Phytochemical Analysis And Characterisation Of Pinus Radiata Biology Essay

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Study was focused on plant Pinus Radiata which is an important class of Coniferous plants. Pinus Radiata mainly known to consists of Terpenoids and Caryophyllene, which contributes various properties such as antibacterial, anticancer and antioxidant. It is known to have anti oxidant activity like other Flavonoids. Which is proved qualitatively by both TLC (Thin Layer Chromatography) and Spectrophotometric methods, these results are compared with the known anti oxidant compound i.e. Quercetine. Study was later focused on phytochemical analysis of Pinus Radiata plant extract by GC/MS (Gas chromatography-Mass spectroscopy), HPLC (High performance liquid chromatography) and NMR (Nuclear magnetic resonance) studies. GC/MS study revealed presence of important chemical constituents such as A-Pinene and B-Pinene. HPLC study is useful in obtaining chromatograms with different peaks. Selected peaks were collected, dried and sent to NMR (Nuclear Magnetic Resonance) study, which was not much successful because of small amount of samples obtained from HPLC chromatograms. So, further work is mainly focused on development of prep HPLC to collect sufficient amount of samples for studying "H" Spectra by NMR.

Keywords:

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Pinus radiata, terpenes, pinenes, quecertin, antioxidant activity, DPPH, TLC plate, HPLC, spectrophotometer, GC-MS and NMR.

Abbreviations:-

DCM - Dichloromethane, TLC- thin layer chromatography, GC-MS, gas chromatography mass spectrometry, DPPH - 2, 2-Diphenyl-1-picrylhydrozyl, HPLC- high performance liquid chromatography, H1-NMR- Nuclear Magnetic Resonance.

Aim: - Phytochemical investigation of conifer plant cones of P. radiata by reflux extraction then analysis by using Thin layer chromatography (TLC), Gas chromatography - Mass spectrometry (GC-MS), high performance liquid chromatography (HPLC), GC-MS-NMR and Spectrophotometric analysis for investigation of anti-oxidant activity in the cone extracts.

INTRODUCTION:

Pinus Radiata evolved about 100 million years ago, pine bark was an inconvenient residue for the wood industry. Pine bark is a rich source of natural polyphenols compounds which have attracted increasing attention on the field of nutrition, health and medicine. Flavonoids and other plant phenolics, such as phenolic acids, stilbenes and tannins , are important in the plants for normal growth development and defence against infection and injury (Ka¨hko¨nen et al., 1999). Polyphenolic extracts and fractions have a potential use in the food industries as alternativs to synthetic BHT (butylated hydroxytoluene), BHA (butylatedhydroxyanisole) and TBHQ (tert-butyl hydroquinone).

Terpenoids are common constituents of the resins of higher plants and they are useful chemostatics characteristics of extant plants. (Hegnauer, 1962, 1992;Langenheim, 1969; Erdtman and Norin, 1966;Thomas, 1986; Otto and Wilde, 2001).

Most of the compounds identified in the fossil resins are the diagenetic products (biomarkers) of terpenoids which were synthesized by living organisms (simoneit 1986).

The conifer plants contain terpenes and flavonoids which display the antioxidant activity. When free radical with single electron uses the other electron and become unpaired, this process is called as oxidation which can result into the aging, immunity problems, heart disease. (Kinderlehrer, 2007) The diterpenoid, totarol have shown anti-oxidant and antibacterial activity. (Bernabeu et al, 2002). Many pines have been used to produce turpentine, a semi-fluid, yellow or brownish resin (oleoresin). (Moussouris and Regato 1999).

Studies suggests that generation of free radicals has important role in the progression of pathological disturbances such as cardiac diseases, cancer etc. These free radicals such as superoxide anion (O2-), hydroxyl radical (OH) destroys the enzyme activity by causing tissue damage normally through covalent bonding and lipid per oxidation. Antioxidants are known to protect cell membrane against free radicals. Synthetic antioxidants causing serious health problems for this reason an extensive search for different types of plant based antioxidants is taking place. Natural antioxidants have proved to be less toxic than Synthetic molecules such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene(BHT), which are highly volatile and unstable at room temperatures. Therefore investigations for various extracts of the plant were screened for antioxidant activity using DPPH (2, 2-Diphenyl-1-picrylhydrazyl), method.

