In this research, we aim to explore the reactive chemical components of the essential oil of the methanol extracted C. pycnocephalus that are considered as the major factors affected on the biological aptitudes such as antioxidant, cytotoxic, and antibacterial activities. Each type of biological character is affected by a significant class of compounds that control its biological behavior i.e. the phenolic contents are crucial for potent antioxidant characters.
Gas-chromatography Mass Spectroscopy “gc-ms”
The components of the essential oil of the extracted C. pycnocephalus were elucidated by Gas-Chromatography Mass Spectroscopy “GC-MS” (c.f. Fig. 2 & Table 1). The results revealed that the extract of C. pycnocephalus includes about mainly six characterized components. In general, 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one located the major component with 57.43% composition which was identified after 9.11 min. Accordingly, other constitutes were characterized with high composition % such as (E)-4-(((2-methoxyoctadec-4-en-1-yl)oxy)methyl)-2,2-dimethyl-1,3-dioxolane (11.53%), methyl (E)-octadec-11-enoate (10.98%), and ethyl iso-allocholate (11.89%), which were identified after 4.92, 29.18, and 35.71 minutes. Additionally, 2-(hept-6-yn-1-yl)malonic acid (3.53%), and methyl 11-((2R,3S)-3-pentyloxiran-2-yl)undecanoate (4.64%) were recorded after 4.15, and 25.89 minutes of retention time. The compounds were classified under several types of naturally occurring components, in which the class of ester of fatty acid has the majority of these components. Therefore, the esters of fatty acids includes (E)-4-(((2-methoxyoctadec-4-en-1-yl)oxy)methyl)-2,2-dimethyl-1,3-dioxolane, methyl 11-((2R,3S)-3-pentyl-oxiran-2-yl)undecanoate, and methyl (E)-octadec-11-enoate. Another class of compounds was identified as hydrocarbons including two components namely, 2-(hept-6-yn-1-yl)malonic acid, and 3,5-dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one. Ethyl iso-allocholate followed the steroid class was found with 11.89% of composition (c.f. Supplementary file).
Alternatively, Al-Shammari et al. (2012), have interpreted the chemical constitutes isolated from the essential oil of C. pycnocephalus L. that grown in Saudi Arabia as basically nineteen components. Thus, the interpreted GC/MS of the plant extract indicated tetradecanoic acid (2.74%), hexadecanoic acid ethyl ester (4.81%), hexadecanoic acid (39.62%), phytol isomer (6.31%), linolenic acid ethyl ester (4.84%), 9,12-linoleic acid (19.46%), 1,2-benzendicarboxylic acid iso-octyl ester (7.11%), and heptacosane (2.34%). Therefore, the major component of this plant was related to hexadecanoic acid (39.62%), which followed the fatty acid class of compounds. On the other hand, the GC-MS spectroscopic analysis of C. pycnocephalus that grown in Iran revealed that the essential oil of this extract contains twenty-nine components such as hexadecanoic acid (23.3%), dibutyl, 1,2-benzene dicarboxylate (8.2%), 6,10,14-trimethyl-2-pentadecanone (7.4%), 1-pentyl octylbenzene (3.7%), and tetradecanoic acid (4.3%). The major component was found to be hexadecanoic acid (23.3%), which is related to the fatty acid class. The established components are terpenes with low composition percentages, fatty acids, and their esters, hydrocarbons, and alkylbenzenes.
Additionally, twenty components of the esters of fatty acids isolated from C. pycnocephalus that grown in Saudi Arabia including 1,2-benzene dicarboxylic acid, dimethyl ester (31.08%), azelaic acid dimethyl ester (7.6%), and palmitic acid methyl ester (20.08%) as the major constitutes of this plant extract. Other researches have been reported the composition of the components of the volatile oils of C. pycnocephalus grown in Iran (Esmaeili et al. 2005), and the identification of the terpenes, and flavone glycoside of C. pycnocephalus (Al-Shammari et al. 2015).
Table 1
The interpreted components of the essential oil of the extracted C. pycnocephalus.
