Exploration of phytochemical and in-vitro antioxidant and antidiabetic properties of Aerva sanguinolenta (L.) Blume

 

Nilanjan Sarkar^1-2, Sara Farheen¹, Mainak Chakraborty¹, Swarupananda Mukherjee¹,

Pallab Kanti Haldar²*

¹Department of Pharmaceutical Technology, NSHM Knowledge Campus, Kolkata - Group of Institutions. Kolkata, West Bengal, India.

²Department of Pharmaceutical Technology, Jadavpur University, Kolkata, West Bengal, India.

 

ABSTRACT:

The present study was aimed to investigate the phytochemical Characterization, in vitro antioxidant and antidiabetic activity of Aerva sanguinolenta (L.) Blume. This is an herb belonging to the family Amaranthaceae. Traditionally the plant is rich sources of the constituents like tannins, flavonoids, terpenoids, sphingolipids, polyphenolic compounds etc. The extraction was carried out using chloroform and methanol in subsequent order of polarity. The phytochemical study was done for the identification of different bioactive compounds present in extract through the HR-LCMS study. For this study it is reported that various compounds like Thalidasine, Alpinine, Compactindiol lactone, 9Z- Octadecene dioic acid, Salviafiaside Methyl Ester, Capreomycin, Para Trifluoromethylphenol, Punicacortein B, α-Linolenic acid, Dicumarol, Euphorbia factor Ti2, Calotropin, Kaempferol 3-(2”,6”-di-(E)-p-coumarylglucoside), 6-Methylthiopurine ribonucleotide, Azelaic acid, Kaempferol 3-rhamnoside 7-xyloside, Quinolin-2-ol, Tiliroside, Amicinonide, Indoleacryli Acid, Benzocaine, Bellendine, Vindoline, Betamethasone, Fabianine, Hypercalin B, Icaceine, Canthin-6-one, Irinotecan, Convallasaponin A are present in this plant which are responsible for anti-inflammatory, anti-tumar, anti-malarial, anti-bacterial, anti-fungal, anti-cholesteremic, antiatherosclerotic, transdermal, antioxidant, anti-tubercular, anti-cancer, anticoagulant, antiangina, antihypertension, anti-microbial, antiparasitic, antifungal, antiplatelet, antidiabetic, hepatoprotective, antiviral, antiallergic, immunosuppressive, antianalgesic, antidiabetic, antimitotic activities respectively. From this study it is revealed that leaves extract of Aerva sanguinolenta (L.) Blume exhibits potent antioxidant activity. The plant extract shows substantial DPPH radical scavenging, Hydroxyl radical scavenging, Nitric oxide radical scavenging, Superoxide radical scavenging activity compared to standard (Ascorbic acid).  From this study it is reported that plant extract is a potent inhibitor of two key enzymes which are linked to type-2 diabetes mellitus like α-amylase and α-glucosidase.

 

 

INTRODUCTION:

Diabetes Mellitus (DM) is a complex and diverse group of disorders characterized by chronic hyperglycemia which affects the metabolism of the biomolecules such as carbohydrates, fats and proteins. DM are of two types, Insulin dependent DM (Type-1) and Non-insulin dependent DM (Type-2)^1.

 

 

About 5-10% of DM is identified as Type-1 Diabetes Mellitus and is characterized by autoimmune destruction of insulin producing beta cells in the islets of Langerhans of the pancreas, causing absolute deficiency of insulin. Around 90-95% of DM is identified as Type-2 Diabetes Mellitus, characterized by peripheral insulin resistance and / or reduced insulin secretion². About 366 million people are suffering from Diabetes Mellitus across the world and by the year 2030, this disease is portended to double³. In many countries of the world, obesity is found to be the most common risk factor for development of Diabetes Mellitus⁴. Long term complications of DM predominantly affects the eyes, kidneys, cardiovascular and nervous system. Natural products, specially from plant sources are traditionally used for the treatment of DM due to their comparable pharmacological potential with lesser side effects⁵. As per the assessment by WHO (World Health Organization), 25% of the currently available drugs are derived from plants⁶. The plant Aerva sanguinolenta (L.) Blume is an erect, perennial herb, 30-150 cm tall, arising from a woody base. This plant belongs to the family Amaranthaceae⁷. The roots of this plant is used to treat dysentery and the paste of the root is applied topically on the affected area as a treatment for headache. Various parts of the plant are used as a tonic, sedative and in the management of dermatitis. The decoction of young branches has been used against haematuria and dysmenorrhea. The ethanol extract of whole plant possesses neuroleptic activity. The whole plant is used as diuretic and demulcent. The flowers of this plant are useful in wound healing and as an anti-inflammatory agent for treatment of injuries from falls, rheumatic arthritis and myalgia. Bark is used as a treatment of hematuria^{8, 9}.

