Encapsulation of Rosmarinus officinalis essential oil (EO) on chitosan and gum Arabic matrix were prepared at different ratios with various concentrations of essential oil. The capsules were characterized by their encapsulation efficacy (EE%) and loading capacity (LC%). The cumulative release profiles were evaluated by UV/Vis spectroscopy. The chemical compounds for free and encapsulated essential oil were determined by GC-MS technique and the insecticidal toxicity against Tribolium castaneum and Oryzeaphilus surrinamensis was determined by fumigant bioassays. Results showed that capsules with ratio chitosan: gum arabic: EO (1: 1: 0.5) revealed the highest EE (45.8%) and LC (2.31%). In vitro release profiles demonstrated that encapsulated essential oil showed a sustained release especially for the ratio with high EO concentration. The major constituents of the rosemary essential oil were 1,8-cineole (39.67%), Camphor (18.04%), followed by borneol (10.52%) and α-Pinene (6.33%). After encapsulation numerous minor compounds have been missing during storage periods until identifying only 1, 8-cineole, α-terpineol and camphor after 60 days of storage. The fumigant tests demonstrated that encapsulated EO exhibited an effective control against insect pest during storage periods namely 30, 45, 60 days with 99%, 66%, 46% for T. castaneum and 100%, 84%, 82% for O. surrinamensis. This work supports the hypothesis that major essential oil components were responsible for its pesticidal activities over storage. Besides, toxicities against T. castaneum and O. surinamensis adults was mainly attributed to 1, 8-cineole.
Research Article
Microencapsulation of rosemary Rosmarinus officinalis essential oil by Chitosan - gum Arabic and its application for the control of two secondary pests of stored cereals
https://doi.org/10.21203/rs.3.rs-1854496/v1
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Microencapsulation
Rosmarinus officinalis
Chitosan
Insecticidal activity
Tribolium castaneum
Oryzeaphilus surrinamensis
Rosemary (Rosmarinus officinalis L.) grows in the Mediterranean region and is mainly identified by its culinary and folklore medicinal uses [1]. Rosemary leaves are a very common spice and its oil is largely used in fragrance flavor industry aromatherapy, antioxidant activity, antimicrobial and antitumor properties [2, 3]. Furthermore, they are recognized for their biological activities including their insecticidal properties [4]. Thus, R. officinalis essential oil (EO) has been previously investigated for its insecticidal efficacy against a wide range of insect pests [5]. Rosemary essential oil composition has been explored and reported in numerous works (Napoli et al. 2010; Jiang et al. 2011.). It varies depending on the geographical region, season, climate, age and plant genetic [6]. In Tunisia, previous investigations conducted on R. officinalis revealed that 1,8-cineole is the omnipresent compound in the essential oil. Additionally, camphor, α-pinene, Borneol, camphene, transcaryophyllene and α-thujone were also reported [7–9]. Indeed, EO including its major constituents has the potential to be used as an environmentally compatible alternative to synthetic pesticides and herbicides [10]. Similarly, Bachrouch et al. [11] and Dugrand et al. [12] demonstrated that the high toxicity effects of EO could be attributed to its richness with major compounds. In the same context, previous research reported that 1,8-cineole is the major component of R. officinalis [13]. Besides, [14] revealed that rosemary EO has insecticidal properties due to their major components such as 1,8-cineole and camphor. Furthermore, due to the strong fumigant toxicity against insect pests of stored products such as Sitophilus oryzae and Oryzaephilus surinamensis, rosemary essential oil has been recommended as an ecofriendly alternative to chemical treatment [15]. However, essential oils are well known by their high susceptibility to degradation upon exposure to light, temperature, and oxygen [16]. Additionally, the direct application of essential oils into foods can affect their perception due to their pungent flavor and odor [17]. In order to overcome these problems, alternative methods are well needed, such as encapsulation [18, 19]. Additionally, Raza et al. [20] revealed that encapsulation is an emerging technique that can be used in food agriculture such as insecticide, pesticides and insect repellants. According to Gonzalez et al.[21], essential oils formulation enhances the efficacy of pest control. Chitosan is a talented biopolymer for active packaging [22] as well as, is a non-toxic, biodegradable and bio-compatible polysaccharide [23]. For these reasons, encapsulation using chitosan as a matrix being an emerging technology has been shown as an effective solution to control insect pests [24].Accordingly, our work aims to : (i) Evaluate the fumigant toxicity of the rosemary EO against Tribolium castaneum and Oryzeaphilus surrinamensis adults (ii) Assess the insecticidal effect of encapsulated EO in chitosan matrix (iii) Determine the major active component loaded in the chitosan and gum arabic responsible for the toxicity potential of EO during different storage periods.