DESCRIPTION OF SPECIES:

It is a species of pine native to coastal California is three very limited areas in Santa Cruz, Monterey and San Luis Obispo Countries, and Guadalupe Island and Cedros Island off the west coast of Baja California, Mexico. It is also extensively cultivated in many other warm temperate parts of the world. It was first introduced into New Zealand in the 1850's; today, over 90% of the country's plantation forests are of this species. This includes the Kaingaroa Forest on the central plateau of the North Island which is the largest planted forest in the world. Australia also has large Radiata Pine plantations. The plant prefers light (sandy) and medium (loamy) soils, requires well-drained soil and can grow in nutritionally poor soil. The plant prefers acid and neutral soils. It cannot grow in the shade. It requires dry or moist soil and can tolerate drought. The plant can tolerate maritime exposure. (Hogan, C. Michael & Frankis, et.al (2009).

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The shape of the tree has a pointed top, which is commonly seen on young trees. The needles are shiny green colour of 4-6 inches in length. The bark of the tree is thick, and deeply furrowed into scaly ridges, and is dark reddish brown in colour. The cones are 3-6 inches in length and exhaled with a pointed tip. The cones are shiny brown in colour and grow clustered in rings on short stalks. "The scales of the cones are raised and rounded, larger on the outer sides and ending in tiny prickle: small, long winged seeds (Little, 1980)". 

The trees have closed cones. This means they need fire to reproduce, which makes them pyrophytes trees. Only two other pines are also pyrophytes the Bishop (Pinus muricata) and the Beach (Pinus contorta).

C:\Users\SONY\Desktop\10007594.JPG

Ref: www.dkimages.com/.../Pinus-radiata-2.html

FIGURE 1

Pinus radiata (Monterey pine) 

USES:

It has vanillin flavor obtained as a by-product of other resins that are released from the pulpwood. The turpentine obtained from the resin of all pine trees is antiseptic, diuretic, rubefacient and vermifuge. It is also very beneficial to the respiratory system and so is useful in treating diseases of the mucous membranes and respiratory complaints such as coughs, cold, influenza and TB. It is a valuable remedy when used internally in the treatment of kidney and bladder complaints and is used both internally and as a rub and steam bath in the treatment of rheumatic affections. Externally it is a very beneficial treatment for a variety of skin complaints, wounds, sores, burns, boils etc and is used in the form of liniment plasters, poultices, herbal steam bath and inhalers. (Frankis, M.P,(1992)).

MATERIALS AND METHODS:

CHEMICALS :-

Solvent hexane, dichloromethane and methanol were purchased from Fisher Scientific (Loughborough, UK). DPPH powder (2, 2-Diphenyl-1-picrylhydrazyl), standard quercetin and TLC plate was purchased from the Sigma Aldrich (Dorset, UK).

EXTRACTION BY REFLUX EXTRACTION METHOD USING SOLVENT OF INCREASING POLARITY:

The dried plant material of Pinus radiata was separated and weighed to 28.45g. The material was transferred to 1000 ml round bottomed flask to this 600 ml of hexane was added to the flask and mouth was fitted to bottom of the reflux condenser. Small amount of anti-bumping granules were added to round bottom flask to avoid bubbling. The whole apparatus was kept in a fume cupboard and connected to the electric heating mantle on 60°C. Flow of water was kept constant.

After running it for one hour the flask was cooled for 15 min and filtration was carried out using Buchner funnel under vacuum by using whatman filter paper. (Sterile Pyrogenic,Puradisc 25AS polyehersulfone membrane 0.2 μm pore size and 25 mm diameter).

Filtrate was transferred to 500 ml round bottomed flask and connected to the Buchi rota vapour R-200 and temperature of the Buchi heating bath was set to 39°C to remove organic solvent by evaporation. The concentrated extract was transferred to small vials using a sonicator to solubilise the extract in hexane and was weighed. The experiment was repeated with more polar solvent dichloromethane then methanol. Small portion of solvent was evaporated using nitrogen gas.

CHROMATOGRAPHY:

Chromatography technique involves separation of mixture into individual compounds using a mobile phase which passes through or over a stationary phase. Substances which have strong affinity towards stationary phases will be eluted slowly, substances which have less affinity towards stationary phase eluted rapidly with the mobile phase.

The components in the mixture distributed differently between the mobile and stationary phase have different interactions with each phase. By changing the composition of the mobile phase, it is possible to analyze a wide variety of compounds.

