manuscript thesis

8/10/2019 Manuscript Thesis

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ABSTRACT

In the Philippines, Euphorbia hirta, known locally as

tawatawa, is used in folk medicine to cure dengue fever by

people in rural areas. Scientific studies show that tawa-tawa

(Euphorbia hirta L.) contains phytol, a chemical substance

known for its anti-dengue properties. However, another plant

is also dominant in the Philippine area which has also

similarities on the physical appearance of tawa-tawa. With the

growth of this plant also known as Alligator weeds

(Alternanthera philoxeroides) in the locale, confusion between

the two plant species arises. The study aimed to identify the

substances contained in the extracts of Tawa-Tawa (Euphorbia

hirta L.) leaves and Alligator weeds (Alternanthera

philoxeroides) by the aid of gas chromatography. The

researchers used 95% ethanol to extract the organic substances

in the leaves. The crude extracts were then machine processed,

namely Rotary Evaporation, in order to remove the impurities

of the crudes. Gas chromatography (GC) was then used for

separating and analyzing compounds that can be vaporized

without decomposition. Findings showed the different

substances that can be found on the two said extracts. The

presence of phytol in the Alligator weeds extract was

positively determined.


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Introduction

The use of herbal-based medicine and medicinal plants to

treat many diseases is growing worldwide as they have few or

no adverse effects. According to a World Health Organization

(WHO) fact sheet dated December 2008, 80% of the population in

some Asian and African countries depends on traditional

medicine as their primary health care due to economic and

geographical constraints. Natural products have become the

main source of test material in the development of antiviral

drugs based on traditional medical practices. Traditional

medicines are based on knowledge, experience and practices

based on indigenous cultural beliefs and knowledge, and are

used to maintain health, prevent, treat and diagnose physical

or mental illness. Traditional medicinal plants have been

reported to have antiviral activity and some have been used to

treat viral infections in animals and humans.

To date, 31 different species have been found to have the

potential to treat dengue; some of these have not yet been

investigated scientifically. In the Philippines, Euphorbia

hirta, known locally as tawatawa, is used in folk medicine

to cure dengue fever by people in rural areas. Practitioners

of traditional medicines believe that decoction of tawatawa


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Statement of the Problem

This study was conducted in order to compare the gas

qualitative characteristics of the extracts of Tawa-tawa

(Euphorbia hirta L.) leaves and Alligator weeds (Alternanthera

philoxeroides). Specifically, it attempted to answer the

following questions:

1.

Is the chemical substance phytol also present in

Alligator weeds extract?

2.How may the extracted Tawa-tawa leaves and Alligator

weeds be described and characterized using gas

chromatography?


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REVIEW OF RELATED LITERATURE

Fig 1. Euphorbia hirta L.

Scientific Name: Euphorbia hirta Linn.

Genus: Euphoribia

Family: Euphorbiaceae

Other Scientific Name: E. piluliferaLinn. Chamaesycepilulifera

LinnE. Capitata

Common Names: Bobi( Bis.), Magatas (Pamp.), Australian asthma

weed (Engl.), Tairas (lv.), Golandrina (Tag.), Cats hair

(Engl.), Tauataua (P. Bis), Snake weed (Engl.), Tawa- tawa

(Tag.).


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Botany

This erect or prostrate annual herb can get up to 60 cm

long with a solid, hairy stem that produced abundant white

latex. There are stipules present. The leaves are simple,

elliptical, hairy (on both upper and lower surfaces but

particularly on the veins on the lower leaf surface), with a

finely dentate margin. Leaves occur in opposite pairs on the

stem. The flowers are unisexual and found in axillary cymes at

each leaf node. They lack petals and are generally on a stalk.

The fruit is a capsule with three valves and produces tiny,

oblong, four-sided red seeds. It has a white or brown taproot.

Euphorbia is the largest genus of the family Ephorbiaceae

with about 1600 species. All species of Euphorbia exude a

milky juice when broken, and Euphoribahirtas local name

gatas-gatas or Tawa-tawa derive from this.

The Philippine Council for Health Research and

Developmentt (PCHRD), the health research arm of the

Department of Science and Technology (DOST) spearheads studies

to assess the curative component of Tawa-tawa plants for

dengue. Asied from Tawa-tawa the PCHRD-DOST also tookinto

other indigenous plants that has components possible for

dengue cure this includes kamote(Ipomeabatatas), bawang

(Allium sativum), papaya (Carica Papaya), tanglad (Cymbopogon


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citrates), luyangdilaw (Curcuma longa), ampalaya

(Momordicacharantia) and oregano (Coleus aromaticusBenth).

As evidence of its continuing support and commitment to

advance herbal medicine research in the country, PCHRD-DOST

recognizes researchers with excellent research on herbal

medicine through the PCHRD GruppoMedica Award. One of which

were the study tawa-tawa conducted by the University of Sto.

Tomas.

In Indonesia, papaya leaves are made into tea

traditionally to cure dengue fever. Mixed messages have been

presented by representatives of the Philippine government. The

Department of Health (DoH) says tawa-tawa is not enough for

critical dengue patients, and urges oral rehydration therapy.

Others, like former Health Secretary Jaime Galvez Tan, are

actively promoting the herbal medication. There is a large

difference between cases of dengue without hemorrhagic fever

and those with, along with the 4 serotypes, recommended

treatment may vary depending on the specific presentation of

dengue.


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Fig 2. Alternanthere philoxeroides

Kingdom: Plantae

Order: Caryophyllales

Family: Amaranthaceae

Genus: Alternanthera

Species: A. philoxeroides

Botany

Alternanthera philoxeroides, commonly known as Alligator

weed, is an emergent aquatic plant. It originated in South

America, but has spread to many parts of the world and is

considered an invasive species in Australia, China, New

Zealand, Thailand and the United States.


