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Study was focused on works Pinus Radiata which is an of import category of Coniferous workss. Pinus Radiata chiefly known to consists of Terpenoids and Caryophyllene, which contributes assorted belongingss such as antibacterial, anticancer and antioxidant. It is known to hold anti oxidant activity like other Flavonoids. Which is proved qualitatively by both TLC ( Thin Layer Chromatography ) and Spectrophotometric methods, these consequences are compared with the known anti oxidizer compound i.e. Quercetine. Study was subsequently focused on phytochemical analysis of Pinus Radiata works infusion by GC/MS ( Gas chromatography-Mass spectrometry ) , HPLC ( High public presentation liquid chromatography ) and NMR ( Nuclear magnetic resonance ) surveies. GC/MS survey revealed presence of of import chemical components such as A-Pinene and B-Pinene. HPLC survey is utile in obtaining chromatograms with different extremums. Selected extremums were collected, dried and sent to NMR ( Nuclear Magnetic Resonance ) survey, which was non much successful because of little sum of samples obtained from HPLC chromatograms. So, farther work is chiefly focused on development of homework HPLC to roll up sufficient sum of samples for analyzing “ H ” Spectra by NMR.

Keywords:

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

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Abbreviations: –

DCM – Methylene chloride, TLC- thin bed chromatography, GC-MS, gas chromatography mass spectroscopy, DPPH – 2, 2-Diphenyl-1-picrylhydrozyl, HPLC- high public presentation liquid chromatography, H1-NMR- Nuclear Magnetic Resonance.

Purpose: – Phytochemical probe of conifer works cones of P. radiata by reflux extraction so analysis by utilizing Thin bed chromatography ( TLC ) , Gas chromatography – Mass spectroscopy ( GC-MS ) , high public presentation liquid chromatography ( HPLC ) , GC-MS-NMR and Spectrophotometric analysis for probe of anti-oxidant activity in the cone extracts.

Introduction:

Pinus Radiata evolved about 100 million old ages ago, pine bark was an inconvenient residue for the wood industry. Pine bark is a rich beginning of natural polyphenols compounds which have attracted increasing attending on the field of nutrition, wellness and medical specialty. Flavonoids and other works phenoplasts, such as phenolic acids, stilbenes and tannic acids, are of import in the workss for normal growing development and defense mechanism against infection and hurt ( KaA?hkoA?nen et al. , 1999 ) . Polyphenolic infusions and fractions have a possible usage in the nutrient industries as alternativs to man-made BHT ( butylated hydroxytoluene ) , BHA ( butylatedhydroxyanisole ) and TBHQ ( tert-butyl hydroquinone ) .

Terpenoids are common components of the rosins of higher workss and they are utile chemostatics features of extant workss. ( Hegnauer, 1962, 1992 ; Langenheim, 1969 ; Erdtman and Norin, 1966 ; Thomas, 1986 ; Otto and Wilde, 2001 ) .

Most of the compounds identified in the dodo rosins are the diagenetic merchandises ( biomarkers ) of terpenoids which were synthesized by populating beings ( simoneit 1986 ) .

The conifer workss contain terpenes and flavonoids which display the antioxidant activity. When free group with individual negatron uses the other negatron and go odd, this procedure is called as oxidization which can ensue into the ripening, unsusceptibility jobs, bosom disease. ( Kinderlehrer, 2007 ) The diterpenoid, totarol have shown anti-oxidant and antibacterial activity. ( Bernabeu et al, 2002 ) . Many pines have been used to bring forth gum terpentine, a semi-fluid, xanthous or chocolate-brown rosin ( oleoresin ) . ( Moussouris and Regato 1999 ) .

