In-Vitro Studies on Antidiabetic and Antioxidant Activity of Hydroalcoholic Extract of Barleria Prionitis Linn

Diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose levels due to defects in insulin production, insulin action, or both. It is one of the most prevalent and serious health conditions affecting millions of people worldwide. The disease can lead to severe complications if not managed properly, affecting various organs such as the heart, kidneys, eyes, and nerves^[1,2]. Insulin and glucagon hormones both are secreted by the pancreas. Insulin is secreted by the beta (ß) cells and glucagon is secreted by the alpha (α) cells both are located in the islets of Langerhan’s. Insulin decreaes the blood glucose level by the glycogenesis and transport glucose into the muscles, liver and adipose tissue. Neural tissue and erythrocytes do not required insulin for glucose utilization whereas alpha (α) cells plays an important role in controlling blood glucose by producing the glucagon and it increases the blood glucose level by accelerating the glycogenolysis^[3,4]. In addition to increased risk of obesity, metabolic and cardiovascular disorders, and malignancy in future life of fetus after delivery^[5]. Type II diabetes mellitus comprises 80% to 90% of all cases of diabetes mellitus. Geographical variation can contribute in the magnitude of the problems and to overall morbidity and mortality^[6,7]. Moreover, people with diabetes who undertake moderate amounts of physical activity are at inappreciably lower risk of death than inactive persons^[8] It is now well established that a specific genetic constitution is required for such an event to cause^[9] The growing burden of diabetes and other noncommunicable diseases is one of the major health challenges to economic developments bedevilling WHO African Region states^[10].  Anti- Diabetic and Antioxidant activities of hydroalcoholic extract of barleria prionitis linn. To study the Anti-Diabetic and Antioxidant activities of the hydroalcoholic extract of barleria prionitis Linn.,

• To prepare hydroalcoholic extract of Barleria prionitis Linn., using Soxhlet apparatus.
• To perform preliminary phytochemical screening of barleria prionitis extract.
• To evaluate the in-vitro.

Plant Profile:

Barleria Prionitis L, a perennial, acanthaceous, barbed, bushy medicinal plant, including in Barleria genus containing 300 species is famous for its medicinal value from ancient time.

Scientific Name: Barleria prionitis L.

Common name: Porcupine flower.

Synonyms: Barleria coreacea, Barleria hystix.

Barleria Prionitis L

Toxonimical Classification:

Domain: Eukaryote

Kingdom: Plantae

Phylum: Spermatophyta

Subphylum:    Angiospeae

Class: Dicotyledonae

Order: Scrophulariales

Family: Acanthaceae

Genus: Barleria

Species: Barleria Prionitis ^[2,12]

Vernacular Name:

Manjachemulli (Tamil) Manjakkanakambaram ( Malayalam) Gorante (Kanada)

Vajradanti (Hindi, Sanskrit) Kalsunda (Marathi)

Plant Type:

Perennial, Seed propagated, Shrub, Vegetatively propagated.

Morphological Characters of the Plant

Plant: B. prionitis is a small, erect, spiny shrub, up to 1.8 m tall with spines in lower leaf axils, branched

Stem: Stems and branches terete, smooth, lenticellate , glabrous. Petiole 1-2.5 cm; leaf blade elliptic to ovate, 4-10.5 × 1.8-5.5 cm, both surfaces pubescent when young but soon glabrescent, sparsely strigose along midvein, base attenuate and decurrent onto petiole, margin entire, apex acute.

Flower: clustered in axils of upper leaves and/or bracts; bracts linear-oblong, 1.2-2.2 × 0.2-0.8 cm.

Seed: Seeds ovate in outline, approximately 7 × 5 mm.^[1,2]

Geographical Distribution:

Native to island and Southeast Asia, China, The Arabian Peninsula and Northeastern Africa.

