PHARMACOGNOSTICAL OVERVIEW ON TERMINALIA ARJUNA: A JUSTIFICATION OF FOLKLORIC BELIEF

PHARMACOGNOSTICAL OVERVIEW ON TERMINALIA ARJUNA: A JUSTIFICATION OF FOLKLORIC BELIEF

Alisha, Myrene R. Dsouza ^* and Ningthoujam Sonia

Department of Chemistry (P.G. Biochemistry), Mount Carmel College Autonomous, Bengaluru - 560052, Karnataka, India.

ABSTRACT: The application of medicinal plants such as Terminalia arjuna to maintain health and treat disease started since time immemorial and still is a part of medical practice. The phytochemical analysis of bark of T. arjuna in methanolic, hydromethanolic (1:1), aqueous and petroleum ether extracts revealed the presence of carbohydrates, amino acid, alkaloids, saponins, tannins, flavonoids, terpenoids, glycosides, xanthoproteins and phenols. In-vitro antioxidant capacity by linear regression analysis was measured by assaying DPPH radical and H₂O₂ scavenging capacities. Their respective IC₅₀ values were found to be 336.89 μg/ml and 253.01 μg/ml. The hydromethanolic extract gave total phenolic, total flavonoids and FRAP values of 754.16 ± 16.26 mg GAE/g, 247.02 ± 8.81 mg/g QE/g and 1.36 ± 0.03 mM FeSO₄ respectively. The IC₅₀ values for in-vitro anti-inflammatory activities were evaluated by the following assays: lipoxygenase inhibition (IC₅₀ = 480.5 μg/ml), heat induced protein denaturation (IC₅₀ = 282.32 μg/ml), proteinase inhibition (IC₅₀ = 1339.3 μg/ml) and RBC membrane stabilization (IC₅₀ = 192.81 μg/ml) at different concentrations using aspirin as control. Studies were also carried out to assess the hypoglycemic potential by assaying the ability of the plant to inhibit alpha amylase (IC₅₀ = 210.91 μg/ml) and pancreatic lipase (IC₅₀ = 51.52 μg/ml) activities. In addition, glucose binding capacity and glucose diffusion was also monitored). Antimicrobial activity of the extracts was studied against common pathogens. The zone of inhibition was observed using well diffusion method.

Anti-inflammatory, Antioxidant, Hypoglycemia, Lipoxygenase, Proteinase, T. arjuna

INTRODUCTION: In India, over 7500 species of medicinal plants are used by various ethnic communities and play an integral part of their culture ^{1, 2}. The use of such herbal medicines and nutraceuticals is versatile and has been utilized in various national healthcare settings.

An estimated 80% of the world’s population living in the developing world rely on herbal medicinal products as a primary source of healthcare ³. One such enthanomedicinally important plant is Terminalia arjuna, belonging to the family Combretaceae. Common folkloric uses of the bark include anti-dysentric, antipyretic, astringent, cardiotonic, lithotriptic, anticoagulant, hypo-lipidemic, antimicrobial and antiuremic agent ⁴.

Studies on the bark of T. arjuna have shown high activities of superoxide dismutase, glutathione and catalase levels, demonstrating effectiveness against oxidative stress ⁵. It was found to decrease LDL, HDL and total cholesterol levels thereby lowering blood pressure and frequency of angina-attacks. In addition, it possesses hepatoprotective, anti-mutagenic, antiviral and antibacterial activities ⁶. Hence the present study was intended to evaluate the presence of phytoconstituents, pharmaco-gnostical and antimicrobial activity of the bark of T. arjuna.

MATERIALS AND METHODS:

Collection and Preparation of Samples: The bark samples of Terminalia arjuna were collected from Bengaluru, India in 2016. The bark was cleaned, rinsed in distilled water, sun dried and ground into a fine powder. Crushed samples were extracted using three different solvent systems: distilled water (aqueous), methanol (MeOH) and hydro-methanolic extract (1:1 v/v). Extraction was carried out on an orbital shaker for 24 h at room temperature. Solvents were evaporated under vacuum and the resulting extracts were stored at 4ºC.

Fluorescence Analysis and Phytochemical Screening: Fluorescence characteristics of the powdered seed with different chemicals were observed in daylight and ultraviolet light ⁷. Phytochemical examinations were carried out for all the extracts as per the standard methodology to confirm the presence of phytoconstituents ^{8, 9}.

Detection of Carbohydrates: Extracts were dissolved individually in 5 ml distilled water and filtered. The filtrates were used to test for the presence of carbohydrates as follow:

Molisch’s Test: Formation of the violet ring at the junction with 2 drops of alcoholic α-naphthol solution indicates the presence of carbohydrates.

Benedict’s Test: Orange red precipitate with Benedict’s reagent indicates the presence of reducing sugars.

Fehling’s Test: Filtrates were hydrolyzed with dil. HCl, neutralized with alkali and heated with Fehling’s A and B solutions. Presence of reducing sugars was indicated by the formation of a red precipitate.

Detection of Alkaloids: Extracts were dissolved individually in dilute HCl and filtered. Tests for the presence of alkaloids were as follows:

Mayer’s Test: Formation of a yellow coloured precipitate with Mayer’s reagent indicates the presence of alkaloids.

Wagner’s Test: Formation of brown / reddish precipitate with Wagner’s reagent indicates the presence of alkaloids.

Dragendroff’s Test: Formation of red precipitate with Dragendroff’s reagent indicates the presence of alkaloids.