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Phytol

Phytol is an acyclic diterpenealchohol that can be used

as a precursor for the manufacture of synthetic forms of

vitamin E and vitamin K1. Intruminants, the gut fermentation

of ingested plant materials liberates phytol, a constituent of

chlorophyll, which is then converted to phytanic acid and

stored in fats.

Phytol is derive from phytyl, the major side chain off of

the chlorophyll- a molecule. When phytol undergoes diagnosis

and catagenesis, pristane and phytane are two of the major

biomarkers that are produced, though most regurlarisoprenoid

alkanes containing 25 or less carbon atoms appear to originate

from phytol.

Name: Phytol

Formula:C20H40O

Contributor: Philip Morris R&D

Foreign Studies

According to Wilkins, (2001) entitled, Clinical

Pharmacokinetic Studies of Pharmaceuticals. Clinical

pharmacokinetic studies are performed to examine the

absorption, distribution, metabolism and excretion of a drug

under investigation (investigational drug and approve drug) in


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system for tuberculosis: pharmacokinetics and therapeutic

effects. Alginate microparticles were developed as ral

sustained delivery carriers for antituberculosis drugs in

order to improve patient compliance. In the present study,

pharmacokinetics and therapeutic effects alginate

microparticle encapsulated antitubercular drugs, i.e.

isoniazid, rifampicin and pyrazinamide were examined in guinea

pigs. Alginate microparticles containing antitubercular drugs

were evaluated for in vitro and in vivo release profiles.

These microparticles exhibited sustained release of isoniazid,

rifampicin and pyrazinamide for 3-5 days in plasma and up to 9

days in organs. Peak plasma concentration (C max), T max,

elimination half-life (t 1/2e) and AUC0- of alginate drugs

were significantly higher than those of free drugs. The

encapsulation of drug in alginate microparcticles resulted in

up to a nine-fold increase in relative bioavailability

compared with free drugs. Chemotherapeutic efficacy of

alginate drug microspheres against experimental tuberculosis

showed no detectable cfu values at 1:100 and 1:100 dilutions

of spleen and lung homogenates. Histopathological studies

further substantiated these observations, thus suggesting that

application of alginate-encapsulated drugs could be in the

effective treatment of tuberculosis.


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According to Onkologie, (2003) on their study SOP 13:

Pharmacokinetic Data Analysis. Pharmacokinetic studies are an

integrated part of the development program of a new drug. They

are intended to define the time course of drug and major

metabolite concentrations in plasma and other biological

fluids in order to obtain information on extraction depends on

corolla and bill morphologies, and the extraction efficiencies

depend, in addition, on the bird weight and nectar

concentration.

According to Vijaya, (2004) on their study Antibacterial

effect of the flavin, polyphenon 60(Camellasinensis) and

Euphorbia hirtaon Shigella spp.-a cell culture study,

antibacterial effect of compounds extracted from

Camellasinensis L. and the methanol extract of Euphorbia Hirta

L. were studied against dysentery causing Shigella spp. using

the Vero cell line. Cytotoxicity studies of the extracts were

performed using the cell line and the non-cytotoxic

concentration of the extract was tested for antibacterial

activity against the cytophatic dose of the pathogen. These

extracts were found to be non-cytotoxic and effective

antibacterial agents.

Study conducted by Wakeham, (2004) on their study

Phytol-substituted chlorophyll from Emilianiahuxleyi


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(prymnesiophyceae). A nonpolar chlorophyll c-like pigment was

isolated bysemi-preparative high performance liquid

chromatography from the coccolithophoroid Emilianiahuxleyi Hay

and Mohler. The visible absorption spectrum of this pigment

was similar to that of chlorophyll C2. However, its nonpolar

chromatographic properties were quite different from those of

the relativity polar chlorophylls C2 and C3 in E.huxleyie and

similar to those of chlorophyll a. Analyses by gas

chromatography mass spectrometry showed that the nonpolar

character of this new chlorophyll c was due to the presence of

phytol, most likely on the acid side chain of the porphyrin

macro-cycle.

This is the first example of a phytol-substituted

chlorophyll c-like pigment.

According to Baxter, (2005) on his study, Absorption of

chlorophyll phytol in normal man and in patients with Refsums

disease, this study was made to determine the extent of

absorption of chlorophyll phytol from the intestine of man,

and the importance of chlorophyll as a source of the phytanic

acid that accumulates in Refsums disease. Uniformly C-labeled

pheophytina was fed to normal human subjects to patients with

Refsums disease. Feces were collected and analyzed. It was

concluded that not more than about 5% of the ingested


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chlorophyll phytol is absorbed by man, whether normal or

afflicted with Refsums disease. On this basis we concluded

that the major portion of phytanic acid that accumulates in

Refsums disease could not be derived from dietary

chlorophyll.

Study conducted by Johnson, (2005) on their study

Euphorbia hirta leaf extracts increase urine output and

electrolytes in rats. Euphrobiahirta is locally used in

Africa and Australia to treat numerous diseases, including

hypertension, and edema. The diuretic effect of the E. hirta

leaf extracts were assessed in rats using acetazolamide and

furosemide as standard diuretic drugs. The water and ethanol

extracts of the plant produced time-dependent increase in

urine output. Electrolyte excretion was also significantly

affected by the plant extracts. This study suggests that the

active component(s) in the water extract of tawa-tawa leaf had

similar diuretic spectrum to that of acetazolamide.