Surveies suggests that coevals of free groups has of import function in the patterned advance of pathological perturbations such as cardiac diseases, malignant neoplastic disease etc. These free groups such as superoxide anion ( O2- ) , hydroxyl extremist ( OH ) destroys the enzyme activity by doing tissue harm usually through covalent bonding and lipid per oxidization. Antioxidants are known to protect cell membrane against free groups. Man-made antioxidants doing serious wellness jobs for this ground an extended hunt for different types of works based antioxidants is taking topographic point. Natural antioxidants have proved to be less toxic than Man-made molecules such as butylated hydroxyanisole ( BHA ) and butylated hydroxytoluene ( BHT ) , which are extremely volatile and unstable at room temperatures. Therefore probes for assorted infusions of the works were screened for antioxidant activity utilizing DPPH ( 2, 2-Diphenyl-1-picrylhydrazyl ) , method.

DESCRIPTION OF SPECIES:

It is a species of pine native to coastal California is three really limited countries in Santa Cruz, Monterey and San Luis Obispo Countries, and Guadalupe Island and Cedros Island off the west seashore of Baja California, Mexico. It is besides extensively cultivated in many other warm temperate parts of the universe. It was foremost introduced into New Zealand in the 1850 ‘s ; today, over 90 % of the state ‘s plantation woods are of this species. This includes the Kaingaroa Forest on the cardinal tableland of the North Island which is the largest deep-rooted wood in the universe. Australia besides has big Radiata Pine plantations. The works prefers light ( flaxen ) and medium ( loamy ) dirts, requires well-drained dirt and can turn in nutritionally hapless dirt. The works prefers acid and impersonal dirts. It can non turn in the shadiness. It requires dry or damp dirt and can digest drouth. The works can digest nautical exposure. ( Hogan, C. Michael & A ; Frankis, et.al ( 2009 ) .

The form of the tree has a pointed top, which is normally seen on immature trees. The acerate leafs are glistening green coloring material of 4-6 inches in length. The bark of the tree is thick, and profoundly furrowed into lepidote ridges, and is dark ruddy brown in coloring material. The cones are 3-6 inches in length and exhaled with a pointed tip. The cones are glistening brown in coloring material and grow clustered in rings on short chaffs. “ The graduated tables of the cones are raised and rounded, larger on the outer sides and stoping in bantam spine: little, long winged seeds ( Little, 1980 ) ” .A

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

Degree centigrades: UsersSONYDesktop10007594.JPG

Referee: www.dkimages.com/ … /Pinus-radiata-2.html

FIGURE 1

Pinus radiataA ( Monterey pine ) A

Use:

It has vanillin spirit obtained as a byproduct of other rosins that are released from the pulpwood. The gum terpentine obtained from the rosin of all pine trees is antiseptic, diuretic, rubefacient and anthelmintic. It is besides really good to the respiratory system and so is utile in handling diseases of the mucose membranes and respiratory ailments such as coughs, cold, grippe and TB. It is a valuable redress when used internally in the intervention of kidney and vesica ailments and is used both internally and as a hang-up and steam bath in the intervention of arthritic fondnesss. Externally it is a really good intervention for a assortment of skin ailments, lesions, sores, Burnss, furuncles etc and is used in the signifier of liniment plasters, cataplasms, herbal steam bath and inhalators. ( Frankis, M.P, ( 1992 ) ) .

MATERIALS AND METHODS:

Chemical: –

Solvent hexane, methylene chloride and methyl alcohol were purchased from Fisher Scientific ( Loughborough, UK ) . DPPH pulverization ( 2, 2-Diphenyl-1-picrylhydrazyl ) , standard quercetin and TLC home base was purchased from the Sigma Aldrich ( Dorset, UK ) .

Extraction BY REFLUX EXTRACTION METHOD USING SOLVENT OF INCREASING POLARITY:

The dried works stuff of Pinus radiata was separated and weighed to 28.45g. The stuff was transferred to 1000 milliliter unit of ammunition bottomed flask to this 600 milliliter of hexane was added to the flask and oral cavity was fitted to bottom of the reflux capacitor. Small sum of anti-bumping granules were added to round bottom flask to avoid bubbling. The whole setup was kept in a fume closet and connected to the electric warming mantle on 60A°C. Flow of H2O was kept changeless.

