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Cytotoxic activity of extracts of Ixora species and their GC-MS analysis.

Byline: Lubna, Rahat Azher Ali, Faheema Siddiqui, Perveen Fazil, Ahsana Dar Farooq, Amna Jabbar Siddiqui and Syed Ghulam Musharraf

Summary: The current study examined the cytotoxic activity of chloroform flowers extracts of five Ixora species Linn. (Rubiaceae). An in-vitro growth inhibition and cytotoxic effect were demonstrated against three different human cancer cell lines named as, uterine cervical (HeLa), lung (NCI H-460) and breast (MCF-7) cancer cell lines. Among different colored flowers extracts of Ixora species the yellow colored flowers of I. coccinea was more potent against aforementioned human cancer cell lines (GI50: ~15 +- 3.8 ug/ml with LC50: 230 +- 3.9ug/ml). GC-MS technique was used for the identification of chemical constituents present in the extract, which may be responsible for their anticancer effect.

Keywords: Ixora coccinea Linn., GC-MS, Phytochemicals, Cytotoxic, Mass spectrometry, Human cancer cell lines

Introduction

Cancer is one of the most dangerous diseases spread in whole World, in which the cell growth is abnormal, as a result of a solid mass of cells called as cancer or tumor. Among the most common kinds of cancers, named as cervical, stomach, breast, lung and liver cancers, lung cancer has recognized to be the major widespread diseases found in men, whereas, cervical cancer is the third commonly found cancer among women with 86 % global burden occurring in developing regions [1]. National Center for Health Statistics provided the mortality data. In United States, 1,688,780 new cancer patients and 600,920 cancer deaths were reported in 2017. Including all kinds of cancers, the cancer incidence rate is 20 % higher in men than in women, while the cancer death is 40 % higher [2]. Conventional treatments of cancer consist of intrusions like psychosocial support, radiotherapy, surgery and chemotherapy [3, 4].

However, due to the increasing rate of mortality associated with cancer and adverse or toxic side effects of cancer chemotherapy, and radiation therapy, discovery of new anticancer agents derived from nature, especially plants, is currently under investigation. Numerous naturally occurring compounds, having miscellaneous chemical skeleton, have been isolated as anticancer drug. A number of possible lead compounds such as combretastatins, vincristine, camptothecin, taxol, vinblastine, podophyllotoxin, etc. were isolated and identified as plant metabolites and several of them have been modified to products with enhanced solubility, activity or toxicity. More than a few flourishing molecules also have appeared as drugs upon alteration of these naturally occurring compounds and several are forthcoming [5].

Ixora genus is a flowering plant belongs to family Rubiaceae. It consists of tropical evergreen trees and shrubs and holds around 500 species with its center of diversity in tropical Asia. Ixora's plant, not only used for ornamental purpose but also used for medicinal principle. This plant blooms throughout the year and easy to grow. They are different from each other as size of leaf, height of plant, color and size of flowers. The flowers appear in clusters, it comes in a variety of brilliant colors like red, orange, yellow, white and pink [6-8]. Ixora coccinea Linn. a time-honored medicinal plant usually called as Jungle geranium and flame of the woods or Vethi in Ayurvedha [9-11]. It was distributed in the tropical and subtropical regions all over the world [4]. It has been reported in literature to having several biological activities, for example, antimicrobial, anti-bacterial, anti-fungal, antioxidant, and anti-inflammatory, activities.

This plant showed hepatoprotective, cardioprotective, leucorrhoea, dysentery, dysmenorrhoea, hemoptysis, hypertension, anti-ulcer, antihelmintic, antiasthematic, hypolipidemic and hypoglycemic properties [10-16]. Hexane, chloroform, ethyl acetate, methanol and aqueous extracts of the plants were evaluated for cytotoxic potential [17, 18]. The phytochemical analysis of the plant resulted in the isolation of variety of class of compounds, such as, alkaloids, flavonoids, tannins, resins, steroids, carbohydrates, terpenoids and saponins [4, 7, 19]. I. chinensis identified by it's almost stalk less leaves and pink color flowers. It is widespread in Southeast Asian gardens. The plant has been reported for several medicinal purposes like stomach problem, tuberculosis, hemorrhage, tumors and headache. Different compounds were identified from this specie namely, fatty acids, flavonoids, and steroids. I. fulgens is a shrub 1.5-3m tall, herbaceous, having red color flowers.

