Phytochemical screening of extracts
Qualitative phytochemical tests of
Anethum sowa L. were performed for the methanol (MeOH), ethyl acetate (EtOAc), chloroform (CE), pet-ether (PE cold) and hexane (Hex.hot) extracts of the root. The results of various chemical tests for the detection and identification of chemical constituents were summarized in Table
2. In the present study, flavonoids, glucosides, cardiac glycosides and anthraquinone glycosides were present in MeOH, EtOAc and CE extracts, but in addition, the saponins were present only in MeOH extract. Steroids were found in all the extract except MeOH extract. On the other hand, all the groups were absent in PE (cold) and Hex.(hot) extracts except steroids. However, alkaloids, carbohydrates and tannins were highly present in the MeOH extract whereas, EtOAc extract showed slightly positive test for alkaloids and absent in CE extract. The carbohydrates showed slightly positive in CE and EtOAc extract.
Table 2
Phytochemical constituents of different extracts (PE, Hex, CE, EtOAc and MeOH) of Anethum sowa L. root
Saponins | a) Frothing | a) (−) | a) (−) | a) (−) | a) (−) | a) (+++) |
b) Emulsification | b) (−) | b) (−) | b) (−) | b) (−) | b) (+++) |
Flavonoids | a) Alkaline reagent Test | a) (−) | a) (−) | a) (+++) | a) (+++) | a) (+++) |
b) Lead acetate Test | b) (−) | b) (−) | b) (++) | b) (++) | b) (++) |
Alkaloids | a) Mayers’ reagents | a) (−) | a) (−) | a) (−) | a) (+) | a) (+++) |
b) Hager’s Test | b) (−) | b) (−) | b) (−) | b) (+) | b) (++) |
c) Wagner’s Test | c) (−) | c) (−) | c) (−) | c) (+) | c) (++) |
Steroids | a) Salkowski test: | a) (+++) | a) (+++) | a) (+++) | a) (+++) | a) (−) |
b) Liebermann-Burchard’s test | b) (+++) | b) (+++) | b) (+++) | b) (+++) | b) (−) |
Carbohydrates | a) Molisch’s test | a) (−) | a) (−) | a) (+) | a) (+) | a) (+++) |
b) Fehling test for reducing sugar | b) (−) | b) (−) | b) (−) | b) (−) | b) (+++) |
Glucosides | a) General test | a) (−) | a) (−) | a) (++) | a) (++) | a) (+++) |
b) Test for glucosides | b) (−) | b) (−) | b) (+) | b) (+) | b) (+++) |
Cardiac glycosides | a) Keller Killiani test | a) (−) | a) (−) | a) (+++) | a) (+++) | a) (+++) |
b) Baljet Test | b) (−) | b) (−) | b) (+++) | b) (+++) | b) (+++) |
Anthraquinone glycosides | a) Borntrager’s Test |
i) O-glycoside | i) (−) | i) (−) | i) (++) | i) (++) | i) (+++) |
ii) C-glycoside | ii) (−) | ii) (−) | ii) (−) | ii) (−) | ii) (−) |
Tannins | a) Gelatin-salt block test: | a) (−) | a) (−) | a) (−) | a) (−) | a) (+++) |
b) Lead acetate test | b) (−) | b) (−) | b) (−) | b) (++) | b) (+++) |
c) Condensed or Phlobatanins | c) (+++) | c) (+++) | c) (++) | c) (++) | c) (++) |
Cyanogenetic glycosides | a) Sodium picrate test | a) (−) | a) (−) | a) (−) | a) (−) | a) (−) |
The value of medicinal plants is due to phytochemical constituents or secondary metabolites that synthesized by the plant body during the plants normal metabolic processes and plants use them to protect themselves against different pathogenic attack [
27]. The optimal effectiveness of a medicinal plant may not be due to the one main active constituent, but may be due to the combined action of different compounds originally in the plant [
28]. Cardiac glycosides work by inhibiting the Na
+/K
+ pump. This causes an increase in the level of sodium ions in the myocytes, which then leads to a rise in the level of calcium ions. This inhibition increases the amount of Ca
2+ ion available for contraction of the heart muscle, improves cardiac output and reduces distention of the heart [
