INTRODUCTION:

The spread of multi-drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases. Down the ages, medicinal plants have evoked interest as sources of natural products for their potential uses as alternative remedies to heal many infectious diseases¹. Herbal plants play an important role in the development of potent therapeutic agents. Over the years, various medicinal plants and their extracts have been reported to be effective in the treatment of diseases². T. arjuna is distributed throughout India, Burma and Sri-Lanka. It mainly grows along the banks of the river and streams. Terminalia arjuna Roxb. (Combretaceae) commonly known as Arjuna is one of the medicinally important evergreen tree³ which possesses antimicrobial, cytotoxic, antidiabetic⁴, antidiarrheal, antidysentric⁵ and hepatoprotective⁶ activities.

Terminalia arjuna stem bark is reported to contain different groups of chemical constituents viz. hydrolysable tannins triterpene⁷ (arjunetin, arjunic acid, arjunolic acid, and arjungenin, Arjunolic acid (2, 3, 23-trihydroxyolean-12-en-28-oic acid); Flavanoids (arjunolone, arjunon, luteolin), phenolics⁸, and phyto sterols⁷, gallic acid, ellagic acid, oligomeric proanthocyanidins (OPCs), calcium, magnesium, zinc and copper⁹. Arjuna is a good source of phytosterol, namely, sitosterol which lowers down the cholesterol in blood serum mediated through inhibition of cholesterol absorption resulting from the higher solubility of phytosterols than of cholesterol in bile salt micelles^10,11.

Traditionally the drug is widely used in the preparations of Ayurvedic and Unani formulations used in cardioprotection¹². Bark powder is useful to cure headache and to kill worms in the teeth and treatment of heart troubles^13,14. Juice is used as antacid^{15 and
fruits are found useful as tonic. Bark ash is used in the treatment of the
snakebite and scorpion sting in rural/tribal peoples.}

In the present study an attempt has been made to investigate physicochemical, phytochemical and antibacterial activity of T. arjuna to explore its therapeutic potential.

MATERIALS AND METHODS:

Plants collection and identification:

The stem bark of Terminalia arjuna was collected from the forest of Chitrakoot region, Satna. The herbarium of the plant was also prepared for their correct identification. After proper collection, the plant was washed with water to remove dust and after washing this plant was dried, grinded and sieved (sieve no.100) and finally the plant sample was stored in airtight container.

Physico-chemical analysis:

Total Ash:

Accurately weighed 2g of powdered drug was taken in a tarred silica dish and ignited at temperature not exceeding 450°C until it became white (carbon free). Cooled in desiccator and weighed. Finally, percentage of total ash content was calculated with reference to air dried drug^16,17.

Acid Insoluble Ash:

The total ash obtained from 2g of bark and leaf sample were boiled with 25ml of dilute hydrochloric acid for 5 minutes. The insoluble matter was collected on Gooch crucible, washed with hot water and ignited to obtain constant weight. The percentage of amount of acid insoluble ash was calculated with reference to air dried drug^{16, 17}.

Alcohol Soluble Extractive value:

2 g powdered drug was placed in a conical flask and macerated with 100 ml of Alcohol (90% v/v) for 6 hours, with frequent shaking and then allowed to stand for 18 hours. Filtered through Whatmann filter paper. 10 ml of filtrate was transferred to flat bottom dish and solvent was evaporated on a water bath. Cooled it in desiccator for 30 minutes and finally weighed. The content of extractable matter air-dried material was calculated^16,17.

Water Soluble Extractive value:

2 g powdered drug was placed in a conical flask and macerated with 100 ml of water for 6 hours, with frequent shaking and then allowed to stand for 18 hours filtered through Whatmann filter paper. 10 ml of filtrate was transferred to an evaporating dish and solvent was evaporated on a water bath. Cooled it in desiccators for 30 minutes and finally weighed. The content of extractable matter air-dried material was calculated^16,17.

Loss on drying:

Weighed 2 gm of the drug powder in a dried petri dish. The sample was heated in an oven at a temperature 105 degree and this procedure was repeated untill constant weight of sample was obtained. After it was dried, the petri dish was allowed to cool in desiccator. The loss on weight in percentage of air-dried material was calculated^16,17.

Preliminary Phytochemical screening:

Preliminary qualitative phytochemical analysis of aqueous and alcoholic extracts of T. arjuna was carried out by employing standard protocol^18,19 for determining the presence and/or absence of phytochemicals viz : Alkaloids: (Dragendorff’s, wagner, mayer;s test), flavonoids (Shinoda test), Carbohydrate (Fehling, Molisch, Anthrone test), Protein (Biurate test, Millons test), Resin, saponins (Foam tests), Tannin ( ferric chloride), starch and steroid (Salkowski test).