Entry
|
Chemical name
|
Classification
|
Retention time (RT, min)
|
Molecular Weight
|
Molecular formula
|
Composition %
|
1
|
2-(Hept-6-yn-1-yl)malonic acid
|
Hydrocarbon “dicarboxylic acid”
|
4.15
|
198.22
|
C10H14O4
|
3.53
|
2
|
(E)-4-(((2-Methoxyoctadec-4-en-1-yl)oxy)methyl)-2,2-dimethyl-1,3-dioxolane
|
Ester of fatty acid
|
4.92
|
412.66
|
C25H48O4
|
11.53
|
3
|
3,5-Dihydroxy-6-methyl-2,3-dihydro-4H-pyran-4-one
|
Oxygenated hydrocarbon
|
9.11
|
144.13
|
C6H8O4
|
57.43
|
4
|
Methyl 11-((2R,3S)-3-pentyloxiran-2-yl)undecanoate
|
Ester of fatty acid
|
25.89
|
312.49
|
C19H36O3
|
4.64
|
5
|
Methyl (E)-octadec-11-enoate
|
Ester of fatty acid
|
29.18
|
296.50
|
C19H36O2
|
10.98
|
6
|
Ethyl iso-allocholate
|
Steroid
|
35.71
|
436.63
|
C26H44O5
|
11.89
|
|
|
|
|
|
|
Ʃ= 100.0
|
The Biological Characteristics Of The Plant Extracts
Once upon a time, medicinal plant extracts have been applied in the treatment of various diseases, where the discovery of medicines lies in the presence of their active essential components in the contents of the plant extract, which led to an advance in pharmacology. The C. pycnocephalus plant has been widely used in traditional medicine for the treatment of many diseases.
Antioxidant Activity - Dpph Assay
As commonly reported, the antioxidant activities of C. pycnocephalus extracts were evaluated by DPPH• (2,2′-diphenyl-1-picrylhydrazyl radical) colorimetric assay as the most commonly used assay for the extracted plants. The procedure is usually applied for the hydrophilic antioxidant constitutes, while their application for evaluating the antioxidant capacity of the hydrophobic components is limited. In this method, the reactive components that provided potent radical scavenging activities should contribute with a weak A-H bonding, which will increase the possibility for stabilizing or trapping the free radicals of DPPH• at a maximum wavelength 517 nm resulting in a discoloration of the DPPH molecule. The violet color of the DPPH radical will change subsequently to colorless by increasing the concentration of the scrutinized samples. The importance of antioxidants arises from the ability of these components to inhibit the oxidation of lipids. The process followed by scavenging or trapping the free radicals of DPPH, and hence determining the radical scavenging activity, as well as the assay in another route is an indication for reducing the capacity of the antioxidants in their reactions with DPPH radical.
The inexpensive reagents applied to enable to run of the investigated sample by this proficient colorimetric assay (Blois 1958; Bondet et al. 1997; Brand-Williams et al. 1995). The mechanism of DPPH assay is postulated to underwent by a mean of a HAT (Litwinienko & Ingold 2005), SET (Fotiet al. 2004; Huang et al. 2005), or mixed (Schaich et al. 2015) mechanisms as rendering to the succeeding reaction sequences:
DPPH·(violet) + XOH → DPPHH (colorless) + XO•HAT Eq. (1)
DPPH·(violet) + XOH → DPPHˉ (colorless) + XO•+ SET Eq. (2)
The phytochemical constitutes (Kozyra et al. 2019) such as phenolic, and flavonoid contents are proficient components that act as strong scavengers for the DPPH radical increasing the antioxidant potential of these samples to protect the lipoperoxy, and lipid peroxidative from oxidation progression. The plausible mechanism of action was controlled by several beneficial effects. Thus, the presence of antioxidant agents enables the scavenging of the free radicals of DPPH depending on the nature of the source of the free radical components, and the concentration of the inspected sample. Therefore, by increasing the sample concentration, the percent of radical scavenging activity will increase resulting in a release in the color intensity of the DPPH (Siripatrawan & Vitchayakitti 2016).
