Bioprospecting of Natural Compounds from Brazilian Cerrado Biome Plants in Human Cervical Cancer Cell Lines.

Cervical cancer is the third most common in Brazilian women. The chemotherapy used for the treatment of this disease can cause many side effects; then, to overcome this problem, new treatment options are necessary. Natural compounds represent one of the most promising sources for the development of new drugs. In this study, 13 different species of 6 families from the Brazilian Cerrado vegetation biome were screened against human cervical cancer cell lines (CCC). Some of these species were also evaluated in one normal keratinocyte cell line (HaCaT). The effect of crude extracts on cell viability was evaluated by a colorimetric method (MTS assay). Extracts from Annona crassiflora, Miconia albicans, Miconia chamissois, Stryphnodendron adstringens, Tapirira guianensis, Xylopia aromatica, and Achyrocline alata showed half-maximal inhibitory concentration (IC50) values < 30 µg/mL for at least one CCC. A. crassiflora and S. adstringens extracts were selective for CCC. Mass spectrometry (Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometer (ESI FT-ICR MS)) of A. crassiflora identified fatty acids and flavonols as secondary compounds. One of the A. crassiflora fractions, 7C24 (from chloroform partition), increased H2AX phosphorylation (suggesting DNA damage), PARP cleavage, and cell cycle arrest in CCC. Kaempferol-3-O-rhamnoside and oleic acid were bioactive molecules identified in 7C24 fraction. These findings emphasize the importance of investigating bioactive molecules from natural sources for developing new anti-cancer drugs.

Bioprospecting the antimicrobial, antibiofilm and antiproliferative activity of Symplocos racemosa Roxb. Bark phytoconstituents along with their biosafety evaluation and detection of antimicrobial components by GC-MS.

BACKGROUND: Plants provide a ray of hope to combat the ever increasing antibiotic resistance and Symplocos racemosa is a valuable medicinal plant. The study focused on highlighting the importance of this plant's phytoconstituents as potential source of novel antimicrobials against planktonic as well as biofilm forming microorganisms, along with their antiproliferative activity. The biosafety of the phytoconstituents was also established, followed by detection of probable antimicrobial components. METHODS: The best organic extractant and major groups of phytoconstituents were tested for their antimicrobial activity against reference microbial strains and drug-resistant clinical isolates. The anti-proliferative potential of the most active group of phytoconstituents was evaluated against cancerous cell lines. The in vitro biosafety of phytoconstituents was evaluated by Ames and MTT assay, while in vivo biosafety of the most active phytoconstituents, i.e., flavonoids was determined by acute oral toxicity. Further, the probable antimicrobial components in the flavonoids were detected by TLC and GC-MS. RESULTS: Ethyl acetate extract was the most effective among various organic extracts, whereas phytoconstituents such as flavonoids, cardiac glycosides, saponins, tannins, triterpenes and phytosterols were the major groups present, with flavonoids being the most potent antimicrobials. The phytoconstituents displayed a significant antibiofilm potential, as exhibited by inhibition of initial cell attachment, disruption of the pre-formed biofilms and reduced metabolic activity of biofilms. The phytoconstituents were significantly active against the drug-resistant strains of E.coli, MRSA and Salmonella spp. Further, flavonoids showed significant cytotoxic effect against the cancerous cell lines but were non-cytotoxic against Vero (normal) cell line. All the test preparations were biosafe, as depicted by the Ames test and MTT assay. Also, flavonoids did not induce any abnormality in body weight, clinical signs, biochemical parameters and organs' histopathology of the Swiss albino mice during in vivo acute oral toxicity studies. The flavonoids were resolved into 4 bands (S1-S4), where S3 was the most active and its GC-MS analysis revealed the presence of a number of compounds, where Bicyclo [2.2.1]heptan-2-one,1,7,7-trimethyl-, (1S)- was the most abundant. CONCLUSIONS: These findings suggest that the phytoconstituents from Symplocos racemosa bark could act as potential source of antimicrobial as well as antiproliferative metabolites.

Bioprospecting microalgae from natural algal bloom for sustainable biomass and biodiesel production.

Natural algal bloom consists of promising algal species which could be a feasible option for the source of bulk biomass and biodiesel production. It has been found in five natural fresh water algal blooms (Uttar Pradesh, India), containing high nitrogen (N) (4.6 ± 0.32 mg/L) and phosphorus (P) (4.12 ± 0.29 mg/L) concentration during spring (23.9-25.9 °C) and summer season (32.0-35.0 °C). Among the isolated algae from naturally occurring bloom, Chlorella sorokiniana MKP01 exhibited highest biomass (1.02 ± 0.02 g/L) and lipid content (174.1 ± 9.6 mg/L) in untreated tap water and urea/single super phosphate (SSP) in the ratio (2:1). The biodiesel quality was assessed and found to be with the Indian and international standards. Algal bloom was artificially developed in the open pond containing 10,000 l tap water supplemented with Urea/SSP (2:1) for a consistent supply of bulk biomass, yielded 8 kg of total biomass and lipid 1.3 kg.