High Performance Thin-layer Chromatography (HPTLC):

HPTLC of Methanolic extract of stem bark were carried out by using Toluene: Ethyl Acetate: Formic acid (7:2.5:0.5) as solvent system. 5 ul of the test solutions were spotted on pre coated silica gel aluminium plate 60F-254 (5cm×10cm) using Camag Linomat5 sample applicator fitted with 100ul Hamilton syringe. Plates were developed in Camag development chamber followed by photo documentation in Camag photoreprostar with CATS software. Visualization was done under 366nm and after derivatization with 5% Methanolic sulphuric acid reagent. Calculated the R_f value and color of the resolved bands^20,21.

Screening of antimicrobial activity²²:

The in vitro sensitivity of the Pseudomonas aeruginosa isolated in the crude extracts of T. arjuna was determined by disc diffusion method. Dried and sterilized paper discs were treated separately with desired concentration of previously prepared methanol and aqueous solution of the crude extract using a micropipette dried in air under aseptic condition and placed at equidistance in a circle on the seeded plate. The concentrations of crude extract used were 1 mg/ml. These plates were kept for 4-6 hours at low temperature and the test materials diffuse from disc to the surrounding medium by this time. The plates were then incubated at 37^oC for 24 hours and zone diameter was measured in mm.

RESULTS AND DISCUSSION:

In present study the bark of T. arjuna Linn. was evaluated for its physicochemical, phytochemical along with HPTLC determination and antibacterial activity against Pseudomonas aruginosa.

Physico-chemical analysis:

Physicochemical and phytochemical analysis are used to check the genuine nature of the crude drug, thus it plays an important role in preventing the possible steps of adulteration²³. Quality tests for drug powder was performed for moisture content, ash content, acid insoluble ash, water soluble extractive, alcohol soluble extractive, and were compared to API standards and are found to be within ranges. The Results of physicochemical analysis are given in (Table 1). The results are expressed as mean (n=3) ± Standard deviation (SD).

Table 1: Physicochemical analysis

Name of the experiment	Result (w/w)	Standard (API)
Loss on drying 105⁰c	3.4%	-
Water soluble extractive value	54%	Not less than 17%
Alcohol soluble extractive value	18.5%	Not less than 16%
Total ash	16.35%	Not more than 17%
Acid insoluble ash	1.8%	Not more than 2%

*Results are average of three values

Preliminary Phytochemical screening:

The results of preliminary phytochemical screening showed the presence of carbohydrates, phenolic compounds, flavonoids, alkaloids, proteins, saponins, and tannins, in the methanolic extract of the drugs (Table 2). The medicinal properties of plants material are mainly due to the presence of various phytoconstituents²⁴. The presence of different phyto constituents in studied drugs justifies their therapeutic potential^25,26. These phytoconstituents have been reported to have multiple biological effects such as anti-inflammatory, anti allergic, antioxidant, antidiabetic, analgesic, antispasmodic, antibacterial, anti-viral, anti-cancer and aldose reductase inhibitory activities. It is also used for the treatment of diarrhea and dysentery²⁷.

High Performance Thin-layer Chromatography (HPTLC):

Fingerprinting analysis of sample was done through HPTLC method, and the data were presented in the (Table 3, Fig.1). The result indicates the presence of various spots with different color at different R_f values. HPLTC is an important analytical tool in the separation, detection and estimation of different classes of natural products. Developed fingerprint profile would serve as a reference standard for quality evaluation and standardization of the formulation with same drug. In the last few decades, an HPTLC technique has gained much popularity for standardization of the herbal drugs and formulations. Analysis of several samples simultaneously using a small quantity of marker compound and mobile phase with very less time is the major advantage of HPTLC²⁸. TLC and HPTLC techniques have been used as a important analytical tools in pharmaceuticals, medicine, chemistry, food analysis, toxicology and environmental science²⁹.

Susceptibility against T. arjuna extract:

The bark of the Terminalia arjuna constitutes an important crude drug, which contains tannins, triterpenoids saponins, flavonoids, sterols, calcium salts, alkaloidal and glycosidal substances, arjunine and arjunglyciside etc. It stops bleeding and pus formation in the gums and is useful in asthma, dysentery, menstrual problems, pain, leucorrhoea, wounds and skin eruptions¹¹. Due to increased awareness of the importance of traditional medicine in human and animal health care, research into the efficacy of some of the herbs used in the treatment of some illness would be worthwhile.