 

MATERIALS AND METHODS:

Plant material collection:

The plant Aerva sanguinolenta (L.) Blume was collected from 24 Parganas (South) district of West Bengal and authenticated by the Botanical Survey of India, Howrah vide voucher specimen number CNH/Tech.II/2021/2b.

 

Preparation of plant extract:

The leaves of Aerva sanguinolenta (L.) Blume were washed, shade-dried and coarsely powdered. The powdered sample was extracted by cold maceration technique employing chloroform and methanol successively, in order of polarity. The sample was soaked in chloroform for 72 hrs and thereafter the macerate was filtered and evaporated in a carefully regulated water bath, to yield a semisolid mixture. The residue was re-soaked in methanol and the same process was repeated. The methanolic extract (MEAS) obtained was stored in a desiccator^{10, 11}.

 

HR-LCMS study:

The methanolic extract was subjected to HR-LCMS analysis. The HR-LCMS of sample was carried out in Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, Pawai, Mumbai. Chemical finger prints of selected medicinal plant extracts were prepared by Agilent high resolution liquid chromatography and mass spectrometry model- G6550A with 0.01% mass resolution. The acquisition method was set to be MS- minimum range 50 (M/Z) and maximum 1000 dalton (M/Z) with a scanning rate of one spectrum per second. Gas chromatography was maintained at 250°C with gas flow of 13 psi/minute. Hip sampler (model- G4226A) with auxiliary speed of 100 µl/minute, ejection speed of 100 µl/minute, flush out factor of 5µl and injection volume of 8µl was used for HR-LCMS. Within 30 minutes of acquisition time and initial 2 minutes of the flow of the solvent composition Water and Acetonitrile in a ratio of 95:5 was used for HR-LCMS.

 

In-vitro antioxidant activity:

DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay:

DPPH is a stable nitrogen centered free radical and is used to determine free radical scavenging activity of antioxidants present in the plant extract. The plant extract was diluted in methanol at various concentrations (100, 200, 300, 400 μg/ml). The stock solution was prepared by dissolving DPPH in methanol and stored at 20°C. 2 ml aliquot of this solution was mixed with the sample (plant extract) at various concentrations and was incubated at 25°C for 30 mins. Thereafter, the absorbance was measured at 517 nm. DPPH control contained 1ml methanol and ascorbic acid was used as standard¹². Percentage Inhibition (%) = (Abs control- Abs sample/Abs control) ×100

Abs control= the absorbance of the control and Abs sample= the absorbance of the extract.

 

Hydroxyl radical scavenging activity:

The Fe³⁺ -ascorbate-EDTA-H₂O₂ system generated the hydroxyl radical. The assay was based on the quantification of the 2-deoxy-D-ribose degradation product, which produced a pink chromogen upon heating with TBA at low pH. The reaction mixture consists of 0.8 ml of phosphate buffer saline (PBS) (50mmol L^-1, pH 7.4), extract/standard at different concentration (100, 200, 300, 400 μg/ml), EDTA (1.04 mmol L^-1), FeCl3 (1mmol L^-1) and 2-deoxy-D-ribose (28mmol L^-1). The mixture was heated in water bath at 37°C for 1 hour. Ascorbic acid and H₂O₂ (10mmol L^-1) were added to this mixture to start the reaction. The reaction mixture was further incubated at 37°C for 1h. Cold thiobarbituric acid (TBA) was added to the incubated mixture, followed by HCl (25%). The mixture was heated at 100°C for 15 mins and then cooled. The absorbance of solution was checked at 532 nm with a spectrophotometer¹³.