Detection of Saponins:

Foam Test: Persistence of foam after 10 min with 0.5 ml extract and 2 ml water, shaken well indicates the presence of saponins.

Detection of Glycosides: Extracts were hydrolyzed with dil. HCl, and then subjected to test for glycosides.

Modified Borntrager’s Test: Extracts were treated with FeCl₃ solution and immersed in boiling water for about 5 min. The mixture was cooled and extracted with equal volumes of benzene. The benzene layer was separated and treated with ammonia solution. Formation of rose-pink colour in the ammoniacal layer indicates the presence of anthranol glycosides.

Detection of Phytosterols:

Salkowski’s Test: The plant extracts were treated with CHCl₃ and filtered. The filtrate thus obtained was mixed with few drops of concentrated H₂SO₄, shaken and allowed to stand. The appearance of golden yellow colour was indicative of triterpenes.

Detection of Flavonoids:

Lead Acetate Test: Formation of yellow colour precipitate with the extract and a few drops of lead acetate solution indicates the presence of flavonoids.

Detection of Proteins and Amino Acids:

Xanthoproteic Test: Formation of yellow colour with extract and concentrated HNO₃ indicates the presence of proteins.

Ninhydrin Test: Formation of blue colour with extract and 0.25% w/v ninhydrin reagent boiled for a few minutes indicates the presence of amino acid.

Millon's Test: A reddish-brown coloration with the extract and Millon’s reagent gently heated indicates the presence of tyrosine residue

Detection of Tannins:

Ferric Chloride Test: Formation of bluish-black colour with the extract and 3 - 4 drops of FeCl₃ indicates the presence of phenols.

Detection of Terpenoids:

Salkowski Test: Appearance of reddish brown colour with 5 ml of extract, few drops of CHCl₃ and 3 ml concentrated H₂SO₄ revealed the presence of terpenoids.

Total Phenolic, Flavonoid and FRAP Activity: Total phenolic contents were estimated according to the spectrophotometric method using gallic acid as standard ¹⁰ and expressed in terms of gallic acid equivalent (mg of GAE/g of tissue). Aluminum chloride colorimetric method was used for determination of total flavonoids ¹¹ and expressed in terms of quercetin equivalent (mg of QE/g of tissue). The ferric ions (Fe³⁺) reducing antioxidant power (FRAP) method was used to measure the reducing capacity of plant extracts ¹².

Evaluation of Antioxidant Activity using various in-vitro Methods:

DPPH Radical Scavenging Activity: Standard ascorbic acid was pipetted out into different test tubes (100 - 500 μg/ml). 0.1 ml solution of each dilution was taken and made up to 3 ml with DPPH (20 μg/ml). The test tubes were incubated for 10 min at room temperature. The contents of each tube were mixed well, and the absorbance was measured at 517 nm against a blank ¹³. The percentage inhibition of DPPH by the samples was calculated as follows:

% Inhibition = (OD of control – OD of sample) × 100 / OD of control                                ..............(1)

Hydrogen Peroxide Scavenging Activity: Hydromethanolic extract of T. arjuna in 3.4 ml phosphate buffer was added to 0.6 ml of 45 mM H₂O₂ solution and the absorbance of the reaction mixture was recorded at 230 nm against a blank containing only buffer ¹⁴. Ascorbic acid was used as standard. The concentration of H₂O₂ (mM) in the assay medium was determined using a standard curve (y = 0.1223x + 29.62; R² = 0.586). H₂O₂ scavenging ability was calculated as IC₅₀. The percentage inhibition was calculated as in Eq. 1.

Evaluation of Anti-inflammatory Activity using various in-vitro Methods:

Lipoxygenase Inhibition: Soybean lipoxygenase activity was assayed ¹⁵. The reaction contained 2.9 ml 0.1M borate buffer pH 9.0 and 50 μl 10 mM linoleic acid. The reaction was initiated by the addition of 50 μl of the soybean enzyme extract. The enzyme activity was measured by following the formation of the product, 12-HETE at 234 nm for up to 1 min. The enzyme inhibition was determined by pre-incubating the enzyme with the hydromethanolic extract prior to determining its 12-LOX activity. The percentage inhibition was calculated as in Eq. 1. IC₅₀ was calculated from y=0.0839x+8.61; R²=0.788.

Proteinase Inhibitory Action: The test was performed according to the modified method of Sakat ¹⁶. 2 ml of the reaction mixture contains 0.06 mg trypsin, 1 ml 20 mM Tris HCl buffer (pH 7.4) and 1 ml test sample of different concentrations (100 - 500 µg/ml). The mixture was incubated at 37ºC for 5 min and then 1 ml of 0.8% (w/v) casein was added. The mixture was incubated for an additional 20 min. 2 ml of 70% perchloric acid was added to arrest the reaction. Cloudy suspension was centrifuged, and the absorbance of the supernatant was read at 210 nm against buffer as blank. The experiment was performed in duplicate. The percentage inhibition of protein denaturation was calculated using Eq. 1. IC₅₀ was calculated from y=0.0399x+4.428, R²=0.8585.

