Chemical Datasets, Antioxidant, Free Radicals Scavenger activities estimate in Aqueous Garlic (Allium sativum) extract
1Ph.D.in Histopathology, Biology Instructor, The Gifted Students' School- Basra, Ministry of Education Basra, Iraq, 61030.
2Fourth Stage, The Gifted Students School- Basra, Iraq, 61030.
*Corresponding Author E-mail: fjasim119@gmail.com, hameedalhilu1@gmail.com
Garlic is one of many medicinal plants that used over thousands of years due to its organosulfur compounds. Materials and methods: commercial garlic was used for the extraction, phytochemicals detections and Gas Chromatography-Mass (GC- Mass) analysis utilized to determine the main compounds in the aqueous extract. Antioxidant activity was evaluated in the garlic extract using total antioxidant capacity (TAC) and DPPH for detecting the concentration that takes half of the free radicals. The phytochemical tests revealed several compounds in the extract, included alkaloid, glycosides, tannins, and flavonoid and phenol while saponin compound was not detected. GC-Mass spotted 15 different compounds included allyl trisulfide with the concentration (1.89%) followed by ascorbic acid 2, 6-dihexadecanoate (22.24%), 9, 12-Octadecadienoic acid (Z, Z) (11.44), and 6-Octadecenoic acid, (Z) (40, 28). Total Antioxidant Activity of garlic aqueous extract reached 60 mg/ml compared to that of ascorbic acid. IC50 examination observed that the aqueous extract of garlic had 308.57΅g/ml value.
KEYWORDS: Garlic Extract; total antioxidant activity, GC of garlic extract, DPPH for free radical scavenger,
Allium stavium.
Antioxidant composites are found in food to deliver health-protecting roles. All cereals, vegetables, and fruits are the major origin of naturally revolving antioxidants. Oxidation reactions can yield free radicals which sequentially initiated starting chain reactions formation and caused a damage or death of the cell1. Since the herbal medicines are more active, inexpensive, available without difficulty and less poisonous in nature, popular people depend on an old medication for their main health care requirements. The core restriction of the traditional medicine is the reduction of calibration. The plants composites play very important role in antioxidant activities specially if its contained phenolic and flavonoid and thus, effective in blocking diseases related with oxidative stress2.
Garlic (Allium sativum) is a bulb that belongs to the Liliaceae family3,4. Garlic's
originated from central Asia, as well
as Great Britain, places in Europe, the Middle
East, and North Africa. The plant has been cultivated in New Zealand, Australia, and North America. A sativum is widely cultivated for culinary purposes
due to its strong smell,
especially when crushed as a spicing agent in
food, and also for its medicinal properties as an old drug since the ancient times.
Allium represents a great variety as consideration generally different morphological features especially in life cycle form (rhizome or bulb), and diversity in their ecological niche. This genus composes mostly of perennial plants and having a bulb5. Some species such as Allium cepa, Allium sativum, and Allium fistulosum, have been rich in bioactive polysaccharides6.
Several vitamins such as vitamins A, C, and B complex as well as linoleic acid were considered one of the most important composing of garlic. Besides, it consists of many useful components, for example, iodine salts, which possess positive influences on the cardiovascular system, silicates that have an affirmative effect on the skeletal and the cardiovascular system, and sulfur salts with positive effects on the skeletal system, cholesterolemia, and regulated liver diseases7. A. sativum extracts have shown a wide-range as antibacterial8,9 and antifungal activity10. Water-based garlic extract has potential antimicrobial activity for urogenital pathogens;
E. coli, Pr. mirabilis and T. vaginalis, and it can be utilize for blocking a many urogenital tract infections11.
Garlic (A. sativum) is a very commonly utilized
component as a food supplement and has many health benefits
that are related
to its bioactive components, however, the unique flavor and functions
promoted the health is usually caused
by sulfur composites of garlic, specifically γ-glutamyl, alliin, and their derivatives, moreover,
it has been estimated that the cysteine
sulfoxides and γ-glutamylcysteine peptides
are non- volatile over 82% of the total sulfur
content of garlic12. The most characteristic odor of garlic came from the predominant
thiosulphate known as Allicin which has an
antibacterial influence and toxic to insects13. The organosulfur
combinations from A. sativum for example alliin, allicin, and diallyl sulfide
supplied the most powerful of its biological activity in defense
against oxidative damage14.
