Percentage yields of extracts
In the preparation of crude methanol, chloroform and petroleum ether extract from the dried leaves of Artemisia absinthium different percent yields were obtained Among the solvents used for extract methanol gave the highest and petroleum ether has the lowest percent yields. The yield of extraction by maceration in different solvents was calculated by equation shown below respectively and summarized in Table 1.
%Yield of crude extract (w/w) = \(\frac{ \text{m}\text{a}\text{s}\text{s} \text{o}\text{f} \text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t} \text{i}\text{n} \text{g}\text{r}\text{a}\text{m}}{\text{t}\text{o}\text{t}\text{a}\text{l} \text{m}\text{a}\text{s}\text{s} \text{o}\text{f} \text{p}\text{l}\text{a}\text{n}\text{t} \text{m}\text{a}\text{t}\text{e}\text{r}\text{i}\text{a}\text{l} \text{u}\text{s}\text{e}\text{d}}\)X 100%
Table 1
Yield of plant extracts in both crude extract
S.No
|
Types of extract
|
Yield of extract (%)
|
1
|
Petroleum ether Crude extract
|
10
|
2
|
Chloroform Crude extract
|
13
|
3
|
Methanol crude extract
|
17
|
Calculating yield of extracts were serious tasks behind phytochemistry, because biological active component of medicinal plant depends on yield. In the preparation of crude methanol extract from the dried leaves of Artemisia absinthium a yield 13.415% was obtained. According to the previous study, it is agreeing with reported by [45]. A yield of12% [46], which extracted by 80% methanol. But, yield of crude methanol extract of test plant disagree with the previous studies which were reported by a yield of 11.4%. [47]. The observed difference may be due to the amount of solvent used, on previous studies 80% methanol and 20% water was used but, in this study normal methanol without water was used. The duration of maceration, on previous work were socked for 3 days but, in this study were socked for 5 days. This study also disagrees with reported by [48] a yield of 5.44% due to the type of solvent. From this previous study was used acetone as solvent but, in this work used methanol as solvent.
Finally, it disagrees that was reported by a yield of 8.4% [49]. Due to the type of solvent which used water as a solvent. Generally, methanol is good solvent in this study to increase yield of extraction that compared the previous study using 80% methanol, chloroform and aqueous extract. The observed difference may be due to the weight of fresh plant material that was used. The yield solvent maceration also depends on polarity of solvent which showed maximum yield for more non polar and more polar solvent.
Phytochemical Screening Tests Of Extracts
Preliminary phytochemical analysis of the crude methanol extract revealed the presence of alkaloids, flavonoids, terpenoids, phenol, tannins and oxalate in A. absinthium leaves and absence of anthraquinone, saponins, steroids and quinine was investigated in the sample extract by different test methods (Table 2).
The existence of these phytochemicals in the extracts may contribute the plant to be known of its medicinal use especially for antimicrobial activity. Tannin, alkaloids and flavonoids have been reported potential ingredients towards the treatment intestinal disorders [48], pain killer and inflammation, respectively [49].
The findings of the present study agreed with previous studies; for chloroform extracts and Hussein Haji et. al in 2016 for aqueous extracts reported that, leaves of A. absinthium possess tannins and phenols [38, 47]. In addition to the previous work, this study showed the presence of alkaloid, flavonoids, terpenoids and oxalate in methanol extract. This implies that methanol is the best solvent compared to chloroform and water for extraction of A. absinthium.
Phytochemical of determination of A. absinthium showed that the presence of constituents like alkaloids, flavonoids, tannins, quinones, steroids, phenols, terpenoids and absence of constituents such as carbohydrates, saponins, glycosides, anthraquinone, coumarins, gums, protein and amino acid in methanol extract. Chloroform extract showed the presence of alkaloids, flavonoids, carbohydrates, tannins, glycosides, quinones, terpenoids, steroid, coumarins, amino acid, phenols anthraquinones and absence of constituents such as saponins, glycosides, protein and gums.
According to [25] the phytochemical analysis of A. absinthium showed the presence of saponins, flavonoids, phenols, tannins, quinones, steroids methanol ethanol extract and acetone extract showed the presence of phenols, tannins, quinones, alkaloids, carbohydrates. And also according to [49] the presence of alkaloids, flavonoids, carbohydrates, tannins, quinines, proteins, phenols and terpenoids.
