3.1. Extraction of bioactive compounds
The extraction of bioactive compounds is necessarily a complex and delicate operation. Its aim, in fact, is to break down the cell walls to extract the bioactive compounds locked in the cells. For comparison purpose, two different drying methods, different extraction processes and two solvents were used to investigate their extraction and compare their respective yields.
3.1.1. Extraction yields
Extraction yields vary considerably depending on the drying method used and the extraction method adopted. The results of the extraction of azolla samples by the different drying methods and in the different solvents used are represented in tables 1.
Table 1: Extraction yield of azolla samples by the different methods used.
|
Extraction yield (%)
|
Azolla pinnata
|
Azolla microphylla
|
|
Ethanol
|
Water
|
Ethanol
|
Water
|
Lyophilization
|
Maceration
|
24.8 ± 1.02
|
10.2 ± 0.74
|
21.8 ± 1.22
|
11.6 ± 1.04
|
Decoction
|
26.07 ± 0.44
|
11.6 ± 0.68
|
22.03 ± 1.23
|
13.03 ± 1.62
|
Oven dry
|
Maceration
|
19.5 ± 1.24
|
9.7 ± 1.45
|
19.16 ± 1.43
|
10,12 ± 0.88
|
Decoction
|
21.2 ± 0.57
|
11.02 ± 1.13
|
19.8 ± 1.17
|
12.02 ± 0.92
|
Moyen ± SD
|
16.75 ± 0.9
|
16.19 ± 1.18
|
We have found that, for the decoction method, ethanol gives the best extraction yield for both species and for the freeze-dried and oven-dried samples, respectively (26.07% and 22.03%), followed by aqueous extracts. (11.6% and 13.03%),
Lyophilization, also known as freeze-drying, and oven drying are two common methods used for drying and preserving biological materials, including plant extracts. Each method has its advantages and disadvantages, and their impact on extraction yield can vary depending on the specific characteristics of the material being processed. The results of our study have shown that freeze-dried samples in both solvent have lead to higher extraction yields compared to oven drying for both of A. pinnata and A. microphylla. It can be explicated by the fact that samples are containing delicate compounds that may degrade or evaporate at higher temperatures. By preserving the integrity of the sample, lyophilization can facilitate the release of bioactive compounds during subsequent extraction processes (EINaker et al. 2020). Whereas, oven drying may be more suitable for materials that are not sensitive to high temperatures and require rapid drying (Bennour et al. 2020). Mphahlele et al. (2016), demonstrated that the best drying method to preserve total pomegranate phenols is freeze-drying compared to oven drying. Also, according to Galaz et al. (2017), high drying temperatures had reduced the polyphenol content.
In the other hand, samples that are decocted record high values compared to those that are macerated. These results are very close to those obtained by Mahmoudi et al. (2013), who found that the best extraction yields of total polyphenols, flavonoids and condensed tannins from artichoke by four solvents (water, methanol, ethanol and acetone), are recorded by the decoction, an average of 17.34% versus 15.64% for maceration.
3.1.2. CG-MS analysis
The results of previous studies about the qualitative analysis indicated the presence of phenols, flavonoids, alkaloids, steroids, tannins, cardio glycosides, and saponins in the ethanolic extract of azolla (Sreenath et al. 2016). Hence, in the present work, this quantitative analysis was used for the further studies.
The GC-MS analysis of the ethanolic extracts obtained from A. pinnata and A. microphylla conducted to the identification of more than 20 different compounds. The results are presented in table 2.