It is found in abandoned fields, but it is less common at higher elevations. Leaves, roots and flowers were reported as a rich source of phytochemicals. I. polyantha is an ornamental shrub cultivated in gardens for white clusters of flowers and evergreen foliage. It is found throughout the greater part of Asia and has been reported to possess many medicinal properties [6]. The present study was the first of its kind to investigate comparative cytotoxic activity of flowers of five different species of Ixora genus. Chloroform extracts of flowers of these species were examined against NCI H-460, HeLa, and MCF-7 human cancer cell lines and identification of their flowers metabolites in chloroform extracts of following species, I. coccinea having yellow and orange color flowers, I. fulgens, I. chinensis and I. polyantha were also performed.

Experimental

Collection of plant material

I. coccinea (with yellow color flowers), voucher specimen (KUH GH 91566); I. coccinea (having orange color flowers), voucher specimen (KUH GH 91565); I. fulgens (red color flowers), voucher specimen (KUH GH 91567); I. chinensis (having pink flowers) voucher specimen (KUH GH 91566); I. polyantha (with white flowers) voucher specimen (KUH GH 91562), were collected in May, 2015 from University of Karachi. They were identified by Dr. Anjum Perveen, Botany department, University of Karachi and the voucher specimen have been deposited in the herbarium of the same department.

Extraction method

Dried, crushed flowers of Ixora fulgens, I. chinensis, I. polyantha, and I. coccinea (orange and yellow) were subjected to extraction. Flowers were extracted by soxhlet extraction technique with chloroform. Extracts were collected, filtered and evaporated by rotary evaporator to give relevant extracts. Total five different kinds of flowers chloroform extracts were obtained and subjected to cytotoxic activity evaluation and GC-MS analysis.

GC-MS studies of flowers of Ixora species

The chloroform extracts were investigated through gas chromatographic flame ionization detection (GC-FID) and gas chromatographic mass spectrometric (GC-MS) methods. GC-FID spectra were run on a Shimadzu GC-17A [FID mode; column, fused silica capillary column DB-1, OV-1 (30 m x 0.53 mm, 0.5mm film thickness). Carrier gas was nitrogen, at 75 AdegC and programmed to 75 AdegC at 240 AdegC min-1 and 3-5 min hold. Injector and detector were at 240 and 250 AdegC respectively. 2 ml of each sample were triplicate injected and quantities represented as relative area % as derived from integrator. GC-MS system was operated on a JSM 600H (Jeol, Japan) with Agilent 6890N USA [EI mode; ionization potential, 70ev; column DS-6 (0.32 mm x 30 m); column temperature 50-250 AdegC (rate of temperature increases 5 AdegC/min); carrier gas, He; flow rate, 1.8 ml/min; split ratio, 1:30].

Sample injection was carried out with a split ratio of 35:1 at a temperature of 25 AdegC. Data processing including deconvolution of spectrum and peak integration were performed by using the Agilent Mass Hunter Qualitative Analysis (version B.04.00).

Chemicals used for cytotoxic activity

The chemicals such as dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), gentamycin sulphate, L-glutamine penicillin streptomycin solution (GPSS), Roswell Park Memorial Institute-1640 medium (RPMI-1640), trichloroacetic acid (TCA), sulforhodamine B (SRB), trisbase, trypsin-EDTA and trypan blue (Sigma Co St. Louis, Mo, USA), acetic acid (Lab scan, Ireland) and doxorubicin (ICN, USA) were purchased from respective suppliers.

Human cancer cell lines

Cancer cell lines, namely, NCI H-460 (large cell carcinoma, lung), MCF-7 (adenocarcinoma, breast) (Michigan Cancer Foundation-7), were kindly provided by the National Cancer Institute (NCI), Frederick, MD. The HeLa (uterine cervix) was obtained from ATCC (American Type Culture Collection). The cancer cell lines were preserved in culture medium containing L-glutamine (1 % w/v or 200 mM), serum (FBS, 10 % v/v), streptomycin (100ug/ml), penicillin (1 % w/v, 104 U/ml), and antifungal amphotericin-B (10 ug/ml) in culture flasks (75 ml). These were kept in the CO2 incubator (5 % CO2) at 37 AdegC till a confluent monolayer was formed within 3-4 days [20].