29]. Cardiac glycosides were found to be present in MeOH, EtOAc and CE extracts, it can be aided in treatment for congestive heart failure and cardiac arrhythmia. Alkaloids are nitrogen-containing naturally occurring compound, commonly found to have antimicrobial properties due to their ability to intercalate with the DNA of the microorganisms [
30]. Flavonoids and tannins are phenolic compounds and plant phenolics are a major group of compounds that act as primary antioxidants of free radical scavengers [
31]. Flavonoids are also found to be effective antimicrobial substances in vitro against a wide array of microorganisms by inhibiting the membrane bound enzymes. They have been reported to possess substantial anti-carcinogenic and anti-mutagenic activities due to their anti-oxidant and-inflammatory properties. They are also active in reducing high blood pressure [
30]. On the other hand, tannins play a major role as antihaemorrhagic agent and have been shown to have immense significance as antihyper cholesterol, hypotensive and cardiac depressant properties [
32]. Glycosides serve as defense mechanisms against predation by many microorganisms, insects and herbivores [
33]. Moreover, glycosides, flavonoids, tannins and alkaloids have hypoglycemic activities [
34]. Saponins are another type of bioactive chemical constituents which are involved in plant disease resistant because of their antimicrobial activity [
35]. On the other hand, saponins have been shown to possess both beneficial (cholesterol-lowering) and deleterious (cytotoxic; permeabilization of the intestine) properties [
36]. Although, some saponins have been shown to be highly toxic under experimental conditions and acute poisoning is relatively rare both in animals and man [
37]. Hypocholesterolemic and antidiabetic properties of saponins are also reported [
38]. Medicinal plants are the best sources for chemical ingredients. Based on preliminary phytochemical studies, the results revealed that Methanol, ethyl acetate and chloroform crude extracts of
A. sowa root contained larger amounts of bioactive secondary metabolites that may be potential as chemotherapeutic drugs.
Antioxidant activity on DPPH radical
The free radical scavenging activity of the root extracts of
Anethum sowa L. were measured by DPPH assay and the summarized results are shown in Table
3. All the extract showed a significant DPPH scavenging activity in a concentration dependent manner. The lower the IC
50 value is, the greater the free radical scavenging activity is. For each sample eight concentrations (1.562-200 μg/mL) were tested. Methanol extract exhibited considerably higher DPPH radical scavenging activity (96.18 %) with the IC
50 value of 13.08 μg/mL than other extracts and the lowest (33.71 %) DPPH scavenging rate was found in the hexane (hot) extract with the IC
50 value of 8.33 mg/mL. The free radical scavenging activities of the extracts decreased in the order of methanol > ethyl acetate > chloroform > pet.ether(cold)>hexane(hot). The IC
50 values were also shown in Table
3. The ethyl acetate and chloroform extract showed the significant radical scavenging activity of 87.11 and 74.97 % with the IC
50 values of 33.48 μg/mL and 36.42 μg/mL respectively. The IC
50 value of the extract was found to be very fair compared to the IC
50 value of the reference standard ASA (3.74 μg/mL) and BHT (11.84 μg/mL) with the highest inhibition of 99.05 and 93.02 % respectively. Moreover, the methanol extract showed the same radical scavenging activity like a standard at the same concentrations.