Table 2: GC-MS analysis of the ethanolic extracts obtained from A. pinnata and A. microphylla
Compounds
|
A. Pinnata (Area %)
|
A. Microphylla (Area %)
|
Our results
|
(Sreenath et al., 2016)
|
1,3-di-iso-propylnaphthalene
|
C16H20
|
0.5
|
NA
|
0.3
|
3-ethyldibenzothiophene
|
C14H12S
|
0.84
|
0.41
|
0.52
|
Tetradecanoic acid, 12-methyl ester
|
C16H32O2
|
1.03
|
0.33
|
0.51
|
Neophytadiene
|
C20H38
|
0.83
|
1.02
|
1.64
|
2-[(Z)-9-octadecenyloxy] ethanol
|
C20H40O2
|
0.72
|
NA
|
0.23
|
9-hexadecenoic acid, methyl ester
|
C17H32O2
|
2.11
|
1.25
|
1.04
|
Hexadecanoic acid, ethyl ester
|
C17H34O2
|
8.04
|
5.6
|
4.8
|
3-cyano-12-isopropoxy-6,11-methanocyclodeca[g] imidazo[5,1-c](1,2,4) triazine
|
C18H15N5O
|
2.04
|
1.38
|
1.15
|
cis-13-octadecenoic acid, methyl ester
|
C19H36O2
|
0.28
|
0.76
|
1.05
|
Hexadecanoic acid, 2,3-dihydroxypropyl ester
|
C19H38O4
|
0.91
|
NA
|
0.2
|
9-Octadecenoic acid
|
C18H34O2
|
22.16
|
18.12
|
15.04
|
Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester
|
C35H68O5
|
1.81
|
1.03
|
0.5
|
(1R)-2-(1S)-1-[2-(Methoxymethoxy) phenyl] ethyl} amino) oxy]-1-phenylethanol
|
C18H23NO4
|
0.34
|
0.4
|
0.69
|
3-[(tert-Butyldimethylsilyl) oxy]-1,4,4a, 9a-tetrahydro-1-phenyl-9H-xanthen-9-one
|
C25H30O3Si
|
1.22
|
0.52
|
0.76
|
Cholest-2-en-1-ol
|
C27H46O
|
NA
|
0.73
|
0.39
|
Oleic acid, eicosyl ester
|
C38H74O2
|
1.27
|
0.26
|
0.34
|
(2R/S,4S/R,6R/S)-4-Hydroxy-2-tridecyl-1,7-dioxadispiro[5.1.5.2]pentadeca-9,12-dien-11-one
|
C26H42O4
|
1.53
|
4.21
|
2.04
|
3á-(Peroxymethyl)-5-vinyl-A,B-bisnor-5á-cholestane
|
C28H48O2
|
0.74
|
0.57
|
0.34
|
2,3,4,5,2’,6’-Hexamethoxy-4’,5’-methylenedioxychalcone
|
C22H24O9
|
38.32
|
71.04
|
55.47
|
Cucurbitacin B, dihydro-
|
C32H48O8
|
NA
|
NA
|
0.15
|
Chol-8-en-24-al 3-hydroxy-4,4,14-trimethyl-
|
C27H44O2
|
0.93
|
NA
|
0.17
|
NA: Not available
For A. pinnata extracts, the highest area percentage value was noted for the compounds “C22H24O9” with 38.32%, while almost the double of area percentage value was observed for the same compound in A. microphylla extract (71.04%). Sreenath et al. (2016) has reported the same major compound in term of area percentage with (55.47%). The findings for both species confirmed the existence of primarily fatty acid, terpenoid, steroid, coumarin, and flavonoid derivatives compounds.
These compounds have been noted to exhibit a range of biological and pharmacological effects (Sakthivel and Guruvayoorappan 2013).
3.2. Insecticidal test
In order to test the insecticidal effect of the bioactive compounds of Azolla, we have only used the extracts obtained from the freeze-dried dry matter. Therefore, we were interested in studying the effect of the two extraction methods (maceration and decoction) and the polarity of the solvents used. The results obtained made it possible to determine the insecticidal effect of the bioactive molecules present in A. pinnata and A. microphylla evaluated through the mortality recorded in adults and larvae of T. castaneum by a contact test at different periods after treatment. The percentage mortality of larvae and adults was increased according to the increasing concentration of extracts. The results of LC50 for both species are presented in table 3.
Table 3: Mean LC50 efficacy of A. pinnata and A. microphylla extracts after 24 h of exposure on T. castaneum.
|
|
Extracts
|
LD50 (µg/mL)
|
Regression equation
|
A. pinnata
|
Larvae
|
Ma-ethanolic
|
1103,44
|
y = 14,08x + 19,02
|
Ma-aqueous
|
-
|
y = 5,5x + 15,5
|
Dec-ethanolic
|
-
|
y = 5,8x + 16,2
|
Dec-aqueous
|
987,31
|
y = 15,1x + 23,7
|
Adults
|
Ma-ethanolic
|
1000
|
y = 10,71x + 25,78
|
Ma-aqueous
|
1589,25
|
y = 11,5x + 4,5
|
Dec-ethanolic
|
-
|
y = 3,2x + 14,4
|
Dec-aqueous
|
872,42
|
y = 12,7x + 38,9
|
A. microphylla
|
Larvae
|
Ma-ethanolic
|
1165,23
|
y = 11,4x + 23,4
|
Ma-aqueous
|
2500
|
y = 7,5x + 11,5
|
Dec-ethanolic
|
2080,58
|
y = 7,7x + 15,3
|
Dec-aqueous
|
968,12
|
y = 11,6x + 29,2
|
Adults
|
Ma-ethanolic
|
1500
|
y = 6x + 31,2
|
Ma-aqueous
|
2087,46
|
y = 7,6x + 16
|
Dec-ethanolic
|
-
|
y = 4,8x + 11,2
|
Dec-aqueous
|
894,65
|
y = 7,9x + 33,5
|
As it is shown in Fig3, the T. castaneum larvae bioassay demonstrated a significant rise in mortality as the concentration of A. pinnata extract increased. The LC50 of ethanolic and aqueous extracts were found to be 1103.44 µg/mL and 987.31 µg/mL, respectively. Bhattacharjee et al. (2022) have reported the same larvicidal activity exhibited by the A. pinnata extracts. The aqueous extracts by decoction had significant insecticidal toxicity against the adults at 500 µg/mL and above. At 872.42 µg/mL, 50% mortality of the adults was achieved, and at 2500 µg/mL, almost a complete mortality was observed (Fig4). Comparatively, the A. microphylla extracts were less toxic towards the T. castaneum larvae and adults with LC50 of 1165.23 µg/mL and 968.12 µg/mL, respectively (Fig5 and 6).
