DETECTION OF ACTIVE CONSTITUENTS FROM THE LEAF EXTRACT OF LANNEA COROMANDELICA BY GC-MS TESTING AND ASSESSMENT OF ITS PHARMACOLOGICAL ACTIVITY

DETECTION OF ACTIVE CONSTITUENTS FROM THE LEAF EXTRACT OF LANNEA COROMANDELICA BY GC-MS TESTING AND ASSESSMENT OF ITS PHARMACOLOGICAL ACTIVITY

Deepa Selvaraj ^*, Anusha Kotapadu, Bandla Sampurna, Balaji P, Aleena Ann Abraham, Sathesh Kumar Kesavan and Chitra Krishnan

Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai-600116, India

ABSTRACT:  Lannea coromandelica (Anacardiaceae) is a tropical tree commonly called as Indian ash tree. Its various parts is said to possess medicinal property and used in Ayurveda and other ancient system of medicine for the treatment of various ailments including treatment of inflammation, gout, cholera, leprosy, vaginal troubles etc. Its Pharmacological study revealed anti-inflammatory, antimicrobial, hypotensive, anticancerous activities. Leaves of Lannea coromandelica were collected from Azhagar malai, Madurai. The sample specimen of Lannea coromandelica was identified and authenticated by Dr. Jaya Raman, Taxonomist, Chennai. The present work was carried out to identify some of the phytocomponents present in the ethanolic leaf extract of the L. coromandelica by GC-MS technique, to ascertain the medicinal properties of the plant and also to assess its anti-oxidant and anti-arthritic potential of ethanolic leaf extract of Lannea coromandelica. The results revealed that ethanolic extract is mainly composed of oxygenated hydrocarbons and predominantly phenolic hydrocarbons. These different active phytochemicals may be responsible for wide range of activities, which may help in the protection against incurable diseases. The future in vivo investigation should be carried out to confirm the activity in animal models.

Lannea coromandelica,

phytochemical, anti-oxidant,

INTRODUCTION: Since times immemorial nature was being a font of medicinal agents.  India is one of the richest countries in the world in stare to possessions of medicinal plants. As they are short of adequate scientific documentation, the conventional Indian system of medicine chiefly in scientific knowledge. Lannea coromandelica prevalently known as Indian Ash tree was found in isolated places and in jungles all over India principally southern part of India. Lannea coromandelica belongs to Anacardiaceae family which has diversity of medicinal uses.

Linguistic variations show the way in a choice of names to the plant. It is used as folk medicine to treat fever, dyspepsia, general debility, gout, dysentery, sore eyes, leprosy, sprains, ulcers, inflammations, Impotency, wounds and much more disorders. Generally leaves, bark, stem and gum of Lannea coromandelica are used to treat an assortment of diseases. Some say it is as important as Neem as it has amity number of uses. It was expansively analysed for its chemical components and pharmacological actions ¹. Lannea coromandelica was found to be precious anti-inflammatory agent and wound healing agent ² and anti-bacterial ³ as it is assumed in ayurveda.

Since there is no further systematic, scientific, research work has been executed on in vitro            anti-arthritic activity of Lannea coromandelica leaves, this study has been engaged for the existing research work.

MATERIALS AND METHODS:

Collection of Plant:

The plant leaves were collected from Madurai, Tamilnadu, India and it was examined , recognized and autentified by Professor P. Jayaraman , Plant Anatomy Research Centre, Chennai. The Leaf was dried in shade for 15 days and pulverised in plant mill and accumulated in air tight box for future use.

Preparation of Extract:

The Leaf powder was subjected to successive extraction by cold maceration for 72 hours, 48 hours and then for 24 hours at room temperature. The leaf powder was consecutively treated with 200 ml of solvents (petroleum ether, benzene, chloroform, ethyl acetate and Ethanol). Solvent was distilled from the extract under reduced pressure.

Phytochemical screening of the leaf extract:

The primary and secondary metabolites were recognized by performing a variety of biochemical tests. The leaf extract was assessed for the existence of phenols, glycosides, quinones, terpenoids, alkaloids, saponins, flavanoids, triterpenoids, steroids and tannins.

Anti-oxidant activity:

Protocol for Nitric oxide scavenging assay:

Sodium nitroprusside (5M) In phosphate – buffered saline(1×PBS pH 7.4) was assorted in the midst of 3 ml of different plant extracts (different concentrations that is 0.25, 0.50, 1.0, 1.5, 2.0, 2.5 mg  /ml) and incubated at 25°c for 150 min. The samples from them were reacted with 1.5 ml of Griess reagent. The absorbance was interpreted at 546nm. Ascorbic acid was taken as optimistic control. The scavenging percentage of nitric oxide of plant extracts was calculated via the following formula:

NO scavenging (%) = [(absorbance of control-absorbance of test sample)/ (absorbance of the control) ×100

Protocol for reducing power:

Distinct amount of the extract (62.5, 125, 250, 500, 1000, 2000 µg/ml))and standard drug Ascorbic acid at equivalent concentration , 2.5ml phosphate buffer pH 6.6 together with 2.5ml of 1% potassium ferricyanide were incubated at 50°c for 20 minutes, 2.5ml of 10% Trichloroacetic acid (TCA) were supplemented to the mixture. Centrifuge for 10 minutes at 3000 r.p.m. Following the centrifugation, supernatant liquid of  2.5ml were diluted with 2.5ml of water and traumatized with 0.5ml freshly prepared 0.1% ferric chloride. The absorbance was measured at 700nm.