After running it for one hr the flask was cooled for 15 min and filtration was carried out utilizing Buchner funnel under vacuity by utilizing whatman filter paper. ( Sterile Pyrogenic, Puradisc 25AS polyehersulfone membrane 0.2 I?m pore size and 25 millimeters diameter ) .

Filtrate was transferred to 500 milliliters unit of ammunition bottomed flask and connected to the Buchi rota vapour R-200 and temperature of the Buchi heating bath was set to 39A°C to take organic dissolver by vaporization. The concentrated infusion was transferred to little phials utilizing a sonicator to solubilise the infusion in hexane and was weighed. The experiment was repeated with more polar dissolver methylene chloride so methyl alcohol. Small part of dissolver was evaporated utilizing N gas.

Chromatography:

Chromatography technique involves separation of mixture into single compounds utilizing a nomadic stage which passes through or over a stationary stage. Substances which have strong affinity towards stationary stages will be eluted easy, substances which have less affinity towards stationary stage eluted quickly with the nomadic stage.

The constituents in the mixture distributed otherwise between the Mobile and stationary stage have different interactions with each stage. By altering the composing of the nomadic stage, it is possible to analyse a broad assortment of compounds.

Chromatography has broad assortment of utilizations like in chemical science and biochemistry research analyzing complex mixtures, sublimating chemical compound, insulating natural merchandises and besides in foretelling physical belongingss. It is besides used in quality control to guarantee the pureness of natural stuffs, to command and better the procedure outputs, or to measure merchandise stableness and proctor debasement.

Thin Layer Chromatography:

This method is utile for separation of constituents from a mixture. It consists of a stationary stage and nomadic stage were both involve in elution of constituents from a mixture. Mobile stage is allowed to go through from the underside to exceed of the stationary stage. Mixture to be extracted is placed on the stationary stage which travels along nomadic stage and gets separated harmonizing to its mutual opposition. ( Ahuja et al 2003 ) .

TLC analysis was performed on each of the infusions. Thin bed silicon oxide home base was taken and a line was drawn from 1.5 centimeters above the underside of the home base, each infusion was spotted on to the home base utilizing glass capillary tubings. And the home base was carefully dipped in the TLC armored combat vehicle which was antecedently filled with the 100ml of prepared dissolver system.

Different solvent systems were prepared and TLC analysis was performed.

Different dissolver readyings

1 ) Hexane: Dichloro methane

80: 20

2 ) Tea system

Toluene: Ethyl ethanoate: Acetic acid

80: 18: 2

This method is chiefly used for polar compounds.

3 ) Hexane: Dichloro methane

70: 30

The home base was so removed carefully and it was foremost air dried.

These home bases were foremost observed under the UV visible radiation and musca volitanss were observed.

The developed home base 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 utile in happening out interaction of different substances with the surface of the solid. The nomadic stage 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 clip to be carried across the stationary stage.

The GC-ms instrument combines two different techniques to organize one individual method of analysing mixtures. Gas chromatography separates the constituents in a mixture, and the mass spectrometry distinguishes the single component. After separation the constituents enter a sensor where an electric signal is created when a constituent is detected. The signal size is dependent on the constituent ‘s concentration. A keeping clip ( the clip from the injection is made to when elution occurs ) is calculated by instrument computing machine. The keeping clip is same for a compound, every bit long as GC conditions are unchanged. Compound assignment can be made if it`s keeping clip is known. ( Hill, D.W et Al ) .