Habitat: B. prionitis occurs in the wild in thickets at low altitudes (up to 600 m; It is a common weed of disturbed areas, forest edges, waterways, open woodlands, rocky outcrops, waste areas, and overgrazed pastures in tropical and subtropical regions .^[1]

Parts Used: Whole Plant

Uses:

Promote healing of wounds and to relieve joint pains and toothache (Parrotta 2001). The leaves are a diuretic and tonic and chewed for fever, rheumatism, liver diseases, indigestion with constipation, jaundice and urinary infections. An infusion of the roots and leaves is applied to boils and sores to reduce swellings, and also used for earache and headache. Because of its antiseptic properties, extracts of the plant are incorporated into herbal cosmetics and hair products to promote skin and scalp health .^[12]

Pharmacological Activates:

Anti-microbial, Anthelmintic, Anti-fertility, Anti-oxidant, Anti-diabetic, Anti-inflammatory, Anti arthritic, Cytoprotective, Hepatoprotective, Anti-diarrhoel, Diuretic, Enzyme inhibitory and Anti-nociceptive activities.^[2]

Phyto Constituents:

Barleria prionitis contains various phytochemicals, including iridoids, phenolic acids, phenylethanoid glycosides, lignans, flavonoids, phytosterols, tannin, saponin, glycoside, and alkaloids^[1][2][3]. Different parts of the plant, such as flowers, leaves, stems, and roots, contain these secondary metabolites^[1].Specific compounds found in Barleria prionitis:

From aerial parts: Apigenin 7-O-β-D-glucoside^[1]

From leaves: 6-hydroxyflavone, scutellarin, melilotic acid, syringic acid, vanillic acid, p-hydroxybenzoic acid^[1] From roots:β-sitosterol^[1]

Other compounds: Flavonoids, saponins, glycosides, phytosterols, and tannin^[11]

Bioactive compounds such as alpha-amyrin, flavone, phenol, phytol, phytol acetate, squalene, and stigmasterol have also been found in extracts of Barleria species^[5]. Additionally, iridoid glycosides like barlerin and verbascoside have been isolated from Barleria, contributing to its antiviral activity^[12]

MATERIALS AND METHODS:

Collection And Authentication of Plant:

The leaves of Barleria prionitis L were collected from the surrounding areas of sunrise agro nursery Pimpri chinchwad pune maharashta, India. The samples were identified and authenticated (Reg.No: BSI/SRC/5/23/2022/Tech/583 and 587) at Botanical Survey of India, Southern circle, Coimbatore.

Extraction Procedure:

200 g of finely powdered leaf powder was defatted with 1 L of petroleum ether in a soxhlet apparatus for 48 h, obtained marc was further extracted with 1 L of 60% ethanol (600ml of ethanol: 400ml of water) in soxhlet apparatus for 48 h, obtained marc was again extracted with water by cold maceration method for 48 h. After extraction the extracts were separately concentrated by distillation and dried at room temperature until get viscous solid mass. The obtained crude extracts were weighed and stored at 40C for the further analysis

Phytochemical Screening of Hydroalcoholic Extract of Barleria Prionitis L ^[13,14]

The plant extracts were subjected to preliminary phytochemical screening for the detection of various plant constituents present. The term qualitative analysis refers to establishing and proving the identity of a substance. The active ingredients, after isolation, can be incorporated into the modern medicine for the development of newer formulation for therapeutic ailments.

Qualitative phytochemical analysis:

The petroleum ether and ethanolic extracts of Barleria prionitis and Phyllanthus acidus were subjected to qualitative tests for the detection of various plant constituents.

1.Detection Of Carbohydrate:

Small quantity of extract was dissolved in 4ml of distilled water and filtered. The filtrate was collected and subjected for the following tests.

a. Molisch’s test: 1 ml of filtrate was treated with 2-3 drops of 1% alcoholic α-napthol solution and 2 ml of conc. sulphuric acid was added along the sides of the test tube. Appearance of brown to violet ring, indicate the presence of carbohydrate.

b. Fehling’s test: To the Fehling solution A and B extract was added and boiled. The formation of brick red precipitate indicates the presence of reducing sugar.

c. Benedict’s test: 1 ml of extract was added to 5ml of Benedict’s reagent, was added and boiled for 2 mins and cool. Formation of a red precipitate shows the presence of sugars.