In this work, the antioxidant properties of root, stem, leaves, and flower extracts of C. pycnocephalus were in vitro evaluated by DPPH assay. The samples were tested for six diversified concentrations (5, 10, 20, 30, 40, and 50 mg/L). The results in Table 2 verified that flower extract is the most potent antioxidant agent with IC50 = 30.69 mg/L relative to the results of ascorbic acid (IC50 = 13.30 mg/L). The leaves extract of C. pycnocephalus located the second-order of radical scavenging capacity with IC50 = 32.78 mg/L, succeeding by the stem extract (IC50 = 41.31 mg/L), and root extract (IC50 = 46.84 mg/L). Table 2 consistently also presented the radical scavenging activity (%) of the extracted root, stem, leaves, and flower of C. pycnocephalus plant.
Figure 3 indicated the plotted percentages of radical scavenging activity against the various concentrations of each tested sample of C. pycnocephalus extract. A linear correlation between the scavenging activity % and their applicable concentrations (5-50 mg/L). The radical scavenging activity percent increased by increasing the sample concentration in a proportional relationship. Besides, the stem extract has the most potent scavenging activity for DPPH radicals at the lower concentration (5 mg/L) with % scavenging radical activity at 17.14±1.01%, however, the root extract located the second-order of activity with % scavenging radical activity at 12.58 ± 0.74% at the same concentration.
Table 2
The antioxidant results of the extracted root, stem, leaves, and flower of C. pycnocephalus plant.
Sample
|
Conc. (mg/L)
|
R1 [a]
|
R2 [b]
|
Mean value
|
Radical Scavenging Activity (%) [c]
|
IC50 (mg/L) [d]
|
Root
|
5
|
0.644
|
0.662
|
0.653
|
12.58 ± 0.74
|
46.84
|
10
|
0.553
|
0.531
|
0.542
|
27.44±1.61
|
20
|
0.508
|
0.53
|
0.519
|
30.52±1.80
|
30
|
0.457
|
0.475
|
0.466
|
37.62±2.21
|
40
|
0.415
|
0.41
|
0.4125
|
44.78±2.63
|
50
|
0.375
|
0.353
|
0.364
|
51.27±3.02
|
Stem
|
5
|
0.623
|
0.615
|
0.619
|
17.14±1.01
|
41.31
|
10
|
0.575
|
0.564
|
0.5695
|
23.76±1.40
|
20
|
0.465
|
0.457
|
0.461
|
38.29±2.25
|
30
|
0.427
|
0.419
|
0.423
|
43.37±2.55
|
40
|
0.391
|
0.397
|
0.394
|
47.26±2.78
|
50
|
0.338
|
0.331
|
0.3345
|
55.22±3.25
|
Leaves
|
5
|
0.666
|
0.673
|
0.6695
|
10.37±0.61
|
32.78
|
10
|
0.595
|
0.611
|
0.603
|
19.28±1.13
|
20
|
0.439
|
0.431
|
0.435
|
41.77±2.46
|
30
|
0.394
|
0.371
|
0.3825
|
48.80±2.87
|
40
|
0.341
|
0.337
|
0.339
|
54.62±3.21
|
50
|
0.213
|
0.208
|
0.2105
|
71.82±4.22
|
Flower
|
5
|
0.676
|
0.688
|
0.682
|
8.70±0.51
|
30.69
|
10
|
0.579
|
0.591
|
0.585
|
21.69±1.28
|
20
|
0.411
|
0.401
|
0.406
|
45.65±2.69
|
30
|
0.324
|
0.327
|
0.3255
|
56.43±3.32
|
40
|
0.292
|
0.285
|
0.2885
|
61.38±3.61
|
50
|
0.233
|
0.238
|
0.2355
|
68.47±4.03
|
[a], [b]: R1, and R2 referred to the values of the first and second read for the absorbance of the samples at different concentrations; [c]: RSA (%) indicated the Radical Scavenging Activity (%); [d]: IC50 referred to the inhibitive concentrations of the tested samples in mg/L.