Table 3
Antioxidant activity of different extract and standard (Average ± SD)
200 | 2.30 | 33.71 ± 0.33 | 35.91 ± 0.65 | 74.97 ± 0.89 | 87.11 ± 0.61 | 96.18 ± 0.31 | 93.02 ± 0.50 | 99.05 ± 0.21 |
100 | 2 | 22.26 ± 0.97 | 22.09 ± 0.45 | 70.23 ± 0.53 | 79.35 ± 0.44 | 87.34 ± 0.27 | 91.98 ± 0.19 | 98.35 ± 0.17 |
50 | 1.69 | 10.29 ± 0.37 | 13.26 ± 0.33 | 60.72 ± 0.38 | 57.41 ± 0.27 | 79.71 ± 0.60 | 85.51 ± 0.76 | 90.71 ± 0.60 |
25 | 1.39 | 8.57 ± 0.31 | 11.60 ± 0.29 | 44.63 ± 0.19 | 32.27 ± 0.68 | 66.46 ± 0.34 | 69.54 ± 0.60 | 85.31 ± 0.60 |
12.5 | 1.09 | 5.14 ± 0.18 | 8.29 ± 0.20 | 23.80 ± 0.51 | 22.58 ± 0.44 | 47.49 ± 0.93 | 55.87 ± 0.82 | 79.79 ± 0.18 |
6.25 | 0.79 | 1.71 ± 0.06 | 1.65 ± 0.04 | 16.08 ± 0.57 | 14.82 ± 0.54 | 33.53 ± 0.34 | 29.69 ± 0.97 | 69.67 ± 0.68 |
3.125 | 0.49 | 0 | 0 | 10.72 ± 0.38 | 9.01 ± 0.75 | 20.87 ± 0.53 | 25.63 ± 0.51 | 37.26 ± 1.12 |
1.562 | 0.19 | 0 | 0 | 5.36 ± 0.19 | 1.93 ± 0.09 | 12.65 ± 0.26 | 9.05 ± 0.51 | 27.02 ± 1.19 |
IC50 value | 8.33 ± 0.21 mg/mL | 4.74 ± 0.22 mg/mL | 36.42 ± 0.41 μg/mL | 33.48 ± 0.16 μg/mL | 13.08 ± 0.03 μg/mL | 11.84 ± 0.29 μg/mL | 3.74 ± 0.05 μg/mL |
Antioxidant activity depends on their scavenging powers which are useful for the management of many disorders like atherosclerosis, angina pectoris, neurodegenerative diseases and cancer diseases. Many synthetic antioxidants such as butylated hydroxy anisole (BHA) and butylated hydroxyl toluene (BHT) are commercially available and widely used, but they may possess some side effects and toxic properties to human health [
39]. So, attention has been directed towards the natural antioxidants from botanical sources, especially plants. However, a significant correlation is observed between the antioxidant activity of herbs and the phytochemical content.
Plant phenolic compounds or poliphenols are the natural antioxidants which can act as a free radical scavenger, metal chelators and singlet oxygen quenchers and as a multi-functional activity [
40]. The anti-oxidative potential of flavonoid depends on their chemical structure. The hydroxyl groups of phenolic compounds allow them to exert direct anti-oxidative activity and were found to play a vital role in stabilizing lipid peroxidation. The most active antioxidant compound is catechol, which possesses two hydroxyl groups in the ortho position. The first chain carrying peroxyl radical was being trapped by H-atom transfer from the labile phenolic O–H and the second by reaction with the resultant phenoxyl radical [
40] and produced stable quinines upon oxidation. This property renders these phenolic compounds as efficient antioxidants.
The presence of phenolic constituents like flavonoids and tannins in the phytochemical study may have contributed to the observed antioxidant activity of this plant parts. Therefore, phenolic compounds in Anethum sowa root extracts are good electron donors and could terminate the radical chain reaction by converting free radicals to more stable products. The high antioxidant activity shown by the methanolic extract of the root suggests that it is a potential therapeutic agent for the control of oxidative damage caused by reactive oxygen species.