Essential oil extraction
Sampling of Rosmarinus officinalis species was made in February 2021 from Korbous arboretum, Nabeul governorate (846 m 36 ° 48’54”N 10 ° 34’ 14”E). The rosemary leaves were rolled out in the shade in thin layers to dry for one to two weeks at room temperature. The essential oils were extracted by hydrodistillation using a CLEVENGER method [25].
Insect rearing
Rearing colonies of T. castaneum and O. surinamensis were initiated from various infested cereals’commodities and maintained under controlled conditions (25 ± 1 °C, 65 ± 5% relative humidity and 12h Light/12h Dark photoperiod). Glass boxes (Volume 1 L) were observed daily to collect the emerged adults. Newly emerged adults (24h old) were used for the bioassays [25].
Median Lethal concentration bioassay
Fumigant tests were conducted against O. surinamensis and T. castaneum in a glass bottle with a volume of 350 ml glass containing 220 g of semolina (1 adult per 10g of semolina). Using a micropipette, the essential oil was deposed on a 7.5cm diameter filter paper disc (Whatman No. 1 paper). Three different doses were used namely 28.5; 57 and 114µl. Then, the discs were glued to the jar wall which was sealed. Untreated bottles were considered as control. The test was repeated 3 times. Mortality assessment was performed after 10 days of storage. The correct mortality rate was expressed as a percentage based on the mortality rate of the treated sample and the control group [26].
Preparation of the Chitosan-gum Arabic-essential oil encapsulation
The used method according to Keita [1] protocol with modifications consists of diluting 4 ml of R. officinalis oil in 40 ml of acetone. This solution was mixed with 40 g of powder (20 g of gum Arabic and 20 g of chitosan). After 5 min of manual stirring, the mixture was placed in a water bath at 40 ° C until the complete evaporation of acetone (obtaining a dry flavored powder). Likewise, a suspension of the powder mixture (gum Arabic + chitosan) with 40 ml of acetone without essential oil was prepared to serve as a control.
The flavored powders were stored in dark, tightly closed bottles with parafilm and placed in the refrigerator at 4 °C.
Characterization of the formulation
Encapsulation efficiency (EE%) and loading capacity (LC%)
EE (%) and LC (%) parameters were determined according to the method described by Keawchaoon and Yoksan (2011) (Eq. 1, Eq. 2). The amount of essential oil charged was calculated from a calibration curve prepared with R. officinalis oil in 95% ethyl alcohol (Abs = 006 [conc] + 0.220; R² = 0.577). Each sample was measured three times at 280 nm
EE (%) = (mass of loaded oil / initial mass of oil) x 100 (1)
LC (%) = (mass of charged oil / mass of sample) x 100 (2)
Cumulative Release (CR %)
The cumulative release characteristics of R. officinalis essential oil loaded with chitosan and gum Arabic powder were determined according to the method cited by Housseini et al. (2013). Cumulative release (%) was calculated using the formula of Keawchaoon and Yoksan [2] (Eq. 3). Each sample was measured three times at 285 nm.
In Eq. (3), Mt: Amount of Chitosane-EO released at each sampling time (t); M0: Initial amount of Chitosan-EO.
Fumigant test using free and encapsulated essential oil
A fumigation test was performed, using the calculated LC50 (The median lethal concentration). Respective quantities of 0.32 g and 0.53 g of chitosan-gum Arabic-EO capsule were used against O. surinamensis and T. castaneum. Each test has 3 replicates; untreated tests serving as a control were maintained under the same conditions. Mortality assessment was carried out after 30, 45 and 60 days of exposure. Phosphine (PHOSTOXIN®) was a chemical treatment that used as a reference at doses of 3 mg / L air.
