1. INTRODUCTION:

Natural products are well known to prevent the Reactive oxygen Species (ROS) and works as an antioxidant for the system where free radicals generate simultaneously. These constituents (phenolics, flavonoids, alkaloids and terpenoids etc.) are very common in almost every plant species. Generally, higher the number of such components, higher will be the activity as an antioxidant¹. Due to the properties of trapping the free radicals these compounds are used for medicinal purpose as well as for food protection. The present scenario suggests applying more and more natural resources in the field of science and technology particularly in the field of pharmacology and food industry.

Higher activity of plants as a free radical scavenger makes it highly efficient towards various problems such as cancer, diabetes, heart disease, inflammations etc². Due to the reason, research interest moves towards the plants and natural habitats for the development and discovering the new products and novel drugs for such type of diseases. Plants have two types of metabolites.

1. Primary metabolites; highly responsible for the growth and development of the species

2. Secondary metabolites; it is not directly involved in any growth but work as a defence mechanism for the species³.

It is also important for human as well as other herbivores as a food source. These compounds exhibits various biological effects and plays a key role in trapping the free radicals, inhibit the per-oxidation⁴ In the recent years, there are number of plant species studied by the researchers to know the important phytoconstituents present in it. Still, there is need to examine various plants to know their neutraceutical benefits.

Camelina sativa L. is one of those plants belongs to the brassicaceae family and well known for its seed oil, which is generally used for the biofuel purpose. It is very favorable and agro economic short seasoned crop which requires very low nutrients and other sources to grow^{5, 6}. Its defence mechanism is so strong that it generally doesn’t rely on the pesticides for the pathogen inhibition⁷. The stronger defence mechanism is the sign that the plant has high amount of secondary metabolite. However, plant is still not very well explored and needs more and more research to know its various neutraceutical benefits. Therefore, the present study was mainly focus to estimate its secondary metabolites using UV- VIS Spectroscopy, study also includes the GC MS analyses to know the chemical constituents present in the methanolic extract of defatted seed. Efforts were also done to know the in vitro antioxidant potential of different defatted extracts where quercetin was used as a standard.

2. MATERIAL AND METHOD:

Collection and cultivation was done as described previously reported method ⁵. 80 g of powder was filled in manually prepared thimble (Whatman filter 00) for the extraction using hexane as a solvent for oil removal in Soxhlet⁸ extraction unit; defatted powder was collected from thimble and dried.

Again, extraction was done using different polar solvents viz. water, MeOH, IPA and EA based on their polarity order. After completion of three cycles solvents was evaporated in rotary evaporator than viscous extract was lyophilized (LABCONCO) for further analyses.

2.2. Phytochemical Analyses:

2.2.1. Estimation of Total Phenolic Content:

Phenol content was estimated using Folin – Ciocalteu reagent method¹¹. 10mg of lyophilized sample was dissolved in 10ml (1mg/ml) and centrifuged at 10000g for 10 minutes. Supernatant was collected and further reacted with 200µl of 10 % (v/v) Folin – Ciocalteu reagent and 800µl of 70mM Na₂CO₃ and assay tubes were incubated at room temperature for 2 hrs. After the completion of reaction blue colour complex formed (phosphotungstic- phosphomolybdenum complex) and then the absorbance was taken at 765nm. Total phenolics calculated from a standard curve using Gallic acid.

2.2.2. Estimation of Flavonoid Content:

Total flavonoids of different extracts were calculated following the method of Lee and Safinar¹². The absorbance was measured at 510nm and total flavonoid content (µg g-1 dry wt quercetin equivalent) was calculated using quercetin as standard.

2.2.3. Estimation of Terpenoid Content:

Terpenoids are very special class of compounds however they don’t necessarily contain phenols in their structure but they are important secondary metabolites present in almost every species of the plant. In these class of compound basic structural unit is isoprene (C-5). Total terpenoids in plant extracts was estimated by the method of Ghorai¹³. The absorbance for test and standard solutions was measured at 538nm and terpenoid content was calculated using linalool (Lil) as standard and expressed as µg linalool equivalent g-1 dry wt.

