The ancient medical care system relied on the knowing of natural sources of therapeutics, and still today more than half of the clinical drugs are derived or are based on plants. The other sources of therapeutics are animals, minerals, microbiological, and synthetic sources. Pharmacological screening of natural sources of therapeutics in in-vitro and in-vivo models is the crucial pre-clinical phase of the drug development process. In the year 2007, the general assembly of the UN adopted Resolution 61/225 whereby 14 November of every year is designated as World Diabetes Day as a recognition of the urgent need for novel therapeutics to curb the existing rise in DM prevalence. Diabetes is regarded to be main cause of blindness, kidney failure, heart attack, stroke, and limb amputations. The modern lifestyle promotes the development of hyperglycaemia, which is the hallmark symptom of DM. Chapter II of this treatise measures the glucose-utilization activity in L6 skeletal muscle and HEPG3A liver cells., whereby the hypothesis is that increased glucose utilization that is induced by alfalfa leaf extracts could mean physiologic alleviation of hyperglycaemia. Natural non-drug-based therapeutics are more desirable because of their affordability, accessibility, and the lack of negative side effects or addiction (dependence). The therapeutic bioactivity of Lucerne extracts was measure against the clinical standards namely metformin and insulin. Medicago sativa leaf extracts induced considerable glucose use both at liver and skeletal muscle levels under controlled laboratory conditions, thus potentiating in-vivo, anti-diabetic therapeutic activity. Oxidative stress and low-grade chronic inflammation are related systematic conditions that can promote a plethora of medical conditions. Chapter III of this treatise measures the DPPH scavenging actions and anti-inflammatory screening of Medicago sativa L extracts under controlled laboratory conditions to establish therapeutic potential against related medical ailments. Trolox, EGCG, and Aminoguanidine (AG) served as our positive controls (Figures 3.1-3.3). The modern lifestyle is bloated with risk factors that promote excessive production of ROS, consequently promoting uncontrolled oxidative stress that often leads to low grade chronic inflammatory response that is well known to encourage neurodegenerative disorders (Alzheimer’s and Parkinson’s Diseases), cancers, diabetes, and cardiovascular ailments (hypertension, atherosclerosis, and stroke). Sedentary lifestyle, poor diet and malnutrition, environmental pollutants and toxins, excessive narcotic and alcohol use are prevalent risk factors affecting modernized and urbanized people. Cancer is a devastating medical condition that was responsible for approximately 10 million deaths in 2020 (WHO, 2022). According to the WHO 30-50% of cancers can be prevented using science-based approaches that including the regular consumption of alfalfa herbal tea, regular physical activity, maintaining a diet rich in phyto-chemo-preventive nutrients, and regular cancer screening for early detection. Existing cancer care is expensive, toxic, and is not accessible to all. Medicinal plants persist to supply chemotherapeutics, for example, paclitaxel, cannabidiol, noscapine, etoposide, vinblastine, and curcumin. Chapter IV of this treatise measures the growth inhibitory and cytotoxicity of alfalfa leaf extracts against four clinical cancer cells (Figures 4.1-4.12). This treatise records the in-vitro bioactivity that establishes potential anti-microbial, oxidative stress, low-grade chronic inflammation and anti-neoplastic therapeutic relief induced by alfalfa leaf extracts. Antimicrobial drug resistance in pathogenic organisms poses a significant threat to public health and quashes our clinical ability to treat common infections. Clinical interventions such as surgery are threatened by nosocomial spreading of drug resistant infections. The clinical pipeline of new antimicrobial drugs is dry; furthermore, there is also an unequal access to antibiotics globally. Chapter V of this treatise records the antimicrobial activity and the MIC measuring of alfalfa leaf extracts towards clinical pathogens (Figures 5.3-5.7). The cytotoxicity screening of alfalfa extracts in green monkey kidney cells (Vero cells) is recorded in Chapter V (Figures 5.8-5.11). Four manuscripts were submitted and following scientific peer review they were accepted and published. Published manuscripts are attached at the end of the thesis as appendices, including abstract acceptances by national and international conferences. In conclusion, the emerging burden of diseases and resistance to available therapeutics indicates an urgent demand of scientific innovation of healing medicines. The clinical care system is triangular, where clinical symptoms lead to diagnostic testing and then laboratory results will inform the therapeutics to be used. While the traditional healthcare system of times past was circular by nature, healing of ailments or clinical manifestation was more holistic. Prevention of diseases and risk factors is the routine of holistic healing methods and medicinal plants play a central role therein. Our experiments validate scientifically the holistic medicine claims of the therapeutic potency of Medicago sativa. The laboratory testing of medicinal plants is the critical step in drug discovery and development research and this treatise contributes new knowledge that can be used for further testing of active compounds from the alfalfa leaf.