Chromatographic analyzes of free and encapsulated essential oil after different storage periods
Chromatographic analyzes were performed by the combination of chromatography gas phase and mass spectrometry (GC-MS) according to [27].. The equipment used is an Agilent-technology 6890N GC type chromatograph. The system is equipped with detector flame ionization, a sampler in splitless mode and a capillary column HP-5MS. For chitosan-EO, 0.5 ml of capsule was mixed with 5 ml of hexane.. Each 1μl sample of free and encapsulated essential oil was injected using splitless mode. The identification were obtained using an integrated data processing program provided by the manufacturer of the chromatograph
Statistical analysis
Probit analysis [28] was used to calculate CL50 and CL95. Statistical analyses were performed using SPSS statistical software version 20.0. All values given were the mean of three replications, and were expressed as the mean ± standard deviation. Data of mortality percentage were subjected to two-way ANOVA, with treatment, insect species and exposure time as main fixed factors. The means were separated using the Least Significant Difference (LSD) (P≤0.01).
Chemical composition of EO
Chemical composition of R. officinalis essential oil using the technique of gas chromatography coupled with mass spectrometry (GC / MS) was displayed in the Table 1.
Chromatographic analysis identified 23 compounds representing 98.35% of the total essential oil. 1,8-cineole (39.67%), Camphor (18.04%), followed by borneol (10.51%) and α-Pinene (6.33%) were the major components. Thus, the chemotype was defined by 1,8-cineole/camphor. Identified compounds have been grouped into chemical classes (hydrocarbon monoterpenes, oxygenated monoterpenes, hydrocarbon sesquiterpenes and other compounds) (Table 1). Monoterpenes presented the significant fraction of the oil (71.14% including 58.87% oxygenated Monoterpenes)... This result is comparable with those reported by Bannour et al. [29], who demonstrated that chemical composition of essential oils from 4 rosemary Tunisian populations consist mainly of 1,8-cineole, camphor, α-pinene and borneol with different proportions. Napoli et al. [30] described three chemotypes of rosemary essential oil; cineoliferum with high percentage of 1,8- cineole; camphoriferum characterized by camphor with more of 20% and finally verbenoniferum with more than 15% of verbenone. Recent study conducted by, Abada et al. (2019) defined 1,8-cineole/α-pinene/camphor as the chemotype of rosemary essential oil collected from different localities in Tunisia. Likewise, Khalil et al., [31], reported that the chemical composition of rosemary essential oil collected from Hamada (Akouda region) consists mainly of camphor (16.29%), 1,8-cineole (16,21 %), bornyl acetate (14.54%) and borneol (6.02%).
Lethal concentrations LC50 and LC95
Lethal concentrations LC50 and LC95 are shown in Table 2. Probit analysis revealed that R. officinalis essential oil is found to be more toxic against the adults of O. surinamensis (LC50= 124.80 mL/L air) comparing to T. castaneum (LC50= 245.82 mL/L air). Similarly, the estimate of LC95 for T. castaneum was higher than that obtained for O. surinamensis (Table 2).
Encapsulation Efficiency (EE %) and Loading Capacity (LC %)
Table 3 reported the percentages of encapsulation efficacy and loading capacity of rosemary essential oil in the chitosan and gum arabic matrix for the three ratios.
According to Table 3, the highest encapsulation efficiency was registered for the ratio 1: 1: 0.5 chitosan: gum arabic: essential oil. The value was 4 times higher than the EE% for the ratio 1: 1:0.1 chitosan: gum arabic: essential oil. Furthermore, the loading capacity was lesser for the ratios 1: 1: 0.25 (1.49±0.05%) and 1: 1: 0.1 (0.6±0.01%) comparing to ratio 1: 1: 0.5 (2.31±0.34). Previous work reported that best results regarding to maximum loading , encapsulation efficiency, were obtained for formulations with higher essential oil concentration [32, 33]. These findings are highly consistent with our results showing that the highest loading capacity was obtained by encapsulating 50% of essential oil. This work demonstrated that the efficacy encapsulation reached EE%=45.8±0.67 for the ratio 1:1:0.5 chitosan:gum arabic:EO (w/w/w).