Brine shrimp cytotoxicity assay
The brine shrimp cytotoxicity of the plant extract represents a rapid, inexpensive and simple bioassay technique for cytotoxic, anti-tumor and anticancer activity. This test is also considered to be phototoxic, pesticidal, trypanocidal, antitumor, enzyme inhibition and ion regulation activities. The brine shrimp cytotoxicity bioassay of
Anethum sowa L. root extracts and that of the positive control vincristine sulphate are summarized in Table
4. The extract showed lethality in a dose-dependent manner. All the extracts exhibited significant toxicity towards brine shrimps at 24 h. The LC
50 value of the methanol extract was found the highest cytotoxicity at the LC
50 value of 5.03 μg/mL with the 100 % mortality at 400 and 200 μg/mL dose levels followed by ethyl acetate (5.23 μg/mL), chloroform (17.22 μg/mL), pet ether (cold) (48.71 μg/mL) and hexane (Hot) (51.90 μg/mL) extracts. The activity of ethyl acetate extract was found to almost same as methanol extract. In comparison to standard positive control vincristine sulphate (LC
50 = 0.46 μg/mL), the ethyl acetate and methanol extract were found promising. Moreover, chloroform extract found moderate activity. On the other hand, hexane (hot) and pet ether (cold) extract demonstrate the lower activity. Higher mortality was observed in the every extract at 400 μg/mL concentration dose levels.
Table 4
Cytotoxic activity of different extract and standard (Average ± SD)
2.602 | 80.60±1.04 | 80.60±1.04 | 100±0.00 | 100±0.00 | 100±0.00 | 1 | 100±.00 |
2.301 | 70.21±1.11 | 70.21±1.11 | 90.85±0.83 | 100±0.00 | 100±0.00 | 0.698 | 90.85±0.83 |
2 | 59.95±1.60 | 59.95±1.60 | 70.65±1.83 | 91.06±1.15 | 93.17±0.27 | 0.397 | 80.12±1.62 |
1.698 | 48.48±2.62 | 46.08±0.60 | 59.95±1.60 | 81.71±1.66 | 84.55±1.19 | 0.096 | 60.51±0.88 |
1.397 | 39.48±0.88 | 35.13±1.59 | 46.08±0.60 | 81.88±1.91 | 73.48±1.31 | −0.204 | 50±0.00 |
1.096 | 29.34±1.83 | 26.94±1.80 | 38.27±1.82 | 59.95±1.60 | 64.85±1.59 | −0.505 | 40.55±0.95 |
0.795 | 19.39±1.05 | 20.47±0.81 | 30.25±0.43 | 50±0.00 | 52.79±2.44 | −0.806 | 30±0.00 |
0.494 | 9.69±0.52 | 9.69±0.52 | 28.61±1.36 | 38.05±2.17 | 40±0.00 | −1.107 | 20±0.00 |
0.193 | 10±0.00 | 10±0.0 | 20.47±0.81 | 30±0.00 | 30±0.00 | −1.408 | 10±0.00 |
−0.107 | 0 | 0 | 8.58±0.43 | 20±0.00 | 20±0.00 | −1.709 | 10±0.00 |
−0.408 | – | – | – | – | 20±0.00 | – | – |
−0.709 | – | – | – | – | 10±0.00 | – | – |
LC50 value (μg/mL) | 48.71±0.70 | 51.90±0.45 | 17.22±0.14 | 5.23±0.11 | 5.03±0.08 | | 0.46±0.05 |
Bioactive compounds are almost always toxic in high doses. In the brine shrimp lethality assay, the degree of lethality was directly proportional to the concentration of the crude extracts of the
A. sowa root. The cytotoxic activity is recommended weak at the LC
50 is 500–1000 μg/mL, moderate at 100–500 μg/mL and strong at 0–100 μg/mL [
41]. However, according to Mayer [
17], extracts derived from natural products which have LC
50 ≤ 1.0 mg/mL are known to possess toxic effects. Conclusively, our results showed that all the extracts could be regarded as toxic to brine shrimp larvae cells based on the LC
50 values in the current study. The
A. salina embryos are highly vulnerable to toxins at early developmental stages [
42]. A dose dependent relationship was observed where the percent of toxicity increased with increase in the concentration of the extracts. Bioassay-guided fractionation offers special advantages for identification of medicinal plant extracts. Most often, a desired biological response is not due to one component but rather due to a mixture of bioactive plant components [
43]. Özçelik reported [
44] the cytotoxic effect of plants is principally contributed by the presence of secondary metabolites like alkaloid, glycosides, steroid, tannin and flavonoids. Steroids, flavonoids and saponins which may also responsible for the prominent cytotoxic effect in brine shrimp lethality bioassay [
45]. It has been established that the cytotoxic compounds generally exhibit significant activity in the brine shrimp lethality assay. Preliminary phytochemical screening of the extracts showed the presence of alkaloids, tannins, saponins and flavonoids type compounds in methanol, ethyl acetate and chloroform extract prominently. This assay can be recommended as a guide for the detection of antitumour and pesticidal compounds because of its simplicity and low cost. Further toxicity studies on individual cell line are also suggested to confirm the anticancer effect of the
Anethum sowa L. root extracts.