Determination of growth inhibition and cytotoxicity

Extracts growth inhibition and cytotoxicity were examined by using sulforhodamine-B assay [21, 22] against the subjected human cancer cell lines i.e. breast (MCF-7), lung (NCI H-460) and uterine cervix (HeLa) cancer cell lines.

Preparation of stock solutions

The stock solutions of flowers of chloroform extracts including I. chinensis, I. polyantha, I. coccinea yellow and orange color flowers, and I. fulgens, (40 mg/ml) were prepared in sterile DMSO (100%) with a final conc. of DMSO without exceeding 0.5 % (v/v). The stock solutions for doxorubicin (1 mM) was constituted in sterile distilled water and kept at -80 AdegC until further use. On the day of experiment, all the dilutions were prepared in RPMI-1640 containing gentamicin (50 ug/ml).

Sulforhodamine-B assay

All three cell lines were trypsinized and seeded in 96-well plate at a density of 10,000 cells/well/100 ul and incubated in CO2 incubator at 37 AdegC to allow monolayer formation (24 h) followed by the addition of various concentrations of all extract (10, 50, 100, 200, 250 ug/ml). Appropriate controls and blanks (drug and extract) were also prepared. The time zero-1 (Tz1 plate) and -2 (Tz2 plate) plates were fixed with gentle addition of cold TCA (50 % w/v, 50 ul /well) before and after the addition of test agents in experimental plates. These were left at room temperature (30 min) followed by washing with distilled water (3x) and drying overnight. Similarly, after 48 h of incubation, experimental plates were also fixed. All the plates (experimental, TZ1 and Tz2) were stained with sulforhodamine solution (100 ul, 0.4 % w/v in 1 % acetic acid) for 30 min. The unbound stain was removed by washing (5 x s) with acetic acid (1 %) and air-dried at room temperature.

The protein bound stain was solubilized in tris base solution (pH 10.2, 100 ul/well, and 10 mM) and absorbance was recorded at 515 nm. The absorbance values in the presence of the test agents were subtracted from blank values. If the absorbance value of the test well was greater than Tz plates, the percent growth was calculated as:

Cell growth (%) = [(T-Tz)/(C-Tz)] x 100

Tz and T represent the absorbance before and after the addition of test agents, respectively. Tz was calculated as the mean Tz1 and Tz2. Control is represented by C. However, if the absorbance value was less than Tz plates, the percent growth was calculated as:

Cell growth (%) = [(T-Tz)/Tz] x 100

The GI50 (growth inhibition of 50 % of cells) was obtained from dose-response curves prepared by plotting the percentage of cell growth versus the concentrations of test agents. All the experiments were repeated three times and conducted in triplicates as emphasized by the NCI, Frederick, USA laboratory.

Results and Discussion

Phytochemistry and pharmacology on medicinal plants named as, I. polyantha, I. fulgens, I. chinensis, and I. coccinea (yellow and orange) showed great medicinal importance in traditional and folk medicines [23]. In the current studies different colored flowers extracts of aforementioned species were extracted with chloroform to investigate their phytochemical effects against HeLa, MCF-7 and NCI H-460 cancer cell lines (Tables 1-3). Most of the tested samples showed promising cytotoxic activity, which were phytochemically studied through GC and GC-MS analysis. Among chloroform extracts of flowers of all subjected five kinds, I. coccinea (orange and yellow), the yellow colored flowers, I. fulgens, I. chinensis, and I. polyantha, of I. coccinea exhibited the most potent growth inhibition (GI50: ~15 +- 3.8 ug/ml) and cytotoxic effect LC50: 230 +- 3.9 ug/ml) against HeLa, NCI H-460 and MCF-7 cancer cell lines.

On the contrary, its orange colored flowers were displayed ~4x lowered growth inhibitory effect (GI50: ~60 +- 2.7ug/ ml). Likewise, I. fulgens flowers also displayed equipotent growth inhibitory effect (GI50: 10 +- 2.9ug/ml) to I. coccinea with variable cytotoxic effect (LC50: 165 +- 3.1 ug/ml) against cervix uterine cancer cell line however, their effect was ~4x lower against breast and lung cancer cell lines (~48 +- 2.2 ug/ml). Whereas, I. polyantha and I. chinensis, flowers extracts exhibited weak growth and cytotoxic effects against all the three tested human cancer cell lines. However, the reference drug doxorubicin was potent against all aforementioned cancerous cell lines. These outcomes are in agreement with earlier reported work that I. coccinea flowers showed cytotoxic activity, the flower extract was reported to contain terpenoids, flavonoids, phenols and tannins [18, 23-25]. Lupeol has been isolated from extract and showed cytotoxic activity [19, 11, 26, 27].