Cumulative release (CR %)
In vitro release studies on capsule profiles of three ratios of chitosan: gum arabic: essential oil (ratios: 1: 1: 0.1; 1: 1: 0.25 and 1: 1: 0.5) were evaluated for 45 days at pH=4. Results are shown in Figure 1. The amount of R. officinalis EO released was measured at various times at 275 nm. For the first day, an initial release reached 10.4%, 11.8% and 13.4 % for ratio 1:1:0.1; 1:1:0.25 and 1:1:0.5 respectively. After three days, the release has been accelerated to reach its maximum 62.3% for the ratio 1:1:0.1. However, for both ratios 1:1:0.25 and 1:1:0.5 the release attained its maximum after 21 days (Figure 1).
The CR% of encapsulated rosemary essential oil was determined and values were ranged from 10.4% to 64.6% for Ratio 1; from 11.8 to 73.4% for Ratio 2 and finally from 13.4 to 75.3% for Ratio 3. Thus, the potential of the capsules, could offer a continuous release of the essential oil of R. officinalis during the different storage periods. These results were found to be in agreement with the findings of Khoobdel et al. [4] which showed that encapsulation technique can produce a pesticide with controlled-release properties and induce the decrease of the number of applications and the applied doses.
Mortality assessment
Figure 2 illustrated the evolution of corrected mortality of T. castaneum and O. surinamensis adults during three storage periods, namely 30, 45 and 60 days. Results pointed out that mortality rate varied depending on the insect species and storage period. The mortality rate of T. castaneum treated with free essential oil varied between 14.53 and 8.2% against 45.23 and 31.18% for O. surinamensis after 30 and 45 days of storage respectively. Meanwhile, the reference treatment (Phosphine fumigation) achieved complete mortalities (100%) after 30 days of storage for both insects. Efficacy of chitosan-gum arabic-EO capsule was stronger toward O. surinamensis than T. castaneum (Figure 2). Rosemary essential oil has been shown to be an effective fumigant against insect pests with 67% for T. castaneum. Conversely, this work demonstrated a decrease of mortality percentages that has been observed after 45 days with 42% followed by 31% after 60 days of storage. Our results, can be supported with those reported by Isikber [34] that pointed out an efficacy control of rosemary oil against stored product pests. However, previous work showed that essential oils are very susceptible to degradation during long period of storage [35]. In this regard, El Asbahan et al. [36] and Ben Abada et al. (2019) indicated that alternative methods such encapsulation enhance the efficacy and the insecticidal toxicity of essential oils during long storage periods . In addition, Zuldigar et al. (2020) indicated that microencapsulation can be considered as one of effective methods in food agriculture and other numerous sectors. In this respect, various industry wastes have been used to overcome this problem such as chitosan (Hosseini et al. 2013). Thus, various research are focused on the management and valorization of food wastes [3]
Statistical analyzes indicate highly significant differences among storage periods (df = 2; F = 70195.54; P ≤ 0.000) and among insect species(df = 1; F =82406.73; P ≤ 0.000). Furthermore, the different treatments differed significantly (df = 2; F =14948.37; P ≤ 0.000).
The mortality percentages for rosemary free oil was 31% for T. castaneum and 62% for O. surinamensis comparing to chitosan: gum arabic: EO with 46% and 82% for T. castaneum and O. surinamensis, respectively after 60 days. It can be seen that encapsulated essential oil exhibited the highest toxicity against insect pests. Additionally, the results showed that free EO lost its toxicity 2 times higher than the lost for chitosan: EO. These results were in accordance with those reported by previous works [24]. Similarly, Ahsaei et al. [37]proved the efficacy of encapsulated R. officinalis against numerous insect pests of stored products.
