A. Shukla, P. Pokhriyal*
Department of Chemistry, Kanya Gurukul Campus, Gurukul Kangri Vishwavidyalaya,
Haridwar-249404, Uttrakhand, India.
Background: This study aimed to examine the anti-inflammatory and antidiabetic effects of the green solvent extract from F.subincisa Buch-Ham.ExSm.This paper also explains how bio-based products have been highly safe and alternative to the use of organic solvent. Methods: The serial extraction was carried out with increasing polarity using a magnetic stirrer: d-limonene, Isopropyl alcohol, Ethyl lactate, and Hydroalcohol.The concentrated and dried extracts were subjected to analysis the antidiabetic activity and anti-inflammatory activity were assessed by employing standard in vitro techniques. Result: In vitro anti-inflammatory assays were performed using heat-induced bovine serum albumin (BSA) denaturation and erythrocyte membrane stabilization assays.α-amylase and α-glucosidase were used to evaluate the anti-diabetic activity of F.subincisa bark in vitro. Diclofenac sodium, Acarbose, and aspirin were used as reference drugs. The in vitro method showed significant anti-inflammatory and anti-diabetic properties of different bio-based extracts tested. Conclusions: Findings from this study showed that F.subincisa exhibited substantial anti-inflammatory and antidiabetic actions in vitro models.
The aversion towards green solvents has generated great interest and growing demand, as an alternative to organic solvents which have several major disadvantages such as their high volatility, flammability, and toxicity1. The ideal alternative solvents suitable for green extraction should be easy to recycle without any deleterious effect on the environment, great solvency, high flash points with low toxicity and low environmental impacts, be easily biodegradable, obtained from renewable resources at a reasonable price2.
Overweight and obesity are major risk factors for several chronic diseases, including diabetes, cardiovascular diseases, and cancer. It leads to the enlargement of adipocytes along with infiltration of immune cells which causes persisting low-grade inflammation, it further increases the number of inflammatory cytokines and production of free fatty acids in body fluid3. The concept and the molecular pathways that link inflammation and insulin have been demonstrated for more than two decades4, 5. Obesity and inflammation are interrelated to each other as in a cycle that develops insulin resistance in various tissues and cells that are insulin sensitive like liver cells, skeletal muscles, and adipocytes which lead to type 2 diabetes (T2D)6. T2D is a chronic illness that is caused due to insufficient insulin secretion that is distinguished by an abnormal increase in glucose level in blood due to lessening insulin production or insulin sensitivity. About 25% of the world population including developing or developed countries suffering from Diabetes mellitus. In China with some estimated that 110 million persons are diabetic which is the highest number of diabetics worldwide, followed by India (70million)and the USA(30million)persons. According to WHO diabetes will be the 7th leading cause of death in 20307. Thus, continuous research with natural aliquots from plants with hypoglycemic effects is vital. Such research will offer new and more constructive therapeutic approaches for the treatment of diabetes and its imminent complications8, 9.
Inflammation is a response of living tissues towards injury and it involves foundational and nearby responses. Regardless of our reliance on neighborhood medication and the huge advances in engineered drugs, an enormous number of the world populaces (80% of individuals) can't bear the cost of the results of the western pharmaceutical industry and need to depend upon the utilization of customary prescriptions, which are principally gotten from plant material10, 11. The truth of the matter is very much perceived by the WHO which has as of late conformed to a stock of therapeutic plants posting more than 20000 species. The family Moraceae comprises a few significant therapeutic plants with a wide scope of pharmacological, natural exercises, and intriguing phytochemical constituents 12, 13. It is the body's reaction to inactivate or decimate the attacking life forms, to evacuate the aggravations, and set up for tissue fix. It is activated by the arrival of compound go-betweens from harmed tissue and relocating cells. The regularly utilized medication for the administration of incendiary conditions is non-steroidal calming drugs, which have a few antagonistic impacts particularly gastric disturbance prompting arrangement of gastric ulcers14. Towards the expansion of modern medicine, natural products have contributed significantly. Recently established medicine globally is being reexamined by extensive investigation on different plant species and their vital curative principles. The wealthy plant kingdom can represent a refreshing source of noble compounds with notable anti-inflammatory activities15. The excellence of herbal medicine seems to be theirs recognize effectiveness, low incidence of serious adverse effects, and low cost.
The plant under study is F.subincisa growing in the Uttrakhand Garhwal region. In the way of penetrating for natural curing agents, the authors did not find reported data about any research of this species, so the present work was carried out to standardize its biological activity to help with its common use as a medicinal plant.
MATERIALS AND METHODS:
Collection and Identification of Plant Material:
The selected plant F.subincisa was collected from the Tehri Garhwal regions of Uttrakhand. The plant was identified and authenticated with the help of the department of Botanical Survey of India and Voucher specimens (N0-31, 02/2019) of the plants were deposited in the herbarium center of the Department of Botanical Survey of India, Dehradun, Uttrakhand, India. The shade dried and powdered plant samples were preserved for further experimentations.
