Evaluation of Phytocomponents from Ether Extract of Azdiracthaindicaby HPLC-Fame Method

 

V. Krishna Murthy Naik¹, K.SudhakarBabu¹, P. Malleswara Reddy¹,J.Latha²

¹Department of Chemistry, Sri Krishnadevaraya University, Anantapuramu, A.P, INDIA

²Department of Environmental Studies, Sri Krishnadevaraya University College of Engineering & Technology, S.K. University, Anantapuramu – 515003, A.P, INDIA

*Corresponding Author E-mail: vadethek@gmail.com

In this Present studies the biochemically active components of extract from Stem bark of Azadiracthaindicaobtained from Aantapur, Andhra Pradesh, India, being reported. The HPLC-FAME (Sillylation technique) analyses showed that the presence of 14 Phytocomponents in the Ether extract of Stem bark of Azadiracthaindica viz D-lactic acid, Hydroxy acetic acid, Succinic acid, 2,3-di hydroxyl Propanoic acid, Fumaric acid, Hydro quinone, Malic acid, 5-oxo-L-Proline, Pyrogallol, Levoglucosan, 3,4-di hydroxy benzoic acid, Beta –D-Glucopyranose, Sucrose, Myo-Inositol. All these Phytocomponents are Pharmacologically important bioactive compounds.

 

 

 

INTRODUCTION:

Plants have always played a major role in the treatment of human traumas and diseases worldwide. The demand for medicinal plant is increasing in both developed and developing countries due to growing recognition of natural product. Herbal medicine is an important part of both traditional and modern system of medicines [1]. Today natural products derived from plants are being tested for presence of new drugs with new modes of pharmacological action [2]. Plants are used as medicine in many countries and also act as a source for many potent drugs [3]. A large number of medicinal plants and their purified constituents have shown therapeutic activities [4]. Natural remedies from medicinal plants proved as safe and effective. Many plant species have been used in folklore medicine to treat various ailments [5].

 

Phytocompounds from medicinal plants are important in pharmaceutical industry for drug development and preparation of therapeutic agents [6]. The development of pharmaceuticals begins with identification of active principles, detailed biological assays and dosage formulations followed by clinical studies to establish safety, efficacy and pharmacokinetic profile of the new drug [7].

 

In this present studies we are concentrated on Extraction of Phytocomponents Neem (Azadirachtaindica A. Juss) is a plant of the Meliaceae family belonging to the Indian subcontinent [8]. It is host of so many therapeutic drugs and natural products. There are as many as Procedures to extract these valuable Phytocomponents among this our Ether extraction Procedure is simple and accurate. Identification of compounds is done By HPLC-FAME (Silylation Method) in this procedure we identified 15 most valuable Phytocomponents and Reported along with their uses.

 

 

MATERIALS AND METHODS:

Collection of plant material:

The fresh Stem of Azadirachtaindica Linn (Neem) were purchased from local nursery garden during the month of April 2016. The plant material was identified and authenticated at, Sri Krishna devaraya University, Botany Department by Dr. S. Thimma Naik. The fresh plant material was dried under shade. Dried plant material was powdered using mechanical grinder and passed through sieve no.60 to get the powder of desired coarseness. Powdered material was preserved in an air tight container.

 

Preparation of Plant extracts:

The bark was dried in the shed and coarsely powdered. The powder was extracted with ethanol in a soxhalet apparatus for 72h. The Ether extract was evaporated in vacuo giving the residue. It was extracted successively and exhaustively with petroleum ether, the extract and fractions were concentrated in a rotary evaporator at reduced pressure.  It was filtered through Whatman no 1 filter paper and then the volume of supernatant was concentrated at 40ºC using hot air oven.

 

Identification of Phytocompounds:

The concentrated Ether extracts stem of Azadiractha Indicawere used for HPLC-FAME analysis. During this analysis instrument was standised by blank (Fig-1 chromatogram). Then direct injection of Sample in to instrument gives (Fig-1) Chromatogram no data was obtained then we opted Silylation Method this technique gives good results with 15 peaks (Fig-2).  Interpretation of Chromatogram of plant extract was conducted using the database of National Institute of Standard and Technology (NIST) library having more than 62,000 spectral patterns. The spectrum of the compounds was compared with the spectrum of NIST library database. The identity of the spectra above 95% was needed for the identification of compounds. The name, molecular weight and structure of the components of the plant extracts were ascertained. The relative percentage amount of each component was calculated by comparing its average peak area with the total area. The Chromatogram of the unknown components were compared with the Chromatogram of the component stored in the NIST library.