Augmented absorbance of the response of mixture is a sign of amplify in reducing power, which is calculated using the formula,

% increase in Reducing Power = [   A _test / A _blank    - 1] X 100

Anti-arthritic activity:

Inhibition of protein denaturation method:

0.5ml of Test solution, Test control solution, Product control solution, Standard solution was prepared. Various concentrations (50, 100, 250, 500, 1000, 2000µ/ml) of test dugs and standard drug diclofenac sodium (50, 100, 250, 500, 1000, 2000µ/ml) were prepared. 1N HCl was used to adjust the pH to 6.3 for all the above solutions. The samples were incubated at 37°c for 20 minutes and the temperature was increased to keep the samples at 57°c for 3 minutes. After cooling, 2.5 ml of phosphate buffer was added to the above solutions. The absorbance was measured at 416nm ⁴. The control represents 100% protein denaturation. The percentage inhibition of protein denaturation can be calculated as

Percentage Inhibition =

100 – [{(optical density of test control – optical Density of product control)/optical density of test solution} × 100

The control represents 100% protein denaturation.

Gas chromatography-mass spectroscopy (GC-MS) analysis:

The additional defined information in qualitative examination can be attained by Gas-chromatography together with mass spectrometry (GC-MC) ⁵. The GC-MS analysis of the Lannea coromandelica leaf extract within absolute alcohol was achieved using a Clarus 500 Perkin Elmer gas chromatography along with a Elite-5 capillary coloumn (5%phenyl 95% dimethyl polysiloxane) (30nm × 0.25mm ID × 0.25 µmdf). Mass detector of Turbo mass gold company was functioning in EI mode. Carrier gas used was Helium at a run time of 1 ml/min. The injector was controlled  at 250°c and the oven temperature was automated as follows;50°c at 8°c/min to 200°c(5min) at 7°c/min to 290°c(10min) at 110°c(2min)to 200°c at 10°c/min to 280°c(9min) at 5°c/min. Total  operation time is 36 minutes. The effective volatile constituents present in the extract was achieved using GC-MS and the interpretation on mass spectrum of GC-MS was concluded using the Database of Indian Institute of Crop Processing Technology (IICPT) having more than 75,000 patterns. The plant extract was liquefied in ethanol and pass through a filter with polymeric solid phase extraction (SPE) column and investigated in GC-MS for different components.

RESULTS AND DISCUSSION:

Preliminary phytochemical studies:

First round of phytochemical learning of the successive extracts of the leaves of Lannea coromandelica was made. The extracts exposed the occurrence of metabolites like flavonoids, steroids, saponins, terpenoids, triterpenoids, glycosides, alkaloids, phenols and tannins, which is specified in Table 1.

 TABLE 1: PRELIMINARY PHYTOCHEMICAL ANALYSIS OF LANNEA COROMANDELICA

Anti-oxidant activity:

Nitric Oxide Radical Scavenging Activity:

Nitric Oxide (NO) is a forceful pleiotropic negotiator in nomerous physiological process like smooth muscle relaxation, inhibition of platelet aggregation, neuronal signalling and cell mediated toxicity parameter. This free radical is a diffusible one, which plays a lot of roles as an effect or molecule in many parts biological systems together with neuronal messenger, anti-microbial, anti-inflammatory and anti-tumor activities ⁶. Furthermore Nitric Oxide plays a key position in repairs of wound by influence angiogenesis and inflammation ⁷. Hence this study will assist us to considerate the role of our extract in various physiological scheme such as diminution of inflammation, smooth muscle relaxation and platelet aggregation.

The scavenging activity of the extract in opposition of nitric oxide released by sodium nitroprusside was inspected. Nitric oxide (NO) scavenging assay is based on the scavenging capability of the extracts as well as Ascorbic acid, which is used as standard. The Scavenging of NO was found to amplify in dose dependent manner. Maximum inhibition of NO was observed in the extracts of uppermost concentration (25 mg/ml) for ethanol extract. At this highest concentration, inhibition was found to be 83.43 for extract and 82.12 for Ascorbic acid which serve up as standard. The results are presented in Table 2.