Isolation of kaempferol with anti-platelet aggregation potential has been reported in literature [28]. Moreover, Ixora peptide I, chemical constituents Identified from I. coccinea showed cytotoxicity against the Hep3B liver tumor cell line [28].

Table-1: Growth inhibitory and cytotoxic effects of chloroform extracts of flowers of Ixora against MCF cell lines (Breast cancer cell lines).

S. No.###Breast cancer cell line(MCF-7)

###Plant name###Doses###% Cell growth###(ug/ml)

###(Codes)###(ug/ml)###inhibition/ cytotoxicity###GI50###LC50

###10###+04 +- 1.2

###1###I. fulgens(C1)###50###+53 +-2.2***

###100###+71 +- 1.8***###48 +- 2.2c###>250

###200###+80 +- 1.5***

###250###+87+- 2.7***

###10###+01 +- 0.4

###2###I. polyantha(C2)###50###+09 +- 1.1

###100###+37 +- 2.5**###220 +- 4.3d###>250

###200###+43 +- 3.5***

###250###+55 +- 4.1***

###3###I. chinensis(C3)###250###+02 +- 2.4###>250###>250

###10###+45+- 1.3***

###4###I. coccinea(Y)(C4)###50###+92 +- 1.5***

###100###-26 +- 1.9***###12 +- 1.4b###205 +- 4.5b

###200###-49 +- 2.1***

###250###-64 +- 0.8***

###10###+24 +- 1.2*

###5###I. coccinea(O)(C5)###50###+41 +- 2.4***

###100###+72 +- 3.1***###58 +- 3.7c###>250

###200###+89 +- 2.7***

###250###-38 +- 4.5***

###6###Doxorubicin ug/ml(uM)###0.1###+37+-5.0**###0.17+-0.03a###5.8+-0.01a

###0.5###+71+-2.0***###(0.3+-0.05)###(10+-0.02)

###5.0###-18+-3.0***

###10.0###-50+-2.0***

Table-2: Growth inhibitory and cytotoxic effects of chloroform extracts of flowers of Ixora against Hela cell lines (Cervical uterine cancer).

S. No.###Cervical uterine cancer cell line

###Plant name###Doses###(HeLa)

###(Codes)###(ug/ml)###% Cell growth###(ug/ml)

###inhibition/cytotoxicity###GI50###LC50

###I. fulgens###10###+51 +- 2.9***

###1###(C1)###50###+62 +- 1.5***

###100###+92 +- 2.8***###10 +- 2.9b###165 +- 3.1b

###200###-80 +- 1.6***

###250###-87 +- 2.3***

###10###+18 +- 1.5*

###2###I. polyantha###50###+32 +-2.4**###>250

###(C2)###100###+43 +- 3.1***###140 +- 3.7c

###200###+61 +- 2.7***

###250###+69 +- 3.4***

###3###I. chinensis###250###+26 +- 2.4*###>250###>250

###(C3)

###10###+45 +- 2.2***

###4###I. coccinea(Y)###50###+99 +- 3.1***

###(C4)###100###-13 +- 2.4***###15 +- 3.8b###230 +- 3.9c

###200###-36 +- 1.9***

###250###-54 +- 2.5***

###10###+13 +- 1.4

###5###I. coccinea(O)###50###+49 +- 2.4***

###(C5)###100###+68 +- 3.5***###60 +- 2.7c###>250

###200###+89 +- 3.4***

###250###-31 +- 4.1***

###6###Doxorubicin###0.006###+5.0 +- 3.0

###ug/ml###0.06###+7.0 +- 3.0###0.5+-0.02a###5.8+-0.1a

###(uM)###0.6###+60 +- 3.0***###(0.88 +-0.04)###(10+-0.1)

###6.0###-52 +- 7.0***

Table-3: Growth inhibitory and cytotoxic effects of chloroform extracts of flowers of Ixora against NCI H-460 (Lung cancer cell line).