Sequential extraction of the plant samples:
Bark was washed under a running tap, followed by rinsing with distilled water, shade dried, and pulverized in a mechanical grinder to obtain coarse powder. The powdered material was stored in an airtight container until use The shed dried powdered plant samples (50 g, soaked overnight) were sequentially extracted in d-limonene, isopropyl alcohol, ethyl lactate, and hydro alcohol up to 2 h using a flat bottom flask attached to water condenser is placed on the magnetic stirrer. The samples were evaporated under reduced pressure in a vacuum oven. The dried extracts were preserved at 40C in the refrigerator for further analysis16.
Chemicals and reagents:
Analytical grade chemicals were used in the study. Bovine serum albumin was obtained from Sigma-Aldrich, Tris buffer (Merck), p-Nitrophenyl-α-Dglucopyranoside (SRL Pvt., Ltd.,), α-amylase (SRL Pvt., Ltd.,),3,5-dinitrosalicylic acid (DNSA) (SRL Pvt., Ltd.,), α-glucosidase (SRL Pvt Ltd),d-limonene (Lobachemie), ethyl lactate (Lobachemie), ethanol (Merck), isopropyl alcohol (Merck), sodium carbonate (CDH), Sodium citrate, Citric acid, Sodium chloride, Sodium hydroxide, and Dihydrogen phosphate was purchased from the vendors as listed. Reference standard Sodium Diclofenac, Acarbose, and Aspirin were obtained from Cipla Ltd, Bangalore. UV-Visible spectrophotometer (Systronics 118) was used for the estimation of antidiabetic and anti-inflammatory activity.
Assessment of antidiabetic activity by using in vitro assays:
Alpha-amylase inhibitory assay:
In vitro amylase inhibition was studied with some modification(17). In brief, 1 ml of the test extract was allowed to react with 1 ml of α-amylase enzyme and 1 ml of 20 mM of phosphate buffer (pH-6.9). After 30-minute incubation at 37 °C, 1 ml of 1% starch solution was added. After incubation for15 minutes at 37 °C, 1 ml of 96 Mm dinitrosalicylic acid reagent was added to both control and test. The absorbance was recorded at 540 nm using a UV-Visible spectrophotometer
α-Glucosidase inhibitory assay:
Plant aliquots were evaluated for α-glucosidase inhibitory activity according to the method with slight modifications(8). Plant extracts (1 mL) at varying concentrations were incubated with 1 ml of the α-glucosidase enzyme solution (1 U/mL) for 30 min at 37 °C with an additional 1ml of 100 mM phosphate buffer (pH 6.8). After 20 min, the reaction was started with the addition of 1mL of 5 mM p-NPG, and the mixture was incubated for 15 min. The reaction was terminated with the addition of 0.5 M of Tris buffer (4 ml) and final absorbance was measured at 410 nm using a UV-Visible spectrophotometer. Acarbose was used as a standard at varying concentrations.
Evaluation of anti-inflammatory by using in vitro assays:
Protein denaturation method:
The assay method was carried out with some modifications18). The plant extracts were prepared at different concentrations (μg/ml). The assay mixtures were prepared by adding 2.8 ml of 50 mM potassium phosphate buffer (pH 7.5), 2 ml sample solutions, 0.2 ml of freshly prepared egg albumin solution. The mixtures were incubated at 70°C for 10 min. The inhibitory activities were assayed spectrophotometrically at 660 nm using a UV-Visible spectrophotometer.
HRBC Membrane stabilization method:
The anti-inflammatory activity of various extracts of Bark of F.subincisa was assessed by in vitro HRBC membrane stabilization method19. Blood was collected from healthy volunteers. The blood was mixed with an equal volume of Alsever’s solution, and centrifuged with isosaline. To 1 mL of HRBC suspension, an equal volume of test drug in different concentrations, 50,100, 500, and 1000 μg/mL, was added. All the assay mixtures were incubated for 30 minutes and centrifuged at 37°C. The hemoglobin content in the supernatant solution was estimated by using a UV-Visible spectrophotometer at 560 nm.
RESULTS AND DISCUSSION:
α-amylase and α-glucosidase assays:
The inhibitory effects of different green extract fractions on α-amylase enzyme were evaluated by the in-vitro method. All the fractions exhibited inhibitory action against the enzymes in a concentration dependant manner. The limonene, isopropyl alcohol, ethyl lactate, and hydro alcohol fractions exhibited the highest α-amylase inhibitory activity of 63.67, 86.67, 90.34, and 62.85 % respectively at a concentration of 1500μg/ml (Figure 1). The alpha-glucosidase inhibitory activity of different fractions of F.subincisa bark was evaluated by the in-vitro method. All the fractions exhibited inhibitory action against α- glucosidase in a dose-dependent manner. The limonene, isopropyl alcohol, ethyl lactate, and hydro alcohol fractions exhibited the highest α-glucosidase inhibitory activity of 59.09, 52.59, 79.22, and 87.66% respectively at a concentration of 1000μg/ml (Figure 2). Results of the study showed that the hydro alcohol extract has the strongest α-glucosidase activity as compared to other fractions at all tested concentrations. A standard reference drug was used as acarbose for both α-amylase and α-glucosidase enzyme inhibitory assay. The standard enzyme inhibitor showed α-amylase inhibitory activity with an IC50 value of 154.87±2.33 µg/ml and α-glucosidase inhibitory activity with an IC50 value of 105.63±1.71 µg/ml. Among all, ethyl lactate and hydro alcohol fraction have shown the highest α-amylase and α-glucosidase enzyme inhibitory activity with an IC50 value of 418.34±2.73 and 265.92±0.67 µg/mL respectively which were comparable with that of acarbose. Highest inhibitory activity indicative Lowest IC50 values.