Antimicrobial activity of Anethum sowa L. root extracts
The antibacterial activity of
Anethum sowa L. root extracts was tested in vitro by using diffusion and liquid dilution method. The extracts were tested against 10 pathogenic bacteria. The antibacterial activity of the extracts was assessed by determining their ZI, MIC and MBC values and shown in Tables
5,
6,
7 and
8. The results revealed that all the Gram-positive organisms were resistant to the PE (Cold), Hex (Hot), CE and MeOH extracts at 400 μg/well concentration except
B. cereus in CE extract. It was observed that EtOAc extract produced the highest zone of inhibition against the Gram-positive bacteria (12–26 mm) which was followed by PE (Cold) (8–18 mm), MeOH (10–15 mm), Hex (Hot) (10–12 mm) and CE extract (10–11 mm) at the dose of 2000 μg/well. At the dose of 1200 μg/well, EtOAc extract showed the highest activity against the Gram-positive bacteria (7–14 mm), PE (Cold) extract showed moderate (5–13) activity whereas, Hex (Hot), CE and MeOH extracts showed weak activity against all the Gram-positive bacteria. However, PE (Cold), Hex (Hot) and CE extracts did not produce any zone of inhibition against
Enterococcus faecalis. Similarly, CE and MeOH extracts were also inactive against
Staphylococcus aureus. Moreover, MeOH extract had no effect against
Bacillus subtilis. Whereas, the standard drugs ciprofloxacin (5 μg/disc) and tetracycline (30 μg/disc) inhibited the growth of entire Gram-positive bacteria with a significant zone of inhibition 21–29 mm and 16–26 mm respectively.
Table 5
Summary of antimicrobial activity of Anethum sowa L. root extracts by diffusion method on Gram-positive bacteria
PE (Cold) | 400 | – | – | – | – |
1200 | 13.03±0.15 | 5.03±0.05 | 11.0±0.10 | – |
2000 | 18.06±0.20 | 8.0±0.10 | 15.0±0.10 | – |
Hex (Hot) | 400 | – | – | – | – |
1200 | 7.03±0.15 | – | 7.1±0.10 | – |
2000 | 10.0±0.20 | – | 12.0±0.10 | – |
CE | 400 | – | 6.06±0.05 | – | – |
1200 | 7.03±0.20 | 8.06±0.05 | – | – |
2000 | 10.06±0.05 | 11.03±0.25 | – | – |
EtOAc | 400 | 10.03±0.15 | 8.06±0.15 | 6.06±0.11 | – |
1200 | 14.03±0.11 | 11.06±0.15 | 8.03±0.11 | 7.13±0.05 |
2000 | 26.0±0.26 | 15.03±0.20 | 15.03±0.15 | 12.03±0.25 |
MeOH | 400 | – | – | – | – |
1200 | – | 7.06±0.11 | – | 8.0±0.10 |
2000 | – | 15.06±0.15 | – | 10.03±0.25 |
Std. CP | 5 μg/disc | 27.0±0.10 | 29.0±0.20 | 25.10±0.10 | 21.13±0.11 |
Std. TE | 30 μg/disc | 24.0±0.10 | 21.03±0.15 | 26.03±0.11 | 16.03±0.15 |
Table 6
Summary of antimicrobial activity of Anethum sowa L. root extracts by diffusion method on Gram-negative bacteria
PE (Cold) | 400 | – | 7.03±0.29 | 9.0±0.26 | – | – | – |
1200 | 12.06±0.25 | 8.0±0.20 | 10.0±0.15 | 6.03±0.05 | 6.03±0.05 | 8.10±0.10 |
2000 | 15.03±0.20 | 13.10±0.42 | 12.0±0.11 | 9.03±0.05 | 11.0±0.20 | 10.06±0.11 |
Hex (Hot) | 400 | – | – | – | – | – | – |