Test For Glyceriods:

a. Legal’s test:

The filtrate was hydrolyzed with dilute hydrochloric acid and heated on water bath. Then added 1ml of pyridine and few drops of sodium nitroprusside solution, made alkaline with sodium hydroxide solution. Appearance of pink to red colour shows the presence of glycosides.

b. Borntrager’s test: Filtrate was hydrolyzed with dilute hydrochloric acid on water bath, then treated with chloroform and shake well. After that separates the chloroform layer and added equal volume of dilute ammonia solution. If ammonia layer acquire pink or violet colour, indicates the presence of glycosides.

c.Keller killiani test: The ethanol extract 0.5ml of strong solution of lead acetate was added and filtered. The filtrate is shaken with 5ml of chloroform. The chloroform layer is separated in a porcelain dish and removes the solvent by gentle evaporation. Dissolve the cool residue in 3ml of glacial acetic acid containing 2 drops of ferric chloride solution carefully transferred the solution to the surface of 2ml of concentrated sulphuric acid. A reddish brown layer form at the junction of the 2 liquid and the upper layer slowly becomes bluish green, darkening the withstanding

Detection Of Alkaloids:

Small quantity of extract was treated with few drops of dilute hydrochloric acid and filtered it. The filtrate was collected and subjected for tests with following reagents.

A.Mayer’s reagent: To the filtrate potassium mercuric iodide was added. The formation of cream colour precipitate, it shows the presence of alkaloids.

B.Dragendroff’s reagent: To the filtrate potassium bismuth iodide was added. If it shows reddish brown precipitate, indicates the presence of alkaloids.

C.Wagner’s reagent: To the filtrate iodine in potassium iodide solution was added. If it shows reddish brown precipitate, indicates the presence of alkaloids.

Detection Of Phytosterol and Steroids:

Small quantity of extract was dissolved in 5ml of chloroform and then subjected to the following tests.

a.Salkowski test: To the above solution 1 ml chloroform and few drops of concentrated sulphuric acid was added. The test tube was shaken for few minutes. The development of red colour in chloroform layer indicates the presence of steroids. Liebermann- Burchard reaction: To the above solution 1 ml of chloroform and few drops of concentrated sulphuric acid and 1-2 ml of acetic anhydride were added. Development of red colour first, then blue and finally green colour, indicates the presence of steroids.

Liebermann’s reaction: To 3 ml of extract in a test tube, 1 ml of acetic anhydride was added and gently heated. The contents of test tube were cooled. Few drops of concentrated sulphuric acid was added from the side of test tube. Appearance of blue shows the presence of steroids.

Detection Of Proteins and Amino Acid:

Small quantity of the extract was dissolved in few ml of water and filtered. The collected filtrate was used for following tests.

a.Millon’s test: To the filtrate Millon’s reagent was added. If white precipitate slowly turns to red colour, indicates the presence of proteins.

b.Biuret test: Filtrate was treated with 5% sodium hydroxide and few drops of 1% copper sulphate solution. Formation of violet or pink colour indicates the presence of proteins.

c.Ninhydrin test: To the filtrate Ninhydrin reagent was added. Development of violet or purple colour indicates the presence of amino acids.

Detection Of Tannins:

The test extract was dissolved in water, warmed and filtered. The filtrate was used for the following tests.

a.Ferric chloride test: 5 ml of filtrate was allowed to react with 1 ml of 5% ferric chloride solution. If dark green or deep blue colour is obtained, tannins are present.

b.Lead acetate test: 5 ml of filtrate was treated with 1 ml of 10% lead acetate solution. Yellow colour precipitation, indicates the presence of tannins.

c.Potassium dichromate test: 5 ml of filtrate was treated with 1 ml 10% aqueous potassium dichromate solution. If yellowish-brown precipitate formed it suggest the presence of tannins.

Dtection Of Flavonoids:

a.Shinoda test: The small quantity of extract was dissolved in alcohol, to that piece of magnesium followed by concentrated hydrochloric acid were added by drop wise and heated. Appearance of magenta colour shows the presence of flavonoids.

b.Pew’s test: A pinch of zinc powder and about 5 drops of 5N hydrochloric acid were added to the test solution. The formation of deep purple red or cherry red colour indicates the presence of flavonoids. Extract was treated with aqueous sodium hydroxide solution. Development of yellow colour indicates the presence of flavonoids.

Detection Of Saponins

a.Foam test: The extract was diluted with distilled water to 20 ml and shaken in a graduated cylinder for 15 minutes. Development of stable foam suggests the presence of saponins.

b.Lead acetate test: 1ml of sample solution was treated with 1% of lead acetate solution, formation of a white precipitate indicates the presence of saponins. 