At the concentration of 10 mg/L, the root extract was found with the most potent antioxidant activity (% RSA= 27.44±1.61%), and the stem extract has the second-order of antioxidant capacity (% RSA= 23.76±1.40%). The concentration of 20 mg/L of the investigated sample is more applicable for the comparison of the antioxidant scavenging radical activities for all the tested samples relative to the results of ascorbic acid. Thus, the flower extract revealed the potent antioxidant capacity of the other plant extracts with % RSA at 45.65±2.69%, this result is comparable with that of ascorbic acid (% RSA= 64.97%). Leaves extract situated the second-order of antioxidant potency (% RSA= 41.77±2.46%), then stem extract (% RSA= 38.29±2.25%), and finally the root extract (% RSA= 30.52±1.80%) at a concentration of 20 mg/L. In the same sequence, flower extract is the most potent antioxidant agent at the concentrations of 30, and 40 mg/L with % scavenging radical activity at 56.43±3.32, and 61.38±3.61%, respectively. The order of activity of the other plant constitutes is followed the extract of leaves (% RSA= 48.80±2.87 & 54.62±3.21%), stems (% RSA= 43.37±2.55 & 47.26±2.78%), and to end with the root (% RSA= 37.62±2.21 & 44.78±2.63%). Unexpectedly, at the higher concentration (50 mg/L), we noticed that leaves extract has the most potent antioxidant activity with % scavenging radical activity at 71.82±4.22% than the flower extract with % scavenging radical activity at 68.47±4.03%. The behavior of stem and root extracts at the concentration of 50 mg/L followed the same sequence of the lower concentration (20, 30, and 40 mg/L) with % scavenging radical activity at 55.22±3.25%, and 51.27±3.02% (Table 2). The values of inhibitive concentrations “IC50” is the half-maximal inhibitory concentration expressed the potential concentration of the tested sample that achieves scavenging for the radicals of DPPH by 50%. The values of IC50 were calculated from the exponential curve (Parejo et al. 2000) plotting the sample concentrations against the remaining percent of DPPH• radical applying linear regression analysis.
The relationship between the IC50 values and the free radical scavenging activity percentages are inversely proportional. As specified from the results of IC50 values in Table 2, and Fig. 4, the flower extract is the most potent antioxidant agent with IC50 = 30.69 mg/L, relative to the IC50 value of ascorbic acid (IC50= 13.30 mg/L). The order of antioxidant capacity of the other plant materials is found in the following order: leaves (IC50= 32.78 mg/L) > stem (IC50= 41.31 mg/L) > root (IC50= 46.84 mg/L). The significant antioxidant potency of the samples depended in this scale of comparison on the nature of the chemical components contained in the distinct extract as the presence of electron sources such as reactive oxygen or nitrogen sorts (Tiwari et al. 2013; Singh et al. 2021) such as hydroxyl, phenoxyl, alkyl peroxyl, linoleic acid, peroxyl, and glutamyl radicals that can stabilize or trap the DPPH free radical improve the antioxidant aptitude (Kwak et al. 2009; Mishra 2016).
Potential Antibacterial Activity
The antibacterial activity of the extracted botanical ingredients of C. pycnocephalus from methanol was assessed by a disc diffusion technique as an in vitro antimicrobial susceptibility testing. The tested samples were prepared in a concentration of 10 mg/L from the root, stem, leaf, and flower extracts. The results are shown in Table 3, and Figure 5 revealed that the four samples are potent antibacterial agents against E. coli, P. aeruginosa, S. typhimurium, and B. cereus bacterial species. Particularly, potent antibacterial activities were recorded for leaf, and flower extracts against E. coli species with inhibition zones equivalent to that of antibiotic standards (20 mm). The root and stem extracts revealed remarkable antibacterial activities against P. aeruginosa species with inhibition zones 22, and 20 mm, respectively, with high potency than the antibiotic standard “Azithromycin” (13 mm), along with good activities of the leaf, and flower extracts with inhibition zones at 10 mm.