 

Nitric oxide scavenging activity:

2 ml of 10 mM sodium nitroprusside, diluted in 0.5 mM PBS, was mixed with 1 ml of the sample at various concentrations (100, 200, 300, 400 μg/ml). The mixture was incubated at 25°C for 150 mins. 0.5 ml of the mixture was mixed with 0.5 ml of Griess reagent. The mixture was further incubated for 30 mins and measured at 540nm¹⁴. The quantity of nitric oxide radical was calculated using the following equation:

 

                                             Abs control-Abs sample

NO radical scavenging activity = -------------------- X100

                                                            Abs control

Where, Abs control is the absorbance of NO radical with methanol and Absorbance of sample is the absorbance of NO radical with sample or Standard

 

Superoxide radical scavenging:

The superoxide radicals were produced by the non-enzymatic phenazine methosulfate- nicotinamide adenine dinucleotide (PMS) system. PBS (20mM), NADH (73μM), NBT (50μM), PMS (15μM) and various concentrations (100, 200, 300, 400μgml) of sample solution were mixed to make 1ml of reaction mixture. The reaction was initiated by PMS (Phenazine methosulphate) and the reaction mixture was incubated at 25°C for 5 mins. The absorbance was measured at 562 nm¹⁵.

 

Test for in-vitro antidiabetic activity:

α-amylase inhibitory assay:

0.5 ml of MEAS was mixed with 0.5 ml of α-amylase solution (0.5mg/ml) to which 0.02M sodium phosphate buffer was added. The mixture was then incubated at room temperature for 10 mins. 0.5 ml of Starch solution in 0.02M sodium phosphate buffer was mixed with the incubated mixture. The resultant mixture was further incubated at room temperature for 10 mins and 1ml of dinitro salicylic acid was added to stop the reaction. The final reaction mixture was heated for another 15 mins at 100°C and dissolved in 10 ml of distilled water and cooled at room temperature. The absorbance was measured at 540 nm. The absorbance of blank and control samples was determined. Acarbose was used as standard drug ^[16]. Percentage inhibition was calculated as follows:

          Absorbance of Control-Absorbance of sample

% Inhibitory activity =-------------------------- X 100

                            Absorbance of control  

 

α-glucosidase inhibitory assay:

At first α-glucosidase was incubated with 50 μl of extracts for 10 mins. Then 50μl of substrate containing 3mM pNPG diluted in 20mM phosphate buffer at pH 6.9 was mixed to start the reaction. The mixture was incubated at 37°C for 20 mins and the reaction was terminated by adding 2 ml of 0.1M Na₂CO₃. α-glucosidase activity was observed by measuring the yellow-colored p-nitrophenol, released from pNPG (4-nitrophenyl β-D-glucopyranoside) at 405 nm. Using a similar procedure, the blank control (20mM PBS instead of α-glucosidase) was prepared. The inhibition of α-glucosidase was calculated using the following equation¹⁷.

                     Abs control- Abs sample

% Inhibition =-------------------------------× 100

                              Abs control

 

Table-1 HR-LCMS Study

Bioactive compounds identified in the methanol extract of Aerva sanguinolenta (L.) Blume: HR-LCMS

 

Figure-1 Antioxidant Activity

The data represent the percentage inhibition of DPPH, Nitric oxide, Superoxide and Hydroxyl radical by MEAS against ascorbic acid (standard). Each point represents the values obtained from experiments, performed in triplicate (mean ± SEM)

 

Figure-2 In vitro Anti-diabetic activity:

The data represent the percentage inhibition of α-amylase and α-glucosidase by MEAS against acarbose (standard). Each point represents the values obtained from experiments, performed in triplicate (mean ± SEM).

 

DISCUSSION:

The phytochemical characterization of Aerva sanguinolenta (L.) Blume demonstrated the presence of flavonoids, alkaloids, saponins, tannins and phenols. Phenolic compounds are the most abundant of the plant secondary metabolites. Among the various classes of phenolic compounds, flavonoids and tannins are the main dietary phenolic compounds. Various phenolic groups neutralize free radicals by donating a hydrogen atom, electrons or chelate metal ions in aqueous solution. Sevaral studies have demonstrated the protective effect of flavonoids against microbial pathogens, oxidative cell injury and degenerative diseases like cardiovascular diseases, cancers³³ etc. Number of studies reveal that tannin obstruct HIV replication³⁴ also possess antiviral, antibacterial³⁵ and anticancer activity³⁶. Alkaoids are naturally occurring chemical compounds consisting of a ring structure and a nitrogen atom. It is the most essential active constituents in natural herbs and is used as chemotherapeutic agents. From the data of the High Resolution-Liquid Chromatography mass spectrometry (HR-LCMS) study, it is revealed that there is a plethora of various bioactive compounds present in Aerva sanguinolenta (L.) Blume plant possessing multiple types of pharmacological activities as depicted in the Table no. 1.

