Phytochemical Analysis, Antioxidant Activity, Fatty Acids Composition, and Functional Group Analysis of Heliotropium bacciferum

Heliotropium bacciferum is paramount in medicinal perspective and belongs to Boraginaceae family. The crude and numerous fractions of leaves, stem, and roots of the plant were investigated for phytochemical analysis and DPPH radical scavenging activity. Phytochemical analysis of crude and fractions of the plant revealed the presence of alkaloids, saponins, tannins, steroids, terpenoids, flavonoids, glycosides, and phenols. The antioxidant (free radical scavenging) activity of various extracts of the Heliotropium bacciferum was resolute against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical with the avail of UV spectrophotometer at 517 nm. The stock solution (1000 mg/mL) and then several dilutions (50, 100, 150, 200, and 250 mg/mL) of the crude and fractions were prepared. Ascorbic acid was used as a standard. The plant leaves (52.59 ± 0.84 to 90.74 ± 1.00), stem (50.19 ± 0.92 to 89.42 ± 1.10), and roots extracts (49.19 ± 0.52 to 90.01 ± 1.02) divulged magnificent antioxidant activities. For the ascertainment of the fatty acid constituents a gas chromatograph hyphenated to mass spectrometer was used. The essential fatty acids for growth maintenance such as linoleic acid (65.70%), eicosadienoic acid (15.12%), oleic acid (8.72%), and palmitic acid (8.14%) were found in high percentage. The infrared spectra of all extracts of the plant were recorded by IR Prestige-21 FTIR model.


Introduction
Medicinal plants and their therapeutic values are extensively used for an array of diseases all over the world. Divergent chemical constituents isolated and characterized from plant species of Boraginaceae family include flavonoids, pyrrolizidine alkaloids, naphthoquinones, phenols, and terpenoids. From different parts of various plants significant pharmacological and biological activities have been reported previously. The biological activities of constituents revealed antitumor, anti-inflammatory, antiviral, antiplatelet, cardiotonic, wound healing, contraceptive, prostaglandin, and wound healing properties [1]. Among foremost health problems, infectious diseases account for 41% of the global disease burden along with noninfectious diseases (43%) and injuries (16%) [2]. A rich source of pyrrolizidine alkaloids is present in Heliotropium bacciferum of family Boraginaceae, some of which have antihyperlipidemic, antitumor, antidiabetic, and antimicrobial properties [3]. Due to the biological activities of the plant antioxidants against reactive oxygen species, such as hydrogen peroxide and superoxide, they have profound significance. Reactive oxygen species (ROS) induce oxidative damage to biomolecules such as carbohydrates, lipids, proteins, and nucleic acids. The oxidative damage causes many diseases such as arteriosclerosis, rheumatoid arthritis, ageing, cancer, and cirrhosis [4]. Because of radiations, chemicals, environmental pollutants, toxins, spicy and deep fried food, and physical stress, free radicals cause change in gene expression, depletion of immune system antioxidants, and abnormal proteins induction. For the production of 2 The Scientific World Journal free radicals in food, living systems, and drugs, oxidation process is one of the most significant routes. Hydroperoxidase and catalase enzymes convert hydroperoxides and hydrogen peroxides to nonradicals and in human body act as natural antioxidants [5]. Several biological mechanisms of polyphenolic substances have been credited to the metal chelating properties or reducing properties of antioxidants [6,7]. In food nutrition assessment, fatty acids have gained significance in the diagnosis of various diseases and pharmacology [8][9][10] due to biological importance [11,12]. In lowering risks of inflammation, heart diseases and, for immunity enhancement, saturated fatty acids either monosaturated or polysaturated have been used [13][14][15][16][17][18]. For fatty acids determination different analytical techniques have been used which contain spectrophotometric, HPLC [19][20][21], enzymatic, and gas chromatography (GC) [22,23]. For the analysis of fatty acids, GC-MS, due to different reasons such as resolution, sensitivity, and speed, was the scheme of choice [24,25]. The present study was therefore designed to investigate the phytochemical and GC-MS analysis, antioxidant activities, and FTIR spectra of methanol, -hexane, ethyl acetate,butanol, and aqueous extracts of the plant Heliotropium bacciferum.

Plant Collection and
Identification. Heliotropium bacciferum was collected from district Karak, Khyber Pakhtunkhwa, Pakistan, and then was identified by plant taxonomist in the Department of Plant Sciences, Kohat University of Science and Technology (KUST), Pakistan.