According to Ogunseli, (2006) on their study Analysis of

the essential oil from dried leaves of E. hirta as a

phytomedication for the management of asthma adopt oral

administration of the aqueous extract.

The increases in oxidative burst found after phytol

injection was found not be restricted to one anatomical


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compartment and was observed in the blood, bone marrow,

spleen, and lymph mode. For example, prevention mediated by

phytol was found to be more pronounce than that of etanercept

and while no significant reduction of disease severity was

after a single etanercept administration at a certain stage of

the disease, phytol was found to be valid as a potential

therapeutic agent.

Study conducted by Sahalan (20070 on their study

Antibacterial Activity of Androgaphispaniculata and

Euophorbiahirta Methanol Extracts. Androgaphispaniculata and

Euphorbia hirta were screened for antibacterial activities

against three Gram positives and Gram negatives. The leaves

from both plants were extracted by methanol extraction. The

antibacterial activity was detected with spread plate

diffusion method.

According to Kumar, (2008) on their study Antibacterial

Evaluation of Snake Weed (Euphorbia hirta), ethanol,

methanol, chloroform and Aqueous extracts of leaf, stem, root

and whole plant of Euphorbia hirta were used to evaluate

antibacterial activity. Among these extracts ethanol and

methanol extracts of leaf and whole plant were more effective

and significant than aqueous and chloroform extracts in

inhibiting the growth of the pathogenic bacteria under the


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study, but were less potent when compared to that of

tetracycline used as positive control.

Study conducted by Bhagwat, (2009) on her study

Pharmacognostic study of plant Euphorbia hirta is a folklore

medicine in India used as antiasthmatic, anthelmetic, wound

healing, antibacterial, antioxidants, and diuretics. Powdered

leaves of Euphorbia hirta were subjected to soxhelate

extraction with petroleum ether, chloroform, acetone, and

finally macerated with water so as to get respective extracts.

The extracts were screened for its physiochemical

characteristics, preliminary phytochemical study of extracts

and quantitative estimation of total phenolic and flavonoid

content.

According to Geetha, (2009) on their study, In Vitro

responses of Encapsulated shoot tips of SpilanthesAcmella.

Multiple shoots were achieved through axillary bud explant of

Spilanthesacmella on MS medium supplemented with 0.5 mg/ 1 BA.

In vitro developed axillary buds and tiny shoots isolated from

the multiple shoots were encapsulated in different strength of

sodium alginate. The present work explains the role of

components of culture medium on morphogenic response of

encapsulated shoot tips to various types of growth regulators

in different concentrations, MS strength on storage time and


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investigated in canine infected incised wounds. The gross

appearance and histopathological reactions of the lesions were

evaluated at 72h after treatment. It was concluded that

infected wound would benefit from treatment with ethanolic

extract of E. hirta and its use in surgical site preparation

is thus recommended.

Study conducted by V. Goetting, (2011) on their study,

Pharmacokinetics of veterinary drugs in laying hens and

residues in eggs. Drug residues in chicken eggs are of

concern because relatively few drugs are labeled for laying

hens, although several medications are approved for other

production classes of poultry (Hofacre, 2006; Castanon, 2007).

Drug residues in eggs may arise when laying hens are

mistakenly given medicated feed, when is feed is contaminated

at the mill during mixing, or when drugs are given off-label

(Kennedy et al, 2000; Donoghue, 2003). While a chicken lays an

egg roughly every 24 hours, each egg takes several days to

develop in vivo, and some egg components are in existence

months before the fully developed and shelled egg containing

them is laid (Etches, 1996; Whittow, 2000). Because of the

protracted nature of egg development, many weeks may be

required following treatment or exposure before eggs are free

of drug residues.


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Jiang et al (2005) investigated the antiviral activity of

four extracts (petroleum ether, ethyl acetate, ethyl ether and

coumane of A. philoxeroides. Their results indicated that all

extracts possess anti-dengue activity but highest inhibition

of dengue virus was observed with petroleum ether extract.


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Methodology

Research Method

The true experimental and analysis type of research

method was used in this study.

Research Locale

The experimental method was conducted at the College of

Arts and Sciences Chemistry Laboratory of Nueva Ecija

University of Science and Technology while the rotary

evaporation and gas chromatography were done at the De La

Salle University, Taft Avenue, Manila. The taxonomical

identification of the plant species was administered at the

National Museum of the Philippines, Taft Avenue, Manila.

Procedural Steps

The study was divided into two phases and the following

procedures were undertaken:

1.

Collection of the Samples

The researchers collected samples of Tawa-tawa (Euphorbia

hirta Linn.) and Alligator Weeds (Alternanthera

philoxeroides) at the researchers town.

2.

Preparation of the Sample

The leaves were separated from the other parts of the

plant. Leaves were washed using water to free it from dirt.


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The leaves were cut into smaller pieces and were air dried

for two days.

3.

Crude Extraction

The researchers used 95% ethanol to extract the

organic substances in the leaves by mixing the dried leaves

with it in glass containers covered with foil. Then, the

mixtures, the submerged leaves in ethanol, were stirred

every five hours so that the leaves in the bottom will be

on the top. After two days, the substances was filtered

with the use of cheesecloth and poured in amber bottles.

4.

Rotary Evaporation

The crude extracts were then machine processed, namely

Rotary Evaporation, in order to remove the impurities of

the crudes.

5.

Gas Chromatography

Gas chromatography (GC) was used in analytical

chemistry for separating and analyzing compounds that can

be vaporized without decomposition. This was done at the

De La Salle University, Taft Avenue, Manila. This is with

the assistance and supervision of the Laboratory

Technician of the facility.


