Phytochemical Screening using GC-MS and study of Anti-Oxidant Activity of two species of Portulaca

 

Trupti P. Durgawale^1*, Chitra C. Khanwelkar², Pratik P. Durgawale³

¹Ph.D. Candidate, Department of Pharmacology, Krishna Institute of Medical Sciences Deemed to be University, Karad, Maharashtra, India

²Professor and Head, Department of Pharmacology, Krishna Institute of Medical Sciences Deemed to be University, Karad, Maharashtra, India

³Research Officer, Department of Molecular Biology and Genetics, Krishna Institute of Medical Sciences Deemed to be University, Karad, Maharashtra, India

 

ABSTRACT:

Portulaca oleraceae and Portulaca quadrifida have been used as a part of diet and in traditional systems of medicine in Asia and Africa. Recent studies have shown that they may also be rich sources of beneficial polyunsaturated fatty acids. The present study was aimed at comparing the phytochemical constituents of ethanolic extracts of the P. oleraceae and P. quadrifida and studying the anti-oxidant activity of these plants collected from western part of India which could further enhance their nutritive value. The different parts of the plants were extracted using microwave-assisted technique, screened for phytochemicals using GC-MS, and anti-oxidant activity was studied using DPPH, ABTS, superoxide radical scavenging assays and ferric reducing anti-oxidant assays. The results of antioxidant assays were compared to standards ascorbic acid and butylated hydroxytoulene for each assay. All the extracts exhibited antioxidant activity comparable to the standards. Amongst the samples studies, the ethanolic extract of the P. oleraceae fresh whole plant showed the maximum activity in most of the assays. The phytochemicals detected in the two species were markedly different from each other but were found to be rich in unsaturated fatty acids and other pharmacologically beneficial compounds. The extracts exhibited anti-oxidant activity in-vitro which could further help investigators study their in-vivo anti-oxidant activity.      

 

 

 

 

 

INTRODUCTION:

Portulaca oleraceae commonly known as Purslane (Family: Portulacaceae) can be found growing as a weed or turfgrass in almost all the regions of Asia, Africa and Mediterranean. It grows as a small shrub with a woody stem and succulent leaves. Owing to its mildly sour taste, it is consumed in the form of pickles, salads, soups, etc. Traditional systems of medicine in Asia and Africa indicate that the plant can be used as a anticancer, antidiabetic, hypocholesteremic, neuroprotective, hepatoprotective, nephroprotective, anti-inflammatory, antiulcer, antimicrobial^1-4. Phytochemical analyses report the presence of flavonoids, alkaloids, tannins, polysaccharides, polyunsaturated fatty acids, vitamins, minerals, etc ⁵.

Another species of Portulaca, commonly known as chickenweed is Potulaca quadrifida. Compared to Purslane, it has smaller leaves and bears yellow flowers. Even P. quadrifida has been indicated to possess medicinal properties and is used in treatment of asthma, cough, urinary discharges, inflammations and ulcers, haemorrhoids recent studies have revealed that these two species may be rich sources of beneficial polyunsaturated fatty acids namely omega-3 and omega-6 fatty acids ⁶.

 

Free radicals generated in the human body, as a result of physiological processes, may interact with their surrounding biomolecules leading to lipid peroxidation, formation of protein and nucleic acid complexes if left untreated. All of these deleterious effects can lead to cardiovascular diseases, cancer, diabetes, neurodegenerative diseases, etc^7-9. In order to scavenge these free radicals, anti-oxidants need to be consumed as a part of diet. Plants polyphenolic compounds play a major role as anti-oxidants^9-15. As mentioned above, polyphenols have been reported to be present in both the Portulaca plant species^5-6. Since they are already accepted as a part of diet, a detailed study of its anti-oxidant activity might further enhance its role as a nutritive food source and encourage investigators to conduct such studies in-vivo.     