Chromatography has wide variety of uses like in chemistry and biochemistry research analysing complex mixtures, purifying chemical compound, isolating natural products and also in predicting physical properties. It is also used in quality control to ensure the purity of raw materials, to control and improve the process yields, or to evaluate product stability and monitor degradation.

Thin Layer Chromatography:

This method is useful for separation of components from a mixture. It consists of a stationary phase and mobile phase were both involve in elution of components from a mixture. Mobile phase is allowed to pass from the bottom to top of the stationary phase. Mixture to be extracted is placed on the stationary phase which travels along mobile phase and gets separated according to its polarity. (Ahuja et al 2003).

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TLC analysis was performed on each of the extracts. Thin layer silica plate was taken and a line was drawn from 1.5 cm above the bottom of the plate, each extract was spotted on to the plate using glass capillary tubes. And the plate was carefully dipped in the TLC tank which was previously filled with the 100ml of prepared solvent system.

Different solvent systems were prepared and TLC analysis was performed.

Different solvent preparations

1) Hexane : Dichloro methane

80 : 20

2) TEA system

Toluene : Ethyl acetate : Acetic acid

80 : 18 : 2

This method is mainly used for polar compounds.

3) Hexane : Dichloro methane

70 : 30

The plate was then removed carefully and it was first air dried.

These plates were first observed under the UV light and spots were observed.

The developed plate was stained with spraying solution of the vanillin/H2SO4/IMS and dried with the sprayer under hot air.

Preparation of staining solution:

Vaniline 4gm

Ethanol 90ml

Sulphuric acid 10ml.

GAS CHROMATOGRAPHY:

Gas chromatography is useful in finding out interaction of different substances with the surface of the solid. The mobile phase here was a gas, which is why it is called as gas chromatography. A substance that interacts more strongly with the surface of the solid will take more time to be carried across the stationary phase.

The GC-ms instrument combines two different techniques to form one single method of analyzing mixtures. Gas chromatography separates the components in a mixture, and the mass spectroscopy distinguishes the individual constituent. After separation the components enter a detector where an electric signal is created when a component is detected. The signal size is dependent on the component's concentration. A retention time (the time from the injection is made to when elution occurs) is calculated by instrument computer. The retention time is same for a compound, as long as GC conditions are unchanged. Compound assignment can be made if it`s retention time is known. (Hill, D.W et al).

The eluted samples from GC enter the Mass Spectrometer`s electron ionization detector. The compounds are electrically charged when accelerated by magnetic field and broken into charged fragments. The different charges are detected, and the mass of each fragment of the compound is plotted on the spectrum. By GC-MS, solution's content can be separated into individual components, and be identified, which makes it a powerful tool.

GC-MS analysis was performed on Thermo Electron Corporation instrument coupled with Trace GC Ultra fitted Triplus auto sampler. The hexane extract was dissolved in DCM, the DCM and methanol extracts were dissolved in their respective solvents. All extracts were prepared at a concentration of 10 mg/ml. 4μL of each extract sample was injected into the injection port at 250°C of GC-MS. The capillary column used was Varian VF-5ms fused silica (30mÃ-0.25mm, film thickness 0.25 μm). Helium gas was used as carrier gas at flow rate of 0.7ml/min. Split ratio was 1/14 and temperature of oven was 4°C for 5minutes then it was increased to 220°C at 3°C/min. Then it was held for 5 minutes at 220°C. Electron impact used was 70eV as ionization mode at 200°C ion source temperature. Samples were scanned from 50-650 mass scan range with scan time of 1.22 second. Thermo Electron Corporation X calibre v 1.4 software was used. The graph, molecular formula and molecular structure were searched and compared in NIST Library version 2 search libraries.

COMPOUND IDENTIFICATION:

"Library search" method is use full in identifying the compound by comparing reference library spectra with the submitted (user) spectra. Spectra should resemble closely in both spectra. Spectra originated from GC/MS data file was a single mass spectral scan or an average, with or without simple background subtraction, it produces a "hit list of library spectra, which is ordered by similarity to the target spectra according to computed "match factor." (Skoog, Hooler, et al(1998)).

HIGH PERFORMANCE LIQUED CHROMATOGRAPHY:

Chromatography is defined as a procedure by which inert materials and drug principle encountered in pharmaceuticals preparations are separated by fractional extraction, adsorption, or in ion exchange on a porous solid. It is defined as means of separating and purifying complex and closely related chemical substance which are difficult to distinguish.(Quantitative Pharmaceutical Chemistry by Glenn-L.Jenkins, Digangi, pg 425-428).