Table 2
Phytochemical test result of leafs extracts of A. Absinthium
Secondary Metabolites
|
Petroleum ether
|
chloroform
|
Methanol
|
Alkaloid
|
+
|
+
|
+
|
Flavanoid
|
+
|
+
|
+
|
Carbohydrates
|
-
|
+
|
-
|
Tanins
|
-
|
+
|
+
|
Saponins
|
-
|
-
|
-
|
Glycosides
|
-
|
-
|
-
|
Quinones
|
+
|
+
|
+
|
Anthroquinone
|
-
|
+
|
-
|
Steroid
|
+
|
+
|
+
|
Coumarins
|
-
|
+
|
-
|
Amino Acid
|
-
|
+
|
-
|
Protein
|
-
|
-
|
-
|
Gums
|
-
|
-
|
-
|
Phenol
|
+
|
+
|
+
|
Terpenoid
|
+
|
+
|
+
|
+ = present - = absent
In the course of this study, the essential oil obtained from A. absinthium was studied. The oil was obtained using hydro distillation and the yields was 0.5% and the oil was aromatic. The major components of the oil were identified. The essential oil was analyzed by GC and constituents of the oil were identified by using RT and NMR. The identified components account for 56% of the oil. The oil contains camphor as a major constituent and account for 41% of the oil. Figure 1 shows the GC of was overlapped with camphor and essential oil A. annua.
From column chromatography two compounds, chamazulene and davanone were isolated and identified on the bases of their 1H NMR spectra. The spectra obtained were comparable with literature values (Table 3) [50].
Table 3
1HNMR spectra of chamazulene and davanone compared with literature value
Position
|
1HNMR, δ (ppm)
|
Chamazulene
|
[50]
|
Davanone
|
[50]
|
1
|
-
|
-
|
5.19, 4.98 (trans)
4.98 (trans)
|
5.04
|
2
|
-
|
-
|
5.88
|
5.78
|
3
|
6.90
|
6.55
|
|
|
4
|
7.38
|
7.15
|
|
|
5
|
-
|
-
|
|
|
6
|
8.14
|
7.93
|
3.26
|
3.88
|
7
|
-
|
-
|
2.68
|
2.60
|
8
|
-
|
-
|
|
|
9
|
7.20
|
7.05
|
|
|
10
|
7.60
|
7.45
|
|
|
11
|
-
|
-
|
|
|
12
|
-
|
-
|
1.64
|
1.57
|
1’
|
2.87
|
2.71
|
1.27
|
1.17
|
2’
|
1.38
|
1.33
|
0.99
|
0.88
|
3’
|
2.67
|
2.55
|
1.77
|
1.70
|
4’
|
2.85
|
2.75
|
|
|
The GC of these two isolated compounds were overlapped with the oil (Fig. 1). The oil is composed of 0.6% chamazulene and 14% davanone.
Antibacterial activity
Determination of zone of inhibition for the extract of the plant material against clinical pathogenic bacteria was effective. Crude methanol and chloroform extract of this plant was active both gram positive bacteria and gram negative bacteria due to the nature of cell wall of bacteria. The anti-bacterial activity of crude methanol extract results is presented in Tables 3. The antibacterial activity of plant extracts was performed at concentrations of 60, 130 and 200mg/mL. The maximum zone of inhibition for the crude methanol extract was achieved for S. aureus (11.77 ± 0.86) at the concentration of 200mg/ml, while the minimum antibacterial activity was obtained (0) at a concentration of 60 mg/ml and 130mg/ml in the same organism. L. monocytogen showed antibacterial activity both the concentration of 60mg/ml,130mg and 200 mg/ml respectively (12.89 ± 0.62, 11.62 ± 0.6 and 11.22 ± 0.36). and gram negative E. coli did not show antibacterial activity at any concentration shown in Table 4. K. pneumonia showed antibacterial activity both the concentration of 60mg/ml and 200 mg/ml respectively (12.9 ± 0.56, and 12.26 ± 0.1)
Table 4
Mean inhibition zone of methanol extract of Artemisia absinthium at different concentration and different test bacteria
Extracts
|
Concentration (mg/ml)
|
Zone of inhibition in Mean ± SD
|
S. aureus
|
L. monocytogen
|
E. coli
|
K. Pneumonia
|
Methanol
|
60
|
-
|
11.22 ± 0.36-
|
-
|
-
|
130
|
-
|
11.62 ± 0.6
|
-
|
12.26 ± 0.1
|
200
|
11.27 ± 0.89
|
12.89 ± 0.63
|
-
|
12.9 ± 0.56
|
Chloroform
|
60
|
-
|
11.33 ± 0.33
|
-
|
-
|
130
|
-
|
11.36 ± 0.67
|
-
|
10.77 ± 0.34
|
200
|
13.23 ± 0.35
|
12.02 ± 0.79
|
-
|
12.37 ± 79
|
Chlora.