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Flowchart of the Activity

Preparation of

Tawa-tawa leaves

Rotary Evaporation

Collection of

Samples

Gas Chromatography

Comparison of the Data

Preparation of Alligator

Weeds (leaves)


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Presentation, Analysis and Interpretation of Data

Gas Chromatography Results

Tawa-tawa leaves extract (Euphorbia hirta L.)

Fragmentation pattern (mass spectral data) of each peak on the

chromatogram.

Comparison from library ( library match )

, 06-Mar-2014 + 12:46:33

7.50 12.50 17.50 22.50 27.50 32.50 37.50 42.50 47.50Time0

100

%

tawa tawa 1 Scan EI+TIC

6.17e78.34

7.01

5.17

3.38

16.78

15.24

10.97

9.48

13.63

18.50 19.73 21.73

24.78 27.75

, 06-Mar-2014 + 12:46:33

4.74 6.74 8.74 10.74 12.74 14.74 16.74 18.74 20.74 22.74 24.74 26.74 28.74Time4

100

%


5.99e78.34

7.01

5.17

3.38

5.61

16.78

15.24

10.97

9.48

13.6313.10

11.83 14.21

16.04

18.5019.73

21.7324.78

23.17 27.7525.8929.13

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55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%

tawa tawa 1 808 (7.040) Scan EI+4.46e6

100.9017

54.9340

83.8836

143.9802

114.8857144.9877

486.7261420.2899240.5111175.5415 225.6131

313.3270275.6084 371.0624356.1564

457.2353


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Name: 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-

Formula: C6H8O4

MW: 144 CAS#: 28564-83-2 NIST#: 4159 ID#: 1856 DB:replib

Other DBs: RTECS

Contributor: UNILEVER RESEARCH, DUIVEN, HELHOEK 30, GROESSEN,

HOLLAND

10 largest peaks:

43 999 | 44 755 | 144 372 | 101 313 | 45

253 |

55 252 | 72 197 | 73 143 | 42 90 | 41 45 |

Synonyms:

1.3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one #

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methHeadtoTailMF=546RMF=920

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

55

61

72

72

84

85

101

101

115

115130

144

144

165 180 194 209 223 237 251 266 281 295 310 324 339 353 368 382 397 411 426 440 455 469 484 498

MW:144(replib)4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

55 72

85

101

115 130

144

OHO OH

O


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Name: 2-Furancarboxaldehyde, 5-(hydroxymethyl)-

Formula: C6H6O3


Other DBs: Fine, TSCA, RTECS, HODOC, NIH, EINECS

Contributor: D.HENNEBERG, MAX-PLANCK INSTITUTE, MULHEIM, WEST

GERMANY

10 largest peaks:

97 999 | 126 710 | 41 668 | 39 336 | 69

289 |

29 173 | 125 136 | 53 131 | 109 131 | 38

100 |

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100

%


96.8667

68.8746

80.8556

125.9078

108.9029 126.9829150.8311

281.3128245.0743215.0095200.5126

417.6703375.0914353.6722

309.2326 482.9643469.6631

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 2-Furancarboxaldehyde,5-(hydroxymethyl)-HeadtoTailMF=756RMF=930

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

53

53

69

69

81

81

97

97

109

109

126

126

148 162 177 191 206 220 235 249 264 278 293 307 322 336 352 366 380 394 409 424 439 453 467 481 496

MW:126(replib)2-Furancarboxaldehyde,5-(hydroxymethyl)-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

5369

81

97

109

126

OH

OO


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Synonyms:

1.2-Furaldehyde, 5-(hydroxymethyl)-

2.5-Hydrxoymethylfurfural

3.Hydroxymethylfurfurole

4.HMF

5.5-(Hyddroxymethyl)Furfurole

6.5-(Hydroxymethyl)-2-formylfuran

7.5-(Hydroxymethyl)-2-furaldehyde

8.5-(Hydroxymethyl)-2-furancarbonal

9.5-(Hydroxymethyl)-2-furfural

10.5-(Hydroxymethyl)-2-furfuraldehyde


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Name: 1-Butene, 4-isothiocyanato-1-(methylthio)-

Formula: C6H9NS2

MW: 159 CAS#: 51598-96-0 NIST#: 5649 ID#: 48848 DB:mainlib

Other DBs: None

Contributor: D.H.WILLIAMS CHEMISTRY DEPT., UNIVERSITY OF

CAMBRIDGE

10 largest peaks:

87 999 | 45 950 | 72 400 | 39 340 | 27

330 |

159 270 | 53 200 | 28 190 | 47 190 | 59

190 |

Synonyms:

1.4-Methylthio-3-butenyl isothiocyanate

2.(1E)-4-Isothiocyanato-1-(methylsulfanyl)-1-butene #

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100

%


86.9113

71.8368

158.8899

125.840696.8667181.8520

253.1272209.8418 464.6248270.1054

428.0816376.0315321.65023 54. 74 65 4 13 .6 40 3

487.1292

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 1-Butene,4-isothiocyanato-1-(methylthio)-HeadtoTailMF=532RMF=894

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

53

53

72

72

87

87

97

98 112

126 152

159

159

182 196 210 225 239 254 268 283 297 312 326 341 355 370 384 399 413 428 442 457 471 486

MW:159(mainlib)1-Butene,4-isothiocyanato-1-(methylthio)-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

53

72

87

98 111

159 N

SC

S


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Name: Ethyl iso-allocholate

Formula: C26H44O5

MW: 436 NIST#: 43053 ID#: 6556 DB:mainlib

Contributor: R RYHAGE MS-LAB KAROLINSKA INSTITUTET STOCKHOLM

SWEDEN

10 largest peaks:

43 999 | 55 914 | 41 867 | 57 797 | 69

609 |

81 547 | 44 492 | 29 476 | 17 469 | 83

460 |

Synonyms:

no synonyms.