High performance liquid chromatography (HPLC) is known to be one of the most effective methods for analysis of mixtures of complex composition. . A great number of various organic substances of different nature and properties required various technical approach and instrumentations to separate complex mixtures, identify and determine their components. Thus, HPLC is of immense importance not only for pharmaceutical purposes but also for

other analytical purposes in various fields.

The principle objectives obtained through the chromatography including HPLC are:

1. Resolution of mixtures into constituent parts. 2. Determination of homogeneity. 3. Comparison of substances suspected of being identical. 4. Purification of any substance or compounds. 5. Quantitative separation from complex mixtures. 6. Indication of molecular structure.

HPLC development led to stationary phases that are chemically bonded to solid support. Covalent bond between the stationary phase and solid substance makes the chemically bonded liquid chromatography columns stable. In column liquid chromatography high pressures drive the mobile phase through a column of stationary phase allowing separation of complex mixtures with high resolution. (IV.Remington the Science and Practice of Pharmacy, pg 615-626).

Reversed phase chromatography consists of a non-polar stationary phase and an aqueous polar phase.

FIGURE 2

REF: /www.chemguide.co.uk/analysis/chromatography/hplc.html

Normal phase HPLC:

HPLC analysis of methanol extract was carried out on the Varian series HPLC with the Varian 2010 pump and Varian 2050 detector. The pump was attached with column, online degasser and detector. 40 mg of fraction five was dissolved in 5 ml of methanol then it was sonicated for 5 minutes in sonicator to remove the gas from the fraction which was performed in the normal phase HPLC with analytical column Waters Spherisorbᴿ 5 μm silica column (4.6 X 250mm). An injection loop of 20 μL was injected into the injector. Then column was calibrated with TEA-1(80:18:2) solution with 1.5ml/min flow rate for 50 minutes to remove impurities from the column. The detector wavelength was set to 280nm in UV-visible detector. The normal phase mobile phase was used similar to TLC plate developing solvent system-2 called TEA (toluene: ethylacetate: acetic acid). To obtain a better separation on trial and error basis different proportion of mobile phase TEA (toluene: ethylacetate: acetic acid in the ratio of 80: 18: 2 was used with 1ml/min flow rate and then 20 μL sample was injected with the same solvent system with 1ml/min was carried out for 25 minutes. To separate the peaks the flow rate was decreased to 0.7ml/min.

ANTI-OXIDANT ASSAY:

PREPARATION OF PLANT MATERIAL:

The various parts of the plants were dried at room temperature and was defatted by extraction with Hexane, Dichloromethane and methanol. Extracts were concentrated to dryness under reduced pressure and controlled temperature. All extracts were preserved in a refrigerator till further use.

PROCEDURE:

Determination of the antioxidant activity by DPPH method of the plant extracts was determined using a method based on the reduction of methanolic solution of colour free radical DPPH solution. A methanolic DPPH solution was mixed with methanolic solutions of extract concentrations. The stock solutions were diluted with methanol to obtain lower dilution. Then samples were shaken vigorously and kept in dark for 30min at room temperature, sample absorbance was measured at 517nm with spectrophotometer. A methanolic solution of DPPH was employed as blank. Antioxidant activity of each extract was determined according to the percentage of DPPH decoloration. Inhibition percentage = [1-(absorbance with compound)/ (absorbance of the blank)] Ã- 100. ( Free Radic Biol Med. 2000; 28: 1538-1546.)

Thermo Electron Corporation (Nicolet evolution 100) spectrophotometer was used to check antioxidant activity of all extract. 4 mg/ml concentrated stock solution of extract was used to make the serial dilutions of (2mg/ml, 1mg/ml). Quercertin (1mg/ml concentration) was used as control.

Calculation: Absorption-DPPH-Absorption sample/ Absorption DPPH *100.

NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (NMR):

NMR phenomenon is based on the fact that nuclei of the atoms have magnetic properties that can be utilized to yield chemical information. Subatomic particles like protons, neutrons and electrons have spin. In some atoms these spins are paired and cancel each other out so that the nucleus of the atom has no overall spin.