2.2.4. Estimation of Alkaloids Content:

Alkaloids are another very important class of secondary metabolites compounds contains nitrogen atom in their basic structures. For the estimation of total alkaloids, each sample extract (1ml) was mixed with 1 ml of 2 N HCl and filtered. 1ml of this solution was then transferred to the tube which already contains 5ml of bromocresol green and 5ml of 0.1 M phosphate buffer (pH 7.4) this reaction mixture was mixed well with 4ml of chloroform incubated at room temperature for 30 minutes. The absorbance was measured at 470nm and total alkaloids content was calculated using atropine (AE) as standard, and expressed as µg/g AE dry wt).

2.3. Constituents Characterization Using Gas Chromatography Mass Spectrometry:

GC MS is mainly used for the separation and identification of multi component mixtures such as hydrocarbons, essential oils, fatty acids and solvents. For GC MS analyses lyophilized methanolic sample was taken and then mg/ml solution was prepared for injecting in the instrument.

GC MS was done at Advanced Instrumentation Research Facility, JNU, New Delhi. Sample was placed in GC–MS QP-2010 model (Shimadzu Scientific Instruments, Kyoto, Japan) equipped with a CombiPALAOC-20i+s] autosampler (CTC Analytics, Zwingen, Switzerland). The compounds were separated on a Rxi®-5Sil-MS capillary column (30 x 0.25mm ID and 0.25μm film thickness). The carrier gas was helium, split ratio 10, injector temperature 260.00°C. The column oven temperature program used was: 50.0°C (hold for 4 min) to 280.0°C at 10°C/min hold for 23 min. The ion source temperature and interface temperature were set at 230.00°C and 270.00°C respectively and the MS mode was electron impact (EI). The compounds were separated by GC and further fragmented by Mass spectrometer and identified by comparing the mass spectra obtained with NIST14 and WILEY8.LIB from the US National Institute of Technology and Standards (NIST) mass spectra libraries. For calculating the total area percentage of peak and relative percentage, we considered the peaks which were repeatedly present in at least two TIC.

2.4. Antioxidant Assay:

2.4.1. DPPH radical Scavenging Activity:

2 ml of DPPH (2,2-Diphenyl-1-picrylhydrazyl) solution (0.1 mM) was added in different sample (0 to 50 µlit) and prepared the volume up to 1ml with adding the respective solvents. Finally, kept all the mixtures in dark place for 40 minutes and then absorbance were taken at 517nm.

2.4.2. ABTS·+ radical cation scavenging activity:

ABTS cation radical was produced by the reaction of 7mM ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in water and 2.45mM Potassium persulfate in equal proportion, mixture kept in dark for overnight. ABTS·+ solution was then diluted with methanol to obtain an absorbance of 0.700 (±0.002) at 734 nm. After this, 1 ml of this solution added to the 1 ml sample (0 to 50 µlit) and kept the mixtures in dark for 5 to 7 minutes. Absorbance was taken at 734 nm.

FRSA (%) calculated with the formula in both DPPH and ABTS activity:

Abs (control) - Abs (test)

FRSA%= ––––––––––––––––––––––––– x 100

Abs (control)

2.5. Statistical analyses:

The treatments and controls of the experiments were carried out in triplicates. SPSS Inc. (IBM 2009) was used for analysis of variance (ANOVA). The treatment means were compared by least significant difference (LSD) test at a significance level of 0.05.

3. RESULT AND DISCUSSION:

3.1. Phytochemical analyses:

In this experiment, four different solvents were taken for extracting the compounds from defatted seeds of Camelina. In each extract, a fine amount of natural product was estimated. Extract percentage was highest in case of methanol (12.51 %) followed by water, IPA and Ethyl acetate respectively. Phenolic compounds of the plants have been well known for their properties as an antioxidant and many reports suggest that it is basic component for curing most of the human disease. Methanol based defatted extract of Camelina shows highest phenolics (19µg/mg GAE dry wt.) followed by IPA (15.82), water (11.39) and ethyl acetate (8.42) respectively. Flavonoids are the special class of compound contains C-15 in its basic structure and responsible for the plant growth and plays key role in its defence mechanism. MeOH based extract showed highest flavonoid component (4.75µg/mg QE dry wt.) followed by IPA, EA and water (3.24, 2.319, 1.465µg/mg QE dry wt.) respectively. Terpenoids are very special class of compounds however they doesn’t necessarily contains phenols in their structure but they are important secondary metabolites present in almost every species of the plant. In these class of compound basic structural unit is isoprene (C-5); they support the basic mechanism of the plant like growth, repair and development. Methanolic extract exhibit (2.32µg/mg Lil Eq Dry wt.) followed by EA, IPA and water (1.516, 1.368, 1.032µg/mg Lil Eq Dry wt.) respectively. Alkaloids are nitrogen containing compounds and was highest in case of IPA (7.245µg/mg AE dry wt.) followed by MeOH, EA and water (5.475, 4.418, 2.436µg/mg AE dry wt.) respectively. Result revels that the most appropriate solvent for extracting the components is methanol.