Development of a Phage Display Panning Strategy Utilizing Crude Antigens: Isolation of MERS-CoV Nucleoprotein human antibodies.

Antibody phage display has been pivotal in the quest to generate human monoclonal antibodies for biomedical and research applications. Target antigen preparation is a main bottleneck associated with the panning process. This includes production complexity, downstream purification, quality and yield. In many instances, purified antigens are preferred for panning but this may not be possible for certain difficult target antigens. Here, we describe an improved procedure of affinity selection against crude or non-purified antigen by saturation of non-binders with blocking agents to promote positive binder enrichment termed as Yin-Yang panning. A naïve human scFv library with kappa light chain repertoire with a library size of 109 was developed. The improved Yin-Yang biopanning process was able to enrich monoclonal antibodies specific to the MERS-CoV nucleoprotein. Three unique monoclonal antibodies were isolated in the process. The Yin-Yang biopanning method highlights the possibility of utilizing crude antigens for the isolation of monoclonal antibodies by phage display.

The Ethnopharmacologic Contribution to Bioprospecting Natural Products.

Descriptions of the use of natural products in traditional medicine have served as starting points for new therapeutics. The details of the traditional use of these organisms can provide important information for future drug discovery and development efforts. Recent technologic advances provide the framework to leverage ethnopharmacologic data in the drug discovery process. Information on the traditional harvest, preparation, storage, and administration of the organisms, and the natural products they contain, provides valuable details regarding characteristics of the active compounds. Importantly, researchers can now rapidly analyze and identify the multiple, and often synergistic, compounds contained in these natural products. Although we are entering the acme of ethnopharmacology, where information regarding the traditional use of organisms can provide valuable natural product leads and accelerate the identification of new therapeutics, this ethnopharmacologic resource is threatened by the loss of traditional medicine knowledge and extinction of organisms.

Endophyte bioprospecting in South Asian medicinal plants: an attractive resource for biopharmaceuticals.

From the last several years, there has been an increasing interest in plant-associated bacteria commonly referred to as endophytes that reside asymptomatically in the internal plant tissues. This interest peaked since the last two decades due to the recognition that endophytes within medicinal plants have the capability to mimic and produce the bioactive metabolites of the host plant. A number of medicinal plants have been used for centuries by the people of South Asia to cure various diseases and this has led to abundant usage experience. Relating to prior ethanopharmacological experiences, scientists have searched for medicinal plants that could be valued sources for endophytes yielding novel metabolites of pharmaceutical importance. This review is therefore an effort to encompass the most recent efforts in the exploration of medicinal plants of South Asia and their endophytes.

Ethnobotanical Study.

Ethnobotany is the study of interrelationships between human cultures and plants, animals, and other organisms in their environment. It also creates an awareness of the link between biodiversity and cultural diversity. From the beginning of civilization, people have been using plants for various purposes like food, shelter, medicines, etc. Ethnobotanists play a key role in exploring these kinds of information from indigenous people which creates a gateway for formulating a novel drug. The content in this chapter deals with these aspects in an approachable manner.

Pharmacognostical Studies.

The chapter deals with tools and techniques employed in pharmacognosy. Pharmacognostic evaluation helps to screen the commercial varieties, substitutes, adulterants and any other quality control of the drugs. It is a simple and reliable tool, helps to obtain information about biochemical and physical properties of crude drug. Methods such as macroscopic and microscopic analysis, maceration, histochemical colour reaction, photomicrography, organoleptic character of plant powder and extracts, fluorescence analysis of plant powder with different chemical reagents, determination of pH of plant powder, water solubility index (WSI) and water absorption index (WAI) and acid value are discussed.

Extraction of Bioactive Compounds.

A bioactive compound influences the health of living organisms and it has extranutritional constituents that typically occur in low quantities in foods, which helps to enhance or boost the immune system. Plants and their products possess bioactive compounds, i.e., secondary metabolites. Here, extraction is an important process to isolate the bioactive compounds. Biological activities of the extract show a significant variation depending on the extraction methods and this also opens a gateway for selecting suitable extraction methods. Hence, different extraction methods have been discussed in this section, which influences the extraction of phytochemicals.

Preliminary Phytochemical Studies.

Plants are the natural producers of medicinal agents like alkaloids, flavonoids, tannins, and phenolics. These phytocompounds alone or in combination act as a therapeutic agent in various disease complications. Various chemical reagents are used to determine the major phytochemicals present in plant parts. Protocols involved in screening of alkaloids, carbohydrates, glycosides, saponins, phytosterols, fixed oils, and fats are shown in this chapter.