RBC Membrane Stabilization Activity: Various concentration of hydromethanolic extract, reference sample and control were separately mixed with 1 ml of phosphate buffer, 2 ml of hyposaline and 0.5 ml of hRBC suspension. Diclofenac sodium (100μg/ml) was used as a standard drug. All the assay mixtures were incubated at 37 ºC for 30 min and centrifuged at 3000 rpm. The supernatant was used to estimate the haemoglobin content using a spectrophotometer at 560 nm ¹⁷. The percentage of hemolysis was estimated assuming that the control produced 100% haemolysis. The percentage inhibition of protein denaturation was calculated using Eq. 1. IC₅₀ was calculated from y=0.1525x+39.78, R²= 0.5502.

Evaluation of Hypoglycemic Activity using various in vitro Methods:

Alpha Amylase Inhibition Assay: The procedure described by Shai et al., ¹⁸ with slight modifications was used to determine the α-amylase inhibitory activity of the fractions. A volume of 250 µl of each hydromethanolic extract fraction or acarbose at different concentrations (100 - 500 µg/ml) was incubated with 500 µl of porcine pancreatic amylase (2 U/ml) in phosphate buffer (100 mM, pH 6.8) at 37 °C for 20 min. Thereafter, 250 µl of 1% starch dissolved in 100 mM phosphate buffer (pH 6.8) was further added to the reaction mixture and incubated at 37 °C for 1 h. Dinitrosalicylate colour reagent (1 ml) was then added and boiled for 10 min. The absorbance of the resulting mixture was read at 540 nm the inhibitory activities of the fractions on α-amylases were calculated by using the following formula:

% Inhibition = 1 – B-b /A-α × 100

Where ‘A’ is the activity of the enzyme without inhibitor, ‘a’ is the negative control without the inhibitor, ‘B’ is the activity of the enzyme with inhibitor, and ‘b’ is the negative control with inhibitor.

Pancreatic Lipase (PPL) Inhibition Assay: This was performed as described by Bustanji et al.,¹⁹ with minor modification. The enzyme solution was prepared immediately before use, by suspending crude porcine pancreatic lipase powder in 0.1 M phosphate buffer (pH 7.6) (100 U/ml). The solution was then centrifuged at 2000 rpm for 10 min and the clear supernatant was recovered. Triolein (1% v/v) was used as the substrate for PLL. The hydromethanolic extract of T. arjuna (5, 12.5, 25, 100, 125, 250, 500 μg/ml) was preincubated with 200 μl of PPL solution for 5 min at 37 °C, before the addition of 800 µl triolein substrate solution. The absorbance was measured at 450 nm against blank using denatured enzyme in an ELISA reader. The denatured enzyme was prepared by boiling the enzyme solution for 5 min. Orlistat was used as a reference drug.

The extract was dissolved in DMSO at a final concentration not exceeding 1% (v/v). The activity of the negative control was checked in the presence and absence of the inhibitor. The % inhibition was calculated according to the formula (2).

Determination of Glucose Adsorption Capacity: Glucose adsorption capacity of the samples was determined by the method of Ou et al.,²⁰. The hydromethanolic extract of T. arjuna bark (1%) was added to 25 ml of glucose solution of increasing concentration (5, 10, 20, 50, and 100 mmol/l). The mixture was stirred well, incubated in a shaker at 37 °C for 6 h, centrifuged at 5000 rpm for 20 min and the glucose content in the supernatant was determined. Metronidazole was used as control. The concentration of bound glucose was calculated using the following formula:

Glucose bound = [Initial Glucose] – [Glucose after 6h] × Volume of Solution / Weight of Sample

In-vitro Glucose Diffusion Inhibitory Assay: To study in-vitro effects of the hydromethanolic extract of T. arjuna bark, on the movement of glucose, a simple model system was adapted as mentioned by Ahmed et al., ²¹ This model involved the use of dialysis tubes that were sealed containing 25 ml of 20 mM glucose and 1% T. arjuna bark extract against 200 ml of distilled water at 37 °C in a shaker water bath. The amount of glucose in the external solution was measuredat 30, 60, 120, and 180 min. Metronidazole was used as the control. Glucose dialysis retardation index (GDRI) was calculated using the following formula:

GDRI% = Glucose content with addition of sample × 100 / Glucose content of control

Screening for Antibacterial Activity of Methanolic Extract:

Agar Well Diffusion Assay: The hydromethanolic, hot and cold aqueous T. arjuna bark extracts were used for evaluation of the antimicrobial activity by the agar well diffusion method ²². Pure isolates of each microorganism (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae) was subcultured on the agar media plates at 37 ºC for 24 h. One plate of each microorganism was taken and a minimum of four colonies were touched with a sterile loop and transferred into normal saline (0.85%) under aseptic conditions. Density of each microbial suspension was adjusted equal to that of 10⁶ cfu/ml (standardized by 0.5 McFarland standards) and used as the inoculum for performing agar well diffusion assay. 100 µl of inoculum of each test organism ± standard deviation were calculated was spread onto the agar plates so as to achieve a confluent growth.

A well of 6 mm diameter was made using a sterile cork borer. 100 µl volume of each extract was placed directly into the wells (in triplicates) of the inoculated agar plates for each test organism, allowed to stand for 1 h for diffusion of extract into the agar and then incubated at 37 ºC for 24 h. Ciprofloxacin (5 μg/disc) was used as a positive control for antibacterial activity. After 24 h of incubation at 37 ºC, zone of inhibition (ZOI) was observed and diameter measured. The experiments were performed in triplicates and the mean values of the diameter of inhibition zones with

Statistical Analysis: Data of in-vitro assays recorded were analyzed using Microsoft Excel to determine IC₅₀. One-way analysis of variance (ANOVA) were conducted and P<0.05 was considered significant.