The eluted samples from GC enter the Mass Spectrometer`s electron ionisation sensor. The compounds are electrically charged when accelerated by magnetic field and interrupt 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 single constituents, 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 car sampling station. The hexane infusion was dissolved in DCM, the DCM and methanol infusions were dissolved in their several dissolvers. All infusions were prepared at a concentration of 10 mg/ml. 4I?L of each extract sample was injected into the injection port at 250A°C of GC-MS. The capillary column used was Varian VF-5ms fused silicon oxide ( 30mA-0.25mm, movie thickness 0.25 I?m ) . Helium gas was used as bearer gas at flow rate of 0.7ml/min. Split ratio was 1/14 and temperature of oven was 4A°C for 5minutes so it was increased to 220A°C at 3A°C/min. Then it was held for 5 proceedingss at 220A°C. Electron impact used was 70eV as ionisation manner at 200A°C ion beginning temperature. Samples were scanned from 50-650 mass scan scope with scan clip of 1.22 2nd. Thermo Electron Corporation X quality V 1.4 package was used. The graph, molecular expression and molecular construction were searched and compared in NIST Library version 2 hunt libraries.

COMPOUND IDENTIFICATION:

“ Library hunt ” method is use full in placing the compound by comparing mention library spectra with the submitted ( user ) spectra. Spectra should resemble closely in both spectra. Spectra originated from GC/MS informations file was a individual mass spectral scan or an mean, with or without simple background minus, it produces a “ hit list of library spectra, which is ordered by similarity to the mark spectra harmonizing to computed “ match factor. ” ( Skoog, Hooler, et Al ( 1998 ) ) .

High PERFORMANCE LIQUED CHROMATOGRAPHY:

Chromatography is defined as a process by which inert stuffs and drug rule encountered in pharmaceuticals readyings are separated by fractional extraction, surface assimilation, or in ion exchange on a porous solid. It is defined as agencies of dividing and sublimating complex and closely related chemical substance which are hard to separate. ( Quantitative Pharmaceutical Chemistry by Glenn-L.Jenkins, Digangi, pg 425-428 ) .

High public presentation liquid chromatography ( HPLC ) is known to be one of the most effectual methods for analysis of mixtures of complex composing. . A great figure of assorted organic substances of different nature and belongingss required assorted proficient attack and instrumentalities to divide complex mixtures, identify and find their constituents. Therefore, HPLC is of huge importance non merely for pharmaceutical intents but besides for

other analytical intents in assorted Fieldss.

The rule aims obtained through the chromatography including HPLC are:

1. Resolution of mixtures into constitutional parts. 2. Determination of homogeneousness. 3. Comparison of substances suspected of being indistinguishable. 4. Purification of any substance or compounds. 5. Quantitative separation from complex mixtures. 6. Indication of molecular construction.

HPLC development led to stationary stages that are chemically bonded to solid support. Covalent bond between the stationary stage and solid substance makes the chemically bonded liquid chromatography columns stable. In column liquid chromatography high force per unit areas drive the nomadic stage through a column of stationary stage leting separation of complex mixtures with high declaration. ( IV.Remington the Science and Practice of Pharmacy, pg 615-626 ) .

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

FIGURE 2

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

Normal stage HPLC:

HPLC analysis of methyl alcohol infusion was carried out on the Varian series HPLC with the Varian 2010 pump and Varian 2050 sensor. The pump was attached with column, online degasser and sensor. 40 milligram of fraction five was dissolved in 5 milliliter of methyl alcohol so it was sonicated for 5 proceedingss in sonicator to take the gas from the fraction which was performed in the normal stage HPLC with analytical column Waters Spherisorba?? 5 I?m silicon oxide column ( 4.6 X 250mm ) . An injection cringle of 20 I?L was injected into the injector. Then column was calibrated with TEA-1 ( 80:18:2 ) solution with 1.5ml/min flow rate for 50 proceedingss to take drosss from the column. The sensor wavelength was set to 280nm in UV-visible sensor. The normal stage nomadic stage was used similar to TLC plate developing solvent system-2 called TEA ( methylbenzene: ethylacetate: acetic acid ) . To obtain a better separation on test and error footing different proportion of nomadic stage TEA ( methylbenzene: ethylacetate: acetic acid in the ratio of 80: 18: 2 was used with 1ml/min flow rate and so 20 I?L sample was injected with the same dissolver system with 1ml/min was carried out for 25 proceedingss. To divide the extremums the flow rate was decreased to 0.7ml/min.

