Investigation of biomolecules from the fractions root extract of Medicago sativa using GC-MS

Vaibhavi Savalia, Devang Pandya

Background: According to Ayurveda, Homeopathic, Chinese traditional system of medicine & modern research, Alfalfa (Medicago sativa, Family Leguminosae) is used as hypocholesterolemic, antioxidant, hypolipidemic, and in atherosclerosis, heart disease, cancer and in stroke. The root of Alfalfa can be used to prepare different formulation like powder, pill, or decoction for lowering cholesterol & lipid. Moreover, methanol extract of roots of M. sativa demonstrated significant inhibition of Angiotensin-converting enzyme.

Objective: So, the present study was carried out to identify the phytoconstituents present in the fractions of methanolic extract of the root of M. sativa by GC-MS analysis.

Materials and methods: Successive methanol extract was prepared by maceration using toluene, chloroform, and methanol. Fractions were isolated by flash chromatography from methanol extract. Chemo- profiling of selected fractions based on TLC was done using GC-MS. The interpretation of the results of GC-MS was ascertained using the database of the National Institute Standard and Technology (NIST) library. A detail literature survey was performed to find therapeutic use of phytoconstituents detected by GC-MS analysis.

Result: GC-MS of fractions of M. Sativa root methanol extract revealed the presence of a total of 28 phytoconstituents out of which, 16 were found medicinally important. Two of those phytoconstituents, didodecyl phthalate, and lupeol, as supporting evidence of antihypertensive activity. Another important phytoconstituent found was Cis-tris methoxy resveratrol, a potent anticancer agent, reported for the first time in M. sativa root.

Conclusion: The results suggest that the root of Medicago sativa contains the potential medicinal value, yielding various phytoconstituents that confirm the application of this plant in various ailments.

The most effective way to reduce the worldwide burden of liver cancer is to prevent it from happening in the first place. The current treatment of liver cancer has significant side effects. Hence, there is a need to develop anti-liver cancer agents of plant origin, which are less toxic, more efficacious and cost-effective. The present study has been performed experimentally by in vitro to examine the anti-liver cancer activity of roots of Bauhinia tomentosa L (Fabaceae). The roots of B. tomentosa was tested for its anti-cancer activity against HUH-7 human liver cancer cell lines by MTT assay. The standard used in this assay was Camptothecin (CPT) at 25µG concentration. Plant extract was tested at 25μg/mL, 50 μg/mL,100μg/mL, 200 μg/mL and 400 μg/mL concentrations. The percent cell viability of standard drug was found to be 49.59% and plant extracts at 25 μg/mL, 50 μg/mL,100 μg/mL, 200 μg/mL and 400 μg/mL concentrations were found to be 93.82%, 86.21%, 74.48%, 63.04%, 45.71% respectively. The cell morphology was observed and recorded under a microscope. The results clearly indicated that B. tomentosa shows a dose-dependent activity and it was maximum at 400μg/mL concentration where it shows 45.71% of liver cancer cell viability and it was comparable to the standard drug where it shows 49.59% of viability.

ABSTRACT

The stem barks of Securidaca longepedunculata are used traditionally across Africa for the treatment of diabetes, cancer, metabolic diseases and asthma. This study was aimed at investigating the potential enzyme activities of the S. longepedunculata on the small intestine. Aqueous extracts of S. longepedunculata were tested in vivo on animal models: A total of 12 Wister rats were assigned into four (I–IV) groups of three animals each. Group I served as the control and was administered 0.5mL of distilled water. Groups II–IV were given 0.5, 1 and 2mg/kg body weight of S. longepedunculata stem bark extracts. The activities of the following enzymes, Alanine transaminase (ALT), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP), Aspartate transaminase (AST), were assayed in the small intestine. The result revealed a significant reduction in ALP and LDH. This gives an indication that the administration of aqueous extract of securidaca longepedunculata can elicit detrimental effect in the small intestine of the albino rat. Also the result obtained in the qualitative analysis shows the presence of phytochemicals such as: saponin, flavonoid and terpernoid.

Polyketide synthases type I (PKS-I) gene cluster is responsible for the synthesis of highly assorted group of secondary metabolites such as antimicrobial and anticancer agents. In our study, screening was carried out using degenerate primers to determine the presence of PKS-I gene cluster in endophytic actinomycetes isolated from two medicinal plants Ocmium teniflorum (Tulsi) and Azadirachta indica (Neem). A total of 28 endophytes that were isolated and identified from our previous study were further confirmed through 16S rRNA gene sequencing to exhibit a 99% similarity with Streptomyces sp. The molecular screening using PCR revealed the presence of PKS- I gene with a product size of 750bps in the isolates, FHK-1, FHK-2, FHK-3, FHK-4, FHK-5, FHK-6, FHK-7, FHK-8, FHK-9, FHK-11, FHK-13, FHK-16, FHK-18, FHK-20, FHK-21, FHK-23, FHK,25 and FHK-28. These isolates were further checked for their antimicrobial potential using their crude extracts. They displayed prominent bioactivity against ATCC pathogens, Escherichia coli, Proteus vulgaris, Rhodococcus equi, Staphylococcus epidermidis, Enterococcus faecalis, and Acinetobacter baumanii. Our study revealed that the endophytes from O. tenuiflorum and A. indica are bioactive and versatile harboring the PKS-I gene cluster.