3.2.1. Impact of the extraction method on the insecticidal capacity of the extracts
We have conducted two extraction techniques in our study to determine the effectiveness of the extracted compounds. It should be noted that mortality was observed in batches of up to 3/10 individuals. Therefore, the toxicity of crude phytopreparations was estimated by evaluating the corrected mortality. The results are presented in table 4 below.
Table 4: Mortality rate of larvae and adults of T. Castaneum treated with both species extracts (2500 µg/mL) after 24 hours.
|
Mortality (%)
|
Larvae
|
Adults
|
|
Ethanol
|
Water
|
Ethanol
|
Water
|
A. pinnata
|
Maceration
|
84,44 ± 1.35
|
40 ± 3.74
|
75,55 ± 1.22
|
60 ± 3.54
|
Decoction
|
43 ± 1.3
|
96 ± 2.68
|
30 ± 3.23
|
96 ± 6.62
|
A. microphilla
|
Maceration
|
76 ± 1.24
|
50 ± 1.45
|
60 ± 2.43
|
55 ±2.88
|
Decoction
|
55 ± 1.57
|
84 ± 2.13
|
38 ± 1.17
|
70 ± 1.92
|
The observation of the petri dishes of the larvae treated with the different extracts (2500 µg/mL) obtained from A. pinnata by maceration revealed that the ethanolic extract gave a very high mortality of 84.44%, comparing with the aqueous extract (40%). Identical observation was noted in adult’s petri dishes (75.55% and 60%, respectively) While, for extracts obtained by decoction we were able to see that the aqueous extracts caused significantly higher mortality, reaching 96% against both of larvae and adults.
The same principles were followed for the A. microphylla extracts, but they exhibited lower mortality rates (76% ethanolic extract and 84% for aqueous extract) compared to A. pinnata extracts. These results are in concordance with the previous study, mentioned that azolla plant has potential as a bioinsecticide to address single chemical compound resistance issues (Ravi et al. 2018).
The insecticidal power of this fern is due to the presence of phenolic compounds, tannins and saponins (Ekanayake et al. 2007). The insecticidal effect of these constituents has been mentioned by several authors. Phenolic compounds have both pesticidal and fungicidal properties. Tannins have insecticidal, larvicidal and repellent properties (Wardell 1987).
Previous studies have shown that these natural compounds can cause symptoms indicative of neurotoxic activity, such as hyperactivity, convulsions and tremors followed by paralysis and death of the insect which are very similar to the effects produced by pyrethroid insecticides (Richardson et al. 2019).
The comparative analysis of these results shows that there is a positive correlation between polarity of the extract tested and the stage of development of the killed insect. According to Hinwood (1997), who states that a polar solvent dissolves a polar solute better than a non-polar solvent. One might think that this difference in biological activity linked to the polarity of our various biocidal products could only be due to a quantitative and/or qualitative difference in the active compounds present there.
3.2.2. Impact of exposure time on the insecticidal activity of extracts
We tried to study the effect of the contact time of the extracts to larval and adult individuals during this work. Therefore, the mortality reading was made after every 8 hours up to 24 hours. The results are presented in the Figure 5. It was found that the average mortality of adults and larvae of T. Castaneum increases depending on the duration of exposure to the extracts used by contact, since an increase in mortality was recorded as we move forward in the exposure time.
The effectiveness of the extracts began from the first reading (after 8 hours) for all the extracts with the exception of the ethanolic extract by decoction which only gave effect after 10 hours. On the other hand, the aqueous extract by decoction was very effective with a mortality rate of 60% from the first reading. In fact, the mortality of individuals increased mainly after 16 hours of exposure.
The findings of the present study may suggest that the dry matter of A. pinnata and A. Microphylla gave a good result for its toxicity on individuals of T. castaneum. This effectiveness is confirmed by the death of larvae and adults of this pest. The results for both species confirmed the existence of primarily fatty acid, terpenoid, steroid, coumarin, and flavonoid derivatives compounds. Activity was always positively associated with an increase in concentration of the bioactive compound. These biologically active molecules have been noted to exhibit a range of biological and pharmacological effects. Ongoing research is being carried out to clarify the elements accountable for the insecticidal effects, along with any potential pharmacological or toxicological properties of such extracts.