Chemical components of free and encapsulated essential oil
Table 4 showed the chemical fractions of essential oil before and after encapsulation. Chitosan: gum arabic showed an effective release of R. officinalis essential oil after 2 h of formulation. The follow-up of the diffusion of rosemary EO chemical components revealed that monoterpenes presented the highest fractions (53.61%) followed by sesquiterpenes (14.06%). Abundance of monoterpenes can be dispensed to the fact that they are the major components in rosemary EO. The results demonstrated a low qualitatively modification to the free EO. In fact, it has been observed a reduction on the quantity of the 18 identified compounds. The formulation released an important quantity of major compounds after 1day. 1,8-cineole was still the major one with 31.25% followed by the camphor and borneol (Table 4). The fraction of the major identified components showed significant difference between free and encapsulated essential oil (df=1; F=460.56; P≤0.00).
Chromatographic analysis of encapsulated essential oil during different storage periods
Figure 4 compares the stability of formulation concentrations of the monoterpenic components during various storage periods by GC-MS analysis. Results revealed 7 compounds that represent 44.15% of the total constituent after 30 days. The major compounds were 1, 8-cineole (23.05%), Camphor (9.06%) and followed by Borneol (7.33%). However, 5 and 4 compounds were identified after 45 and 60 days respectively. The quantity of 1,8-cineole as a major compound, after 60 days was 3 times lesser than those registered after one day. This research reported that, the number and the fraction of identified chemical components in the formulation decreased to reach only 4 components after 60 days. We assume that the decrease of chemical concentrations is mainly due to the degradation of monoterpenic compounds during the exposure time. In this context, other works conducted by Noudjou et al. [4]; Kouninki et al. [5] revealed that that a decrease in chemical components of formulated essential oil induces a decrease in mortality percentages. The one-way ANOVA test followed by Duncan's multiple range test classification revealed high significant differences between storage periods for the identified component (df=2; F=290.25; P≤0.00).
Correlation
Figure 5 reported the essential oil components and insects’ mortalities as a function of the storage periods and chemical fractions during various storage periods. Results revealed a decrease on monoterpenic compounds identified after 30, 45 and 60 days of storage. Besides, mortality percentages were 99% after 30 days to reach 46% after 60 days. The obtained results suggest that 99% and 100% of T. castaneum and O. surinamensis mortalities respectively were clearly linked to the 7 identified components especially 1,8-cineole (23.05%). The decrease on mortality percentages to 46% after 60 days of storage could be assigned to the fact that monoterpenes components fractions revealed an important reduction to 10.75% for 1,8-cineole. Thus, insects’ mortality percentages could be attributed to the presence of 1,8-cineole, Camphor and α-terpineol. This research reported that 1,8-cineole was the major compound with 23.05 % followed by camphor 9.14 % for chitosan-EO capsule after 30 day of storage. Furthermore, after 60 days of storage, results showed that 1,8-cineole and camphor were 10.55% and 5.66% respectively in the chitosan-EO capsules. In a related study 1,8-cineole had high insecticidal toxicity against insect pests such as Tetranychus urticae koch [38].
Moreover, matrix illustrated in Table 5, indicated high significant correlations between 1, 8-cineole fraction and mortality percentages [O. surrinamensis (r=0.98, P≤0.05); T. castaneum (r=0.99, P≤0.05)]. However, no significant correlations have been observed between the other compounds and mortality percentages during different storage periods at (P≥0.05). Thus, the hydrocarbons monoterpenes and sesquiterpenes are not efficient comparing to oxygenated monoterpens against T. castaneum and O. surrinamensis.
Likewise, Nguemtchouin et al. [39] indicated high correlation between many components such as 1,8-cineole and insects’ mortality. Even though, monoterpenes like 1,8-cineole and terpinen-4-ol exhibited high toxicity against Sitophilus oryzae and Sitophilus zeamais [39, 40]. In the same context, Isman et al. [14]revealed that R. officinalis especially 1,8-cineole is the most responsible compound for mortality of larvae of Pseudaletia unipuncta and Trichopulsiani.