S. No.###Lung cancer cell line

###(NCI H-460)

###Plant name(Codes)###Doses(ug/ml)###% Cell growth inhibition/cytotoxicity###(ug/ml)

###GI50###LC50

###10###+07 +- 1.9

###1###I. fulgens###50###+56 +- 1.7***

###(C1)###100###+63 +- 1.4***###46 +- 1.9c###>250

###200###+94 +- 2.8***

###250###-12 +- 2.1***

###250###+01 +- 1.1

###2###I. chinensis###250###+01 +- 3.8###>250###>250

###(C3)###10###+40 +- 2.4***

###3###I. coccinea(Y)###50###+98 +- 2.9***

###(C4)###100###-31 +- 3.9***###12 +- 3.1b###200 +- 4.7b

###200###-51+- 4.2***

###250###-62 +- 4.6***

###10###+20 +- 1.6*

###4###I. coccinea(O)###50###+46 +- 2.7***###60 +- 3.1c

###(C5)###100###+58 +- 2.9***###>250

###200###+78 +- 3.0***

###250###+88 +- 3.9***

###5###Doxorubicin###0.1###+42+-5.0**

###ug/ml###0.5###+76+-2.0***###0.17+-0.05a###5.4 +- 1.2a

###(uM)###5.0###-18+-3.0***###(0.26 +-0.08)###(9.3 +- 1.2)

###10.0###-53+-2.0***

Bis (2-ethyl hexyl) 1,2-benzene dicarboxylate (10), n-tetradecanoic acid (43), n-eicosane (57), n-hexadecane (54), and methyl octadecanoate (65), were recognized as the chemical compounds present in flowers of chloroform extracts of all Ixora species named as I. fulgens, I. chinensis, I. polyantha, and both species of I. coccinea (yellow and orange color flowers). Some of identified chemical compounds have been reported in literature to give cytotoxic effect. Bis (2-ethylhexyl) 1, 2-benzene dicarboxylate (10), [29, 30], n-tetradecanoic acid (43) [31], n-hexadecane (54), n-tridecane (51), n-eicosane (57) [32], were reported to have anticancer activity. Methyl octadecanoate (65), methyl hexadecanoate (67), and methyl tetracosanoate (71), exhibited potential anticancer activities against MCF-7 cancer cell line [33]. Moreover, 6,10,14-trimethyl-2-pentadecanone (31), has been isolated from extract of Viscum album L., possessed effective cytotoxic activity against tumour cells line [34].

Chloroform extracts of I. coccinea (yellow), and I. fulgens, flowers exhibited strong activity against cervical (HeLa), lung (NCI H-460), and breast (MCF-7) cancer cell lines, these cytotoxic effects may be due to the synergistic result of phytochemicals identified by GC-MS examination (Table-4). The structures of compounds were recognized by GC and GC-MS studies and verified through mass library search software were given in Figs 1 and 2. Hierarchical clustering was carried out through applying Euclidean distance metric, whole connection to create a dendrogram for clustering of different flower species using normalized intensities of important compounds (p-value <0.05) by using online software, biit.cs.ut.ee/clustiv. Vertical lines length in the dendrogram is a evaluation of difference, whereas shorter lines reveal close association of the groups. This approach clustered the five different plant species.

Two plant species phytochemicals namely I. fulgens and I. coccinea (orange) flowers were the most similar from other species. Moreover, the most unlike species were examined of dissimilarity index according to plant metabolite, named as I. coccinea flowers (orange) and I. chinensis flowers (Fig. 3). Five Ixora species flowers chloroform extracts obtained were investigated for their growth inhibitory and cytotoxic activity as well as phytochemicals was identified by using GC-MS technique. Present investigation showed that I. fulgens flowers extracts were found to be most significant against HeLa cell lines. I. coccinea flowers extract was as good as against MCF-7, HeLa, and NCI H-460 human cancer cell lines. 33 Chemical compounds were recognized from I. fulgens flowers whereas, 17 metabolites were identified from I. coccinea flowers (yellow), which are mostly responsible for its cytotoxic and growth inhibitory actions preferably towards cervical cancer cell line.