RESULTS AND DISCUSSION: Ethnomedicinal plants have been documented in a number of ancient texts for their ability to treat various common ailments as they are a rich source of bioactive molecules. The utilization of these substances as phytochemical agents in the treatment of inflammation has picked up significance all through the world. In the present research work hydromethanolic extract of T. arjuna was evaluated for the antimicrobial, antioxidant, anti-inflammatory and antidiabetic properties.

Fluorescence and Phytochemical Analysis: The bark of T. arjuna produced different colours and fluorescence under UV light and day light when treated with various reagents Table 1.

TABLE 1: FLUORESCENCE ANALYSIS OF POWDERED BARK OF T. ARJUNA

TABLE 2: PHYTOCHEMICAL INVESTIGATION OF T. ARJUNA

- indicates absence, + denotes average, ++ means abundance of phytochemicals.

Phytochemicals are secondary metabolites produced by many plants possessing medicinal uses. The analysis of the phytochemical results revealed greater presence of saponins, flavonoids, terpenoids, coumarins, quinines, cardiac glycosides, xanthoproteins, steroids, phenols, carboxylic acid group alkaloids, tannins, terpenoids, glycosides, resins in hydromethanolic extract of T. arjuna Table 2.

Total Phenolic, Flavonoid and FRAP Activity: Phenolics are known to balance carbohydrate and lipid metabolism, weaken hyperglycemia, dyslipidemia and insulin resistance, invigorate insulin discharge, enhance fat tissue digestion and ease oxidative stress. In addition, they possess physiological properties, such as anti-allergenic, anti-atherogenic, anti-inflammatory, anti-microbial, antioxidant, anti-thrombotic, cardioprotective and vasodilatory effects ^{23, 24, 25}. The total phenolic content calculated using the standard curve of gallic acid (y = 0.0096x, R² = 0.997) was found to be 754.16 ± 16.26 mg GAE/g of fresh weight tissue respectively Table 3.

Flavonoids, the most common secondary metabolite provides health benefits through their antioxidant activity. Their levels as estimated spectrophotometrically using a standard curve of quercetin (y = 0.0014x + 0.0267, R² = 0.9988) was found to be 247.02 ± 8.81mg/g fresh weight tissue Table 3. The antioxidant potentials of the extracts of the T. arjuna bark was estimated from their ability to reduce ferric tripyridyltriazine (Fe³⁺ -TPTZ) complex to a coloured ferrous tripyridyltriazine (Fe²⁺ -TPTZ). The results showed that FRAP values were higher in methanol: water extracts compared to the standard i.e., ascorbic acid Table 3. The antioxidant activity of the plant extract is dependent on storage time, geographic origin, harvesting, temperature and light during the storage and solvent used ²⁶.

TABLE 3: QUANTITATIVE ANALYSIS OF TOTAL PHENOLS, TOTAL FLAVONOIDS AND FRAP ACTIVITY OF T. ARJUNA BARK

All values are represented as mean ± SD (n=3). GAE - Gallic acid equivalents; QE - Quercetin equivalents.

In-vitro Antioxidant Activity:

DPPH Radical Scavenging Activity: The total antioxidant capacity of the extracts was calculated using % inhibition against concentration of ascorbic acid (y = 0.1529x + 13.49; R² = 0.853) Fig 1A. At 500 µg/ml concentration, T. arjuna extract exhibited maximum DPPH radical scavenging activity that was found to be 70.3+3.3 %. The IC₅₀ values calculated from graph and were found to be 238.78 µg/ml (Ascorbic acid, y = 0.1529x + 13.49), and 336.89 µg/ml (T. arjuna, y = 0.1365x + 4.014, R² = 0.986) Table 4. The results of the present investigation demonstrate that T. arjuna can significantly decrease in-vitro DPPH radical levels. The antioxidative activities observed is attributed to mechanisms exhibited by different polyphenolic compounds such as tocopherols, flavonoids and other organic acids ²⁷. Due to their redox properties, they serve as highly effective free radical scavengers causing the quenching of singlet and triplet oxygen or decomposing peroxides.

FIG. 1: ANTIOXIDANT ACTIVITIES OF HYDROMETHANOLIC EXTRACT OF BARK OF T. ARJUNA AND ASCORBIC ACID; DPPH RADICAL SCAVENGING (A) AND H₂O₂ SCAVENGING ACTIVITIES (B)

H₂O₂ Scavenging Activity: H₂O₂ scavenging assay was used to monitor the decrease in the absorbance of H₂O₂ upon its oxidation. The H₂O₂ scavenging activity of T. arjuna was determined and compared with ascorbic acid Table 4, Fig. 1B. The IC₅₀ values are calculated from graph and were found to be 215.68 µg/ml (Ascorbic acid, y = 0.1394x + 20.0; R² = 0.7914) and 253.01 µg/ml (T. arjuna, y = 0.1822x + 3.9, R² = 0.9761).

H₂O₂ crosses cell membranes rapidly where it reacts with Fe²⁺ and Cu²⁺ ions to form hydroxyl radicals resulting in various toxic effects. H₂O₂ is a weak oxidizing agent causing enzymatic inactivation by oxidation of essential thiol (-SH) groups. It degrades hemeproteins, such as hemoglobin, to release Fe ions. The ability of the extract to scavenge H₂O₂ is indicative of high antioxidant potential.