Organosulfur compounds like allicin and diallyl disulfide (DADS are produced by garlic (belongs to the family of Allium) which give its bitter as well as spiced aroma. Its benefits have been recognized to hypotensive and vasorelaxant activities. Garlic preparations are widely available as garlic powder (GP) or garlic oil (GO). Epidemiological researches have shown a close bond between stomach cancer, Helicobacter pylori infection and the consumption of Allium vegetables. A long time ago, garlic has been used as herbal medications for treating a range of diseases like hypertension, rise blood cholesterol and thrombosis. Altogether, it can also yield opposite effects such as cutaneous agitation, edema and allergic interaction dermatitis15.
Both medicinal and economical depended
on herbal medication because it has become an item of global importance. Herbal therapies are successes and increasing
patient defiance as they are lacking of usual
side effects of allopathic medicines16. The subject of this work is to determine the composites of the aqueous
garlic extract with the evaluation of the antioxidant capacity and free radical scavenging activity of the extract.
Fresh garlic bulbs were purchased from a local store (Basra governorate, Iraq) in December 2019. Garlic bulbs were cleaned with distilled water, and aqueous extract was prepared according to Martha et al. methods17, 50g of garlic was homogenized in 100ml of cold distilled water, which gives the solution a concentration of 500mg/ml. The homogenized mixture was filtered two times through gauze. The clear solution was collected and then put in containers were can be dry out. Then storage for later analysis.
Functional groups in garlic extract were determined by using chemical indicators like Dragendroff reagent for alkaloid, Benedict's reagent for glycoside, Ferric Chloride for phenolic compounds, Lead Acetate and Potassium Hydroxide for flavonoid, and Mercury Chloride for saponin18.
The sample of the dried aqueous extracts of garlic was subjected to GC-MS Analysis in the College of Agriculture- the University of Basrah to chemical compound detection
Total antioxidant activity was mounted by using TAC reagent, which consists of 7.45ml of 0.9942g of Sodium Sulfate (28mM solution), Sulfuric acid (0.6mM solution), and 1.2359g of Ammonium Molybdate (4mM solution). These components were blended with 250ml distilled water. 300΅l of the extract was dissolved in 3 ml of TAC reagent. Blank was maintained with distilled water replacing the TAC reagent. All sample mixtures absorbance was measured at 695nm. Total antioxidant activity has appeared as the number of amounting to ascorbic acid19. The standardization curve was set by dissolving ascorbic (500, 400, 300, 200 and 100μg/mL) in methanol20.
The aqueous garlic extract antioxidant activity was evaluated based on the scavenging activities of the demonstrated 1, 1-diphenyl-2-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO) free radical. Various concentrations of 1000΅l of the test extract were added to 3ml of a methanol solution of DPPH (0.004%, w/v). Then lifted in the dark place at room temperature for 30 min, the absorbance has amounted against a blank at 517 nm with a spectrophotometer21. Free radical DPPH inhibition of in percent (%) was calculated by the formula:
DPPH inhibition Percentage (%) = [(ab blank ab sample)/ab blank)] *100
Where: ab blank is the control reaction (included all elements without test compound) absorbance, and ab sample is the test compound absorbance.
IC50 values (concentration of sample required to scavenge 50% of free radicals) were numbered from the recession equation, prepared from the concentrations of the extract, and percentage inhibition of free radical formation/percentage inhibition DPPH was assayed. Synthetic antioxidant component L-ascorbic acid (Sigma-Aldrich, St. Louis, MO), were used as positive controls and all tests were carried out in duplicate22.