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100

%


59.9186

80.8556125.8406

94.8490

107.8945

133.0301167.0148

181.4493206.0162

248.9666 329.7726302.1852265.0052

438.7620416.7299358.0364 402.5582

479.9413

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . Ethyliso-allocholateHeadtoTailMF=666RMF=781

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100 55

60

69

81

81

95

95107

108

121

126

133

145159

167181

185

206 221

235

249

253

265

272

279

287

293

313

330

339

358

367382

394

400

417

418

439453 480

MW:436(mainlib)Ethyliso-allocholate

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100 55

6981 95 107

121145

159 185 213227 253 272

287 301 316 339 367382 400

418

HO OH

OH O

O


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Name: 7,8-Epoxylanostan-11-ol, 3-acetoxy-

Formula: C32H54O4


Contributor: Chemical Concepts

10 largest peaks:

44 999 | 43 978 | 57 781 | 69 751 | 55

702 |

95 623 | 83 522 | 41 501 | 81 499 | 71

465 |

Synonyms:

no synonyms.

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100

%


92.831154.9340

82.9416

108.0289

109.0374134.9785

138.0688

151.0326177.0856

209.9760

260.0392225.5459

318.0927281.1786

354.2765 475.9778448.1664 489.4132

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 7,8-Epoxylanostan-11-ol,3-acetoxy-HeadtoTailMF=738RMF=760

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

57

69

69

83

83

93

95

108

109123

135

149

150167

173 191

210226

227

241

244

260

269

281

287

295

304

318

3 20 3 44

354

362 377

382

393

398

409 424

427 441

442 466

476

484 502

MW:502(mainlib)7,8-Epoxylanostan-11-ol,3-acetoxy-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

57 6983

95109

123149

173 191 219

244

269 287304

320 344358 372 393 409 424 442457 484 502

OO

O

HO


31/60

31 | P a g e

Name: 2-Cyclohexen-1-one, 4-hydroxy-3,5,5-trimethyl-4-(3-oxo-

1-butenyl)-

Formula: C13H18O3


Other DBs: None


10 largest peaks:

124 999 | 43 408 | 166 186 | 41 105 | 125 93

|

69 88 | 95 82 | 39 80 | 55 79 | 123 68 |

Synonyms:

1.4-Hydroxy-3,5,5-trimethyl-4-[(1E)-3-oxo-1-butenyl]-2-

cyclohexen-1-one #

, 06-Mar-2014 + 12:46:33

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


123.9590

54.9340 78.9041

110.8525 125.0342

150.0251181.9863206.0162

398.5957296.8159231.7200 317.7570 348. 6368

4 25 .7 97 8 4 77 .4 55 7461.4675

MW:222(replib)2-Cyclohexen-1-one,4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl)-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

55 69 95 109

124

149166

180 204 222

O

OHO

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 2-Cyclohexen-1-one,4-hydroxy-3,5,5-trimethyl-4-(3HeadtoTailMF=573RMF=750

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

55 69

79 95

95 109

111

124

124

135

149

150166

166180

182196

204

210

222

225 239 254 268 283 297 312 326 341 355 370 384 399 413 428 442 457 471 486


32/60

32 | P a g e

Name:Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester

Formula: C35H68O5


Other DBs: HODOC

Contributor: R.T.HOLMAN,UNIVERSITY OF MINNESOTA

10 largest peaks:

43 999 | 57 952 | 55 801 | 41 641 | 98

613 |

73 603 | 60 500 | 71 490 | 69 481 | 239

433 |

Synonyms:

1.Palmitin, 1,2-di-

2.Dipalmitin

3.Glycerol 1,2-dipalmitate

, 06-Mar-2014 + 12:46:33

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


72.9138

59.9186

96.8667 128.9987

114.8857 256.0799212.9962184.9400171.0433

227.0224284.2657

305.9438385.8358

371.3982478.9337412.7671435.3362

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . Hexadecanoicacid,1-(hydroxymethyl)-1,2-ethane HeadtoTailMF=693RMF=775

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

10057

6073

73

83

83

97

98

115129

129

143

143

157

157

171

171

185

185

213

213

227

239

241

256

256270

276291 306

313

320

331

334 349

354

377

382 401

405

415

419 434

437

448

451

463 479494

507

MW:568(mainlib)Hexadecanoicacid,1-(hydroxymethyl)-1,2-ethanediylester

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100 57

73

83

98

129

157171 185 213

239

256270 285

3133 31 3 54368 382 401 415 437 451 507

OOHO O

O


33/60

33 | P a g e

4.1,2-Dipalmitin

5.1,2-Dipalmitoylglycerol

6.2-Hydroxy-1-[(palmitoyloxy)methyl]ethyl palmitate #


34/60

34 | P a g e

Name: 7,8-Epoxylanostan-11-ol, 3-acetoxy-

Formula: C32H54O4



10 largest peaks:

44 999 | 43 978 | 57 781 | 69 751 | 55

702 |

95 623 | 83 522 | 41 501 | 81 499 | 71

465 |

Synonyms:

no synonyms.