TABLE 4: EVALUATION OF IN-VITRO ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES OF HYDROMETHANOLIC T. ARJUNA BARK EXTRACTS

Results are mean ± SEM (P < 0.05), obtained from three replicates

In-vitro Anti-inflammatory Activity: Inflammation is a complex physiopathological response to different stimuli. The inflammatory process involves the activity of inflammatory mediators such as neutrophil derived free radical, reactive oxygen species (ROS), nitric oxide (NO), prostaglandins and cytokines. Over production of these leads to tissue injury by damaging macromolecules, lipid peroxidation of membrane and tissue damage play important role in pathogenesis of many inflammatory diseases ²⁸.

Thus, free radicals are important mediators that provoke or sustain inflammatory processes and consequently, their neutralization by antioxidants and radical scavengers can attenuate inflammation.

Lipooxygenase Inhibition: Lipoxygenases include non-heme iron-containing dioxygenases that involved in the biosynthesis of leukotrienes, needed for the pathophysiology of several inflammatory and allergic diseases ²⁹. The flavonoid quercetin, abundant in plants behaves as an antioxidant due to its lipoxygenase-inhibitory activities and free radical-scavenging properties ³⁰. It does so by binding covalently to iron or by forming molecular complexes blocking access to iron. In general, antioxidants inhibit lipid hydroperoxide formation by scavenging lipidoxy or lipid peroxy- radical formed in course of enzyme peroxidation, thus limiting the availability of lipid hydroperoxide substrate necessary for the catalytic cycle of LOX. The plant lipoxygenase pathway is in many respects equivalent to the ‘arachidonic acid cascades’ in animals. Hence, in-vitro inhibition of lipoxygenase constitutes a good model for the screening of plants with anti-inflammatory potential. Methanolic extract of T. arjuna was assayed at 100 - 500 μg/ml, and the inhibition obtained at different concentrations is as shown in Table 4. The standard diclofenac exhibited 60.3 ± 3.3% inhibitions at a concentration of 500 μg/ml. The IC₅₀ values are calculated from graph and were found to be 315.5 µg/ml (Diclofenac, y= 0.0971x + 19.36, R² = 0.6764), and 480.5 µg/ml (T. arjuna, y = 0.0838x + 9.73; R² = 0.8528) Fig. 2A.

Inhibition of Heat Induced Protein Denaturation: Protein denaturation refers to the loss of protein secondary and tertiary structure by the application of external stress or compounds, such as strong acid or base, a concentrated inorganic salt, an organic solvent or heat. One cause of inflammation is the denaturation of proteins leading to the loss their biological function. As part of the investigation on the mechanism of the anti-inflammation activity, ability of T. arjuna bark extract to inhibit protein denaturation was studied. Maximum inhibition of 72.1% was observed at 500 μg/ml by T. arjuna. Diclofenac, a standard anti-inflammatory drug showed the maximum inhibition 92.1% at the concentration of 500 μg/ml compared with control Table 4. The IC₅₀ values are calculated from graph and were found to be 222.85 µg/ml (Diclofenac, y=0.1715x+11.78, R² = 0.945) and 282.32 µg/ml (T. arjuna, y = 0.1369x + 11.35, R² = 0.914) Fig 2B.

Proteinase Inhibitory Activity: Activated leukocytes are widely implicated in cardiovascular disease (CVD). Mononuclear cells are recruited to sites of vascular injury thus contributing to foam cells within atherosclerotic plaques ³¹. Leukocyte activation occurs in all the conditions associated with an increased CVD risk: infection, hypertension, hyperlipidemia, hyperglycemia, obesity, and atherosclerosis ³². Activated WBCs discharge into the surrounding milieu reactive oxygen species (ROS) and a variety of proteolytic enzymes, particularly serine proteases. Proteinases have been implicated in arthritic reactions while leukocytes proteinase plays an important role in the development of tissue damage during inflammatory reactions. Proteinase inhibitors were found to provide protection against such inflammation-induced damage ³³. T. arjuna extract and diclofenac exhibited maximum inhibition of 29.9% at 500μg/ml while diclofenac showed a maximum inhibition 20.7% and 29.9% at 500 μg/ml respectively Table 4. The IC₅₀ values calculated from the graph were found to be 899.1µg/ml (Diclofenac, y = 0.0595x + 3.054, R² = 0.937) and 1339.3 µg/ml (T. arjuna, y = 0.0376x + 0.359, R² = 0.907) respectively Fig. 2C.

RBC Membrane Stabilization Activity: The erythrocyte plasma membrane resembles the lysosomal membrane and hence the stabilizing effect of drugs on erythrocyte membrane may correlate with its lysosomal membrane stabilizing effect ³². Any factor contributing to the lysosomal membrane stabilization leads to the inhibition of release of the inflammatory mediators and consequent inhibition of the process of inflammation. The mechanism of inflammation injury is attributed to the release of ROS from activated neutrophil and macrophages causing tissue injury through lipid peroxidation of membranes. In addition, ROS propagate inflammation by stimulating the release of the cytokines such as Il-I, TNFα, and INFγ, which further stimulate recruitment of additional neutrophil and macrophages. Thus, free radicals are important mediators that provoke or sustain inflammatory processes and consequently their neutralization by antioxidants and radical scavengers can attenuate inflammation. Maximum inhibition was observed at 500 μg/ml, where Diclofenac and T. arjuna exhibited 99.36 and 88.98% inhibition respectively Table 4. The IC₅₀ values for the standard drug and T. arjuna was found to be 121.16 (y = 0.172x + 29.16, R² = 0.728) and 192.81 (y = 0.1685x + 17.515, R² = 0.8671) μg/ml respectively Fig. 2D.