ANTI-OXIDANT ASSAY:

Preparation OF PLANT MATERIAL:

The assorted parts of the workss were dried at room temperature and was defatted by extraction with Hexane, Dichloromethane and methyl alcohol. Infusions were concentrated to dryness under decreased force per unit area and controlled temperature. All infusions were preserved in a icebox boulder clay further usage.

Procedure:

Determination of the antioxidant activity by DPPH method of the works infusions was determined utilizing a method based on the decrease of methanolic solution of color free extremist DPPH solution. A methanolic DPPH solution was assorted with methanolic solutions of extract concentrations. The stock solutions were diluted with methyl alcohol to obtain lower dilution. Then samples were shaken smartly and maintain in dark for 30min at room temperature, sample optical density was measured at 517nm with spectrophotometer. A methanolic solution of DPPH was employed as space. Antioxidant activity of each infusion was determined harmonizing to the per centum of DPPH decoloration. Inhibition per centum = [ 1- ( optical density with compound ) / ( optical density of the space ) ] A- 100. ( Free Radic Biol Med. 2000 ; 28: 1538-1546. )

Thermo Electron Corporation ( Nicolet development 100 ) spectrophotometer was used to look into antioxidant activity of all infusion. 4 mg/ml concentrated stock solution of infusion was used to do the consecutive 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 karyon of the atoms have magnetic belongingss that can be utilized to give chemical information. Subatomic atoms like protons, neutrons and negatrons have spin. In some atoms these spins are paired and cancel each other out so that the karyon of the atom has no overall spin.

NMR measures the soaking up of electromagnetic radiation in the frequence part of approximately 4 to 900 MHz ( Skoog et al 2007 ) . Normally there are two types of NMR spectrometers in usage 1 ) Pulse or Fourier transform ( FT-NMR ) , 2 ) continuous-wave ( CW ) spectrometers. NMR usually involves Deuterated dissolver systems for sample readying. Some common deuterated dissolvers are dimethyl sulfoxide ( DMSO ) , trichloromethane, H2O, propanone, methyl alcohol and acetonitrile. DMSO is a “ cosmopolitan dissolver ” giving first-class solubility and high boiling point. ( Ahuja et al 2003 ) . The chemical construction of the molecule can be made by agencies of scope of frequences vital for orientation and excitement of molecule ( Watson, 2005 ) .

Instrument used AVANS Bruker Ultrashield 500.13 MHz magnet was used for analysis of sample. Solvent used MeOD ( deuterated methyl alcohol ) to fade out all samples. Tetramethyl silane was used as a mention.

Consequence:

Extraction BY REFLUX EXTRACTION METHOD USING SOLVENT OF INCREASING POLARITY:

Table-1

Type of infusion

Sum of infusion ( g )

Hexane infusion

0.71

Dichloromethane infusion

0.27

Methanol infusion

0.75

Reflux extraction method removes foreign stuff from petroleum works stuff, and compounds solubilised in the dissolver used harmonizing to their mutual opposition they are Hexane ( H ) , Dichloromethane ( D ) and Methanol ( M ) .

2 ) TLC PLATE WITH HEXANE, DICHLOROMETHANE AND METHANOL

Bespeaking detached compounds.

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

Thin bed chromatography home base ( TLC ) , demoing midst separated sets of three infusions used ( Hexane, Dichloromethane and Methanol.Violet color musca volitanss observed under UV visible radiation indicated by side pointers.

FIGURE 3

MORE COMPUNDS SEPERATED BY TEA SYSTEM.

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

Figure: 4

Thin bed chromatography home base ( TLC ) , demoing midst separated sets of three infusions used ( Hexane, Dichloromethane and Methanol.