Chitosan-EO capsule revealed the highest EE% (45.8%) for the ratio 1:1:0.5 and a cumulative release (75.3%) after 21 days. In particular, capsule chitosan: gum arabic: EO caused 100% mortality of O. surrinamensis against 42% caused by free essential oil after 45 days of storage. According to these results, we can conclude that capsule chitosan: gumarabic: EO can be designed as an effective treatment against pests that present sustained release features. However, the reduction of mortality percentages after 60 days of storage can be explained by the loss of volatile compounds, which may be affected by various conditions such as preparation, conservation, etc.. Thus, all these factors should be taken in consideration into future research.
Ethical Approval
Not applicable, because this article does not contain any studies with human or animal subjects.
Consent to Participate
All participants consent to participate of the present study.
Consent to publish
All participants consent to publish the present study.
Authors Contributions
Abir Soltani wrote the manuscript with input from all authors. Jouda Mediouni-Ben Jemâa devised the project, the main conceptual ideas and proof outline. IslemYangui,Sarra Ncibi, Tasnim Djebbi, Insaf Ajmi Sadraoui, Maha Ben Abada, Hadhami Chargui, Khaoula Hassine, Amina Laabidi, Hela Mahmoudi, Hatem Majdoub provided critical feedback and helped shape the research, analysis and manuscript. All authors discussed and commented on the manuscript.
Funding
This research was funded by the program of Valorization of Research Results (VRR), the General Directorate of Research Promotion, The ministry of Higher Education and Scientific Research of Tunisia.
Competing Interests
The authors declare that they have no conflict of interest.
Informed consent Informed consent was obtained from all individual participants included in the study.
Availability of data and materials
The authors confirm that the data supporting the finding of this study are available within the article and its material. Raw data that support the findings of this study are available from the corresponding author, upon reasonable request.
Novelty statement
The novelty of our work entitled “Microencapsulation of rosemary Rosmarinus officinalis essential oil by Chitosan - gum Arabic and its application for the control of two secondary pests of stored cereals.” can be summarized as The Major essential oil components were responsible for its pesticidal activities over storage. Identified chemical components in the formulation decreased to reach only 4 components after 60 days. We assume that the decrease of chemical concentrations is mainly due to the degradation of monoterpenic compounds during the exposure time. In addition, high correlation has been determined between insects' mortality and chemical component fractions.
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Table 1 LC50 and LC95 values of Rosmarinus officinalis from the North West of Tunisia after 10 days of storage against Tribolium castaneum and Oryzeaphilus surrinamensis.
|
Insects |
CL50 |
CL95 |
Regression equation |
χ ²value |
|
Tribolium castaneum |
245.82 |
496.66 |
Y=0.012 X-2.24 |
9.23 |
|
Oryzaephilus surinamensis |
124.80 |
231.30 |
Y=0.02 X-2.2 |
5.85 |
Data tested by χ2-test for homogeneity of 1:1 ratio;