FIG. 2: ANTI-INFLAMMATORY ACTIVITIES OF STANDARD DRUG DICLOFENAC AND HYDRO-METHANOLIC EXTRACT OF T. ARJUNA BARK; LIPOXYGENASE INHIBITION ASSAY (A), INHIBITION OF HEAT INDUCED PROTEIN DENATURATION (B), PROTEINASE INHIBITORY ACTIVITY (C) AND RBC MEMBRANE STABILIZATION ASSAY (D)

In-vitro Hypoglycemic Activity: Diabetes mellitus is a metabolic disorder characterized by hyper-glycaemia and alterations in carbohydrate, lipid, and protein metabolism, associated with absolute or relative deficiencies in insulin secretion and/or insulin action ³¹. The World Health Organization (WHO) has estimated that major burden of diabetes will occur in developing countries, and by 2025, there will be 42% increase (from 51 to 72 million) in the developed countries and 170% increase (from 84 to 228 million) in the developing countries ³⁴. It is estimated that by 2025, India, China, and the United States will be the top three countries to have large number of diabetics ³⁵. The high prevalence of diabetes as well as its long-term complications has led to an ongoing search for hypoglycaemic agents. Over the years, various medicinal plants and their extracts have been reported to be effective in the treatment of diabetes ³². Plants are rich sources of antidiabetic, anti-hyperlipidemic and antioxidant agents such as flavonoids, gallotannins, amino acids, and other related compounds.

Alpha Amylase Inhibition Assay: The digestion of starch takes place over several stages. Salivary amylase partially digests starch into shorter oligomers. This is followed by pancreatic amylase in the gut into maltose, maltotriose and small malto-oligosaccharides. Inhibition of alpha amylase can lead to reduction in post prandial hyperglycemia in diabetic condition. The IC₅₀ values for the standard drug Acarbose and T. arjuna was found to be 324.81 (y = 0.0832x + 23.02, R² = 0.9072) and 210.91 (y = 0.072x + 34.35, R² = 0.9908) μg/ml respectively Table 5, Fig 3A. Our results showed that the bark extract of T. arjuna possess stronger antidiabetic activity when compared with acarbose standard. This shows that the bark of the medicinal plant T. arjuna could be a good alternative to other medicines used for treating diabetes.

Pancreatic Lipase Inhibition Assay: This enzyme is required for dietary triacylglycerol absorption catalyzing its hydrolysis to 2-monoacylglycerol and fatty acids prior to absorption. Natural compounds such as proteins and saponins are known to inhibit gastrointestinal lipases ³⁰. In addition, polyphenolic extracts from a number of plants have been shown to be effective inhibitors of the intestinal pancreatic lipase enzyme ³⁶.

A dose-dependent manner of inhibition was exhibited by the bark extract of T. arjuna Table 5. Orlistat, the standard drug and a pancreatic lipase inhibitor, exhibited maximum inhibition of 75.71% at 500 g/ml while T. arjuna exhibited 66.45% at the same concentration. The IC₅₀ values for Orlistat and T. arjuna was found to be 88.07 (y = 0.0587x + 44.83, R² = 0.9556) and 55.52 (y = 0.0458x + 47.64, R² = 0.48) μg/ml respectively Fig. 3B.

FIG. 3: ANTI-DIABETIC ACTIVITIES OF HYDROMETHANOLIC EXTRACT OF T. ARJUNA BARK; ALPHA AMYLASE INHIBITION ASSAY WITH ACARBOSE AS CONTROL (A) AND PANCREATIC LIPASE INHIBITION ASSAY WITH ORLISTAT AS CONTROL (B)

TABLE 5: EVALUATION OF IN-VITRO ANTI-DIABETIC ACTIVITIES OF HYDROMETHANOLIC EXTRACT OF T. ARJUNA BARK

Results are mean ± SEM (P < 0.05), obtained from three replicates.

In-vitro Glucose Adsorption Capacity: The glucose adsorption capacity of the methanolic bark extract of T. arjuna at different glucose concentration was investigated in this study, and the results are presented in Fig. 4.

FIG. 4: GLUCOSE BINDING CAPACITY OF HYDRO-METHANOLIC EXTRACT OF BARK OF T. ARJUNA COMPARED TO THE CONTROL METRONIDAZOLE

It was observed that glucose binding capacity was directly proportional to the molar concentration of glucose. The bark sample was found to be effective in adsorbing glucose at both lower and higher concentrations. This could be attributed to the insoluble and soluble constituents and fibers present. Absorption of glucose would thus reduce the amount of glucose available for transport across the intestinal lumen, consequently reducing postprandial hyperglycemia event.

In-vitro Glucose Diffusion Inhibitory Assay: The methanolic extract of T. arjuna bark was subjected to this assay to determine glucose diffusion and GDRI across the dialysis membrane and the results are shown in Table 6. The rate of glucose diffusion across the membrane in the control was found to increase from 30 to 180 min however, inhibitory effects on movement of glucose across the dialysis membrane was noted in the sample. GDRI was calculated to predict the effect of a fiber on the delay in glucose absorption in the gastrointestinal tract ³⁷. The GDRI was found to be a decrease in an increase with time.

