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Identification of Fatty Acids in Launaea Procumbens

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Identification of fatty acids in leaves and roots of Launaea procumbens by GC-MS and assessment of flavonoid and phenolic content and antioxidant activity

This study presents an analysis of fatty acid composition of in root and leaves of Launaea procumbens by GC-MS and hexane extract were studied for the determination of total flavonoid and phenolic content along with scavenging assays by using the 2, 2-diphenyl-1-picrylhydrazyl (DPPH). This analysis enabled the identification of 22 fatty acid in root and 18 in leaves of this plant representing 88.48 and 88.44 % of total extract respectively. The major fatty acid in root and leaves are myristic acid, palmitic acid, margaric acid, oleic acid, linoleic acid, linolenic acid, behenic acid, cerotic acid, montanic acid. Quantification data revealed that total flavonoid and phenolic was higher in leaf than root.

Keywords: Launaea procumbens; fatty acid; GC-MS; soxhlet extraction; DPPH, antioxidant activity, flavonoid, phenol

1. Introduction

L. procumbens, genus, Launaea Cass. belongs to the tribe Lactuceae (Cichorieae) (Tom et al. 1977) family of Asteraceae consists about 40 species (El-Bassuony et al. 2006) Nearly 20 species of this genus are growing in Pakistan (Krishnamurti A. 1969; Nasir E et al. 1972) growing in dry, saline and sandy habitats (Ozenda P. 2004). L. procumbens is a decumbent herb with a tuft of simple or branched flowering stems having yellowish flower. It occurs as a weed throughout the plains of India (The wealth of India, 1962).

Many plants of this genus are used in the folk medicines in the treatment of various skin problems, dysentery and tumors (El-Bassuony et al. 2006), antitumor, antioxidant, insecticidal and cytotoxic activities (Rasid S et al, 2000).

Ayurvedic preparations prepared from this plant are used in wound healing, sound health and longevity, as well as used as a food supplement (Wazir et al. 2007). Traditionally, it has been used in the treatment of rheumatism (Parekh et al. 2006), painful urination, liver dysfunctions, reproductive disorders (Ahmad et al, 2006) and hormonal imbalance in male (Qureshi et al, 2008), fever, itches, ulcers, cuts swellings, eczema eruptions (Bhandari MM, 1988). Previous chemical analysis of L. procumbens showed salicylic acid, vanillic acid, synergic acid, 2-methylresercinol and gallic acid (Shaukat et al, 2003). These compounds have spasmogenic, cardiovascular, anticarcinogenic, anti-inflammatory, and antioxidant properties to scavenge reactive oxygen species (Singh et al, 2007). In indigenous system of medicine the leaves of Launaea procumbens were reported to be useful in the treatment of urinary stones (Nadkarni, 2000; Sukhdev, 2006).

Naturally occurring sunstances in higher plants with their antioxidant property attention has increased on the protective effect of these natural antioxidants against chronic disorders caused by oxidative process (8, 9).  L. procumbens exhibited antioxidant and radical scavenging property.

Phytochemical studies of flavonoid and phenolic compounds with a broad spectrum of biological activity are important to understanding the practical uses of L. procumbens.

It is worthy to be note that there is almost no report on the chemical analysis of fatty acids of Launaea procumbens. The aim of this study is to identify fatty acids with their content in different parts of Launaea procumbens using GC-MS and determination of total flavonoid and phenolic compounds, including assessment of antioxidant property.

2. Results and Discussion

In this study the fatty acids composition in root and leaves of L. procumbens were compared by GC-MS and assessment of flavonoid and phenolics content in hexane extract. The antioxidant activity of the leaf and root extracts was evaluated by spectrophotometry of the presence of the DPPH radical which is often used to compare the activity of plant extracts. This analysis enabled the identification of 22 fatty acid in root and 18 fatty acid in leaves of total extract. Comparative study of identified fatty acids in root and leaves of L. procumbens are listed in Table 1. The composition percentage of fatty acids in root follows oleic acid (12. 68 %), linoleic acid (14. 84), behenic acid (6. 38%), lignoceric acid (3. 83%) and Montanic acid (3. 69 %) whereas in leaves oleic acid (12. 68%), linoleic acid (14. 84), behenic acid (6. 38%), lignoceric acid (3. 83%) and Montanic acid (3. 69%). In leaves myristic acid (6. 47%), palmitic acid (43. 78%), margaric acid (3. 22%) linolenic acid (4. 26%) are in higher amount than root. The main free fatty acids in root and leaves are myristic (5. 52 and 6. 47%), palmitic (30. 80 and 43. 78%), margaric (1. 52 and 3. 22%), oleic acid (12. 68 and 8. 33%), Linoleic acid (14. 84 and 11. 12%), linolenic (2. 47 and 4. 26%), behenic (6. 38 and 1. 41%), lignoceric (3. 83), cerotic (1. 20 and 1. 31%), montanic (3. 69 and 2. 14%) respectively. The minor fatty acids are caprylic, pelargonic, capric, lauric, pentadecanoic, palmitoleic, arachidic, henicosanoic, tricosanoic, pentacosanoic acid. The proportions of saturated fatty acid (55. 76 – 63. 84%) were higher than unsaturated fatty acid (32. 82-24. 82%) in root and leaves by GC-MS analysis respectively. GC-MS confirmation of fatty acids by mass spectral structure elucidation represented saturated, unsaturated and branched chain fatty acid. Therefore these results revealed both root and leaves have almost similar fatty acid but in different amount.