Each value expressed as mean ± S.E.

Fig 2

3.2. Important phytoconstituents p/r in defatted seeds of Camelina using GC – MS:

GC MS analysis of the MeOH based extract showed almost 30 compounds in which approximately 13 constituents were in noticeable amount. In all the volatile constituents, highest reported compound was 10- Undecenenitrile (23.64%) which is nitrogen containing cyano compound and present in castor oil and other brassicaceae plants works as fragrance agent. 1, 19 –Eicosadiene (19.43%) is the second most potent compound comes in the GC MS analysis. 9 – (Methylsulfanyl) nonanenitrile (11.9%) is volatile cyano compound firstly isolated from watercress (Nasturtium officinale) and generally found in green vegetables, It is very important chemical substance and utilized by an organism for its growth and reproduction¹⁴. Other important component which found in the GC MS report is ß- Monolinolein (4.34%) this compound is acyl monoglyceride a derivative of linolic acid. It involves in number of biological processes such as lipid transport, lipid metabolism and cell signaling; played an important biological role includes energy source, storage and as a nutrient, in industrial application it is used as emulsifier and surfactant¹⁵.

Fig 3: GC MS Chromatogram

Table 2: Showing Phytochemical constituents P/r in Camelina defatted cake

Sr. No	R.T.	Peak Area (%)	Compound	MF	MW	Nature of Compound	Pubchem CID
1.	4.99	23.64	10- Undecenenitrile	C₁₁H₁₉N	165	Cyano compound	104424
2.	24.57	19.43	1,19 -Eicosadiene	C₂₀H₃₈	278	Unsaturated acyclic compound	519006
3.	16.32	11.9	9 – (Methylsulfanyl) nonanenitrile	C₁₀H₁₉NS	185	Cyano compound	93987
4.	22.00	7.9	Pentanoic acid, 10-undecenyl ester	C₁₆H₃₀O₂	254	monounsaturated Ester	543363
5.	23.53	6.66	cis,cis,cis-7,10,13-Hexadecatrienal	C₁₉H₃₄O	234	Aldehyde	5367366
6.	34.59	4.34	ß- Monolinolein	C₂₁H₃₈O₄	354	Monoglyceride	5365676
7.	9.00	4.33	3',5'-Dimethoxyacetophenone	C₁₀H₁₂O₃	180	Ketone	95997
8.	30.83	3.69	2-Diethylaminoethylsulfanyl (methyl) phosphinic acid	C₇H₁₈NO₂PS	211	Phosphorous oxy acid	167960
9.	19.37	3.21	n-Hexadecanoic acid	C₁₆H₃₂O₂	256	Saturated Fatty acid	985
10.	13.56	3.08	8-(Methylsulfanyl)octanenitrile	C₉H₁₇NS	171	1Cyano compound	187924
11.	22.18	2.71	Sinapic acid methyl ester	C₁₂H₁₄O₅	238	MUFA	5321318
12.	18.89	1.95	9-(Methylsulfanyl)nonanenitrile	C₁₀H₁₉NS	185	Cyano compound	93987
13.	26.62	1.69	Chloromethyl 6-chlorododecanoate	C₁₃H₂₄Cl₂O₂	283	Ester	543311
14.	10.19	1.50	Z-dodec-9-en-1-ol	C₁₂H₂₄O	184	Hydroxy compound	6431342
15.	27.81	1.34	cis-13-Eicosenoic acid	C₂₀H₃₈O₂	310	MUFA	5312517
16.	39.28	1.12	Phytylpalmitat	C₃₆H₇₀O₂	534	Long chain esters	6437053
Others	-	1.51	-	-	-	-	-
Total (in %)		100

Palmitic acid (3.21%) is another important class of compound found in report, it is a saturated fatty acid which have antimicrobial properties in very high manner, it is precursor of long chain fatty acid and also one of the major fatty acid present in the human breast milk. It also exhibit high anti-inflammatory activities by reducing the impact of inflammatory activators such as prostaglandin E2, IL‐1β, TNF α, IL‐6 and nitric oxide¹⁶. A potent bioactive compound i.e. Sinapic acid (2.71 %) was also found in noticeable amount, this phenolic component is derivative of Cinammic acid generally found in ester form and exhibit various bioactive, antimicrobial, anticancer, anti-anxiety and anti-inflammatoryactivities^{17, 18}.