Proximate Composition Analysis.

The proximate composition of foods includes moisture, ash, lipid, protein and carbohydrate contents. These food components may be of interest in the food industry for product development, quality control (QC) or regulatory purposes. Analyses used may be rapid methods for QC or more accurate but time-consuming official methods. Sample collection and preparation must be considered carefully to ensure analysis of a homogeneous and representative sample, and to obtain accurate results. Estimation methods of moisture content, ash value, crude lipid, total carbohydrates, starch, total free amino acids and total proteins are put together in a lucid manner.

Mineral Quantification.

Optimal intakes of elements, such as sodium, potassium, magnesium, calcium, manganese, copper, zinc and iodine, can reduce individual risk factors including those related to cardiovascular diseases among humans and animals. In order to meet the need for vitamins, major minerals, trace minerals, fatty acids and amino acids, it is necessary to include a full spectrum programme that can deliver all of the nutrients in the right ratio. Minerals are required for normal growth, activities of muscles, skeletal development (such as calcium), cellular activity, oxygen transport (copper and iron), chemical reactions in the body, intestinal absorption (magnesium), fluid balance and nerve transmission (sodium and potassium), as well as the regulation of the acid base balance (phosphorus). The chapter discusses the chemical and instrumentation techniques used for estimation of minerals such as N, P, Ca, Mg, K, Na, Fe, Cu, Zn, B and Mb.

Anti-nutritional Factors.

Anti-nutritional factors such as trypsin inhibitor, phytic acid and cyanogen are as important as nutritional content of any edible plant part. The anti-nutritional factors can be defined as those substances generated in natural food substances by the normal metabolism of species and by different mechanisms (e.g. inactivation of some nutrients, diminution of the digestive process or metabolic utilization of feed) which exert effects contrary to optimum nutrition. Hence, trypsin inhibitor, phytic acid and cyanogens present in edibles with the methods in the chapter would be helpful.

Quantification of Secondary Metabolites.

Plants are a rich source of secondary metabolites that have medicinal and aromatic properties. Secondary metabolites such as alkaloids, iridoids and phenolics generally produced by plants for their defence mechanisms have been implicated in the therapeutic properties of most medicinal plants. Hence, quantification of these metabolites will aid to discover new and effective drugs from plant sources and also to scientifically validate the existing traditional practices. Quantification of large group of phytochemicals such as phenolics and flavonoids is quantified in this context.

In Vitro Antioxidant Assays.

Various chemical in vitro assays have been developed to measure antioxidant capacities of plant products. Despite the recent popularity in the antioxidant research, the lack of standardized assays to compare research results from different research groups has been a major challenge. The examination of various antioxidant assays is required for the development of standard methods that are broadly applicable by researchers and industry. However, due to the complex nature of biological systems, there is no single universal method for measuring antioxidant capacity. Antioxidant methods such as DPPH*, ABTS+, nitric oxide, super oxide, metal chelating confirming the free radical scavenging property of the plants with widely used methods are simplified in this chapter.

In Vitro Antihaemolytic Activity.

In this assay, the rat erythrocytes are induced with hydroxyl radical damage by the addition of H2O2. This causes the rupture of the RBC and the contents leak out giving the red colour of the haemoglobin to the medium. Hence, anti-haemolytic activity has been presented here in a brief and easy manner using the rat erythrocytes.

Antimicrobial Activity.

Natural products of higher plants may possess a new source of antimicrobial agents with possibly novel mechanisms of action. They are effective in the treatment of infectious diseases while simultaneously mitigating many of the side effects that are often associated with conventional antimicrobials. A method using scanning electron microscope (SEM) to study the morphology of the bacterial and fungal microbes and thus determining antimicrobial activity is presented in the chapter.

In Vitro Anthelmintic Assay.

Nematodes are believed to have originated 1000 million years ago as free living anaerobic benthic organisms. Modern anthelmintic drugs have been in use but the worms have created resistance to most of the broad spectrum anthelmintics such as benzimidazoles, levamisole, avermectins, etc. However, these drugs cause side effects. More recently, plant extracts have been reported as having developed resistance against anthelmintics. A study using earthworms to determine the activity has been developed and explained in a well-defined manner in this chapter.

Toxicity Studies.

Toxicity studies in the animal models are done to determine the dose level recommended for the treatment of disease as drug. This guideline enables the characterization of adverse effects following repeated daily inhalation exposure to a test. This chapter includes oral and dermal toxicity studies which are discussed as per OECD guidelines. Both acute and subacute toxicity studies are given special emphasis.