The higher GDRI value indicates higher retardation index of glucose by the sample. The inhibition in movement could occur due to the presence of fiber resulting in decrease in concentration and encapsulation of enzyme and starch.

TABLE 6: EFFECT OF HYDROMETHANOLIC EXTRACT OF T. ARJUNA BARK ON GLUCOSE DIFFUSION COMPARED TO THE CONTROL METRONIDAZOLE

Values in parenthesis indicate GDRI. Values are expressed by mean ± SD of five samples in each group

Antimicrobial Activity of T. arjuna: Most of the bioactive medicinal metabolites are synthesized via plant secondary metabolic pathways during the vegetative stage of plant's life cycle and these compounds are responsible for the therapeutic properties of the plant tissue chosen. Chemical or synthetic drugs and antibiotics have been used for treatment of human diseases and infections. In general, these compounds effectively inhibit and/or stop microbial growth via disruption of the synthesis of microbial nucleic acids, proteins and cell walls ³⁸. It was found that all extracts of T. arjuna tested showed antibacterial activity against S. aureus with highest zone of inhibition in hot aqueous extract (28 mm) followed by cold aqueous extract (26 mm) Table 7. The zone of inhibition produced by the methanolic bark extract against E. coli was 17 mm, however, both hot and cold aqueous extracts lacked antibacterial activity. In general, except for S. aureus, it was observed that methanolic extract possessed slightly higher antibacterial activity than aqueous extracts. The limited spectrum of antimicrobial activity could be due to the polarity of antibacterial compounds which make them more readily extracted by methanol when compared to aqueous extract. In addition, antagonistic effects of other constituents may exert an influence on the bioactive compounds.

TABLE 7: ANTIBACTERIAL ACTIVITY OF HYDROMETHANOLIC EXTRACT OF T. ARJUNA BARK

All values are represented as mean ± SD (n = 3)

CONCLUSION: The present study, showed the presence of various bio active metabolites confirming it as a potent source for modern drugs. In addition, pharmacognostical characters and physicochemical parameters studied prove that the hydromethanolic extract of T. arjuna bark possesses potential antioxidant, anti-inflammatory, anti-diabetic and anti-microbial activities. Ethanomedicinal use of T. arjuna as a useful remedy in arthritic disorders could possibly be because of its excellent anti-inflammatory and antioxidant potential. These assays provide a scientific authentication to the traditional claims of its use in arthritis and other anti-inflammatory disorders. The hypoglycemic activity mediated by increasing glucose adsorption and decreasing glucose diffusion rate is suggestive of its anti-diabetic role. However, these results should be confirmed by in-vivo models and clinical trials for their effective utilization as therapeutic agents.

ACKNOWLEDGEMENT: The authors are grateful to Mount Carmel College, Autonomous, Bengaluru, India for provision of facilities needed for conducting this project.

CONFLICT OF INTEREST: The authors declared no competing interests.

REFERENCES:

1. AICEP: Summary report of the all India coordinated ethanobiological project (AICP), Ministry of Environment and Forest, Government of India 1994.
2. Anthropological survey of India. People of India project Report for 1994.
3. Bandaranayake WM: Quality control, screening, toxicity, and regulation of herbal drugs. Modern Phytomedicine. Turning Medicinal Plants into Drugs. Weinheim: Wiley-VCH GmbH & Co. KGaA 2006.
4. Mandal A, Das K and Nandi DK: In-vitro bioactivity study of bark extract of Terminalia arjuna on probiotics, commercially available probiotic formulation. International Journal of Phytopharmacology 2010; 1(2): 109-113.
5. Karthikeyan K, Bai BRS, Gauthaman K, Sathish KS and Devaraj SN: Cardioprotective effect of the alcoholic extract of Terminalia arjuna bark in an in-vivo model of myocardial ischemic reperfusion injury. Life Sciences 2003; 2727-2739.
6. Manna P, Sinha M and Parames CS: Aqueous extract of Terminalia arjuna prevents carbon tetrachloride induced hepatic and renal disorders. BMC Complement Altern Med 2006; 6: 33.
7. Chase CR and Pratt RJ: Fluorescence of powdered vegetable drugs with particular reference to development of a system of identification. J Amer Pharm Assoc 2002; 38: 324-331.
8. Harborne JB: Phytochemical Methods, A Guide to Modern Techniques of Plant Analysis, 3^rd, Springer (India) Pvt. Ltd., New Delhi 1991.
9. Khandelwal KR: Practical Pharmacognosy, 19th Ed., Nirali Prakashan, Pune 2009.
10. Slinkard K and Singleton VL: Total phenol analysis: automation and comparison with manual methods. Am. J. Enol. Vitic 1997; 28: 49-55.
11. Chang CC, Yang MH, Wen HM and Chern JC: Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food Drug Anal. 2002; 10: 178-82.
12. Oyaizu M: Studies on products of browning reactions: antioxidative activities of products of browning reaction prepared from glucosamine. Japanese Journal of Nutrition 1986; 44: 307-315.
13. Braca A, Sortino C and Politi M: Antioxidant activity of flavonoids from Licanialicaniae flora. J. Ethnopharmacol 2012; 79: 379-81.
14. Ruch RJ, Cheng SJ Licanialicaniaeflora Klaunig JE: Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 1989; 10: 1003-8.
15. Axelrod BC, Cheesbrough TM and LaaskoSL: Lipoxygenase from soybean. Methods Enzymol. 1981; 71: 441-51.
16. Sakat S, Juvekar AR and Gambhire MN: In-vitro antioxidant and anti-inflammatory activity of methanol extract of Oxalis corniculate Int. J. Pharm. Pharmacol. Sci. 2010; 2: 146-55.
17. Azeem AK, Dilip C, Prasanth SS, Junise V and Hanan S: Anti-inflammatory activity of the glandular extracts of Thunnusala longa. Asia Pac. J. Med. 2010; 3: 412-20.
18. Shai LJ: Yeast alpha glucosidase inhibitory and antioxidant activities of six medicinal plants collected in Phalaborwa, South Africa. South African Journal of Botany 2010; 76: 465-470.
19. Bustanji Y, Mohammad M and Hudaib M: Screening of some medicinal plants for their pancreatic lipase inhibitory potential. Jordan Journal of Pharmaceutical Sciences 2011; 4(2): 81-88.
20. Ou S, Kwok K, Li Y and Fu L: In-vitro study of possible role of dietary fiber in lowering postprandial serum glucose. J Agric Food Chem 2001; 49(2): 1026-9.
21. Ahmed F, Sairam S and Urooj A: In-vitro hypoglycemic effects of selected dietary fiber sources. J Food Sci Technol 2011; 48(3): 285-9.
22. Ahmad I and Beg AZ: Antimicrobial and phytochemical studies on 45 Indian medicinal plants against multi-drug resistant human pathogens. J Ethnopharmacol. 2001; 74(2): 113-23.
23. Benavente-Garcia O: Uses and properties of citrus flavonoids. Journal of Agricultural and Food Chemistry 1997; 45: 4505-4515.
24. Sonia N, Myrene RD and Alisha: Pharmacological evaluation of Parkia speciosa for antioxidant, anti-inflammatory, anti-diabetic and antimicrobial activities in-vitro, International Journal of Lifesciences 2018; Special Issue, A11: 49-59.
25. Ashmita M, Myrene RD and Vaishnavi B: Evaluation of pharmacological activities of seed and pericarp of Litchi chinensis International Journal of Engineering & Scientific Research 2018; 6(1): 25-35.
26. Moure A, Franco D, Sineiro J, Domĭngguez H, Nūňez MJ and Lema JM: Antioxidant activity of extracts from Gevuina avellana and Rosa rubiginosa defatted seeds. Food Research International 2001; 34(2): 103-109.
27. Tahareen S, Shwetha R and Myrene RD: Potential antioxidant, anti-inflammatory and antibacterial evaluation of extracts of Leucas aspera using in-vitro International Journal of Pharmacy and Pharmaceutical Sciences 2016, 8: 11, 292-297.
28. Khalid MN, Al-Shaibani ES, Alhadi FA, Al-Soudi SA, Dsouza MR: Hepatoprotective and antioxidant effects of single clove garlic against CCl₄-induced hepatic damage in rabbits. BMC Complementary and Alternative Medicine 2017; 17: 411.
29. Ghosh MN: Fundamentals of Experimental Pharmacology. Scientific Book Agency, Calcutta 1998; 174-179.
30. Sadik CD, Sies H and Schewe T: Inhibition of 15-lipoxygenases by flavonoids: structure-activity relations and mode of action. Biochemical Pharmacology 2003; 65(5): 773-81.
31. Mandal S, Patra A, Samanta A, Roy S, Mandal A, Mahapatra TD, Pradhan S, Das Koushik and Nandi DK: Analysis of phytochemical profile of Terminalia arjuna bark extract with antioxidative and antimicrobial properties. Asian Pacific Journal of Tropical Biomedicine 2013; 3(12): 960-966.
32. Debnath S: Antibacterial and antifungal activity of Terminalia arjuna Wight &Arn. bark against multi-drug resistant clinical isolates. Journal of Coastal Life Medicine 2013; 1(4): 315-321.
33. Das SN and Chatterjee S: Long term toxicity study of ART-400. Ind Indg Med 1995; 16: 117-123.
34. Hebbani AV, Reddy VD and Nallanchakravarthula V: In-vitro anti-hemolytic activity of Terminalia arjuna (Roxb.) Wt. & Arn. Bark Powder Aqueous Extract. Ind. J. Adv. Chem. Sci. 2014; 3: 02-108.
35. Akhter K, Dockray S and Simmons D: Exploring factors influencing non-attendance at the diabetes clinic and service improvement strategies from patients' perspectives. Practical Diabetes International 2012; 29(3).
36. Harborne JB: Phytochemical methods- A guide to modern techniques of plant analysis. Springer (India) Pvt. Ltd, New Delhi 1998; 5-32.
37. Basha S and Kumari VS: In-vitro antidiabetic activity of Psidium guajava leaves extracts. Asian Pac J Trop Dis 2012; 98-100.
38. Randhir R, Lin YT and Shetty K: Phenolics, their antioxidant and antimicrobial activity in dark germinated fenugreek sprouts in response to peptide and phytochemical elicitors. Asia Specific Journal of Clinical Nutrition 2004, 13(3): 295-307.
    

    ---

    How to cite this article:

    Alisha, Dsouza MR and Sonia N: Pharmacognostical overview on Terminalia arjuna: a justification of folkloric belief. Int J Pharm Sci & Res 2018; 9(12): 5411-23. doi: 10.13040/IJPSR.0975-8232.9(12).5411-23.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---