FIGURE 5:

Table 2

4 ) Pinus radiata ( hexane ) infusion 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 find the good lucifer with the compound library. Assortment of compounds were obtained from Hexane infusion like I± pinene, I? pinene, caryophyllene, cis-verbenol etc which proven to be a ground to analyze in deepness.

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 chiefly obtained from methane infusion like pimaric acid and caryophyllene oxide with lucifer factors 795 and caryophyllene oxide severally.

FIGURE 7:

Table 4

Pinus radiata ( Di chloro methane infusion ) 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 spectrometry ) .Match factor help full in indentifying the compound from the library spectra. ( MF ) Molecular expression.

I±- pinene, I?- pinene and caryophyllene were chiefly obtained from the Dicholoromthane infusion with 927, 932 and 896 lucifer fatcors severally.

5 ) Calculation:

% of Antioxidant activity:

With Quercetine criterion: 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 infusion:

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 infusion:

Optical density 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 %

Methylene chloride:

Optical density 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 musca volitanss bespeaking presence of antioxidant activity.FIGURE: 4HEXANE ( H ) METHANOL ( M ) DICHLOROMETHANE ( D )

TLC ( thin bed chromatography ) home base, DPPH ( 2, 2-Diphenyl-1-picrylhydrazyl ) used to stain the home base, Hexane ( H ) , METHANOL ( M ) , DICHLOROMETHANE ( D. ) . Yellow musca volitanss were observed on the home base for all three infusions, bespeaking that they all contain compounds with anti-oxidant activity.

6 ) HPLC RESULTS:

When works infusion is subjected to HPLC under given conditions it gave chromatogram screening different extremums at different clip intervals which indicates the presence of constituents in the mixture.

Weights of the gathered extremums were measured and were found to be in little amounts.100Aµg, 250Aµg and170g.

Table 9:

Valuess OBTAINED FROM THE CHROMATOGRAM.

Extremum

NO

Retention clip

( min: sec )

Area

[ AµVa?™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 extremums 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.

Discussion:

1 ) The TLC home base ( FIGURE 3 ) consists of three different works infusions i.e. Hexane, Dichloromethane and Methanol, were separated into single compounds as they moved with nomadic stage and identified as thick sets. It was observed that among the three works infusions Hexane and Dichloromethane was separated good instead than methanol which is extremely non-polar. Harmonizing to the image it was assumed that polar constituents moved probably with the dissolver system and non polar constituents that is methanol infusion still staying stick to the silicon oxide home base because it can non be removed with the dissolver used and therefore the process was repeated with more polar dissolver.

The TEA system ( Toluene: Ethyl ethanoate: Acetic acid / 80: 18: 2 ) used was more polar system than the dissolver used before and the constituents were better separated than with the dissolver used before.So polar constituents are separated with TEA dissolver system. FIGURE 4 shows thick sets and resolved into separate compounds with TEA system. TLC is use full merely in dividing the mixture into single compounds but can non place the compound so work is carried in the way to place the detached compounds.

3 ) The antioxidant activity is of premier importance since different O species are deleterious to wellness. The usage of DPPH extremist as TLC spray reagent proposed for testing antioxidants. It is good suited for sensing of antioxidants in petroleum works extracts or pure compounds isolated from works stuff. It allows the sensing of active musca volitanss and avoids the non utile isolation of common phenolic antioxidants. The consequences of experiments demonstrated that works infusion posses a powerful antioxidant activity as home base is stained with xanthous musca volitanss ( FIGURE 5 ) , which indicates the presence of antioxidant activity in the compound. The constituents separated on the TLC home base were observed under UV visible radiation and besides stained bluish with DPPH solution in methyl alcohol at concentration of 80Aµg/ml. By TLC experiment it is proved qualitatively and for qualitative analysis spectrophotometric check is conducted.

4 ) The DPPH check showed that all infusions exhibited minimum antioxidant activity even at the lowest trial concentration of 0.005gm./ milliliter.

