Keywords:
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| phenol, terpenoids, flavonoids, alkaloid, protein, secondary metabolites |
Introduction
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| Medicinal plants are rich source of novel drugs that forms the ingredients in traditional systems of medicine, modern medicines, nutraceuticals, food supplements, folk medicines, pharmaceuticals intermediates bioactive principles and lead compounds in synthetic drugs [1]. One of the most important medicinal plants, which are widely used in the traditional system of medicine, is Terminalia chebula [2]. This drives the need to screen medicinal plants for novel bioactive compounds as plant based drugs are biodegradable and safe [3]. A natural product plays an important role in the field of new drugs research and development because of their low toxicity, easy availability and cost effective [4]. The primary metabolite like chlorophyll, amino acids, nucleotides, simple carbohydrates or membrane lipids, play recognized roles in photosynthesis, respiration, solute transport, translocation, nutrient assimilation and differentiation [5]. Secondary metabolites are synthesized by the plants as part of the defense system of the plant [6]. The plant contains chebulinc acid, tannic acid, gallic acid, resin, anthroquinone and sennoside. It also contains glycosides, sugar, triterpenoids ,steroids and small quantity of phosphoric acid these compounds were proven to exhibit anti-bacterial, anti-fungal, anti-viral and anti-carcinogenic [7]. The Terminalia chebula shows antioxidant, adaptogenic and anti-anaphylactic, hypolipidemic, hepato protective, cardio protective, anti-diabetic, wound healing, immuno-modulatory and chemo preventive [8, 9]. Terminalia chebula is rich in tannin, which is hydrolysable to pyrogallal was found in fruits [10]. |
Materials & Methods
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| Collection of Plant |
| Herbal Plant Terminalia chebula was collected from the dense forest at Bengali, Karnataka state, India, based on its ethno medical importance. |
| Preparation of powder |
| The leaves, fruits, seed, stem and root powders were prepared [11] by washing with distilled water, surface sterilized with 10% sodium hypochlorite solution, rinsed with sterile distilled water and air dried at room temperature under shadow and then milled to fine powder. |
| Extraction of plant material |
| Methanolic extract |
| The methanol extract was prepared [4] by taking 10grams of powdered sample, were soaked in 50ml of methanol and it was kept in Soxhled apparatus at 80 degree Celsius for 48 hours. This extraction was taken and allowed for evaporation and it was concentrated with Dimethyl Sulfoxide (4.64g). |
| Phytochemical activity |
| The phytochemical analysis of Terminalia chebula was carried out [12] by adopting the following tests |
| Test for alkaloids |
| The Terminalia chebula extracts of leaves, fruits, seed, stem and root were filtrated then treated with Potassium mercuric iodide (Dragendroffs reagent) and observed for the colour change in the test tubes. |
| Test for flavonoids |
| 0.2 grams of plant extracts such as leaves, fruits, stem and root was added into test tube containing 2ml of diluted sodium hydroxide and mixed well. After mixing 2ml of diluted hydrochloride was added into the test tubes and observed for colour change. |
| Test for terpenoids |
| 0.5grams of plant extracts such as leaves, fruits, seed, stem and root was added to test tubes containing 2ml of chloroform and content was mixed well. Then 2ml of concentrated sulphuric acid was added carefully and observe for presence of reddish brown colour. |
| Quantities assay for tannin |
| The quantities assay for tannin acids was carried [6] out by taking the 500mg finely dried plant extract was added into a glass beaker containing 5ml of 70% aqueous acetone. The content solution was uniformly mixed and gently boiled in a water bath for 30 minutes. The solution was centrifuged at 3000 rpm for 10 minutes at 40C and supernatants were collected and stored in freezing condition. The pallet were dissolved in 5ml of 70% aqueous acetone and recentrifuged at 3000rpm for 10 minutes at 40C. The supernatants were collected and mixed with freezing stored supernatants. To this supernatants 1 ml of Folindenis reagent, 3ml of Sodium carbonate solution was added and solution was diluted to 20 ml by using distilled water. The solution was mixed well and incubated at room temperature for 30 minutes. The absorbance was measured in a spectrophotometer at 700nm. |
| Extraction of Reducing sugar |
| Extraction of sugars from the sample was usually carried [9] out by using 80 per cent ethanol. The crushed material is refluxed in a Soxhlet's apparatus for about 30 minutes. After refluxing, alcohol was wiped out from the extract with the addition of distilled water. The extract was centrifuged at 3000 rpm .To the supernatant, 1 ml of saturated lead acetate solution and 1 ml of saturated Di-sodium hydrogen phosphate solution was added. After centrifugation, the clear supernatant was collected and made up to a known volume with distilled water. To make standard, 1ml aliquots are taken from the supernatant, respectively in hard-glass test tubes. The 5ml of Somogyi's reagent and distilled water are added so that final volume of the solution in each test tube comes to 15ml. The tubes covered with lids are heated in a boiling water bath for about 15 minutes. These are then cooled to room temperature. The tubes, in which the solutions have reprecipitated, are discarded. 1ml of 2.5 per cent potassium iodide solution was added to each test tube. Then 3ml of 1.5N Sulphuric acid was added to this and shaken well till the golden yellow colour is formed. A burette was filled with 0.005 N sodium thiosulphate solutions. It was titrated with the sample on addition of 1-2 drops of starch solution to the latter. The end point was determined by the complete disappearance of the colour from the solution. A blank was also prepared by mixing 5ml of Somogyi's reagent and 10ml of distilled water. It was heated in a boiling water bath for about 15 minutes. After cooled and 1 ml of 2.5 per cent potassium iodide solution and 3ml of 1.5 N sulphuric acid are added. Titration was done using 0.001 N sodium thiosulphate solution containing starch solution and colorless end-point was determined. |