Detection Of Terpenoids:

Knoller’s Test: In a test tube, 2 or 3 granules of tin was added and dissolved in 2 ml of thionyl chloride solution. Then, test solution was added. A deep purple colour that changes to red indicates the presence of terpenoids.

Test For Fixed Oil and Fats

a.Spot test: Pressed a small quantity of extract between the two filter papers, the strain on the filter paper indicates the presence of fixed oils.

b.Saponification test: Added a few drops of 0.5N of alcoholic potassium hy

droxide to small quantity of various extract along with a drop of phenolphthalein separately and heat on a water bath for 1 to 2 hours. The formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats.

Test For Gums and Mucilage

10ml of ethanol extract was slowly added 25 ml of absolute alcohol with constant stirring filter the precipitate and dried in air. The precipitate for its swelling property indicates the presence of carbohydrates.

In-Vitro Antidiabetic Activity of Hydroalcoholic Extract of Barleria Prionitis:

In vitro Alpha Amylase Assay ^[15]

Different concentration of extract (10, 20, 30, 40, 50 μg/ml) was taken into different test tubes. Made the volume to 0.5 ml with phosphate buffer of pH 6.9; Control was prepared by taking 0.5 ml of phosphate buffer. The solutions were then treated with 0.5 ml of alpha amylase (0.5mg/ml). The solution was incubated at 25 ?C for 10 minutes. Added 0.5 ml of 1% starch solution in 0.02 M sodium phosphate buffer of pH 6.9 to all the tubes, and then incubate at 25 ?C for 10 minutes. The reaction was stopped by adding 1.0 ml of DNSA and the reaction mixture was kept in boiling water bath for 5 minutes, cooled to room temperature. The solution was mixed with 8 ml distilled water. Blank was measured by taking 1 ml of phosphate buffer. The absorbance of the solution in colorimeter at 540 nm against blank solution was taken. Standard acarbose was prepared in the same manner at different concentrations and absorbance was measured. The results were calculated and expressed in the basis of percentage inhibition.

In Vitro Antioxidant Activity of Hydroalcoholic Extract of Barleria Prionitis:

DPPH radical scavenging assay ^[16]

The effect of the extracts on DPPH radical was estimated using the method of Liyana- Pathiranan and Shahidi. A solution of 0.135 mM DPPH in methanol was prepared and 1.0 ml of this solution was mixed with 1.0 ml of extract in methanol containing 0.02–0.1 mg of the extract. The reaction mixture left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm.

RESULT AND DISCUSSION

Extractive Yield of Hydroalcoholic Extract of Barleria Prionitis

The percentage yield of extract was found to be 14%.

Preliminary Phytochemical Analysis of HALEBP

In the phytochemical studies, hydro-alcoholic extracts of leaves of Barleria prionitis showed the presence various phytoconstituents. Preliminary phytochemical screening of Barleria prionitis revealed the presence of bioactive compounds such as carbohydrates, glycosides, alkaloids, tannins, flavonoids, phenols and saponins in hydroalcoholic extract.

Pharmacological studies

Invitro antidiabetic activity of HALEBP Alpha   amylase inhibition assay

In the present study, the hydroalconolic extracts of Barleria prionitis Linn showed α-amylase inhibitory activity. The search for a new α-amylase inhibitor from medicinal plants is a striking method for the management of postprandial hyperglycemia. The concentration-dependent α-amylase inhibitory activities and the IC50 value. were estimated as indicated in Figure 2 and Table 2 respectively. Anti-diabetic activity was evaluated by using alpha amylase inhibitory assay and IC50 values for HALEBP and Acarbose are found to be 43.04 μg/ml and 24.81 μg/ml respectively.

Data showing the preliminary phytochemical screening of the HALEBP

Percentage alpha amylase inhibition of HALEBP and Acarbose

Invitro antioxidant activity of HALEBP DPPH radical scavenging activity

The free radical scavenging activity of the extract was estimated by comparing the %inhibition of HALEBP with standard Ascorbic acid. IC50 was also calculated to determine the amount of extract needed to quench 50% of radicals. Hydroalcoholic extract of Barleria prionitis exhibited a dose dependent scavenging activity with IC50 values of 39.29 µg/ml. Where the IC50 for standard was found to be 10.91 µg/ml. The concentration-dependent DPPH radical scavenging activities and the IC50 values.

DPPH scavenging activities of HALEBP and Ascorbic acid