Considerable amount of flavonoid and phenolic content were found in hexane extract and exhibited efficient scavenging of DPPH are enlisted in Table 2. The hexane extract of leaves possessed the highest total flavonoid and phenolic content than root. Result showed that leaves have highest antioxidant activity as compared to root.

Table 1. Fatty acid composition in root and leaves of Launaea procumbens

Compounds

RT

Molecular

formula

MW

Peak area %(Root)

Peak area % (Leaf)

Caprylic acid

10. 88

C8H16O2

144

0. 34

0. 07

Pelargonic acid

13. 78

C9H18O2

158

0. 09

0. 13

Capric acid

16. 45

C10H20O2

172

0. 04

­_

Lauric acid

21. 38

C12H24O2

200

0. 26

1

Myristic acid

25. 82

C14H28O2

228

5. 52

6. 47

Pentadecanoic acid

27. 90

C15H30O2

242

0. 64

_

Palmitic acid

29. 92

C16H32O2

256

30. 80

43. 78

Palmitoleic acid

30. 14

C16H30O2

254

0. 65

0. 89

Margaric acid

31. 78

C17H34O2

270

1. 52

3. 22

Oleic acid

33. 57

C18H34O2

282

12. 68

8. 33

Linoleic acid

33. 85

C18H32O2

280

14. 84

11. 12

Linolenic acid

34. 34

C18H30O2

278

2. 47

4. 26

7, 10, 13-Eicosatrienoic acid

35. 77

C20H34O2

306

0. 07

_

Arachidic acid

36. 99

C20H40O2

312

0. 10

0. 81

11, 14-Eicosadienoic acid

37. 30

C20H36O2

308

2. 01

_

Heneicosanoic acid

38. 62

C21H42O2

326

0. 33

_

Behenic acid

40. 17

C22H44O2

340

6. 38

1. 41

Tricosanoic acid

41. 66

C23H46O2

354

0. 68

0. 31

Lignoceric acid

43. 11

C24H48O2

368

3. 83

0. 94

Pentacosanoic acid

44. 51

C25H50O2

382

0. 34

0. 15

Cerotic acid

45. 86

C26H52O2

396

1. 20

1. 31

Montanic acid

48. 45

C28H56O2

424

3. 69

2. 14

Melissic acid

51. 26

C30H60O2

452

_

2. 10

∑ Saturated Fatty acid

     

55. 76

63. 84

∑ Unsaturated Fatty acid

     

32. 82

24. 82

Total Fatty Acid

     

88. 48

88. 44

Others

     

11. 52

11. 56

3. Expeimental

3. 1. Plant Material

Plant samples was collected from local area of Lucknow (India) in the month of June, 2014 and identified by Dr. Anand Prakash, Botanist, National Botanical Research Institute (NBRI), Lucknow. A voucher specimen (No. 216343) has been deposited in the herbarium of NBRI. Plant was dried out away from direct sunlight and stored in a dried area till the time of the experiment.

3. 2. Soxhlet Extractionof Leaves and Root

The powdered root (20gm) and leaves (15gm) was extracted with 500 ml of petroleum ether (40-60°C) in soxhlet apparatus for 8 hrs. The extracts were filtered, cooled and concentrated under reduced pressure at 40° C to afford 3. 06% and 4. 17% of the extract respectively until dryness. The extracts were stored in dark bottle and kept at 4°C until analysis.

3. 3. Formation of fatty acid methyl ester (FAME)

The crude extract (500 mg) in concentrated sulphuric acid (2 mL) and methanol (20 mL) was heated under reflux on a water bath for 3 h. It was cooled to room temperature and extracted with petroleum ether (3×20 mL) and water in a separating funnel. The petroleum ether extract was dried over Na2SO4. The extract was dried under reduced pressure at 40°C. Prepared fatty acid methyl ester (FAME) was stored for further analysis.