| Identification of amino acid, phenolic and aromatic compounds by using TLC method |
| A sample of 500mg/ml concentration of plant extracts was prepared [13] and from this solution, 4μl of the sample prepared was taken and spotted on the silica coated TLC plate. It was then kept at solvent position with the solvent to run under capillary pressure. Here ethanol, methanol and acetic acid (5ml, 5ml, and 100μl) were used as a solvent. The spots were then identified in the iodine chamber for phenolic compounds, ferric chloride for aromatic compounds and ninhydrin for amino acids. |
| Column chromatography |
| The crude aqueous methanol extract of Terminalia chebula was subjected to column chromatography [1] over silica gel 100-200 mesh. The column was eluted with solvents of increasing order of polarity. The fractions were collected in 10ml each and allowed to evaporate to get the residue. Each fraction was tested for the presence of various constituents by Thin Layer Chromatography. |
RESULT
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| Terminalia chebula |
| Kingdom-Plantae, Subkingdom-Tracheobionta, Superdivision-Spermatophyta, Division- Magnoliophyta, Class-Magnoliopsida, Subclass- Rosidae, Order-Myrtales, Family-Combretaceae, Genus-Terminalia, Species- Terminalia chebula |
| Phytochemical activity: |
| Alkaloid |
| There was a reddish brown colour formation in the test tubes after treating with Potassium Bismuth iodide solution thus indicates the presence of alkaloid in the plant extract (Figure 1). |
| Flavonids |
| The yellow colour was formed in the test tubes after treating with 5% Sodium hydroxide and diluted hydrochloride acid thus indicated the presence of flavonoids (Figure 2). |
| Terpenoid |
| The Terpenoid was present in the plant extract as there was reddish brown colour formation in the test tubes after treating with chloroform and concentrated Sulphuric acid (Figure 3). |
| Quantity of Tannic acids |
| The phenol content was found maximum in root (85.36mg/gdw) followed by seed (78.30mg/gdw), stem (72.46mg/gdw), roots (65.30mg/gdw) and leaves (21.39mg/gdw). |
| Reducing sugar |
| The maximum content of sugar was in leaves (7.12mg/gdw) followed by fruit (5.70mg/gdw), stem (4.80mg/gdw), root (4.10mg/gdw) and seed (3.7mg/gdw). |
| Thin layer Chromatography |
| In thin layer chromatography the amino acids showed pink colour spots were observed after treating with ninhydrin. The sugars showed the purple and black spots after treating with ferric chloride. The phenolic compounds showed blue spots after treating with iodine solution |
| Column chromatography |
| Higher content of protein was observed in fruits of T. Chebula (44.40mg/gdw) followed by seed (42.10mg/gdw), roots (40.60mg/gdw), leaves (36.10mg/gdw) and stem (29.40mg/gdw). The Purification of compounds in various extracts was performed by using column chromatography was presented in (Table 1). |
DISCUSION
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| Terminalia chebula is called the king of medicine in Tibet and is always listed at the top of the listed in Ayurvedic medicine [14]. A diverse arsenal of new antibacterial agents is urgently needed to combat the diminishing efficacy of existing antibiotics [15]. In earlier studies, lipids were reported to be higher in leaves (46.0mg/gdw) of M. oleifera and the roots (39.0mg/gdw) of C. quadrangularis [16, 17]. In modern therapeutic treatments, nanoparticles and liposomes are being used to develop delivery systems that are convenient and effective for tackling problems in disease treatments [18]. Proteins are complex nitrogenous organic substances that are one of the most important plant products to man. A part from this, the protein hydrolytes from various sources are reported to possess antioxidant activity [19, 20]. Plant phenols are groups of natural products with variable structure that are well known for their beneficial effects on health possess significant antimicrobial and antioxidant activities [21, 22]. Terminalia chebula have been noted to possess shikimic acid, gallic acid, B – sitosterol, tannic acid, triethyl ester of chebulic acid, ethylester of gallic acid and ellagic ethaedioic acid [23]. Terminalia plant contains several constituents like tannins, flavonoids, sterols, amino acids, fructose, resin, and fixed oils. It is also found to contain compounds like anthraquinones, 4, 2, 4 chebulyl-dglucopyranose, terpinenes and terpinenols [24]. Primary metabolite also acts as precursors for bioactive compounds used as therapeutic drugs [25]. Phytochemical from medicinal plants serve as lead compounds in drug discovery and design [26]. Aellagitann interchebulin along with punicalagin, terflavin-A, shikimic, tricontanoic, palmitic acids, beta-sitosterol, daucosterol, triterpene chebupentol were found in fruits [27]. The compounds phloroglucinol and pyrogallol, isolated along with ferulic, vanillic, p-coumaric and caffeic acids constitutes for the antioxidant activity of the plant [28]. |
Tables at a glance
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| Table 1 |
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Figures at a glance
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| Figure 1 |
Figure 2 |
Figure 3 |
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