 

MATERIALS AND METHODS:

The required reagents such as sodium carbonate, sodium hydroxide, ethanol, L- ascorbic acid (ascorbic acid), butylated hydroxytoluene (BHT), 2, 2- diphenyl-1- Picrylhydrazyl (DPPH), 2, 2’- Azino- bis (3- ethylbenzothiazoline-6- sulfonic acid) Diammonium salt (ABTS), potassium persulfate, potassium dihydrogen phosphate, di- potassium hydrogen phosphate, potassium fericyanide, tricholoacetic acid, ferric chloride, nitro blue tetrazolium chloride (NBT), β- nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt (β- NADPH), Tris(hydromethyl) amonimethane (Tris) were purchased from Sisco Research Laboratories Pvt. Ltd. Shimadzu spectrophotometer (UV- 1800) present in the Department of Biochemistry  was used for all spectrometric measurements.

 

Collection and authentification of plants:

The protocol followed during the study was approved by the Institutional Protocol Committee and Ethics Committee. P. oleraceae and P. quadrifida plants were harvested from local fields in the Sangli district, Maharashtra where they were growing as weed. The plant specimens were authenticated by Dr. Dhanaji S. Pawar, Associate Professor, Department of Botany, M. H. Shinde Mahavidylaya, Tisangi, Maharashtra. Some of the P. oleraceae plants were dried in shade and their seeds were separated and stored for further use.

 

Extraction:

Microwave Assisted Extraction were carried out in a controlled Catalyst  microwave  system  having maximum power output  800 Watt, 50  gram (g)  sample and 120 milliliters (ml) solvent for 20 minutes (min) to obtain the following extracts seperately-

     I.   Aqueous extract, Ethanolic extract, methanolic extract and Butanolic extract of Portulaca oleraceae whole plant.

   II.   Ethanolic Portulaca oleraceae dry whole plant.

  III.   Ethanolic Portulaca quarifida fresh whole plant.

 IV.   Ethanolic Portulaca oleraceae seed.

The extracts were evaporated to dryness and stored at minus 20 degree Celsius (°C) deep freezer until required. Each of the following experiments was performed in triplicate for three independent experimental repeats.              

 

DPPH radical scavenging assay:

The DPPH free radical scavenging assay was performed to study the free radical scavenging activity of the extracts¹⁶. Ascorbic acid and butylated hydroxytoluene (BHT) were used as standards. The extract dilutions were prepared in ethanol in a final volume of 250 μl. 750 μl of 200 μM DPPH solution was added to each tube, mixed and incubated at room temperature in the dark for 30 minutes. Absorbance was measured at 517 nm. The control tube contained only DPPH solution. The percentage inhibition or scavenging (% scavenging) of the DPPH radical was calculated as follows:

 

% Inhibition = [(Absorbance of control – Absorbance of sample)/ (absorbance of control)]*100

 

ABTS radical scavenging assay:

The ABTS radical scavenging assay was performed with ascorbic acid and BHT as standards¹⁷. 7 mM ABTS solution with 2.4 mM potassium persulfate was incubated at room temperature in dark for 16 hours. The absorbance of the ABTS+ solution at 734 nm was adjusted to 0.7 and 1 ml of this solution was added to 0.2 ml of standard and sample dilutions in ethanol. The solutions were incubated at room temperature for 30 minutes and the shift in absorbance maxima was detected at 734 nm. The control tube containing the ABTS+ solution was used to calculate the percentage inhibition or scavenging (% scavenging) of the ABTS+ radical as follows:

 

% Inhibition = [(Absorbance of control – Absorbance of sample)/ (absorbance of control)]*100

 

 

Superoxide radical scavenging assay:

As mentioned earlier, the NADPH- PMS method was used for the reduction of NBT and for the generation of superoxide free radicals ^18-19. Ascorbic acid and BHT were used as standards. 0.1 ml of 1.13 mM NADPH, 0.25 ml of 500 μM NBT, 0.35 ml Tris-HCl (pH 8.0) were added to a tube containing 0.1 ml of standard or sample dilutions. The solutions were mixed and 0.2 ml of 40.3 μM PMS was added to each tube, incubated at room temperature for 5 minutes and absorbance was measured at 560 nm. The control sample contained 0.1 ml ethanol instead of sample or standard. The superoxide radical scavenging activity was expressed as percentage inhibition as follows:

 

% Inhibition = [(Absorbance of control – Absorbance of sample)/ (absorbance of control)]*100

 

Ferric reducing antioxidant power:

To investigate the total reducing power of the extract, the ferric reducing antioxidant power assay was performed with Ascorbic acid and BHT as standards²⁰. 0.25 ml of phosphate buffer (pH 6.6), 0.25 ml of 1 % potassium ferricyanide were added to 0.1 ml of standard or sample. The mixture was incubated at 50 ᵒC for 20 minutes and after cooling 0.25 ml of 10 % trichloroacetic acid was added. The samples were then centrifuged at 5000 rpm for 10 minutes and the upper layer was added to 0.25 ml water and 0.05 ml 0.1 % ferric chloride. Absorbance was then measured at 734 nm. The control tube contained 0.1 ml ethanol instead of standard or sample. The reducing power was estimated by plotting the concentration of samples against the measured absorbance at 734 nm.

 

Gas chromatography coupled with mass spectrometry:

The gas chromatography (GC) equipment used was Scion 436-GC Bruker with column BR-5MS (5% Diphenyl / 95% Dimethyl poly siloxane) of 30 meter (m) length, 0.25 millimeter (mm) inner diameter and film thickness of 0.25 micrometer (µm). The carrier gas flow rate was set at 1 milliliter (ml) per minute (min) with a split ratio of 10:1. The sample injection volume was 2 microliter (µl) at 280 degree Celsius (°C). The oven temperature program was a hold at 110 °C for 3.5 min, increase up to 200 °C at rate of 10 °C/min, up to 280 °C at rate of 5 °C/min with 12 min hold. The total runtime was 40.5 min. The detector was TQ Quadrapole Mass Spectrometer with MS work station 8 software. The inlet line temperature was 290 °C with source temperature 250 °C. The electron energy was 70 eV and mass scan with mass by charge ratio of 50 – 500 atomic mass unit. The solvent delay was 0 – 3.5 min with total runtime of 40.5 min²¹.

RESULTS AND DISCUSSION:

Given the increasing number of cases of cancer, diabetes, and other diseases related to sedentary lifestyle, the consumption of anti-oxidant rich diet might help prevent some of the damage caused by free radicals ^7-9. Anti-oxidant rich diet also has lesser side- effects as compared to artificially synthesized anti-oxidants and are more accessible to majority of the population. The anti-oxidant property of vegetables and fruits is contributed by ascorbic acid, flavanoids, alkaloids, tannins, and other polyphenolic compounds^9-15. During this study, the anti-oxidant activity of different extracts of P. oleraceae and P. quadrifida was observed for different stable free radicals such DPPH, ABTS, superoxide and finally ferric reducing capacity. In order to compare the activities of extracts with standards ascorbic acid and BHT, the half inhibitory concentration (IC₅₀) was calculated for each extract.

 

The DPPH radical is a stable free radical with an absorbance maxima at 517 nm. The neutralization of free radicals by polypenolic compounds present in the extract or by standards results in discoloration of the DPPH solution proportional to the amount of radicals neutralized¹⁶. As such the extracts exhibited increasing scavenging of free radical with increasing concentration. The highest activity amongst the extracts was observed for the ethanolic extract of fresh P. oleraceae whole plant while the aqueous extract of the same plant exhibited the least activity Table 1.

 

The ABTS^{· +} cation is produced by the reaction between ABTS and potassium persulfate used in the ABTS free radical scavenging activity assay¹⁷. The reducing compounds present in the extracts reduce the ABTS^{· +} cation which can be detected as a shift in absorbance maxima at 734 nm. In the tested extracts, the percentage of neutralized free radicals was seen to increase proportionally to the increasing concentrations. The ethanolic extract of fresh P. oleraceae whole plant exhibited the highest activity while the aqueous extract of the same plant exhibited the least activity Table 1. 

 

In the superoxide radical scavenging assay, superoxide radical produced by the NADPH/ PMS system reduces the tetrazolium salt NBT to insoluble blue colored formazan crystals. The anti-oxidants prevent the reduction of NBT by scavenging the superoxide radicals which can be observed as a decrease in absorbance measured at 560 nm¹⁸. The extracts exhibited increasing anti-oxidant activity with increasing concentration with the ethanolic extract of fresh P. oleraceae whole plant exhibiting the highest activity Table 1.

 

Table 1: The calculated half maximum inhibitory concentration (IC₅₀) values of extracts and standards for DPPH, ABTS, and superoxide radical scavenging assays.