1200 | – | 6.0±0.10 | 8.0±0.17 | – | – | 7.03±0.15 |
2000 | – | 9.03±0.12 | 11.0±0.11 | – | – | 11.03±0.25 |
CE | 400 | – | 7.0±0.30 | 7.10±0.10 | – | – | – |
1200 | 7.03±0.15 | 10.1±0.10 | 8.07±0.11 | – | – | 7.10±0.17 |
2000 | 9.0±0.30 | 12.0±0.21 | 10.1±0.10 | – | – | 10.06±0.11 |
EtOAc | 400 | 7.06±0.25 | 7.03±0.12 | 9.0±0.10 | – | 6.0±0.10 | 6.03±0.05 |
1200 | 11.03±0.28 | 13.0±0.10 | 12.0±0.05 | – | 8.0±0.17 | 7.06±0.11 |
2000 | 13.06±0.30 | 15.0±0.15 | 14.0±0.17 | – | 16.03±0.20 | 12.0±0.26 |
400 | – | – | – | – | – | – |
1200 | 6.03±0.05 | 7.03±0.06 | 9.03±0.05 | – | – | 9.06±0.20 |
2000 | 8.06±0.20 | 10.0±0.17 | 11.0±0.10 | – | 7.06±0.05 | 12.16±0.15 |
Std. CP | 5 μg/disc | 25.03±0.40 | 23.10±0.17 | 29.0±0.11 | 39.0±0.17 | 35.03±0.37 | 28.03±0.23 |
Std. TE | 30 μg/disc | 9.03±0.30 | 11.0±0.21 | 16.10±0.15 | 23.0±0.10 | 25.03±0.20 | 23.06±0.28 |
Table 7
Minimum inhibitory concentration (MIC) of Anethum sowa L. root extracts
Gram positive bacteria |
Bacillus subtilis
| 125 | 62.5 | 125 | 62.5 | 250 | 1.55 |
Bacillus subtilis
| 250 | 62.5 | 250 | 125 | 125 | 3.125 |
Staphylococcus aureus
| 250 | 250 | 500 | 125 | 250 | 6.25 |
Enterococcus faecalis
| 250 | 250 | 62.5 | 125 | 250 | 0.37 |
Gram negative bacteria |
Escherichia coli 12079
| 500 | 500 | 500 | 62.5 | 250 | 0.75 |
Escherichia coli 2799
| 250 | 250 | 62.5 | 62.5 | 250 | 0.37 |
Pseudomonas aeruginosa, | 1000 | 250 | 250 | 125 | 125 | 3.125 |
Salmonella enteritidis 1375 | 125 | 250 | 250 | 62.5 | 125 | 0.37 |
Salmonella typhi
| 250 | 250 | 250 | 125 | 125 | 0.19 |
Acetobacter aceti
| 125 | 125 | 62.5 | 62.5 | 250 | 0.37 |
Table 8
Minimum bactericidal concentration (MBC) of Anethum sowa L. root extracts
Gram-positive bacteria |
Bacillus subtilis
| 250 | 125 | 250 | 125 | 500 | 12.5 |
Bacillus cereus
| 500 | 125 | 500 | 500 | 500 | 25 |
Staphylococcus aureus
| 500 | 500 | 1000 | 1000 | 500 | 50 |
Enterococcus faecalis
| 1000 | 1000 | 125 | 500 | 500 | 100 |
Gram-negative bacteria |
Escherichia coli 12079
| 1000 | 1000 | 1000 | 125 | 500 | 50 |
Escherichia coli 2799
| 500 | 500 | 125 | 125 | 500 | 6.25 |
Pseudomonas aeruginosa, | 1000 | 500 | 500 | 250 | 250 | 25 |
Salmonella enteritidis 1375 | 250 | 500 | 500 | 500 | 250 | 6.25 |
Salmonella typhi
| 500 | 500 | 500 | 500 | 250 | 3.125 |
Acetobacter aceti
| 500 | 250 | 125 | 250 | 500 | 50 |
On the other hand, there was a significant variation was observed against the Gram-negative bacteria of the root extracts shown in Table
6. The EtOAc extract exhibited the highest activity (12–16 mm) followed by PE (Cold) (9–15 mm), MeOH (7–12 mm), CE (9–12 mm) and Hex (Hot) (9–11 mm) extracts at the dose of 2000 μg/well. Hex (Hot), CE, EtOAc and MeOH extracts had no susceptibility against
Salmonella enteritidis 1375. Similarly, Hex (Hot) and CE extract showed the same against
Salmonella typhi and also Hex (Hot) extract in