Care full surveies of TABLE 5 suggests that the highest concentrations of Quercetine criterion ( 2mg/ml ) demoing highest antioxidant activity ( 97.28 % ) at 517nm, maintaining the wavelength changeless gradual lessening in the Quercetine concentration taking to gradual lessening in antioxidant activity. Table 6 stand foring the antioxidant activity of Methanol Concentration, at 2mg/ml concentration antioxidant activity of methyl alcohol is ( 95.5 % ) and lowest concentration ( 0.0625mg/ml ) screening ( 33.76 % ) . TABLE 7 demoing Hexane activity at 2mg/ml concentration hexane activity is ( 74.81 % ) where every bit at lowest concentration it is ( 23.0 % ) . And TABLE 8 stand foring the antioxidant activity, at 2mg/ml methylene chloride concentration antioxidant activity is ( 81.3 % ) and at lowest concentration 0.125mg/ml activity is 17.6 % . so, it can be concluded as methane infusion has highest antioxidant activity i.e. ( 95.5 % ) which is about equal to the criterion compound used i.e. is Quercetine and activity came diminishing as there is the lessening in concentration of methyl alcohol, where as Hexane and Dichloromethane demoing less antioxidant activity when compared to methanol infusion.

5 ) HPLC method followed here gave different chromatograms under appropriate solvent systems, which posses ‘ different extremums at different clip intervals known as keeping clip. These extremums were collected into the vial to divide the constituents from the prepared sample. These collected samples were so dried under N gas and were weighed. Such gathered samples were sent to farther analysis with NMR method to place the gathered extremums from the mixture.

6 ) A complementary relationship exist between the GC the MS. The MS can supply specific consequences, but non good qualitative findings. The GC is efficient in compound separations, but is non reliable in placing them. The benefits of a two dimensional technique like GC/MS allows for both qualitative and quantitative analysis of a solution. The keeping clip and the mass spectra are both acquired from the GC/MS, which can be used to place the substance. Overall, the GC/MS instrument is a conclusive tool for proof designation.

GC/MS chromatograms revealed that the works consists of chemical constistuents like a-pinene and B- pinene which were normally known as gum terpentines, lucifer factor playing an of import function in placing the compounds obtained by comparing resembling compound in the library spectra. These pinenes are hydrocarbons they come under one of the indispensable greasy components. The other most of import component it posed of is caryophyllene oxide, an oxygenated terpenoid, it acts as an fungicide 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.

Decision:

The above survey reveals that the petroleum works infusions which underwent for extraction and purification procedure, after it is subjected to different techniques like chromatography ( TLC, HPLC, GC/MS ) , NMR for dividing the compounds from mixture and placing them. TLC fundamentally separated the petroleum mixtures of works into different compounds. GC/MS survey revealed the presence of terpenes like Diterpene, Monoterpene and fungicidal agent like caryophyllene in it. The extremums collected by HPLC method are non for sample readying so, there is no much informations collected by HPLC but the gathered extremums were sent for NMR survey, as a consequence with H spectra obtained from NMR there were merely two extremums obtained one is of deuterated methyl alcohol and the other is of TMS internal criterion 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 check was performed by spectrophotometric analysis and it was found that the methyl alcohol infusion was demoing more antioxidant activity so the other two infusions ( Hexane and Dichloromethane ) .

Future work:

Methane infusion has shown highest anti-oxidant activity about near to the criterion compound ( Quercetine ) which has a great range to transport on farther. I± pinene, I? pinene and caryophyllene oxide an anti-fungal agent were normally found in all three infusions i.e. , Hexane, Dichloromethane and Methane because of which it can be applied in medical field for intervention against fungous onslaughts. With H spectra obtained from NMR there were merely two extremums obtained one is of deuterated methyl alcohol and the other is of TMS internal criterion. So, farther work can be done on this, a homework HPLC method has to be carried out in order to roll up adequate sample at least 5mg of works infusion to acquire adequate extremums by NMR from which there is a opportunity of garnering some of import groups present in Pinus radiate works.

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