NMR measures the absorption of electromagnetic radiation in the frequency region of roughly 4 to 900 MHz (Skoog et al 2007). Normally there are two types of NMR spectrometers in use 1) Pulse or Fourier transform (FT-NMR), 2) continuous-wave (CW) spectrometers. NMR normally involves Deuterated solvent systems for sample preparation. Some common deuterated solvents are dimethyl sulfoxide (DMSO), chloroform, water, acetone, methanol and acetonitrile. DMSO is a "universal solvent" giving excellent solubility and high boiling point. (Ahuja et al 2003). The chemical structure of the molecule can be made by means of range of frequencies vital for orientation and excitation of molecule (Watson, 2005).

Instrument used AVANS Bruker Ultrashield 500.13 MHz magnet was used for analysis of sample. Solvent used MeOD (deuterated methanol) to dissolve all samples. Tetramethyl silane was used as a reference.

RESULTS:

EXTRACTION BY REFLUX EXTRACTION METHOD USING SOLVENT OF INCREASING POLARITY:

Table-1

Type of extract

Amount of extract(g)

Hexane extract

0.71

Dichloromethane extract

0.27

Methanol extract

0.75

Reflux extraction method removes foreign material from crude plant material, and compounds solubilised in the solvent used according to their polarity they are Hexane (H), Dichloromethane (D) and Methanol (M).

2) TLC PLATE WITH HEXANE, DICHLOROMETHANE AND METHANOL

Indicating separated compounds.

HEXANE (H) DICHLOROMETHANE (D) Methanol(M)

Thin layer chromatography plate (TLC), showing thick separated bands of three extracts used (Hexane, Dichloromethane and Methanol .Violet colour spots observed under UV light indicated by side arrows.

FIGURE 3

MORE COMPUNDS SEPERATED BY TEA SYSTEM.

HEXANE(H) DICHLOROMETHANE(M) METHANOL(M)

FIGURE: 4

Thin layer chromatography plate (TLC), showing thick separated bands of three extracts used (Hexane, Dichloromethane and Methanol.

FIGURE 5:

TABLE 2

4) Pinus radiata (hexane) extract with GC-MS:

Name

Molecular Formula

Match Factor

Caryophyllene oxide

C15H24O

930

Beta-pinene

C10H16

923

Alpha-pinene

C10H16

924

Bicyclo[3.1.1]hept-2-ene

C10H16

923

Caryophyllene

C15H24

916

Juripine lansifolene

C15H24

911

Cis-verbenol

C10H16O

899

Cyclohexane methyl

C17H14

892

2-pine-10-al myrtenal

C10H16O

872

Alpha caryophyllene

C15H24

864

2-pine-4-one Berbenone

C10H14O

838

MF value was set at 800 or higher to determine the good match with the compound library. Variety of compounds were obtained from Hexane extract like α pinene, β pinene , caryophyllene , cis-verbenol etc which proven to be a reason to study in depth.

FIGURE 6:

TABLE 3

Pinus radiata (methanol extarct) with GC-MS:

Name

Molecular Formula

Match Factor

Caryophyllene oxide

C15H24O

867

1.4-methanoazulene

C15H24

857

Iso pinocarveol

C10H160

829

Pimaric acid

C20H30O2

795

Bicyclo [3.1.1]hept-2-ene

C10H16O

803

Acids were mainly obtained from methane extract like pimaric acid and caryophyllene oxide with match factors 795 and caryophyllene oxide respectively.

FIGURE 7:

TABLE 4

Pinus radiata(Di chloro methane extract) with GC-MS:

Name

Molecular Formula

Match Factor

Beta-pinene

C10H16

932

Ch

C10H14O

931

Caryophyllene oxide

C15H24O

927

Alpha pinene

C10H16

927

Bicyclo [3.1.1]hept-3-ene-2-ol

C10H16O

919

Methanoazulene

C15H24

919

Caryophyllene

C15H24

896

Bicyclo[3.1.1]hept-3-ene

C10H14O

870

Oxabicyclo

C15H24O

855

GC-MS (gas chromatography-mass spectroscopy).Match factor help full in indentifying the compound from the library spectra. (MF) Molecular formula.

α- pinene ,β- pinene and caryophyllene were mainly obtained from the Dicholoromthane extract with 927 , 932 and 896 match fatcors respectively.

5) Calculation:

% of Antioxidant activity:

With Quercetine standard: Table 5:

Absorbence of DPPH=1.143

Quercetine Concentration

Absorption at 517nm

% of Antioxident activity

2mg/ml

0.023

97.28%

1mg/ml

0.166

85.47%

0.5mg/ml

0.192

83.2%

0.25mg/ml

0.234

79.5%

0.125mg/ml

0.319

72.09%

0.0625mg/ml

0.470

58.8%

0.03125mg/ml

0.509

55.4%

.