 (Expressed as mean + standard error of mean, n = 3)

 

The ferric reducing antioxidant assay measures the samples ability to reduce the ferrous (Fe³⁺) ions to ferric (Fe²⁺) ions which can be detected with a change in absorbance maxima²⁰. The standards ascorbic acid and BHT exhibited increasing reduction activity with proportional increase in concentration. A similar trend was observed for all the extracts, thus indicating ferric reducing activity over the tested range of concentration Figure 1.

 

 

(a)

 

 

(b)

 

(c)

 

 

(d)

 

 

(e)

 

 

(f)

 

 

(g)

 

 

(h)

 

 

(i)

Figure 1: The ferric reducing antioxidant power results as observed for (a) Ascorbic acid, (b) BHT, (c) Ethanolic P. oleraceae fresh whole plant, (d) Ethanolic dry P. oleraceae, (e) Ethanolic fresh P.quadrifida, (f) Ethanolic P.oleraceae seed, (g) Methanolic P.oleraceae fresh whole plant, (h) Butanolic P. oleraceae fresh whole plant, (i) Aqueous P. oleraceae fresh whole plant.      

 

Earlier study reported anti-oxidant activity of P. oleraceae extracts at grown under controlled conditions at different stages of growth with IC₅₀ values of DPPH activity ranging from 1.3 mg/ml to 1.7 mg/ml²². Another study also indicated that the nutritive value of the plant depends on its growing conditions²³. Hence, the growing conditions and stage of growth of the plant during the time of harvest may play a major role in the anti-oxidant activity of the plant.

 

The results obtained for GCMS analysis of ethanolic extract of P. oleraceae whole plant show the presence of many bioactive phytochemicals Table 2. A number of unsaturated fatty acids such as Dodecanoic acid, 2,3-bis(acetyloxy)propyl ester, E-9-Tetradeceoic acid, 2-Bromotetradecanoic acid, Hexadecanoic acid, ethyl ester, Cyclopropaetetraecanoic acid, 2-octyl-, methyl were identified which are generally considered to be promote good health by reducing incidences of cardiovascular complications²⁴. Moreover, digitoxin which is a known heat stimulant and cytotoxic agent with proven anticancer activity against a number of cancer cell lines^25-26. The monounsaturated fatty acid Myristoleic Acid, also has been reported to be effective as a cytotoxic agent for the treatment of prostate cancer ²⁷. While, cyclobarbital has been reported to possess to have antipsychotic and sedative hypnotic activity²⁸. 

 

Table 2: GC-MS analysis results of ethanolic extract of P. oleraceae fresh whole plant 

Sr.No.	Retention time (min)	Name of compound	Molecular Formula	Molecular Weight	Peak Area (%)
1	4.94	Nonanal, 3-(metylthiol)-	C10H20OS	188	0.7
2	5.91	Benzene, 1,3-bis(1,1-dimethylethyl)-	C14H22	190	0.75
3	6.6	D- Mannose	C6H12O6	180	0.57
4	8.09	E-9-Tetradeceoic acid	C14H26O2	226	1.41
5	8.2	7-Methyl-Z-tetradecen-1-ol acetate	C17H32O2	268	0.86
6	9.52	D-Gala-1-ido-octonic amide	C8H17NO8	255	1.87
7	9.75	2-Bromotetradecanoic acid	C14H27BrO2	306	0.46
8	10.79	7-Methyl-Z-tetradecen-1-ol acetate	C17H32O2	268	1.12
9	11.99	3-O-Methyl-D-Glucose	C7H14O6	194	85.86
10	13.1	2-Hexadecanol	C16H34O	242	0.52
11	15.17	Cyclobarbital	C12H16N2O3	236	0.55
12	15.58	Hexadecanoic acid, ethyl ester	C18H36O2	284	0.52
13	17.2	1-Dodecanol,3,7,11,- trimethyl-	C15H32O	228	0.95
14	17.97	9,12-Octadecadienoyl chloride, (Z,Z)-	C18H3ClO	298	0.93
15	18.77	Dodecanoic acid, 2,3-bis(acetyloxy)propyl ester	C19H34O6	358	1.33
16	22.36	Cyclopropaetetraecanoic acid, 2-octyl-, methyl	C26H50O2	394	0.64
17	28.34	D-Mannitol, 1-decylsulfonyl-	C16H3407S	370	0.37
18	35.8	Digitoxin	C41H64O13	764	0.6