- median lethal concentration;
- Lethal concentration at 95
- Standard error
Table 2 Chemical fractionof total identified constituents (%) of essential oils extracted from Rosmarinusofficinalis
|
N° |
component |
Fraction (%) |
RIa /RIb |
|
|
Monoterpene hydrocarbons |
12.27 |
|
|
1 |
α-Pinene |
6.33 |
937/913 |
|
2 |
α -Terpinene |
0.39 |
1021/1015 |
|
3 |
b-pinene |
2.84 |
982/965 |
|
4 |
g-Terpinene |
0.7 |
1107/1041 |
|
5 |
ɒ-Terpinolene |
0.62 |
1092/1099 |
|
6 7 8 |
b-Myrcene Bornylacetate α –Phellandrene |
0.93 2.52 0.18 |
992/991 1289/1280 1008/1003 |
|
|
Oxygenated Monoterpenes |
58.87 |
|
|
7 |
p-Cymene |
1.33 |
1028/1026 |
|
8 |
1,8-Cineole |
39.67 |
1036/1041 |
|
9 |
Borneol |
10.51 |
1185/1164 |
|
10 |
Carvacrol |
0.86 |
1433/1306 |
|
11 |
Linalool |
1.48 |
1151/1175 |
|
12 |
Terpinen-4-ol |
1.31 |
1200/1164 |
|
13 |
ɒ -Terpineol |
4.17 |
1208/1200 |
|
|
Sesquiterpenes hydrocarbons |
21.36 |
|
|
14 |
α –Caryophyllene |
0.22 |
1459/1428 |
|
15 |
b-Caryophyllene |
3.1 |
1429/1421 |
|
16 |
Camphor |
18.04 |
1175/1153 |
|
|
Othercompounds |
6.88 |
|
|
18 |
Heptane |
2.58 |
|
|
19 |
Cycloheptasiloxane |
0.33 |
1526.55/ |
|
21 |
1,2 -Phenylene |
1.02 |
|
|
22 |
Tetracyclohexane |
0.25 |
1854.48/ |
|
|
Minorcompounds |
1.83 |
|
|
|
Major compounds |
97.5 |
|
|
|
Total identified compounds (%) |
99.33 |
|
RI a: Retention Index determined according to the homologous series of n-alkanes (C9-C24); RIb; Literature Retention Index
Table 3 Encapsulation efficiency (EE) and Loaded capacity (LC) of Rosmarinus officinalis essential oil loaded in chitosan matrix determined by UV-Vis spectrophotometry
|
Chiosane: gum arabic: R. officinalis EO (w/w) |
EE (%)±SE |
LC (%)±SE |
|
Ratio 1: 1 :0.5 |
45.8±0.67 |
2.31±0.34 |
|
Ratio 1: 1 :0.25 |
25.9±0.51 |
1.49±0.05 |
|
Ratio 1: 1 :0.1 |
12.9±0.82 |
0.6±0.01 |
Table 4 Composition of Rosmarinus officinalis essential oil before and after one day of formulation.
|
|
|
Chemical components (%) on free EO |
Chemical components (%) after formulation |
|
1 |
α-Pinene |
6.33 |
5.6 |
|
2 |
α -Terpinene |
0.39 |
0 |
|
3 |
b-Pinene |
2.84 |
2.11 |
|
4 |
g-Terpinene |
0.7 |
0.56 |
|
5 |
ɒ-Terpinolene |
0.62 |
0.3 |
|
6 |
b-Myrcene |
0.93 |
0.45 |
|
7 |
O-Cymene |
1.33 |
0.12 |
|
8 |
1,8-Cineole |
39.67 |
31.25 |
|
9 |
Borneol |
10.51 |
7.33 |
|
10 |
Linalool |
1.48 |
0.98 |
|
11 |
Terpinene-4-ol |
1.31 |
0.81 |
|
12 |
ɒ -Terpineol |
4.17 |
4.1 |
|
13 |
b-Caryophyllene |
3.1 |
3 |
|
14 |
Camphor |
18.04 |
11.06 |
|
15 |
Heptane |
2.58 |
1.99 |
|
16 |
α –Bornylacetane |
2.52 |
1.53 |
|
17 |
1,2 Phenylene |
1.02 |
0.8 |
Table 5 Pearson correlation matrix between chemical components and mortality percentages of Tribolium castaneum and Oryzeaphilus surrinamensis during different storage periods under ambient condition
|
|
Storage period |
1,8-Cineole |
Borneol |
Camphor |
b-Caryophyllene |
Mortality T. castaneum (%) |
Mortality O. surrinamensis |
|
Storage period |
1 |
-.990 |
-1.000* |
-.995 |
-1.000** |
-0.991 |
-.986 |
|
1.8-Cineole |
-1.000* |
.987 |
1 |
.997* |
1.000** |
0.990* |
.982* |
|
Borneol |
-1.000* |
.987 |
1 |
.997* |
1.000** |
0.930 |
.982 |
|
Camphor |
-.995 |
.973 |
.997* |
1 |
.996 |
0.978 |
.965 |
|
1.2 Penylene |
-.877 |
.935 |
.865 |
.826 |
.871 |
0.854 |
.945 |
|
b-Caryophyllene |
-1.000** |
.989 |
1.000** |
.996 |
1 |
0.993 |
.984 |