HR-LCMS Phytochemical Profiling and Evaluation of Anti-Diabetic Activity of Amphiroa Fragilissima by Key Enzymes Inhibition Assay

Abstract

Introduction: Considering the seaweed extracts and their bioactive constituents which are able to inhibit enzymes involved in carbohydrate metabolism and reduce blood glucose during fasting, random and postprandial and same had been witnessed with in-vivo and in-vitro studies. In addition to their many industrial applications, seaweeds are well known for their ability to improve community nutritional status since they are richer sources antioxidants, macronutrients, and vitamins (B12, A, and K) than other plants. Seaweeds differ in their protein content depending on the species. It is found that the protein content of red and green seaweeds is higher than that of brown seaweeds. The red seaweeds like Palmaria palmata (dulse) and Porphyra tenera (nori) were observed that 36% and 49% of the dry matter consisted of macromolecule like protein respectively. Consequently, there is widespread use and significant exploitation of these two species. Recent investigations highlight novel prospects in the realm of medicine related to bioactive compounds derived from seaweeds. The potential bioactive substances of sea weeds such as alkaloids, flavonoids, polysaccharides, fatty acids, and polyphenols have antibacterial, antioxidant, anticancer, and immune-boosting qualities as functional foods.

Objectives: The current management of diabetes mellitus could be achieved with the help of synthetic hypoglycemic medications; however long-term use of these medications may lead to several side effects. So, there is a need for the natural compounds with anti-diabetic potential without any side effects. The current work intended to screen phytochemicals using High Resolution-Liquid Chromatography Mass Spectrometry and examine the in-vitro anti-diabetic properties for hydro-ethanolic extracts obtained from Amphiroa Fragilissima (HEEAF) by enzyme inhibition method.

Methods: The phytochemical analysis revealed the presence of alkaloids, flavonoids, proteins, free amino acids, gums, mucilage, carbohydrates, sterols, saponins, tannins, and polyphenols in HEEAF. The HR-LCMS study also proved the presence of various biologically active secondary metabolites like, Erucamide, 4-Hydroxybenzocyclobutene-1,2-Dione, Pentadecyl N-pentanoylalaninate, 7- Dehydrodesmosterol, Threo-Sphingosine, (-)-,Diethylene glycol n-butyl ether, Oleamide, sphinganine, Betaine, 2-Aminopalmitic acid, Stearoyl ethanolamide, Undecanamide, Coumarandione, Pentadecyl N-pentanoylalaninate, Octyl 5-O-pentanoyl-1-thiopentofuranoside, DL-Carnitine, Pentadecyl N-pentanoylalaninate, Embelin, Azelaic acid, 4-Caproylresorcinol, Pregna-4,6-diene-3,20-dione, (+)-[6]-Gingerol, Methyl (3E,6E)-3,6-dodecadienoate, BHQ, Brassylic acid and 4-Undecyl benzenesulfonic acid etc., in HEEAF. The anti-diabetic potential of HEEAF was investigated through the inhibitory action on α-amylase and α-glucosidase.

Results: The HEEAF showed significant increased inhibitory effects on α-amylase with IC₅₀ value of 9.14 µg/ml against 3.12µg/ml for acarbose as the reference standard. Similarly, the significant inhibitory effect was showed on α- glucosidase with IC₅₀ values of 65.34 µg/ml against 25.95µg/ml for voglibose as the standard drug. The in-vitro study revealed the possible mechanism of anti- diabetic activity of Amphiroa Fragilissima by enzyme inhibition method and can be carried forward for toxicity study as per OECD guidelines followed by in-vivo studies to prove its safety and efficacy.

Conclusions: It has been concluded that, the important secondary metabolites had been identified in HEEAF by performing the preliminary phytochemical and HR-LCMS analyses. The investigation of inhibitory activities of A. fragilissima on α-amylase and α-glucosidase sheds information on the potential application of this seaweed in the management of diabetes. Subsequent isolation of the crude extracts will be needed to determine the primary compound accountable for its anti- diabetic effect. Further in- vivo research is required to confirm the pharmacological features of Amphiroa fragilissima as promising anti-diabetic drug. Therefore, HEEAF can be applied in the treatment and management of diabetes mellitus after performing toxicity studies as per OECD guidelines to establish their safety.