Escherichia coli 12079. MeOH and Hex (Hot) extract showed registrant of all the Gram-negative bacteria at the dose of 400 μg/well. At the dose of 1200 μg/well, EtOAc (7–13 mm) and PE (Cold) (6–12 mm) extract showed moderate activity to the Gram-negative bacteria. Comparing the results with ciprofloxacin (5 μg/disc) and tetracycline (30 μg/disc), the entire Gram-negative bacteria inhibited to the extracts with a significant zone of inhibition 23–39 mm and 9–25 mm respectively. Again, the better activity was demonstrated with 2000 μg/mL concentration of all the tested extracts. The results demonstrate that Gram-positive bacteria are more sensitive to the extracts than the Gram-negative bacteria.
This higher resistance among Gram-negative bacteria could be due to the differences in the cell membrane of this bacterial group. The Gram-negative bacteria possess an outer membrane and a unique periplasmic space which is not found in the Gram-positive bacteria [
46]. The resistance of Gram-negative bacteria towards antibacterial substances is related to the hydrophilic surface of their outer membrane which is rich in lipopolysaccharide molecules, presenting a barrier to the penetration of numerous antibiotic molecules and it is also associated with the enzymes in the periplasmic space, which are capable of breaking down the molecules introduced from outside [
47]. The Gram-positive bacteria do not have such an outer membrane and cell wall structure. Antibacterial substances can easily destroy the bacterial cell wall and cytoplasmic membrane and result in a leakage of the cytoplasm and its coagulation [
48]. However, MeOH extract did not show the trend completely in this study.
The MIC is the lowest concentration of the agent that completely inhibits visible growth, disregarding a single colony or a thin haze within the area of the inoculated spot [
49].
Anethum sowa L. root extracts were tested with agar-dilution assay and susceptibilities were compared with a ciprofloxacin (positive control) at the different concentration by serial dilution technique. The results are shown in Table
7. The lowest MICs (62.5 μg/mL) was observed in EtOAc extract against
B. subtilis, E. coli 12079, E. coli 2799, S. enteritidis 1375 and
A. aceti. Moreover, the best activity possessed antibacterial activities with MICs of 62.5-125 μg/mL of EtOAc, CE and Hex(Hot) extracts followed by MeOH extract (125–250 μg/mL). However, PE (Cold), Hex (Hot) and CE extracts showed weak inhibition (MIC 500 μg/mL) in the test tubes containing
Escherichia coli 12079. On the other hand, PE (Cold) extract showed the very weak against
Pseudomonas aeruginosa. Overall, the Gram-positive bacteria showed the stronger activity than Gram-negative bacteria of the MIC experiment. However, standard ciprofloxacin showed the complete inhibition (i.e. no turbidity) against all of the test bacteria.