By studying the results of the antibacterial activities of samples as inhibitors of bacterial growth, we found that the four samples had distinct activities in the process of inhibiting the growth of bacterial species of the type S. typhimurium by 14, 13, 26, and 25 mm, respectively, relative to the results of the antibiotic “Tetracycline” (10 mm). Medium to high activities of the four samples was observed against S. epidermidis bacterial strains compared to the results of the four antibiotics, while the highest result of inhibiting the growth of those bacterial species was for the flower extract with an inhibition zone at 15 mm. Looking at the results of the four samples as components of C. pycnocephalus, we found that the activity of methanolic extracts against Gram-positive bacterial species is not good compared to their results with Gram-negative bacteria. This does not indicate the lack of quality of those extracts to inhibit the growth of Gram-positive bacteria, as some extracts have higher results than antibiotics such as extracts of leaves and flowers towards inhibiting the growth of bacterial species of type B. cereus by an amount of 23 and 25 mm, respectively, and these results are higher than that obtained by all the antibiotics (5-20 mm) (Table 3).
Table 3
The antimicrobial activities are represented by the inhibition zone diameter (mm) of the methanol extract of C. pycnocephalus and standard antibiotics.
Microbes
|
Inhibition zone in mm
|
C. pycnocephalus (10 mg L-1)
|
Standard antibiotic (10 mg L-1)
|
Root
|
Stem
|
Leaf
|
Flower
|
Cephradin
|
Tetracycline
|
Azithromycin
|
Ampicillin
|
|
Gram-negative bacteria
|
|
|
|
|
E. coli
|
13
|
10
|
20
|
20
|
15
|
20
|
20
|
20
|
P. aeruginosa
|
22
|
20
|
10
|
10
|
0
|
0
|
13
|
0
|
S. typhimurium
|
14
|
13
|
26
|
25
|
0
|
10
|
0
|
0
|
S. epidermidis
|
10
|
5
|
5
|
15
|
10
|
20
|
23
|
10
|
|
Gram-positive bacteria
|
|
|
|
|
B. cereus
|
11
|
13
|
23
|
25
|
20
|
10
|
20
|
5
|
S. aureus
|
12
|
10
|
10
|
14
|
20
|
20
|
20
|
30
|
S. haemolyticus
|
0
|
0
|
5
|
5
|
25
|
23
|
23
|
20
|
S. xylosus
|
0
|
0
|
0
|
5
|
20
|
20
|
20
|
25
|
K. pneumonius
|
10
|
10
|
13
|
13
|
10
|
20
|
13
|
5
|
LSD0.05 [a]
|
1.61
|
1.32
|
1.47
|
1.50
|
1.71
|
1.84
|
1.41
|
2.31
|
[a] LSD0.05 expressed the calculated least of the smallest significance between two means as each test was run on those two means (calculated by Factorial ANOVA). |
Among the good results are also a high activity of inhibiting the growth of S. aureus of the flower methanolic extract by an amount of 14 mm and the good activity of leaf and flower extracts as inhibitors of K. pneumonius growth with an amount of 13 mm compared to the highest inhibitory activity of the antibiotic "Tetracycline" with an inhibition efficiency of 20 mm. The results also demonstrated that the lowest efficiency to inhibit the growth of bacterial microorganisms was found against S. haemolyticus and S. xylosus using any of the four extracts with inhibition efficiency from inactive to 5 mm (Table 3).
The mechanism of action for the bacterial infections (Sawa, et al. 2019; Bascones-Martínez et al. 2009) is controlled by six main factors: (1) the interface with the bacterial cell wall (inhibit cell wall synthesis) (Mohammad et al. 2017), (2) depolarize the cell membrane (Higgins et al. 2005), (3) inhibition of the protein synthesis (De Vriese et al. 2006), (4) inhibition of nucleic acid synthesis (Srivastava et al. 2011), and (5) metabolic pathway inhibition (Li et al. 2019). Additionally, five basic mechanisms of action for antibiotics (Etebu & Arikekpar 2016) are known including the inhibition of cell wall synthesis, protein synthesis (translation), nucleic acid synthesis, cell membranes alteration, and antimetabolite activity. The action of the tested samples as antibacterial agents is to continually disrupt and prevent the growth of bacterial species. The interpretation of these results is consistent with previous results in deducing the efficacy of the plant extract as an antimicrobial agent (El-Shahaby et al. 2013).