Table 1: Chemical analysis of the garlic extract.
|
Functional group |
Indicator |
Result |
|
Alkaloids |
Dragendroff reagent |
+ |
|
Glycosides |
Benedict's reagent |
+ |
|
Phenol |
Ferric Chloride |
+ |
|
Tannins |
Lead Acetate |
+ |
|
Flavonoids |
Potassium Hydroxide |
+ |
|
Saponin |
Mercury Chloride |
- |
Fig. (1) GC-MS Chromatogram analysis for the Garlic Extract
The result revealed that the extract is composed of alkaloids, glycosides, phenol, tannins, and flavonoids by the positive reaction with the indicator while giving a negative reaction with the saponin indicator. Table (1):
The GC-Mass Spectroscopy chromatogram analysis showed 15 peaks revealed to 15 different compounds, (Fig. 1), as well as the identity and concentration of these compounds, shown in the table (2). The most important compounds included allyl trisulfide (1.89%), ascorbic acid 2, 6-dihexadecanoate (22.24%), 9, 12- Octadecadienoic acid (Z, Z) (11.44), and 6-Octadecenoic acid, (Z) (40, 28) (Fig.2)
Table 2: GC-MS analysis of garlic extract with retention time, identity, and concentration of compounds.
|
Peak# |
Ret. Time |
Area% |
Name |
|
1 |
3.615 |
1.89 |
Trisulfide, di-2-propenyl, Allyl trisulfide, Diallyl trisulfide, 1, 3-Diallyltrisulfane |
|
2 |
21.905 |
22.24 |
Ascorbic acid 2, 6-dihexadecanoate |
|
3 |
22.027 |
4.03 |
d-Glucitol, 2, 5-anhydro-1-O-octyl-, 2, 5-Anhydro-1-O-octylhexitol |
|
4 |
22.073 |
1.57 |
Heptane, 1-(ethenylthio) -, Sulfide, |
|
5 |
22.127 |
2.03 |
1-(.beta.-d-Arabinofuranosyl)-4-O-difluoromethyluracil |
|
6 |
22.193 |
1.00 |
Dodecanoic acid, (4-dodecanoylaminobutyl)amide, |
|
7 |
25.068 |
2.70 |
Hexadecyl heptafluorobutyrate |
|
8 |
26.267 |
11.44 |
9, 12-Octadecadienoic acid (Z, Z), Linoleic acid |
|
9 |
26.433 |
40.28 |
6-Octadecenoic acid, (Z) |
|
10 |
26.607 |
4.87 |
Boron, (2, 6-dimethyl-5-hepten-2-amine)tris(trifluoromethyl)-, (t-4)- |
|
11 |
26.673 |
1.37 |
N-Oleoyl-L-glycine, trimethylsilyl ester |
|
12 |
26.753 |
3.06 |
2-Bromotetradecanoic acid, Tetradecanoic acid, |
|
13 |
26.813 |
1.14 |
Lycophyll, .psi., .psi.-Carotene-16, |
|
14 |
27.093 |
0.79 |
Pyrimidine-5-carboxylic acid, 1, 2, 3, 4-tetrahydro-6-methyl-2-oxo-4-(2-thienyl)- |
|
15 |
27.132 |
1.60 |
Methyl trans-9-(2-butylcyclopentyl)nonanoate |
Fig. (2) The identification with the chemical structure of 1, and 2 peaks.
Fig. (3) the identification with the chemical structure of 8, and 9 peaks.
The results show that the garlic extract has an antioxidant of about 60 mg/ml as it was numerous to ascorbic acid (Fig.4).
Fig. 4. The total antioxidant of garlic extract which detected by
TAC.
Fig. 5. The free radical DPPH inhibition in percent by garlic extract.
The inhibition of free radical DPPH in percent by garlic extract was shown in figure 5. The garlic extract increased as the concentration raised. Also, the results revealed that IC50 of garlic extract was 308.57΅g/ml.
Antioxidant activity was detected by several assay methods in vitro and in vivo, but only a few quick and steadfast methods appropriate to antioxidant activity evaluate for an enormous number of plant extract samples exist2325. Total antioxidant capacity assay, such as the TAC and DPPH are the most common methods for the antioxidant detection for large-scale investigation. The Chemical analysis detected alkaloids, glycosides, phenol, tannins, and flavonoids in the aqueous extract of garlic but without saponin because of saponin was little dissolved in water. Moreover, maybe the storage time led to declining of polyphenol during the storage can be clarified by the possible oxidation of polyphenols to quinones.26,27.