3.3. DPPH· and ABTS·+ scavenging activity:

Oxidation is very essential and basic mechanism for the survival of any cellular organism, the oxygen molecule is very necessary to metabolize proteins, fats, carbohydrates etc. for providing energy to the body, however it does not happened free of cost¹⁹. Oxygen is highly reactive in nature and responsible for producing some active unstable molecule which leads to disturb the mechanism of the body of an organism.

Table3: showing IC50 value for each extract, Quercetin used as a standard

Extracts	For DPPH		For ABTS
	IC50	1/IC50	IC50	1/IC50
Water	0.501	1.996	0.396	2.525
MeOH	0.400	2.500	0.241	4.149
IPA	0.475	2.105	0.339	2.950
EA	1.880	0.532	0.421	2.375
Quercetin (as +ve Control)	0.396	2.525	0.262	3.817

These unstable molecules are commonly called as “Reactive oxygen Species” (ROS) or “Free Radicals”. These molecules are regularly formed in metabolic pathway system, but excess of these free radicals can damage the fat of the cells due to which it loses their structure and function and leads to generate various diseases. Oxidation not only affects the organisms, but also a main cause of deterioration of food stuffs and affects its colour, taste, order, nutritional values etc. To check these adverse effects of oxidation process, body utilizes some potential molecules knows as antioxidants. These antioxidants include potential secondary metabolites, vitamins, fats, lipids etc. The DPPH and ABTS assays are most widely used experiments for knowing the antioxidant capability of any plant extract or any pure compound. Both dissolve in alcoholic solution and their color exhibit a characteristic peak at 517 and 734 nm respectively. When any compound or extract traps these free radicals the color of DPPH and ABTS becomes lighter. This colour dilution is a primary significance that sample has antioxidant property, after taking absorbance results clearly exhibit highest FRSA activity when Methanolic was used as an extracting solvent in both DPPH and ABTS. IC50 was also calculated against all the extract. (Concentration of the compound required to trap 50% of the free radicals generated by oxidative metabolism). IC50 for methanolic extract was very near to the quercetin which was taken as positive control.

CONCLUSION:

Results show that the highest secondary metabolites are extracted when the methanol was used as an extracting solvent. The higher content of phenolics shows the capability of Camelina cake as potent antioxidant product. GC MS report also suggest the presence of important bioactive components in defatted cake, report also suggest a positive side of defatted cake that there is no hazardous or toxic component was present; it shows the capability of this cake as a food additive or it can be used a source of novel drugs or can be used in various therapeutic applications.

ACKNOWLEDGEMENT:

Financial assistance and research facility provided by DIBER DRDO is duly acknowledged.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

ABBREVIATIONS:

MeOH Methyl Alcohol

IPA Iso Propyl Alcohol

EA Ethyl Acetate

ROS Reactive Oxygen Species

PUFA Poly Unsaturated Fatty Acid

GC MS Gas Chromatography Mass Spectrometry

SDW Sterile Distilled Water

DPPH 1, 1-Diphenyl-2-picrylhydrazyl radical

ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt)

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Received on 02.09.2019 Modified on 30.11.2019

Asian J. Research Chem. 2020; 13(1): 38-43.

DOI: 10.5958/0974-4150.2020.00009.7

Sr No.	Solvent	Formula	B. P.(˚C)	Density (g/ml)	Polarity Index	Relative Polarity
1.	Water	H₂O	100	0.998	10.2	1.00
2.	Methanol	CH₄O	64.6	0.791	5.1	0.762
3.	IPA	C₃H₈O	82.4	0.785	3.92	0.546
4.	EA	C₄H₈O₂	77	0.894	4.4	0.228