Cytotoxic Activity
The cytotoxic activity of C. pycnocephalus methanol extract was assessed using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay under distinct conditions. The method is identical to a cell number growth curve. The MTT reagent as a reliable indicator is so sensitive to the light, this engaged the run of the experiments in the dark. Hepatocellular carcinoma (HepG2) tumor cell line was selected to assess the anticancer potency of the investigated extracted medicinal plant. The method was applied for determining the cell metabolic activity based on the aptitude of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzymes to reduce the MTT tetrazolium dye to its formazan “insoluble” that has a purple color. The number of viable cells should increase with growth, decrease with cytotoxic treatments, and remain the same (or plateau) with cytostatic treatments. The IC50 values expressed the concentration that represented 50% of the inhibition of cell growth was calculated by applying the curves obtained from plotted the percentages of cell survival versus drug concentration (µM). Thus, the potency of cytotoxicity will rise by the decrease in the extract concentration and IC50 values. The MTT solution may be affected by the results of cytotoxicity, so we have run a control sample “blank” that was a few "empty" wells containing MTT solution without any of the cell lines. The control sample is a benefit for calculating the cell viability percent, as it produces 100% viability of healthy cells. The experiments were run using five concentrations of each plant extract (31.3, 62.5, 125, 500, and 1000 µg/mL) prepared in a serial dilution (Table 4).
Table 4
Cytotoxic results of the extracted C. pycnocephalus against HepG2 tumor cell line.
Samples | Conc. (µg/mL) | R1 [a] | R2 [a] | IC50 (µg/mL) [b] |
C. pycnocephalus extract | 1000 | 0.714 | 0.755 | 46.2 |
500 | 1.4 | 1.32 |
125 | 2.4 | 1.7 |
62.5 | 1.6 | 1.7 |
31.3 | 1.8 | 1.8 |
0 | 1.3 | 1.3 |
[a] R1, R2 are the absorbance read of the extracts at diverse concentrations. |
[b] IC50 values are the inhibitive concentration expressed sample concentration that contributes roughly 50% of the death of cancer cells. |
Table 4 signified the results of the cytotoxic effects specified by the inhibitive concentration values of the extract of C. pycnocephalus on HepG2 tumor cell line. The results of cytotoxicity revealed that the extract of C. pycnocephalus has a potent cytotoxic effect on HePG-2 cell line with an IC50 value at 46.2 µg/mL. It is worth mentioning that the mechanism of cytotoxicity of the extracted samples as cytotoxic agents on HepG2 tumor cell line is commonly dependent on the structural nature of the components of each extract, and the nature of the cancer cell line. Additionally, the nature of the particles of the extracted plants such as surface morphology, size, and aggregation of the particles might control the results of cytotoxicity. The efficiency of the plant extract as an efficient anti-cancer agent for tumor cell growth depended on several factors as previously noted, including the nature of the chemical components of this extract, the type of cancer cell, and the concentration of the extracted plant used in this assess (Khacha-Ananda et al. 2013).
EC 50 of C. pycnocephalus extract
The dose-response relationship of the assessed C. pycnocephalus extract is plotted in Figure 6. The dose-response curve in Fig. 6a was normalized in the X-axis direction by its EC50 value (Fig. 6b). The value of EC50 of the methanol extract of C. pycnocephalus was initially calculated by plotting the sample absorbance against the log of doses at different concentrations of the serial dilution (Fig. 6). The low concentrations of the extract are not enough to produce a response, while the high doses produce a maximal response, and the vertical point of the curve resembles an EC50 value. The data analysis specified that the higher concentration (dose = 1000 µg/mL) as calculated for EC50 value (2.82 µg/mL) has a cytotoxic effect on HepG2 cell lines.