, 0 6-Mar-2014 + 12:46:33

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


66.922196.9339

82.8743

109.1718

125.9750166.8133

150.2938

182.0534

263.9315222.2576 282.3195

434.2614310.8435

397.2525 451.6596 477.1198

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 7,8-Epoxylanostan-11-ol,3-acetoxy-HeadtoTailMF=744RMF=769

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

57

67

69

83

8395

97

109

109123

126149

149

167

173

182

191

222

227

241

244

264

269

282

287304

320

332

344

347 361

362

376

377 393

397

409 424

434

442

452

466

471

484 502

MW:502(mainlib)7,8-Epoxylanostan-11-ol,3-acetoxy-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

10057 69

8395

109123

149173 191 219

244

269 287304

320 344358 372 393 409 424 442457 484 502

OO

O

HO


35/60


36/60

36 | P a g e

2.Stigmast-5-en-3-ol

3.-Dihydrofucosterol

4.-Sitosterin

5.Angelicin

6.Angelicin (steroid)

7.Cinchol

8.Cupreol

9.Quebrachol

10.Rhamnol


37/60

37 | P a g e

Name:Trilinolein

Formula: C57H98O6


Other DBs: Fine, EINECS

Contributor: Japan AIST/NIMC Database- Spectrum MS-IW-5614

10 largest peaks:

67 999 | 55 839 | 81 829 | 95 610 | 41

528 |

69 448 | 79 409 | 82 385 | 43 350 | 68

324 |

Synonyms:

, 06-Mar-2014 + 12:46:33

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


56.8876

95.0508

148.9505125.6390 166.8805

207.0901181.9192

238.4978 470.1334252.1877

299.0307 334. 0689 376. 3672 426. 4695 484.7108

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . i TrilinoleinHeadtoTailMF=645RMF=746

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

5567

69

81

81

95

95

109123

1 26 1 49

149

159

163

167

177

182

201

207

215 234

238252

262

266

280

290

305

306

321

334

337351

355

365

376390

391405

412426

427

440

447 463

470485

487

MW:878(mainlib)Trilinolein

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

10055

6781

95

109123

149 163 177191 215 234

262280

295 321337

351365 391405 427 445 460 475 491

C57H98O6


38/60

38 | P a g e

1.9,12-Octadecadienoic acid (Z,Z)-, 1,2,3-propanetriyl ester

2.2,3-Bis[(9Z,12Z)-9,12-octadecadienoyloxy]propyl (9Z,12Z)-

9,12-octadecadienoate #


39/60


40/60

40 | P a g e

Alligator weeds (Alternanthera philoxeroides)

Fragmentation pattern (mass spectral data ) of each peak on

the chromatogram..

Comparison from library ( library match )

, 05-Mar-2014 + 18:40:01

7.50 12.50 17.50 22.50 27.50 32.50 37.50 42.50 47.50Time0

100

%


2.98e818.15

18.02

16.323.13

15.4114.11 21.73

, 05-Mar-2014 + 18:40:01

12.85 13.85 14.85 15.85 16.85 17.85 18.85 19.85 20.85 21.85 22.85 23.85 24.85Time0

100

%


2.12e8

18.02

16.32

15.4114.11

15.85

16.79 17.96

21.73

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


86.9113

72.8465

59.9186

102.0448 115.9612

167.0819 181.9863

434.5301369.5852318.0255224.2709 305.7425

253.0601 393.6931 469.8647 485.7185

C:\TurboMass\DEFAULT.PRO\Data\tawa : . i 3-O-Methyl-d-glucoseHeadtoTailMF=646RMF=816

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

57

60

73

73

87

87

102

103

126 144

145 163

167

194

199 216 232 248 264 280 296 312 328 345 361 377 394 410 426 442 459 476 492


41/60

41 | P a g e

Name: 3-O-Methyl-d-glucose

Formula: C7H14O6


Contributor: LAC, NIDDK, NIH, Bethesda, MD 20892

10 largest peaks:

73 999 | 74 900 | 87 743 | 43 411 | 45

363 |

42 298 | 71 290 | 57 262 | 75 259 | 59

239 |

Synonyms:

no synonyms.

MW:194(mainlib)3-O-Methyl-d-glucose

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

57

73

87

103 145 163 194

HOOH

OHO OH

O


42/60

42 | P a g e

Name: 3,7,11,15-Tetramethyl-2-hexadecen-1-ol

Formula: C20H40O


Other DBs: IRDB

Contributor: NIST Mass Spectrometry Data Center, 1990.

10 largest peaks:

81 999 | 82 986 | 43 965 | 95 962 | 123

892 |

55 852 | 41 811 | 57 811 | 71 748 | 68

728 |

Synonyms:

1.(2E)-3,7,11,15-Tetramethyl-2-hexadecen-1-ol #

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


94.983567.9994

81.9996

123.0854

109.0374 124.0262

125.1014

181.7178234.73982 08 .1 63 9 2 62 .85 77

277.5546477.5901320.1064 356.2907

377.1059448.9726

436.3438

C:\TurboMass\DEFAULT.PRO\Data\tawa : . i 3,7,11,15-Tetramethyl-2-hexadecen-1-olHeadtoTailMF=716RMF=905

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100 55

57

68

71

81

82

95

95

109

123

123

137

137 179

182198 214 235 253 269

278

285 301 318 334 351 367 383 399 415 431 447 463 479 496

MW:296(mainlib)3,7,11,15-Tetramethyl-2-hexadecen-1-ol

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100 5571

81 95

109

123

137 179 278OH


43/60

43 | P a g e

Name: 5,10-Pentadecadien-1-ol, (Z,Z)-

Formula: C15H28O


Other DBs: None

Contributor: HCACAV,60(4)1161(1977);13; G. OHLOFF

10 largest peaks:

67 999 | 55 930 | 41 880 | 81 760 | 95

440 |

29 280 | 110 190 | 135 100 | 121 80 | 149 30 |

Synonyms:

1.(5Z,10Z)-5,10-Pentadecadien-1-ol #

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


80.9229

57.0223

94.9835

122.9510

108.9029

124.0262

137.1283 149.2192 208.0968182.0534 278.2257242.1888 316.2132 402.0209387.5818365.0864 423.7827

494.1827473.3579

C:\TurboMass\DEFAULT.PRO\Data\tawa : . i 5,10-Pentadecadien-1-ol,(Z,Z)-HeadtoTailMF=115RMF=921

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100 55

57

67

73

81

81

95

95

110

123

135

137

163

167

192

208 224

224

242 258 278 300316 337 357 402 4 22 4 53 473 4 94

MW:224(mainlib)5,10-Pentadecadien-1-ol,(Z,Z)-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100 556781

95

110135

163 192 224

OH


44/60

44 | P a g e

Name:Hexadecanoic acid, methyl ester

Formula: C17H34O2


Other DBs: Fine, TSCA, EPA, HODOC, NIH, EINECS

Contributor: O A MAMER, MCGILL UNIVERSITY, MONTREAL, CANADA

10 largest peaks:

74 999 | 87 575 | 43 368 | 41 304 | 55

251 |

75 183 | 29 177 | 57 159 | 69 127 | 143

102 |

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


73.9235

86.8440

143.039897.0012

128.9987 227.0224171.1104

185.0742 270.1725

366.6979295.2051 338.7680 453.4734410.3492

C:\T urb oM ass\DEFA UL T.PRO\Data\tawa i Hexadecanoicacid,methylesterHeadtoTailMF=890RMF=914

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

55

74

74

87

87

97

97

111

111

143

143

171

171

199

199

227

227

254270

270

295 339 367 392 409 445 466 482

MW:270(replib)Hexadecanoicacid,methylester

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

55

74

87

97111 143

167 185 227 270

O

O


45/60

45 | P a g e

Synonyms:

1.Palmitic acid, methyl ester

2.n-Hexadecanoic acid methyl ester

3.Metholene 2216

4.Methylhexadecanoate

5.Methyl n-hexadecanoate

6.Methylpalmitate

7.Uniphat A60

8.Emery 2216

9.Radia 7120


46/60

46 | P a g e

Name: n-Hexadecanoic acid

Formula: C16H32O2


Other DBs: Fine, TSCA, RTECS, EPA, HODOC, NIH, EINECS, IRDB


10 largest peaks:

43 999 | 73 905 | 60 838 | 41 749 | 57

634 |

55 616 | 29 414 | 69 310 | 71 285 | 61

218 |

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


72.9138

96.8667

83.0089128.9987

114.9530

256.0799

213.0633171.0433

157.1439

199.0360 227.0895 257.2207

432.7836316.3475 383.6197365.2207

496.2650467.5135

C:\T urboMass\D EFAU LT .PR O\D ata\tawa n-HexadecanoicacidHeadtoTailMF=783RMF=869

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

60

60

73

73

83

83

97

97

115

129

129157

171

185

213

213 239

256

256

281 297 314 330 347 363 379 395 411 429 445 461 477 494

MW:256(mainlib)n-Hexadecanoicacid

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

10060 73

83 97 129

137 157 185 213

239256

OH

O


47/60

47 | P a g e

Synonyms:

1.Hexadecanoic acid

2.n-Hexadecoic acid

3.Palmitic acid

4.Pentadecanecarboxylic acid

5.1-Pentadecanecarboxylic acid

6.Cetylic acid

7.Emersol 140

8.Emersol 143

9.Hexadecylic acid

10.Hydrofol


48/60

48 | P a g e

Name: 7,10-Octadecadienoic acid, methyl ester

Formula: C19H34O2


Other DBs: None

Contributor: R.T.HOLMAN,UNIVERSITY OF MINNESOTA

10 largest peaks:

67 999 | 81 900 | 41 720 | 55 700 | 95

670 |

43 630 | 82 540 | 79 480 | 96 450 | 68

430 |

Synonyms:

1.Methyl (7E,10E)-7,10-octadecadienoate #

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


66.9894 80.8556

94.9835

95.9924

109.0374

123.0182136.1206

293.9970164.0604262.1195220.1771 455.1528400.6777322.5900 357.2307 432.6492 493.0406

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 7,10-Octadecadienoicacid,methylesterHeadtoTailMF=795RMF=874

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

55

67

67

81

81

95

95

109

109

121

135

136

150

164

164 195

206 220

220

235 262

263

276 294

294

309 323 339 355 370 385 401 417 431 446 461 476 491

MW:294(mainlib)7,10-Octadecadienoicacid,methylester

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100

55

6781

95

109135 150

164194 220 235

263

294

O

O


49/60

49 | P a g e

Name: 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-

Formula: C19H32O2


Other DBs: Fine, TSCA, HODOC, EINECS

Contributor: SERL

10 largest peaks:

79 999 | 67 780 | 41 610 | 55 570 | 93

470 |

81 430 | 80 420 | 95 400 | 43 310 | 108

270 |

Synonyms:

1.Linolenic acid, methyl ester

2.Methyl all-cis-9,12,15-octadecatrienoate

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


78.9714

66.9894 94.9835

107.9617

121.0692

135.0457 149.0849

292.1179173.1246

236.0149222.1904 261.1129 427.2755350.3824304.8699

3 93 .1 55 8 4 64 .2 21 7 4 88 .4 05 6

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . 9,12,15-Octadecatrienoicacid,methylester,(Z,Z,HeadtoTailMF=863RMF=928