METHANOL:

With Methanol extract:

TABLE 6

Methanol Concentration

Absorption at 517nm

% of Anti oxidant activity

2mg/ml

0.129

95.5%

1mg/ml

0.189

93.4%

0.5mg/ml

0.357

87.5%

0.25mg/ml

0.389

86.4%

0.125mg/ml

1.060

63.06%

0.0625mg/ml

1.901

33.76%

Hexane:

With Hexane extract:

Absorbance of DPPH : 1.632

TABLE 7

Hexane Concentration

Absorption at 517nm

% of Anti oxidant activity

2mg/ml

0.411

74.81%

1mg/ml

0.675

58.63%

0.5mg/ml

1.145

29.84%

0.25mg/ml

1.206

23.0%

0.125mg/ml

1.256

23.0%

Dichloromethane:

Absorbance of DPPH: 2.499

TABLE 8

Dichloromethane Concentration

Absorption of Di-Chloro methane

% of Anti oxidant Activity

2mg/ml

0.465

81.3%

1mg/ml

0.652

73.9%

0.5mg/ml

1.001

59.9%

0.025mg/ml

1.660

33.5%

0.125mg/ml

2.070

17.16%

Yellow spots indicating presence of antioxidant activity.FIGURE: 4HEXANE(H) METHANOL(M) DICHLOROMETHANE(D)

TLC (thin layer chromatography) plate, DPPH (2, 2-Diphenyl-1-picrylhydrazyl) used to stain the plate, Hexane (H), METHANOL (M), DICHLOROMETHANE (D.). Yellow spots were observed on the plate for all three extracts, indicating that they all contain compounds with anti-oxidant activity.

6) HPLC RESULTS:

When plant extract is subjected to HPLC under given conditions it gave chromatogram showing different peaks at different time intervals which indicates the presence of components in the mixture.

Weights of the collected peaks were measured and were found to be in small amounts.100µg, 250µg and170g.

TABLE 9:

VALUES OBTAINED FROM THE CHROMATOGRAM.

Peak

NO

Retention time

(min:sec)

Area

[µV∙sec]

1

2.974

15422.66

2

10.291

134954.25

3

11.609

113572.96

4

14.719

78800.08

5

15.051

105.41

6

17.558

155222.68

Sample no: 001,Autosampler : SER200, Instrument name: PE HPLC1, Sampling Rate : 10.0000 pts/s, sample volume : 1.000000ul, Dilution Factor: 1.00.

7) H1NMR RESULTS:

The sample collected from HPLC chromatogram peaks were sent for the H1NMR analysis and chromatogram was obtained which indicates the presence of aldehyde between 9 and 8 ppm. Peak of Para substituted aromatic ring appeared between 8 and 7 ppm.

DISCUSSIONS:

1) The TLC plate (FIGURE 3) consists of three different plant extracts i.e. Hexane, Dichloromethane and Methanol, were separated into individual compounds as they moved with mobile phase and identified as thick bands. It was observed that among the three plant extracts Hexane and Dichloromethane was separated well rather than methanol which is highly non-polar. According to the picture it was assumed that polar components moved likely with the solvent system and non polar components that is methanol extract still remaining stick to the silica plate because it cannot be removed with the solvent used and hence the procedure was repeated with more polar solvent.

The TEA system (Toluene: Ethyl acetate: Acetic acid / 80 : 18 : 2) used was more polar system than the solvent used before and the components were better separated than with the solvent used before .So polar components are separated with TEA solvent system. FIGURE 4 shows thick bands and resolved into separate compounds with TEA system. TLC is use full only in separating the mixture into individual compounds but cannot identify the compound so work is carried in the direction to identify the separated compounds.

3) The antioxidant activity is of prime importance since different oxygen species are injurious to health. The use of DPPH radical as TLC spray reagent proposed for screening antioxidants. It is well suited for detection of antioxidants in crude plant extracts or pure compounds isolated from plant material. It allows the detection of active spots and avoids the non useful isolation of common phenolic antioxidants. The results of experiments demonstrated that plant extract posses a potent antioxidant activity as plate is stained with yellow spots (FIGURE 5), which indicates the presence of antioxidant activity in the compound. The components separated on the TLC plate were observed under UV light and also stained blue with DPPH solution in methanol at concentration of 80µg/ml. By TLC experiment it is proved qualitatively and for qualitative analysis spectrophotometric assay is conducted.