Table-4: Identification of chemical compounds of chloroform extract of flowers of Ixora species through GC and GC-MS studies.

S. No.###Compounds name###Retention time###I. fulgens###I. polyantha###I. chinensis###I. coccinea(Y)###I. coccinea(O)

###%

1###1-Tetradecanol(1)###44.7###-###-###6.96###-###-

2###2-Heptenal(2)###6.1###0.7###-###-###-###0.62

3###2-Decenal(3)###20.1###1.67###-###-###-###1.78

4###n-Nonanal(4)###13.9###-###-###-###-###0.36

5###n-Octenal(5)###3.7###-###-###-###0.52###-

6###13-Octadecenal(6)###42.6###-###-###-###26.01###-

7###N,N-Bis(2-hydroxyethyl) dodecanamide(7)###28.9###-###0.42###-###-###0.07

8###Vanillin(8)###24.5###-###-###-###-###0.51

9###Isovanillic acid(9)###29.7###-###4.25###-###-###-

10###Bis(2-ethyl hexyl), 1,2-benzenedicarboxylate(10)###55.6###1.57###1.83###8.29###0.63###1.38

11###Dibutyl 1,2-benzenedicarboxylate(11)###35.3###-###-###1.08###-###1.31

12###(1-Methyldecyl) benzene(12)###31.8###-###-###0.91###-###-

13###(1-Methylundecyl) benzene(13)###34.0###-###-###1.28###-###-

14###Phthalic acid, butyl ester, ester with butyl glycolate(14)###37.3###-###0.53###-###-###-

15###5,7,7-Trimethyl bicyclo [3.3.0] oct-8-en-2-one(15)###53.0###0.33###-###-###-###-

16###1,6,8-Trimethyl-10 -methylene-5-phenyl-1,2,3-###76.6###1.55###-###-###-###-

###triazo[4,5]tricycle

17###1-Methyl-4-isopropyl-bicyclo[2.2.2] octa-5-ene-2,3-###17.3###-###5.19###-###-###-

###dicarboxylic anhydride(16)

18###4-Methyl undecane(17)###17.9###1.77###-###-###-###-

19###5-Methyl undecane(18)###17.9###-###5.70###-###-###-

20###4,7-Dimethyl undecane(19)###29.4###-###0.56###2.31###-###-

21###5,7-Dimethyl undecane(20)###56.6###-###-###-###2.68###-

22###3,3,6-Trimethyl decane(21)###46.7###-###-###0.05###-###-

23###3,8-Dimethyl decane(22)###57.5###-###4.14###-###-###-

24###2,6,10-Trimethyl dodecane(23)###54.6###-###-###-###1.01###-

25###2,5-Dimethyl tridecane(24)###55.5###-###5.44###-###-###-

26###2,5-Dimethyl tetradecane(25)###53.2###-###3.65###-###-###-

27###2,6,10,14-Tetramethyl hexadecane(26)###66###-###0.67###-###1.17###-

28###2,6,10,15-Tetramethyl heptadecane(27)###60.8###-###2.78###-###-###-

29###Isopropyl palmitate(28)###38.8###-###1.43###-###-###-

30###3-Methyl-5-propyl nonane(29)###45.0###-###1.39###-###-###-

31###Pluchidiol(30)###33.9###2.22###-###-###-###-

32###6,10,14-Trimethyl 2-pentadecanone(31)###34.7###1.5###1.1###-###0.75###0.49

33###Branched nonadecane(32)###57.9###-###-###21.17###-###-

34###1,1,4,4-Tetramethyl 2,5-dimethylene cyclohexane(33)###53.0###-###-###-###-###1.19

35###1,5-Diethenyl 2,3-dimethyl cyclohexane(34)###42.8###-###-###-###-###10.6

36###8 Methyl-tricyclo[3.3.0.0(2,8)]octan-3-one(35)###20.6###-###-###-###-###0.28

37###Tricyclo[4.3.1.0(2,5)]decane(36)###21.1###-###-###-###-###0.22

38###1-(2-Methylene-3-butenyl)-1-(1-methylene propyl)###45.4###-###4.45###-###-###-

###cyclopropane(37)