 

 

The GCMS results obtained for the ethanolic extract of P. quadrifida have identified the presence of omega 9 fatty acid oleic acid which has several health benefits such as lowering risk of cardiovascular diseases and have been recommended especially for diabetic patients as they may additionally help in glycemic control Table 3,24,29. Another important phytochemical is medium chain fatty acid 3-hydroxydecanoic acid which is associated with fatty acid metabolic disorders³⁰. Pentadecanol is a very long chain fatty alcohol which is known to lower plasma chloesteol in humans³¹.  

 

 

 

Table 3: GC-MS analysis results of ethanolic extract of P. quadrifida fresh whole plant

Sr. No.	Retention time (min)	Name of compund	Molecular Formula	Molecular Weight	Peak Area (%)
1	4.96	1,8-Nonadien-3-ol	C9H16O	140	6.86
2	5.9	Benzene, 1,5-dimethyl-2,4-bis(1-methylethyl)-	C14H22	190	3.73
3	6.62	Octadecane-12-on-1-ol, TMS	C21H44O2Si	356	12.72
4	8.11	Dodecanoic acid, 3-hydroxy-	C12H24O3	216	9.42
5	8.21	1-Dodecanol,3,7,11,- trimethyl-	C15H32O	228	5.66
6	9.77	1-Flouro-2-methyl-1-(N-methyl-N-phenylamino)-1,3-butadiene	C12H14FN	191	4.05
7	10.8	2H-Oxecin-2-one,3,4,7,89,10-hexahyro-4-hydroxy-10-methyl-,[4S-(4R*,5E,10S*)]-	C10H16O3	184	5.43
8	12.17	1-Gala-1-ido-octose	C8H16O8	240	2.98
9	12.88	α-D-Glycopyranoside, O-α-D-glucopyranosyl-(1.fwdarw.3)-β-D-fructofuranosyl	C18H32O16	504	6.49
10	13.13	1-Dodecanol,3,7,11,- trimethyl-	C15H32O	228	4.71
11	13.62	2,5-Octadecadiynoic acid, methyl ester	C19H30O2	290	3.43
12	13.99	Estra-1,3,5(10)-trien-17β-ol	C18H240	256	5.45
13	14.54	7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione	C17H24O3	276	6.22
14	15.61	2-Undecanone 2,4-dinitrophenylhyrazone	C17H26N4O4	350	2.57
15	17.22	12-Methyl-E,E-2,13-octadecaien-1-ol	C19H36O	280	4.78
16	17.98	Oleic acid	C18H34O2	282	5.52
17	20.4	2-Myristynoyl pantetheine	C25H44N2O5S	484	9.97

 

CONCLUSION:

The extracts tested for free radical scavenging activity and ferric reducing anti-oxidant activity using different assays point to the observation that the extracts have considerable anti-oxidant activity. Among the extracts studied, the ethanolic extract of P. oleraceae fresh whole plant exhibited the highest activity while the aqueous extract of the same plant exhibited the least activity. The trend of anti-oxidant activity for the different extracts was observed to be similar for all the assays. These results may further be utilized by investigators to study the anti-oxidant potential of extracts for in-vivo experiments. The GC-MS analysis of extracts of P. oleraceae and P. quadrifida has shown the presence of many bioactive pharmacologically beneficial compounds which may enhance their role as dietary sources of polyunsaturated fatty acids and antioxidants.         

 

ACKNOWLEDGEMENT:

The authors would like to extend their gratitude towards the Directorate of Research, Krishna Institute of Medical Sciences Deemed to be University, Karad, Maharashtra, India for their continued support and encouragement in carrying out this research.

 

 

 

 

FINANCIAL ASSISTANCE:

This project was financially supported by the Directorate of Research, Krishna Institute of Medical Sciences Deemed to be University, Karad, Maharashtra, India.

 

CONFLICT OF INTEREST:

None to be declared.

 

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Accepted on 24.11.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(12): 5534-5540.