S. aureus and
B. cereus are well-known for being resistant to numerous antibiotics. Moreover, these organisms are capable of producing several types of enterotoxins that can cause septicaemia and several forms of enteritis. These findings are of promising in the case of EtOAc extract that was found to be active against these bacterial species.
In the MBC experiment (Table
8), the most susceptible bacteria to the EtOAc extract were
Bacillus subtilis, Escherichia coli 12079 and
Escherichia coli 2799 followed by CE extract against
Enterococcus faecalis, Escherichia coli 2799 and
Acetobacter aceti and Hex (Hot) extract against
Bacillus subtilis and Bacillus cereus presenting an important growth of inhibition at the lowest concentration of MBC at 125 μg/mL. MeOH extract showed moderate effect against
Pseudomonas aeruginosa,
Pseudomonas aeruginosa and
Salmonella typhi with MBC value of 250 μg/mL. Furthermore, PE (Cold), Hex (Hot), CE and EtOAc extract showed less potent activity at the dose of 1000 μg/mL. against
Staphylococcus aureus, Enterococcus faecalis, Escherichia coli 12079 and
Pseudomonas aeruginosa. On the other hand, standard ciprofloxacin showed the most susceptible to Gram-positive and Gram-negative bacteria than the experimental extracts.
Microbial contamination still poses an important public health and economic concerns for human society. For these reasons, there has been increasing interest in searching of natural antimicrobial agents and it has notably increased for healthy lifestyles and healthy ageing. The screening of the chemical groups found in root extracts showed the presence of tannins, flavonoids and anthraquinone. The presence of these constituents at the high concentration may possess antimicrobial properties in the different extracts. Again, the antimicrobial effects of tannins take place by involving different mechanisms such as inhibition of extracellular microbial enzymes, deprivation of the substrates required for microbial growth or direct action on microbial metabolism through inhibition of oxidative phosphorylation [
50]. Flavonoids and tannins are effective antimicrobial substances possibly due to their capability to form a complex with the extracellular and the soluble protein and to intricate with the cell membrane leading to the death of the bacteria [
51]. Moreover, the plant extract was also positive for steroids which are very important compounds because it has a relationship with compounds such as sex hormone and also reported as antibacterial properties [
52,
53]. The presence of the steroids in PE (Cold) extract justifies the antibacterial property of this plant part. Accordingly, the presence of these molecules in the active fractions might play an important role in the observed antibacterial activity. Their mode of action probably depends on the individual microorganism which could explain the large differences in MIC values between bacteria.
The antifungal activity of
Anethum sowa root extracts were measured and represented in Table
9. The extracts did not show any growth of inhibition against
Aspergillus niger and Tricoderma sp. except
Candida albincas which showed some weak activity to the CE and MeOH extract. The results were compared with the standard drug, Fluconazole (100 μg/disc). The root extracts contain a number of secondary metabolites in the current studies. The isolation and purification of the extract may discover many significant novel antimicrobial lead compounds.
Table 9
Summary of antifungal activity of Anethum sowa L. root extracts by diffusion method
Aspergillus niger
| – | – | – | – | – | – | | | | – | – | – | – | – | – | – | – | – |
Candida albincas
| | – | – | – | – | – | 7 | 250 | 500 | | – | – | | 250 | 500 | | – | – |
Tricoderma sp.
| – | – | – | – | – | – | | | | – | – | – | – | – | – | – | – | – |