39###2-(1,1-Dimethylethyl) anthracene(38)###45.8###-###0.8###-###-###-

40###4,8,12,16-Tetramethylheptadecan-4-olide(39)###50.8###-###-###-###0.6###-

41###7-Hydroxy-6-methoxy- 2H-1-benzopyran-2-one(40)###37.7###-###14.59###-###-###-

42###n-Octanoic acid(41)###17.7###1.77###-###-###-###0.37

43###n-Dodecanoic acid(42)###28.7###1.58###-###-###-###-

44###n-Tetradecanoic acid(43)###33.2###2.39###1.0###5.56###1.06###3.73

45###n-Hexadecanoic acid(44)###37.4###16.14###-###-###17.2###23.83

46###n-Octadecanoic acid(45)###44.0###-###6.82###3.50###-###3.08

47###9-Octadecenoic acid(46)###43.6###1.27###-###-###-###-

48###9-Oxononanoic acid(47)###26.9###1.52###-###-###-###4.03

49###Kaempferol(48)###29.2###1.59###-###-###-###-

50###10-Undecenoyl chloride(49)###29.3###-###-###-###-###1.09

51###2-Fluoro-3,3-dimethylbutanal(50)###50.7###-###0.98###-###-###-

52###n-Tridecane(51)###52.3###-###2.77###14.29###-###-

53###n-Tetradecane(52)###54.6###-###-###-###-###4.06

54###n-Pentadecane(53)###56.8###-###-###8.64###-###-

55###n-Hexadecane(54)###58.5###1.11###1.3###3.6###4.66###5.79

56###n-Nonadecane(55)###59.7###-###-###2.85###-###-

57###1-Nonadecene(56)###33.7###-###-###2.77###-###-

58###n-Eicosane(57)###64.5###2.17###1.02###1.07###20.28###5.07

59###n-Tricosane(58)###60.1###-###2.56###-###0.78###-

60###n-Nonacosane(59)###57.9###-###4.67###-###-###-

61###n-Tritriacontane(60)###62.4###-###-###0.59###-###-

62###Methyl octanoate(61)###14.8###0.78###-###-###-###0.49

63###Methyl 9-oxo, nonanoate(62)###25.2###2.36###-###-###-###2.09

64###Nonanedioic acid, mono methyl ester(63)###29.4###-###-###-###-###0.07

65###Methyl tetradecanoate(64)###32.2###2.46###-###-###-###0.74

66###Methyl octadecanoate(65)###41.9###5.22###1.03###3.14###0.63###4.7

67###Methyl hexadecanoate(66)###36.4###38.1###9.19###8.44###3.8###19.29

68###Ethyl hexadecanoate(67)###37.9###0.87###-###-###-###-

69###Methyl eicosanoate(68)###55.2###2.78###0.3###-###0.25###2.3

70###Methyl docosanoate(69)###55.4###1.57###5.44###-###-###1.13

71###Methyl tricosanoate(70)###57.3###1.89###-###-###-###-

72###Methyl tetracosanoate(71)###59.0###1.23###-###-###-###0.14

73###Methyl 9-octadecenoate(72)###41.0###0.19###-###-###-###-

74###Methyl 9,12-octadecadienoate(73)###40.8###0.54###2.05###1.61###-###-

75###Methyl 9,12,15-octadecatrienoate(74)###42.7###4.2###-###-###-###8.59

76###Tri methyl silyl hexadecanoate(75)###39.3###-###-###-###0.79###-

77###Methyl 10-oxo 8-decenoate(76)###29.3###5.5###-###-###-###-

78###Ethyl 1-methyl hexadecanoate(77)###38.7###-###-###-###-###0.96

79###2-Ethylhexyl tridecyl sulfite(78)###31.8###-###3.17###-###-###-

80###2,3-Dimethylthiirane 1,1-dioxide(79)###57.3###-###-###1.29###-###-

81###4-Vinyl phenol(80)###19.4###0.57###-###-###-###-

82###2,4-Bis(1,1-dimethylethyl) phenol(81)###27.5###-###-###1.6###-###-

83###(-) - Loliolide(82)###33.4###1.09###-###-###-###-

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Author:Lubna; Ali, Rahat Azher; Siddiqui, Faheema; Fazil, Perveen; Farooq, Ahsana Dar; Siddiqui, Amna Jabba
Publication:Journal of the Chemical Society of Pakistan
Article Type:Technical report
Date:Oct 31, 2018
Words:5593
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