Daucus carota seeds are important in the treatment of a broad spectrum of ailments. This study evaluated the phytochemicals status and in vitro antioxidant effects of aqueous and diethyl ether extracts of D. carota seed. 50 g of D. carota seeds were pulverized and dissolved in 200 ml distilled water for 24 hours, the crude aqueous extract obtained (AQE, 11.05g, 22.1% yield) was partition in water/diethyl ether (2:1) to yield the diethyl ether fraction (DEE, 0.45g, 0.90% yield). The extracts were evaluated for quantitative phytochemicals, 2,2-diphenyl-1-picrylhydrazyl (DPPH), total antioxidant capacity and ferric ion reducing effect at 0.4 to 2.0 mg/ml. AQE and DEE contain alkaloids (7.28%, 5.22%), flavonoids (5.56%, 4.03%), saponins (4.68%, 1.52%), steroids (3.23%, 2.71%), tannins (0.40%, 0.27%) and phenol (0.37%, 0.22%) respectively. The DPPH scavenging effect of the extracts was superior to the reference compound butylated hydroxytolune (BHT) with IC₅₀ of 2.84 mg/ml compared to AQE with 2.00 mg/ml and DEE with 1.39 mg/ml respectively. AQE extract when compared with BHT had similar total antioxidant capacity while BHT was significantly (p<0.05) higher than DEE. However, both AQE and DEE express better ferric ion reducing effect compared to BHT at the concentrations range. The results shows that aqueous and diethyl ether extracts of D. carota seed possess good in vitro radical scavenging activities with presence of higher phytochemical content in the aqueous extract.

In current scenario, several surgical braces are available for different body parts like knee, elbow, back etc. for several clinical conditions, especially pain in that part of the body. All these supportive treatments are aided by some medication through oral route, which produces several side effects on prolonged use. Present work endeavors to combine these different treatments: supportive and medicated treatments by incorporating sustained release medicated topical patch into the inner side of surgical braces through specially designed patch holder mechanism for easy incorporation and removal of patch. It not only eliminates need of oral medication but also gives localized administration of medication, thus resulting in more patient compliant treatment with improving overall safety profile. For developing model prototype of the same, combination of surgical knee brace and oral NSAID (diclofenac) was selected. In order to execute the working prototype, a pocket mechanism to incorporate a sustained release patch of 7 cm X 7 cm size was designed. Patch formulation was randomly selected based on trial batches for sustaining drug release up to 12 h, considering put on- put off phases of brace of 12 h each. The pocket was designed in such a way that in case of any discomfort or other unfavorable conditions, it can be easily removed from the brace. Flexibility in terms of size, shape and drug release profiles proves its versatility for other supportive braces and medications. Thus this innovation can be easily extended for other braces like elbow braces, shoulder braces, back braces etc. It not only enhances efficacy of the current treatments but also improves patient compliance significantly.

Cancer is a group of diseases characterized by a wide diversity in location, cellular features and aggressiveness. Nonetheless, a common aspect seen in different types of cancer, namely in carcinomas, is the alteration in post-translational modifications of proteins, particularly in protein glycosylation [1]. Due to increased or decreased expression of particular enzymes participating in glycosylation of proteins, different glycan structures are formed, which are typical of tumoral cells. When the respective glycoproteins are secreted into the blood stream, these aberrant structures can be used as valuable cancer biomarkers, since they are not synthesized by normal cells.

Glycan structures are efficiently and selectively detected by lectins, which are proteins of natural origin with high affinity for a particular or a very small group of glycan epitopes. Lectins are used for glycan detection as antibodies are used for protein recognition. Therefore, they have been employed in diverse analytical techniques when the aim is to selectively detect or capture specific glycans from a complex sample.

Lectin biosensors are attractive devices for the detection of cancer-associated glycobiomarkers in serum since they combine the advantageous aspects of biosensors (portability, easy use in point-of-care analysis, low sample requirement) with the high selectivity of lectin biorecognition. This work presents three lectin-based impedimetric biosensors for the selective detection of specific aberrant cancer-associated O-glycans, namely STn, Tn and T antigens, which are well-established pan-carcinoma biomarkers. For these three biosensors, Sambucus nigra agglutinin, Vicia villosa agglutinin and Arachis hypogeae agglutinin were used as biorecognition elements, with specificity for STn, Tn and T antigens, respectively. The binding event between each lectin and the corresponding aberrant O-glycan was monitored by electrochemical impedance spectroscopy, measuring the increase in the biosensor’s impedance after incubating the samples. The increase in impedance was related to the lectin-glycan complex formation.