4) The DPPH assay showed that all extracts exhibited minimal antioxidant activity even at the lowest test concentration of 0.005gm./ ml.

Care full studies of TABLE 5 suggests that the highest concentrations of Quercetine standard(2mg/ml) showing highest antioxidant activity(97.28%) at 517nm, keeping the wavelength constant gradual decrease in the Quercetine concentration leading to gradual decrease in antioxidant activity. TABLE 6 representing the antioxidant activity of Methanol Concentration, at 2mg/ml concentration antioxidant activity of methanol is (95.5%) and lowest concentration (0.0625mg/ml) showing (33.76%). TABLE 7 showing Hexane activity at 2mg/ml concentration hexane activity is (74.81%) where as at lowest concentration it is (23.0%). And TABLE 8 representing the antioxidant activity, at 2mg/ml dichloromethane concentration antioxidant activity is (81.3%) and at lowest concentration 0.125mg/ml activity is 17.6%. so, it can be concluded as methane extract has highest antioxidant activity i.e. (95.5%) which is nearly equal to the standard compound used i.e. is Quercetine and activity came decreasing as there is the decrease in concentration of methanol, where as Hexane and Dichloromethane showing less antioxidant activity when compared to methanol extract.

5) HPLC method followed here gave different chromatograms under appropriate solvent systems, which posses' different peaks at different time intervals known as retention time. These peaks were collected into the vial to separate the components from the prepared sample. These collected samples were then dried under nitrogen gas and were weighed. Such collected samples were sent to further analysis with NMR method to identify the collected peaks from the mixture.

6) A complementary relationship exist between the GC the MS. The MS can provide specific results, but not good qualitative findings. The GC is efficient in compound separations, but is not dependable in identifying them. The benefits of a two dimensional technique like GC/MS allows for both qualitative and quantitative analysis of a solution. The retention time and the mass spectra are both acquired from the GC/MS, which can be used to identify the substance. Overall, the GC/MS instrument is a conclusive tool for proof identification.

GC/MS chromatograms revealed that the plant consists of chemical constistuents like a-pinene and B- pinene which were commonly known as turpentines, match factor playing an important role in identifying the compounds obtained by comparing resembling compound in the library spectra . These pinenes are hydrocarbons they come under one of the essential oily constituents. The other most important constituent it posed of is caryophyllene oxide, an oxygenated terpenoid, it acts as an antifungal against dermatophytes.

7) Chromatogram obtained from H1NMR indicates the presence of aldehyde between 9 and 8 ppm. Peak of Para substituted aromatic ring appeared between 8 and 7 ppm.

CONCLUSION:

The above study reveals that the crude plant extracts which underwent for extraction and purification process, after it is subjected to different techniques like chromatography (TLC, HPLC, GC/MS), NMR for separating the compounds from mixture and identifying them. TLC basically separated the crude mixtures of plant into different compounds. GC/MS study revealed the presence of terpenes like Diterpene, Monoterpene and antifungal agent like caryophyllene in it. The peaks collected by HPLC method are not for sample preparation so, there is no much data collected by HPLC but the collected peaks were sent for NMR study, as a result with H spectra obtained from NMR there were only two peaks obtained one is of deuterated methanol and the other is of TMS internal standard and the chromatogram obtained from H1NMR indicates the presence of aldehyde between 9 and 8 ppm. Peak of Para substituted aromatic ring appeared between 8 and 7 ppm. And an Antioxidant assay was performed by spectrophotometric analysis and it was found that the methanol extract was showing more antioxidant activity then the other two extracts (Hexane and Dichloromethane).

Future work:

Methane extract has shown highest anti-oxidant activity almost near to the standard compound (Quercetine) which has a great scope to carry on further. α pinene, β pinene and caryophyllene oxide an anti-fungal agent were commonly found in all three extracts i.e., Hexane, Dichloromethane and Methane because of which it can be applied in medical field for treatment against fungal attacks. With H spectra obtained from NMR there were only two peaks obtained one is of deuterated methanol and the other is of TMS internal standard. So, further work can be done on this, a prep HPLC method has to be carried out in order to collect enough sample at least 5mg of plant extract to get enough peaks by NMR from which there is a chance of gathering some important groups present in Pinus radiate plant.