A wide range of composites came from allicin transformed, including S-allylmercaptocysteine, allylmercaptan, allylmethyl diallyl disulfide, vinyldithiins, disulfide, ajoene, and possibly allylsulfonic acid and allylsulfinic28 which detected by GC-MS chromatography but in the low amount due to allicin analysis challenging because of its instability. Direct GC definition of allicin has not been achieved because it undergoes rapid breakdown in the oven of a GC, even at temperate temperatures28. Unsaturated fatty acid which is determined by GC-MS analysis in the aqueous extract has a role in the antioxidant activity because of the existence of the paired bond, they are more susceptible to oxidation than are the saturated fatty acids.
The results showed that garlic has antioxidants activity because of its contents specially Allyl trisulfide and ascorbic acid which were detected by GC-Mass spectroscopy chromatography analysis. Although phenolic-type compounds consider the most antioxidant activities sources deliver from plant29. Also, garlic consists of sulfur, phosphorus, zinc and potassium ions, temperate quantities of vitamin C, vitamin A, selenium, with smaller quantities of, calcium, sodium, magnesium, iron, B complex vitamins, and allicin, a composite to trap free radicals30. The antioxidant activity is directly related to the total phenolic content in herbs and vegetables and fruits which have been proved by previous studies3133. By comparison of antioxidant action and a total phenolic component of the medicinal herbs between aqueous and alcohol extract, there were different values of antioxidant activity and total phenolic content in aqueous extracts were lower than those in alcohol extracts29,34 however, major antioxidative activities refers to be phenolic and flavonoids35.
Aqueous garlic extract revealed its antioxidant action by scavenging some enzymes, likes catalase, superoxidase dismutase, and glutathione peroxidase, and prevents lipid peroxidation36. Antioxidant activity was straight resulted by the matters of phenolic composites33,37. The radical scavenging activity also related positively to the total phenolics of the garlic extract. These accomplishments are interconnected to the thiosulfinates, volatile sulfur compounds, and low-molecular-weight compounds38. Free radical scavenging, inhibition of hydrolytic one of flavonoids properties which are a group of polyphenolic compounds39. Gorinstein et al.40 detected the antiradical action in Allium species garlic, (white, red, and yellow) against onion by DPPH radical. In other research IC50 values estimated in the essential oil and ethanol extract of Tunisian garlic were 300μg/ml and 600΅g/ml respectively41 while the essential oil of Indians garlic has IC50 of 500΅g/ml42. It was clearly explained that the methanolic extract was having higher phenolic content43. The fresh garlic might be a basis of antioxidant properties as detected by DPPH scavenging analysis, There is an agreement with Rahman et.al.28. Some Allium species revealed antioxidant properties, in addition to these activities of bulbs and the parts above ground of garlic (Allium sativum L.), Allium fistulosum L., and other species of the genus, the radical scavenging is the main explanations to bioactivity mechanisms proposed by Allium species6.
Experiences showed that garlic has a very small activity as an antioxidant, only about 14mg/ml as it is calculated to ascorbic acid. IC50 values for aqueous extract of garlic was 308.57΅g/ml. The experiences also determined functional groups in garlic, which are Alkaloids, Glycosides, Tannins, and Flavonoids. The main important compounds which detected by GC-Mass were ascorbic acid, 2, 6-dihexadecanoate, 9, 12- Octadecadienoic acid (Z, Z), and 6-Octadecenoic acid (Z).
The authors are grateful to the authorities of University of Basrah Biology Department/ College of Science and Dr. Ali Al-Sharaa / College of Agriculture - for their help during our experiment.
The authors declare no conflict of interest.
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Received on 05.10.2020 Modified on 18.11.2020
Accepted on 12.12.2020 © RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(10):5157-5162.
DOI: 10.52711/0974-360X.2021.00897