All biosensors were constructed following the same general procedure, demonstrating its high versatility. A thorough characterization and validation of the biosensors’ performance was carried out, evaluation their selectivity, sensitivity and ability to discriminate between samples from healthy donors and from cancer patients with different carcinomas. Using the three lectin biosensors in the analysis of the same sample could also help to characterize the glycosylation profiles of glycoproteins in the diverse types of carcinomas.

[1] Pinho S.S., Reis C.A. Glycosylation in cancer: mechanisms and clinical implications. Nat. Rev. Cancer 15 (2015) 540-555.

The protein coding-information only represents a small portion of the genetic load of a living organism. It is well established that essential information codes functional RNAs, called non-coding RNAs (ncRNAs), which play key roles in the essential biological processes of the cell life. Many mRNAs also act as truly ncRNAs besides being translated into proteins. Therefore, the repertoire of potential drug targets to fight diseases goes beyond proteins. Viral RNA genomes encode all the information for completion of the infectious cycle. They are multifunctional molecules, which act as replication templates and mRNAs. Further, defined structural domains in viral RNA genomes play key functions for the completion of the viral cycle and the regulation of the essential processes; these domains have also been involved in virulence. The West Nile Virus (WNV) genome consists in a single stranded RNA molecule, which contains a single ORF flanked by untranslated regions (UTRs). The 3’UTR is required for efficient translation, but the mechanisms involved in this regulation are still obscure. In this work, we show evidences that the WNV-3’UTR specifically recruits the 40S ribosomal subunit. We have localized two potential binding sites of the 40S. Binding of the 40S induced conformational changes in highly conserved structural domains within the WNV-3’UTR. Functional assays support the hypothesis that recruitment of the 40S particle by the 3’UTR is required for an efficient translation. Interfering with the 40S recruitment, by targeting the WNV-3’UTR binding sites, constitutes a potential antiviral strategy by the development of new therapeutic compounds.

Migraine is one of the most common neurological disorder, which has long been related to brain serotonin (5-HT) depletion and neuro-inflammation. Despite many treatment options are available, the frequent occurrence of unacceptable adverse effects further supports the research toward nutraceuticals and herbal preparations, among which Tanacetum parthenium and Salix alba showed promising anti-inflammatory and neuro-modulatory activities.

The impact of extract treatment on astrocyte viability, spontaneous migration and apoptosis was evaluated. Anti-inflammatory/anti-oxidant effects were investigated on isolated rat cortexes exposed to a neurotoxic stimulus. The lactate dehydrogenase (LDH) release, nitrite levels and 5-HT turnover were evaluated, as well. A proteomic analysis was focused on specific neuronal proteins and a fingerprint analysis was carried out on selected phenolic compounds.

Both extracts appeared able to exert in vitro anti-oxidant and anti-apoptotic effects. S. alba and T. parthenium extracts reduced LDH release, nitrite levels and 5-HT turnover induced by neurotoxicity stimulus. The downregulation of selected proteins suggest a neurotoxic, which could be ascribed to an elevate content of gallic acid in both S. alba and T. parthenium extracts.

Concluding, both extracts exert neuroprotective effects, although the downregulation of key proteins involved in neuron physiology suggest caution in their use as food supplements.

Inflammatory bowel diseases (IBDs) are chronic, relapsing and multifactorial disorders of the colonic mucosa, which show increased and unbalanced intestinal immune response to external stimuli. Plant-derived extracts were described to possess the capability in contrasting IBDs-related oxidative stress and inflammatory pathways. In the present study, we investigated the water extract of Harpagophytum procumbens DC. ex Meisn. in an experimental model of IBD. Additionally, a microbiological investigation was carried out to discriminate the efficacy against bacterial and fungal strains involved in IBDs. An untargeted proteomic analysis was also conducted on more than 100 colon proteins involved in tissue morphology and metabolism. The extract showed the ability to blunt the level of selected biomarkers of oxidative stress and inflammation, including serotonin, prostaglandins, cytokines and transcription factors_. Additionally, the extract inhibited the growth of Candida albicans and C. tropicalis, in vitro. The extract was also able to exert a pro-homeostatic effect on the levels of a wide plethora of colon proteins, thus corroborating protective effects against the burden of inflammation and oxidative stress. On the other hand, the supra-physiological downregulation of cytoskeletal-related proteins involved in tissue morphology and antimicrobial barrier function suggests caution in the use of food supplements enriched with H. procumbens.