Extraction and Fractionation.
The plant leaves, stem, and roots were shade-dried, crushed, and milled into powder form. The coarse power (500 g) of each part was taken and macerated in methanol for 15 days by the same method as that of Allen Jr. et al. [26]. After maceration, the soluble methanol fraction was filtered and concentrated under vacuum as a consequence of Rotary vacuum evaporator (PLC/MBC (Phy. Std.)/011 Eyela) at 40 ∘ C. The crude methanol extract (80 gm) of each part was then suspended in distilled water (500 mL) and partitioned in succession with -hexane, ethyl acetate,butanol, and water.

Ash
Value. The method of Premnath et al. [27] was employed for the determination of ash value of the plant Heliotropium bacciferum. Furnace PLC/MBC/W1/32 was used for the determination of ash value.

Moisture Value.
For the determination of moisture value of the plant, the method of Ashutosh et al. [28] was used. For moisture value determination, Oven PLC/MBC/W1/21 was used.

Extractive Value.
The extractive values of all the five (5) extracts of the leaves, stem, and roots of plant Heliotropium bacciferum were determined by the method of Singh et al. [29].

Preliminary Phytochemical
Screening. Qualitative tests were performed on different extracts of leaves, stem, and roots of the plant by employing standard protocols [30][31][32] for the detection of carbohydrates, saponins, alkaloids, tannins, terpenoids, steroids, flavonoids, and so forth.

Diphenyl Picryl Hydrazine (DPPH) Radical Scavenging
Activity (Antioxidant Activity). The DPPH radical scavenging activity of the crude and various fractions of leaves, stem, and roots of Heliotropium bacciferum were determined by UV spectrophotometer at 517 nm in opposition to 2,2diphenyl-1-picrylhydrazyl (DPPH) radical. The antioxidant activity was resolved by the procedures described in the past [33] with slight modifications. Stock solution (1000 mg/mL) of extracts of Heliotropium bacciferum was prepared; then dilutions of the crude and fractions (50, 100, 150, 200, and 250 mg/mL) were prepared. As a standard, vitamin C (ascorbic acid) was used. For comparison, dilutions (50, 100, 150, 200, and 250 mg/mL) of ascorbic acid were also prepared. Solution of DPPH (0.003 g/100 mL) was prepared and then this solution was added to each of the five dilutions of the plant extracts. The absorbance was calculated after 30 minutes at 517 nm by spectrophotometer. The increase in the DPPH free radical scavenging activity is attributed to the decline in the absorbance of the DPPH solution. Then the percent radical scavenging activity (% RSA) was calculated by the following formula: (1)

Preparation of Standards.
For the preparation of internal standard, in 1 mL hexane, 13.7 mg tridecanoic acid methyl ester was dissolved. 10 mg of -hexane extract was diluted in FAMEs mix standard (10 mL) with dichloromethane (CHCl 2 ) for preparation of external standard.  chromatographic technique. GC-MS procedure was used for fatty acids investigation. Methylation is focal procedure used for the conversion of nonvolatile fatty acids (FA) into volatile fatty acids methyl esters or FAMEs [34]. The standard procedure was used for determination of fatty acid contents [35]. In 25 mg sample, 0.1 mL internal standard and 1.5 mL methanolic NaOH (0.5 N) were added. The solution was heated for 5 minutes on hot plate in boiling water. The sample was then cooled and 10% CH 3 OH and 2.5 mL BF 3 solution were added. Sample solution again was potted and in boiling water on hot plate heated for about 30 minutes. Then cooled and saturated NaCl solution (4 mL) was added to the esterified solution and extracted twice with hexane (1 mL), filtered by 0.45 micrometer ( m) membrane filter and subjected to GC-MS scheme.

FTIR (Fourier Transform Infrared Spectroscopy) Study of Plant
Extracts. IR Prestige-21 (Shimadzu Japan) FTIR model was used with IR Solutions software [36]. The scheme used by Meenambal et al. [37] was carried out for all the plant extracts in dried form by FTIR spectroscopy.

Moisture, Ash, and Extractive
Values. The moisture value of the whole plant was 12% and the ash value was 8.67%. The plant extractive values were calculated separately for all the five (5) extracts of leaves, stem, and roots. Methanol extract of leaves, stem, and roots had high percentage of extractive values shown in Table 1.