Table 2: Preliminary phytochemical screening

Phyto constituents	Observation	Methanol extract	Aqueous extracts
Alkaloids Dragendorff’s test Wagner’s test Mayer;s test	Orange colour appears Brown colour appears Pale yellow colour appears	+ve +ve +ve	-ve -ve -ve
Carbohydrate Anthrone’s test Fehling’s test Molisch test	Brown colour appears Brick red colour appears Ring of red violet colour appears	+ve +ve +ve	+ve +ve +ve
Flavonoids	Light brown colour appears	+ve	+ve
Protein Biuret’s test Millon’s test	Brown colour appears Orange colour appears	-ve -ve	-ve -ve
Resins	Turbidity appears	+ve	+ve
Saponin	Honey comb like froth forms	+ve	+ve
Steroid	Red violet colour ring appears	+ve	+ve
Tannin	Brown colour appears	+ve	+ve
Starch	Yellow colour appears	-ve	-ve

Arjun bark extract was used as a biological tool to resolve the antibiotic resistant Pseudomonas aeruginosa problem. Arjun extract showed promising effect against the isolated Pseudomonas aeruginosa at concentration of 1 mg/ml. The zone diameter of the extract is given in (Table 4). This study revealed that Terminalia arjuna would be a good antibacterial drug in the treatment of Pseudomonas aeruginosa infections, provided if it is found effective and nontoxic through in vivo study. Further study is needed to understand the molecular mechanism of the extract that will helped us to make more effective therapeutics to combat Multi-Antibiotic Resistant (MAR) pathogen. The decrease in percent was found to be more potent in aqueous extract of T. arjuna which is 33% high as compared to other solvents. Hence, it was found that people used chloramphenicol in their dose as an antibiotic is more beneficial for them.

Table 3: R_f values color of the bands resolved in test solutions of T. arjuna Linn.

Mobile phase: Toluene: Ethyl Acetate: Formic acid (7:2.5:0.5)

Wave length

Standard

Test sample

366nm

0.07 (pink), 0.11 (pink) ,0.22 (light blue), 0.31 (light yellow), 0.54 (light pink), 0.59 (light blue), 0.77 (blue), 0.86 (pink) , 0.91(pink)

0.07 (pink), 0.11 (pink), 0.20 (blue), 0.24 (blue), 0.31 (light yellow), 0.43 (sky blue), 0.54 (light pink), 0.54 (light pink), 0.66 (reddish blue), 0.77 (sky blue), 0.82 (blue), 0.86 (pink), 0.91 (pink)

366nm AD

0.08 (light pink) , 0.63 (light brown), 0.92 (light orange)

0.08 (light pink), 0.15 (faint blue), 0.23 (blue), 0.59 (sky blue), 0.63 (light brown), 0.83 (blue), 0.92 (orange color)

Table 4: Zone diameter of Terminalia arjuna extracts:

Pathogen

Zone of inhibition

Chlorom

phenicol

Methanol extract

Aqueous extract

Antibiotics (chloram

phenicol)

Pseudomonas aeruginosa

14 mm

37mm

36mm

20mm

Pathogen

Zone of inhibition

Chlorom

phenicol

Methanol extract

Aqueous extract

Antibiotics (chloram

phenicol)

Pseudomonas aeruginosa

24 mm

37mm

36mm

32 mm

T1 T2 T1 T2

(I) 366nm before derivatization (II) 366nm after derivatization

Fig. 1: HPTLC Chromatographs of Terminalia arjuna- acid- T1- Marker (Ellagic acid and T2 – Sample of Terminalia arjuna (methanolic)

CONCLUSION:

Quality evaluation of medicinal plants is a fundamental requirement of industry and other organization dealing with ayurvedic and herbal products. The bark of Terminalia arjuna contains appreciable amount of secondary metabolite. These phytoconstituents may acts as resource of pharmacologically active agents and natural antioxidants. The present investigations will be helpful while standardizing the drug for its various pharmacological potentials and to check the adulteration in natural valuable drug at the time of consumption for desire pharmacological effect. The additional antibiotic resistance might have contributed to the initial selection of the new strain, since these antibiotics are used to treat patients. Antibiotics clearly influence the prevalence of novel antibiotic-resistant clones in asthmatic areas. It is hoped that this study would lead to the establishment of some compounds that could be used to formulate new and more potent anti-microbial drugs of natural origin against Pseudomonas aeruginosa.

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Received on 24.08.2017 Modified on 18.09.2017

Res. J. Pharmacology & Pharmacodynamics.2017; 9(3): 147-151.

DOI: 10.5958/2321-5836.2017.00025.8