 

Figure-1 demonstrates the DPPH radical scavenging, Hydroxyl radical scavenging, Nitric oxide radical scavenging and Superoxide radical scavenging activity of methanolic extract of Aerva sanguinolenta (L.) Blume, using ascorbic acid as standard. From figure-1 it is observed that Aerva sanguinolenta (L.) Blume has substantial concentration dependent DPPH radical scavenging activity, in comparison with ascorbic acid. Percentage inhibition of DPPH radical scavenging activity of MEAS is similar to 200μg/ml concentration of ascorbic acid. The IC₅₀ value of MEAS and Ascorbic acid is 133 ± 3μg/ml and 109±4μg/ml, respectively. From figure-1 it is further revealed that MEAS has detectable hydroxyl radical scavenging activity compared to ascorbic acid. However, for certain concentration the percentage inhibition of ascorbic acid is greater than that of MEAS. The estimated IC₅₀ value of MEAS and ascorbic acid are 281±6μg/ml and 222±3μg/ml respectively. The results depicted in Figure-1 also illustrates the concentration dependent increase in nitric oxide scavenging activity of MEAS in comparison with ascorbic acid at 546nm. The IC₅₀ value of MEAS and Ascorbic acid are 269±4μg/ml and 204±5μg/ml respectively. Superoxide radical scavenging activity is also increased with the increase in MEAS concentration as depicted in Figure-1. The IC₅₀ value of MEAS is 259±4μg/ml and the IC₅₀ value of ascorbic acid is 154±6μg/ml.

 

Experimental results reveal that the plant extract significantly inhibits the α-amylase and α-glucosidase enzymes. Figure- 2 represents that MEAS has significant antidiabetic activity compared to Acarbose. With increasing concentration of MEAS, increase in the percentage inhibition of α-amylase and α-glucosidase activity was observed.

 

CONCLUSION:

The important medicinal plant Aerva sanguinolenta (L.) Blume is used for many diseases and disorders. In this study in-vitro antioxidant and antidiabetic activity are performed. The HR-LCMS analysis confirms that various compounds with different chemical structures are present in this plant which are responsible for anti-inflammatory, anti-tumor, anti-malarial, anti-bacterial, anti-fungal, anti-cholesteremic, anti-atherosclerotic, anti-oxidant, anti-tubercular, anti-cancer, anticoagulant, anti-anginal, anti-hypertensive, anti-microbial, anti-parasitic, anti-platelet, anti-diabetic, hepatoprotective, anti-viral, anti-allergic, immunosuppressive, analgesic, anti-diabetic, anti-mitotic activities. Any imbalance between the free radicles and physiological anti-oxidants, leads to formation of a condition known as ‘Oxidative Stress’. This oxidative stress precipitates many pathological conditions among which Diabetes Mellitus is an important metabolic disorder. From this study it is revealed that the leaf extract of Aerva sanguinolenta (L.) Blume exhibits significant antioxidant activity by DPPH radical scavenging, Hydroxyl radical scavenging, Nitric oxide radical scavenging and Superoxide radical scavenging. It is further discovered as a potent inhibitor of two key enzymes (α-amylase and α-glucosidase) related to Type-2 Diabetes Mellitus. From this study it may be concluded that Aerva sanguinolenta (L.) Blume possesses potent antidiabetic activity. Further studies employing advanced scientific method on this plant might help discover new entities, which shall facilitate the development of new therapeutic agents for the benefit of the society and mankind.     

 

CONFLICT OF INTEREST STATEMENT:

We declare that we have no conflict of interest.

 

ACKNOWLEDGEMENTS:

The authors acknowledge the support extended by Department of Pharmaceutical Technology, NSHM Knowledge Campus, Kolkata - Group of Institutions and Department of Pharmaceutical Technology, Faculty of Engineering and Technology, Jadavpur University, Kolkata, India for providing necessary infrastructure and financial support. The authors are also thankful to SAIF, IIT Bombay for the analytical service.

 

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Accepted on 14.02.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5267-5272.