Phytochemical
Screening. Phytochemical screening of various extracts of the leaves, stem, and roots of plant Heliotropium bacciferum revealed the presence of steroids, tannins, alkaloids, saponins, glycosides, terpenoids, phenols, and flavonoids (Table 2). In all plant extracts alkaloids were present. Except -hexane fraction, saponins were present in all plant extracts.

Diphenyl Picryl Hydrazine (DPPH) Radical Scavenging
Activity (Antioxidant Activity). Tables 3, 4, and 5 demonstrate the antioxidant activities of the leaves, stem, and roots of plant Heliotropium bacciferum. Standard "ascorbic acid" exhibited significant DPPH radical scavenging activities. The plant leaves extracts revealed excellent DPPH radical scavenging activities ranging from 52.59 ± 0.84 to 90.74 ± 1.00 at concentrations of 50, 100, 150, 200, and 250 mg/mL, respectively (Figures 1, 2, and 3). The Scientific World Journal           Table 6 viewing the names of fatty acids, area of relevant peaks, relative percentage compositions, times of the analysis, and retention time (R. time) was obtained from gas chromatography mass spectrometry (GC-MS) analysis. The percentage concentration and areas are the mean of the 3 measurements shown in Table 6. Figure 5 shows the obtained GC-MS chromatogram of the -hexane extract of the plant Heliotropium bacciferum with regularly labeled signals detected by GC-MS detector (Analytes). In the sample under investigation, the saturated and the unsaturated fatty acids were found ( Figure 4).

Discussion
Plants containing steroids and flavonoid present in fruits and vegetables reduce the risk of atherosclerosis, which is build-up of fatty deposits in the artery walls [38]. Phenols and flavonoids in olive act as antioxidant, anticancer, antimicrobial, and antibacterial agents [39]. For compound identification, FTIR spectroscopy was used and run between the ranges of 400 and 4000 cm −1 under IR region. The peaks revealed that the plant has compounds such as amides, alcohol, aldehyde, ethers, ketone, and carboxylic acid [40].
Many herbs and plant species have been reported to possess DPPH radical scavenging activity. The plant Heliotropium bacciferum revealed significant DPPH radical scavenging activity. Other plants of genus Heliotropium also showed antioxidant activity. Plant aqueous fraction was primarily active. It has an EC 50 value of 20.51 g/mL. Modak isolated three (3) flavonoids, 3-O-methylgalangin, 7-O-methyleriodictiol, and naringenin from the plant Heliotropium taltalense. The isolated flavonoids exhibited DPPH radical scavenging activity which recommends that Heliotropium bacciferum may possess flavonoids accountable for radical scavenging activity [41]. Phenolic compounds, for example, flavonoids, are of fastidious interest because of their antioxidant activity through oxygen radicals scavenging and peroxidation inhibition. Antioxidants that scavenge free radicals have a key role in inflammatory disorders, cancer, aging, and cardiovascular diseases [42]. Many antioxidant activities are due to the presence of coumarin lignans, flavonoids, flavones, anthocyanin, isocatechins, isoflavones, and catechins [43]. Heliotrine alkaloid demonstrated temporary hypotension perse in dogs and extensively condensed the nicotine induced vasopressor spasmogenic responses [44].
Drugs formulations on the basis of antioxidants are mostly used for the treatment and for the prevention of different diseases, such as Alzheimer's disease, stroke, cancer, diabetes, and atherosclerosis [45]. Some bacterial fatty acid profiles vary in composition according to external stimuli (temperature, pH, nitrogen source, salinity, etc.) [46]. In order to use specific fatty acid biomarkers to interpret environmental community structure, microorganisms should be examined for fatty acid patterns and their variation under different conditions. Taylor and Parkes showed that fatty acid profiles in some sulphate-reducing bacteria can be influenced by carbon source; however, in all cases major fatty acid biomarkers were identifiable [47]. Linoleic acid was found in highest percentage (65.70 ± 0.004%) in Heliotropium bacciferum followed by eicosadienoic acid (15.12 ± 0.002%), oleic acid (8.72 ± 0.007%), palmitic acid (8.14 ± 0.005%), stearic acid (1.74 ± 0.003%), elaidic acid (0.58 ± 0.002%), and myristic acid (0.20 ± 0.005%), respectively. In food nutrition evaluation, fatty acids have immense biological importance. In pharmacology and disease diagnosing, fatty acid also has key significance [48]. The unsaturated (monounsaturated or polyunsaturated) fatty acids are frequently used for declining heart disease risks, inflammation and increasing the immunity [14,49].