Fig. 1 Inhibition of a – amylase by F. subincisa extract
Fig. 2 Inhibition of a – glucosidase by F.subincisa extract
HRBC membrane stabilization and inhibition of protein denaturation assays:
The results of anti-inflammatory activity determined by the Bovine Serum Albumin Assay (BSA) and human red blood cell membrane stabilization test were shown in Fig.3 and Fig.4. The hydro alcohol and ethyl lactate of bark showed a concentration-dependent anti-inflammatory activity, and the protection percent increased with an increase in the concentration of the samples. At a concentration of 1000 μg/ml, the hydro alcohol fraction of bark produced 86.65% and 93.81% inhibition of HRBC hemolysis and bovine serum albumin respectively as compared with 90.05±4.56% and 92.56 ± 5.04 % produced by standard drug aspirin and sodium diclofenac. Likewise. It is clear from the data that F.subincisa bark showed a greater response.
Fig.3 Inhibition of Protein Denatutarion by F.subincisa extract
Fig. 4 HRBC membrane stabilization by F.subincisa extract
Biocompatible solvents for the extraction of natural products became a revolution area of research between the manufacturing industry and academics not only research but also education20. It is a new area of innovations that will not end. Efficient extraction of various kinds of phytochemicals by “green” solvents provides a bio-viable alternative to conventional solvents21. Lessor non-toxicity of these solvents makes chemical processes economically as well as ecologically sustainable22, 23. To treat various types of disease herbal medicine is very useful. Restorative plants exist in many regional varieties depending on the topographical flora24, 25. Contemporary medicament was originally extracted from herbal sources, they are now made a facsimile, and many other drugs are subsided from plant substances. With the availability of an improvident amount of medicinal plants, traditional restorative plants are frequently used in rural parts26, 27. Many plant extracts have been reported to have hypoglycaemic activities and are used in Ayurveda for the treatment of diabetes28. In this study, an in vitro inhibitory effect of different extracts of F.subincisa on porcine pancreatic amylase and alpha-glucosidase activities was evaluated. The ethyl lactate and hydro alcohol extracts of the bark show the highest inhibition. These had lowered the blood glucose level successively, which may be due to the increased level of insulin in the blood. In the present research investigation, different solvent extracts of F.subincisa are evaluated for their anti-diabetic activity. Two different in vitro assays were used to evaluate anti-diabetic activities of different solvent extracts of F.subincisa viz., alpha-amylase and glucose uptake It may be due to the presence of chemical constituents such as terpenes, quercetin, rutin, and alkaloids in the extracts. To study anti-inflammatory activity erythrocyte membrane is used which is analogous to the lysosomal membrane. Restraining the inflammatory response by preventing the release of lysosomal constituents of activated neutrophils, such as bacterial proteases, which causes auxiliary tissue inflammation and extracellular release29, 30. In our study, all the hydroalcoholic extracts significantly stabilized. Our investigation showed that hydro alcohol extracts possess significant anti-proteinase activity and it is logical to predict that, the anti-inflammatory activity of the tested drug may be due to the anti-proteinase activity of the extracts 31-34. So in the course of inflammation, any phytoconstituent which has the ability to defend denaturation of protein could contribute significantly.
The potential use of green solvents namely ethyl lactate, isopropyl alcohol, ethyl lactate, and d-limonene in the extraction of F.subincisa bark was presented in this work. The magnetic stirrer is a suitable technology and the green solvents show prominence to extract phytoconstituents from F.subincisa bark, shorter extraction times with lower consumption of extraction solvents than the traditional extraction method like soxhlet extractor. The results also indicate that the green solvent extracts of F.subincisa possess anti-inflammatory and anti-diabetic properties. The extract inhibited the heat-induced albumin denaturation, stabilized the Red Blood Cells membrane, non-enzymatic Glucosidase inhibitory activity, and amylase inhibitory activity. This study gives an idea that the compounds of F.subincisa can be used as a lead compound for designing a potent anti-inflammatory and anti-diabetic drug that can be used for the treatment of various diseases such as inflammation, carcinoma, aging, and neurological disorder.
CONFLICTS OF INTEREST:
There are no conflicts of interest.
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Accepted on 02.07.2021 © RJPT All right reserved
Research J. Pharm.and Tech 2022; 15(4):1732-1736.