 TABLE 2: NITRIC OXIDE SCAVENGING EFFECT OF SUCCESSIVE LEAF EXTRACT OF LANNEA COROMANDELICA

Reducing power activity:

Reducing  power  is  connected  with  antioxidant  activity  which might serve  as  a  considerable  reflection  of  the  antioxidant  activity ⁸. Reducing  power  effect of the components was considered as a sign of  that,  they  are  electron  donors  and  can  trim down  the  oxidized  intermediates  of  lipid  peroxidation  processes,  as a result , they  can  act  as  primary  and  secondary antioxidants ⁹. In  this assay,  the  colour  of  the  test  solution was yellow and it vary  to a variety of shades of blue and green depending on theabilityorthe reducing power of each compound. Existence of reducers causes the translation of the Fe3+/ferricyanide complex used in this method. Among each and every one of the extracts ethanolic leaf extract  of Lannea coromandelica exhibited respectable  activity with utmost inhibition of about 79.3 % at a concentration of 2000 µg/ml when paralled to standard Ascorbic acid at the same concentration for which the percentage inhibition is 86.6%. The results are presented in Table 3.

 TABLE 3: REDUCING POWER ACTIVITY OF ETHANOLIC LEAF ECTRACT OF LANNEA COROMANDELICA

Anti-arthritic activity:

Inhibition of Protein Denaturation Method:

Protease inhibitory activity of ethanolic leaf extract of Lannea coromandelica was estimated as described in the literature ¹⁰. Diclofenac sodium was included as standard. In vitro Anti-Arthritic assay was done by inhibition of protein denaturation method. The highest percentage inhibition of ethanolic extract of Lannea coromandelica was about 81.08% at concentration of 1000µg/ml in contrast to standard which was about 89.36% at same concentration. The results were exposed in the Table 4.

 TABLE 4: ANTI-ARTHRITIC ACTIVITY OF ETHANOLIC LEAF EXTRACT OF LANNEA COROMANDELICA BY PROTEINASE INHIBITION.

Gas Chromatography-Mass Spectroscoy (GC-MS) analysis:

GC-MS chromatogram of the ethanolic leaf extract of Lannea coromandelica (Fig. 1) illustrate peaks indicating the presence of a wide range of compounds. The compound recognized in GC-MS analysis was benzyl alcohol (8.3%) , DL-Arabinitol (5.69%), phenol, 2-methoxy-4-(1-propenyl) (2.84%), phenol, 2, 4-bis(1,1-dimethylethyl) (1.28%), 4H-1-benzpyran, 4, 4, 5, 8 –tetramethyl (1.42%), 2, 6, 10 – dodecatriene - 1- imine - 3, 7, 11 - trimethyl - 1 ( 1%), 1, 6-Anhydro – β – D -glucopyranose (11.38%), 5 – Isopropenyl – 2 - methylcyclopent-1-enecarboxaldehyde (1.28%), Amyl nitrite ( 2.84%) , 1, 1'-bicyclohexyl, 2-methyl-trans - (7.11%), 3 - methyl - 5 - (2, 6 -dimethylheptyl) - 1, 5 - pent - 2 - enolide (2.84%), cyclohexanecarboxylic acid, 4 – propyl – 4 -methoxyphenyl ester (15.65%), 3, 7, 11, 15-tetramethyl - 2 - hexadecen - 1 - ol (1.42%), n-Hexadecanoic acid (21.34%), Hexadecanoic acid, ethyl ester (2.84%), Phytol (11.38%), 1, 2-benzenedicarboxalic acid, bis (4-methylpentyl)ester (1.14%) .

FIGURE 1: GC/MS SPECTROGRAM FOR THE LANNEA COROMANDELICA ETHANOLIC LEAF EXTRACT

Most of them are oxygenated hydrocarbons and primarily phenolic hydrocarbons, these phytochemicals may be accountable for various pharmacological behaviour like anti-oxidant and anti-arthritic activity, etc.

In view of preliminary phytochemical studies, in vitro anti-oxidant and anti-arthritic study ethanolic extract showed more appreciable results, GC-MS examination was carried out to recognize the components responsible for its potency. The present study in Lannea coromandelica recommends that, because of the presence of these components anti-arthritic potency was enhanced, though it should be further evaluated in future by appropriate in vivo studies.

 ACKNOWLEDGEMENT: Authors acknowledge S. Kumaravel (Scientist), IICPT, Thanjavur for his analysis and validation of the results and also for his precious assistence throughout the study.

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    How to cite this article:

    Selvaraj D, Kotapadu A, Sampurna B, Balaji P, Abraham AA, Kesavan SK and Krishnan C: Detection of Active Constituents from the Leaf Extract of Lannea Coromandelica by GC-MS Testing and Assessment of Its Pharmacological Activity. Int J Pharm Sci Res 2015; 6(3): 1217-21.doi: 10.13040/IJPSR.0975-8232.6 (3).1217-21.

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