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480

0

50

100

50

100

55

55

67

67

79

79

93

95108

108

121135

135

149

149

163

163

191

191

205 219 236

236

261276 292

292

307 321 341 358 373 387 403 421 435 452 466 481 495

MW:292(replib)9,12,15-Octadecatrienoicacid,methylester,(Z,Z,Z)-

60 90 120 150 180 210 240 270 300 330 360 390 420 450 4800

50

100

55

67

79

93

108

135 149 163 191 236 292

O

O


50/60

50 | P a g e

3.Methyllinolenate

4.Methyl (9Z,12Z,15Z)-9,12,15-octadecatrienoate #


51/60

51 | P a g e

Name: Phytol

Formula: C20H40O


Other DBs: Fine, TSCA, RTECS, HODOC, EINECS

Contributor: Philip Morris R&D

10 largest peaks:

71 999 | 43 381 | 57 334 | 41 260 | 55

259 |

69 239 | 81 223 | 68 199 | 123 184 | 56

169 |

Synonyms:

1.2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]]-

2.trans-Phytol

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


70.9616

123.0182

80.9902 94.9835

124.0934

196.0827151.0997

278.2257249.0336222.2576 296.1448

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . i PhytolHeadtoTailMF=920RMF=931

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310

0

50

100

50

100

57

57

71

71

81

81

95

95

111

111

123

123

137

137

147

151

157 165 179

179

193

196

207 222 236 249 263 278

278

296

MW:296(replib)Phytol

5 0 6 0 7 0 8 0 9 0 1 00 1 10 1 20 1 30 1 40 1 50 1 6 0 1 70 1 80 1 90 2 00 2 1 0 2 20 2 30 2 40 2 50 2 60 2 70 2 8 0 2 90 3 00 3 1 00

50

100

57

71

8195 111

123

137 151 179 196 278

HO


52/60

52 | P a g e

3.3,7,11,15-Tetramethyl-2-hexadecen-1-ol

4.(2E)-3,7,11,15-Tetramethyl-2-hexadecen-1-ol #


53/60


54/60


55/60

55 | P a g e

Name: Ethyl iso-allocholate

Formula: C26H44O5


Contributor: R RYHAGE MS-LAB KAROLINSKA INSTITUTET STOCKHOLM

SWEDEN

10 largest peaks:

43 999 | 55 914 | 41 867 | 57 797 | 69

609 |

81 547 | 44 492 | 29 476 | 17 469 | 83

460 |

Synonyms:

no synonyms

, 05-Mar-2014 + 18:40:01

55 75 95 115 135 155 175 195 215 235 255 275 295 315 335 355 375 395 415 435 455 475 495m/z0

100

%


69.0093

80.9902

94.9835

109.0374 123.0854

206.8216146.9356 191.3170428.2831281.0443255.0733

218.4322306.8835 370.3239

327.4902393 .357 2 45 0. 7192

465.2294

MW:436(mainlib)Ethyliso-allocholate

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 5100

50

100 55

69 8195 107

121145

185199 229 253 272 289 304 319 339 367

382 400418

HO OH

OHO

O

C:\TurboMass\DEFAULT.PRO\Data\tawatawa : . i Ethyliso-allocholateHeadtoTailMF=681RMF=776

60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510

0

50

100

50

100

55

55

69

69

81

81

95

95107

109

121

137

145

161

171

176191

197

207

213227

232 249

253

264

272

281 299

311

327

339

341

367

370

382

393

400

414

418

444 459 474 489


56/60

56 | P a g e

Summary of Findings

Based on the results of the study, the following findings are

derived:

1.

The tawa-tawa leaves extract contains 4H-Pyran-4-one,

2,3-dihydro-3,5-dihydroxy-6-methyl-, -

Furancarboxaldehyde, 5-(hydroxymethyl)-, 1-Butene, 4-

isothiocyanato-1-(methylthio)-, Ethyl iso-allocholate,

7,8-Epoxylanostan-11-ol, 3-acetoxy-, 2-Cyclohexen-1-one,

4-hydroxy-3,5,5-trimethyl-4-(3-oxo-1-butenyl)-,

Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl

ester, 7,8-Epoxylanostan-11-ol, 3-acetoxy, -Sitosterol,

Trilinolein, and 7,8-Epoxylanostan-11-ol, 3-acetoxy-.

2.The alligator weeds extract (leaves) contains 3-O-Methyl-

d-glucose, 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, 5,10-

Pentadecadien-1-ol, (Z,Z)-, Hexadecanoic acid, methyl

ester, n-Hexadecanoic acid, 7,10-Octadecadienoic acid,

methyl ester, 9,12,15-Octadecatrienoic acid, methyl

ester, (Z,Z,Z)-, Phytol, 1,2-Benzenedicarboxylic acid,

diisooctyl ester, and Ethyl iso-allocholate.

3.

The presence of phytol in the Alligator weeds extract was

positively determined.


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Conclusions

With the data gathered, the researchers can therefore

conclude:

1.

Alligator weeds extract contained the substance phytol.

2.

The components of Tawa-tawa leaves extract and Alligator

weeds extract (leaves) were characterized using Gas

Chromatography.

Recommendations

1.

Further study about Alligator weeds extracts capability

of serving as an anti-dengue medication.

2.MTT assay must be conducted to determine the cytotoxicity

of both plant species.

3.

The use of petroleum ether as a solvent must be taken

into consideration.


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Appendix A

Figure 1 Collection of the Samples

Figure 2 Preparation of the Sample

Figure 3 Crude extraction


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Figure 4 Rotary Evaporation

Figure 5 Gas Chromatography


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APPENDIX B

manuscript thesis

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