 

RESULTS AND DISCUSSION:

HPLC-FAME Chromatogram of the Ether extract of Azadirachtaindicashowed 15 major peaks (Figure-2) and have been identified after comparison of the Chromatogram with NIST library (Table-1), indicating the presence of various phytocomponents. From the results, it was observed that presence of  D-lactic acid, Hydroxy acetic acid, Succinic acid, 2,3-di hydroxyl Propanoic acid, Fumaric acid, Hydro quinone, Malic acid, 5-oxo-L-Proline, Pyrogallol, Levoglucosan, 3,4-di hydroxy benzoic acid, Beta –D-Glucopyranose, Sucrose, Myo-Inositol. The phytocomponents that contribute to the medicinal property of the plant Stem bark is listed in Table. 2.

 

 

Fig: -1 HPLC-FAME Chromatogram of Blank Ether and AzadiracthaindicaDirect injection.

 

 

 

Fig: -2   HPLC-FAME Chromatogram of Ether extractof Azadiracthaindica

Table -1List of Identified compound in Azadiractha Indica Ether extract

 

 

Table -2Bioactivity of Phytocomponents identified in Ether extract of The Stem bark of Azadirachtaindica.

 

 

CONCLUSION:

This study may also be enhances the traditional usage of Azadiracthaindica due to its bioactive compounds identified by HPLC-FAME analysis. Further investigation is required for the pharmacological activity of specific compound of 5-oxo-L-Proline and Levoglucosan, which may lead to the development of new drug for the treatment of specific disease. Thus the HPLC-FAME analysis is the first step towards understanding the nature of active principles in. Azadiracthaindica. We hope this study will be helpful to further pharmacological studies for researchers and drug development in pharmaceutical industry.

 

ACKNOWLEDGEMENTS:

This research work was financially supported by UGC, New Delhi for providing financial assistance as PDF, UGC Post doctoral fellowship. We are also thanks to Dept of Chemistry Sri Krishnadeveraya University, Ananthapuramu, India for supporting this study. The authors are thankful to the Indian Institute of chemical Technology, Hyderabad, for providing the HPLC-FAME instrumental facilities.

REFERENCES:

1.     Kirtikar JD, Basu BD. Indian medicinal plants. Allahabad: Lalit Mohan Basu; 1994. pp. 1229–1231.

2.      Charles A, Leo Stanly A, Joseph M, Alex Ramani V. GC-MS analysis of bioactive components on the bark extract of AlseodaphnesemecarpifoliaNees(Lauraceae) Asian J Plant Sci Res. 2011;1(4):25–32.

3.      Mahesh B, Satish S. Antimicrobial activity of some important medicinal plant against plant and human pathogens. World J Agric Sci. 2008;4(S):839– 843.

4.     Janakiraman N, Johnson M, Sahaya Sathish S. GC-MS analysis of bioactive constituents of Peristrophebicalyculata(Retz.) Nees. (Acanthaceae). Asian Pac J Trop Biomed. 2012; S46– S49.

5.     Balamurugan K, Nishanthini A, Mohan VR. GC-MS analysis of Polycarpaeacorymbosa(L.) Lam whole plant. Asian Pac J Trop Biomed. 2012; S1289– S1292.

6.     Nisha K, Darshana M, Madhu G, Bhupendra MK. GC-MS analysis and anti-microbial activity of Psidium guajava(leaves) grown in Malva region of India. Int J Drug Dev Res 2011;3(4):237– 245.

7.     Ncube NS, Afolayan AJ, Okoh AI. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. Afr J Biotechnol. 2008;7(12):1797–1806.

8.     Chopra I.C, Gupta K.C. and Nazir B.N. Preliminary study of antibacterial substance from Melia azadirachta, Ind. J. Med. Res., 1952; 40: 511-515.

 

 

Received on 30.01.2018         Modified on 12.02.2018

Accepted on 24.02.2018         © AJRC All right reserved