|
1000 µg/ml
|
9.03 ± 0.85
|
8.07 ± 0.1.6
|
9.45 ± 0.44
|
-
|
DMSO
|
100µL
|
-
|
-
|
-
|
-
|
Chlora. = Chloramphenicol DMSO = dimethyl sulfoxide Conc. = Concentration
|
According to Table 3 the extracts isolated from Artemisia absinthium showed antibacterial activity against three of the tested bacteria which was indicated by clear zone of inhibition. These are matched with the previous researches has antimicrobial action. [42]. The results also that showed the anti-bacterial activities of Artemisia absinthium extracts revealed concentration dependent inhibition zone. In this biological active compound, zone of inhibition in all tested bacteria increased with increasing of the extract concentration, this is might be due to the reason that, as increasing the concentration of extract increased activity of secondary metabolites, in which chemicals are depend on concentration behind phytochemistry.
The above Table showed that, A. absinthium extracts showed high antibacterial only L. monocytogen at all concentration, while Staphylococcus aureus showed only at 200 mg/ml which implies it is concentration dependent and highly drug resistance bacteria. Therefore, zone of inhibition of these bacteria is effective at the concentration of 200 mg/ml based on this work. But, E. coli did not show antibacterial activity at all concentration due to their structural membrane. Gram positive bacteria are more susceptible towards plants extracts as compared to gram negative bacteria [49]. This is because the fact that the cell wall in gram positive bacteria is a single layer and the gram negative cell wall is multilayered structure [42, 47].
For the present study, crude extracts of leaves of A. absinthium which showed antibacterial effect against gram positive bacteria (L. monocytogen) at all concentrations. However, methanol extracts of A. absinthium did not show any zone of inhibition for gram negative bacteria (E. coli). This result agrees with the previous reports in which both methanol and chloroform extract could not inhibit the growth of gram negative bacteria. Preliminary qualitative phytochemical screening showed the antibacterial activity of methanol extract due to the presence of alkaloid, terpenoids, flavonoids, tannins and phenol compound by integration.
In Table 3 methanol and chloroform extract had greater zone of inhibition than positive control chloramphenicol. This difference is may be integration effect of different phytochemical of plant extract but, positive control is the most isolated and purified clinical isolated drug those isolated pathogens. The anti-bacterial activity of crude chloroform extract results are presented in Tables 3. The antibacterial activity of plant extracts were performed at concentrations of 60, 130 and 200mg/mL. The maximum zone of inhibition for the crude chloroform extract was achieved for S. aureus (13.23 ± 0.35) at the concentration of 200mg/ml, while the minimum antibacterial activity was obtained (0) at a concentration of 60 mg/ml and 130mg/ml in the same organism. L. monocytogen showed antibacterial activity both the concentration of 60 mg/ml,130 mg/ml and 200 mg/ml respectively (12.02 ± 0.79, 11.33 ± 0.33 and 11.36 ± 0.67). and gram negative E. coli did not show antibacterial activity at any concentration shown in Table 4. K. pneumonia showed antibacterial activity both the concentration of 60mg/ml and 200 mg/ml respectively (12.37 ± 0.79, and 10.77 ± 0.34). Moreover, chloroform extract at a concentration 200 mg/ml showed high inhibition zone than methanol extract at the same concentration towards S. aureus, L. monocytogen and K. Pneumonia, respectively, 13.23 ± 0.35, 12.02 ± 0.79 and 12.37 ± 79.