3. 4. Determination of the total phenolic content

Total phenolics contents (TPC) were estimated using the method of Singleton and Rossi. Two hundred micro liters (1-5 mg/ml; dissolved in respective solvent) of each fraction was added in ten milliliter of 1:10 folin –cicocalteu reagent and incubated for 5 min before the addition of 7 ml of 0. 115 mg/ml Na2CO3. The resulting solution was incubated a further 2 h before absorbance readings were taken at 765 nm. Gallic acid was used in the calibration curve. Results were expressed as mg gallic acid (GAE)/g dried plant extract. Data for each fraction was recorded in triplate.

3. 5. Determination of the total flavonoid content

Total flavonoids content was determined by using a method described by Sakanaka et al . Briefly, 0. 25 ml of each fraction (1-5 mg/ml, dissolved in respective solvent) and rutin standard solution (15-250 µg/ml) was mixed with 1. 25 ml of distilled water in a test tube, followed by addition of 75 µl of a 5%(w/v) sodium nitrite solution . After 6 min, 150 µl of 10%(w/v ) aluminum chloride solution was added , and the mixture was allowed to stand for a further 5 min before 0. 5 ml of 1 M NaOH was added. The mixture was made up to 2. 5 ml with distilled water and mixed well. The absorbance was measured immediately at 510 nm. The results of samples were expressed as mg of rutin equivalents of total dreid fractions. All fractions were run in triplicate.

3. 6. DPPH radical scavenging assays

The free –radical scavenging activity was measured by using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. DPPH assay was performed according to the procedure as reported by Gyamfi et al. DPPH solution was prepared by dissolving 3. 2 mg in 100 ml of 82% methanol. 2. 8 ml of DPPH solution was added to glass vial followed by the addition of 0.2 ml of test sample solution, in methanol, leading to the final concentration of 1 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml and 100 µg/ml. Mixture of DPPH, and each fraction was shaken ewell and kept in the dark al controlled room temperature (25-28 C ) for 1 hr. After incubation change in color was measured at 517 nm. Mixture of 2.8 ml of 82% methanol and 2.8 ml of DPPH solution were taken as control. The test of each fraction was performed in triplicate. Percentage inhibition was measured according to following formula and IC50 value was calculated by graph pad prism software.

% scavenging = Abs. of control- Abs. of fraction x 100 / Abs. of control

Table 2 : Extraction yield, TPC, TFC and % Inhibition of hexane extract of Launaea procumbens

 

Root

Leaves

Extraction yield ( % )

3. 06

4. 17

Total phenolic content

125 mg / g

120 mg / g

Total flavonoids content

14. 3 mg/ g

9. 6 mg / g

% Inhibition at 200 mg / ml

75 %

68 %

3. 7. Gas Chromatography (GC)

Gas Chromatography was accomplished with a Thermo Fisher TRACE GC ULTRA using a TR 50MS column (30m x 0. 25mm ID x 0. 25 µm, film thickness); carrier gas, helium; temperature programming, 2 min. delay for solvent, at 50°C temperature rising at 2°C/min to 120°C and at 3°C/min. to 250°C and finally held isothermally for 15 min. The injector temperature was 230°C and carrier flow was constant flow 1 ml/min, in split mode (1:50) with injector volume 1µl. The relative proportion of the sample constituents were percentages obtained (% area) by FID peak–area normalization, without the use of response factor.

3. 8.  Gas Chromatography-Mass Spectrometry (GC-MS)

GC-MS analysis was performed with a Thermo Fisher TRACE GC ULTRA coupled with DSQ II Mass Spectrometer instrument using a TR 50MS column (30m x 0. 25mm ID x 0. 25 µm, film thickness); carrier gas, helium; temperature programming, 5 min. delay for solvent, at 50°C temperature, hold time 5. 0 min, rising at 4°C/min to 250°C and finally held isothermally for 5 min. The injector temperature was 230°C and carrier flow was constant flow 1 ml/min, in split mode (1:50) with injector volume 1µl. The ion source temperature was set at 220°C, transfer line temperature was 300°C, and the ionization of the sample components was performed in EI mode at an ionization voltage of 70eV. Mass range was used from m/z 50 to 650 amu. The chemical composition of fatty acids was identified by comparing their spectra with those of a NIST library and confirmed by comparing their retention indices with data published in various literatures.

4. Conclusion

As a result of this study plant indicated the types of fatty acids in the extract contribute little to its adverse clinic effect and fatty acid present are beneficial for health. Any one or more compounds present in the plant may be valuable for the many diseses.

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