Many EOs originated from diverse plant families have been considered and studied as potential sources of natural agrochemicals. In this respect, previous research results on Umbeliferae (Apiaceae) family plant materials revealed the significant acaricidal activities of butylidenepthalides isolated from *Angelica acutiloba* Kitagawa var. *sugiyame* Hikino (Kwon & Ahn, 2002) and the similar activity of the EO of *Foeniculum vulgare,* attributed to the presence of *p*-anisaldehyde and (+,−)-fenchone in the EO (Lee, 2004). These EOs were practically inactive in fumigant toxicity tests against *Lycoriella mali* though they are known to contain the active monoterpenes *α*-pinene and *β*-pinene (Choi et al., 2006), which are common constituents of many Umbelliferae EOs. Methanolic extracts of *Angelica dahurica*, *Cnidium officinale*, and *Foeniculum vulgare* were also tested against the Coleoptera *Lasioderma sericorne*, *Sitophilus oryzae* and *Callosobruchus chinensis* exhibiting a moderate activity only the second extract (Kim et al., 2003a; Kim et al., 2003b). Other EOs of this family screened as inactive against coleoptera were originated from the species *Anethum graveolens* L., *Apium graveolens* Houtt., *Coriandrum sativum* L., *Cuminum cyminum* L. and *Petroselinum sativum* L. (Regnault-Roger & Hamraoui, 1994; Papachristos & Stamopoulos, 2002). On the contrary, the EOs of *Pimpinella anisum* L. and *Cuminum cyminum* L. displayed excellent ovicidal and insecticidal activities against the *Tribolium confusum* du Val and the *Ephestia kuehniella* Zeller (Tunc et al., 2000). In addition, the aqueous extract of *Pimpinella anisum* exhibited good repellent effect against the adults of sweet potato

These rather controversial results are not connected with the impressive activities that Umbelliferae EOs were found to exhibit against the Diptera, with the EO of *Ammi visnaga*  displaying -among 19 EOs- the most potent ovicidal activity against *Mayetiola destructor*  (Lamiri et al., 2001). In addition, tests against *Droshophila melanogaster* of furanocoumarins and pthalides isolated from *Angelica acutiloba* Kitagawa var. *sugiyame* Hikino revealed the hypothesis that the insecticidal properties of the plant extracts are connected with the acetylcholinesterase inhibition (Miyazawa et al., 2004). Finally, alkylpthalides originated from *Cnidium officinale* Makino were tested as extremely effective against *Droshophila*

substitutes that also display the major disadvantage of resistance development (Alves et al., 2010; Daaboub et al., 2008; Lima et al., 2011).

Recent research trend on mosquito chemical control mainly focuses on currently used compounds, aiming to enhance their potency and circumvent the problems connected with their application. In this respect, the so far developed pyrazole derivatives are quite efficient exhibiting however adverse environmental effects (Stevens et al., 2011), while the corresponding pyrroles display the desirable efficiency (Raghavendra et al., 2011) but adequate research on their environmental side effects is still underway. Amides, such as methazolamide and acetazolamide, were also evaluated as potent mosquito larvicides but were found to display significant bioaccumulation properties (Del Pilar Corena et al., 2006) which discourage their broad use. Finally, various novel pyrimido-quinolione molecules have been developed and assessed as highly toxic for other organisms (Rajanarendar et al., 2010). Ray (2010) recognized that the insecticide treated nets, in connection with the long lasting insecticidal nets, have resurrected the chemical control of malaria's mosquito vector. This may be rationalized considering that their targeted application resolves the problems connected with the environmental impacts of chemical control agents since limit their expansion, availability and environmental penetration.

Despite the numerous efforts and progress achieved, the efficacy of insecticidal nets in malaria prevention still constitutes a hot issue, since depends strongly upon a plethora of additional factors (Killeen & Smith, 2007). In particular, despite efforts (Pennetier et al., 2010) to overcome the recognized for longtime resistance development issues of insecticidal nets, todate these problems have not been resolved (Yadouleton et al., 2011). An additional drawback derives by the combined impact of herbicide application that promotes the crossresistance to mosquito populations (Boyer et al., 2006; Riaz et al., 2009).

The corresponding biological control has dictated the development of novel-alternative mosquito control tools, including the sterile males technique (Patersson et al., 1968), the genetically modified mosquitoes (Gu et al., 2011; Lavialle-Defaix et al., 2011), the entomopathogenic fungi (Van Breukelen et al., 2011; Kanzok & Jacobs-Lorena, 2006) and bacteria. Among the tools developed the bacterial pathogens application is considered as the most prominent intervention, displaying species selective insecticidal ability (Hayes et al., 2011) which is considered as an efficient means for mosquito control without harmful impacts for the environment (Caquet et al., 2011). Major thresholds limiting the wider application of this technique are related with the induced pathogen introduction among the natural mosquito populations (Hancock et al., 2011) and the threats connected with bioaccumulation and resistance development (Tilquin et al., 2008). In general, the biological control tools are still under development, presenting todate a low degree of maturity for large-scale interventions.

Temephos was considered as one of the most potent-safe insecticides. Its recent exclusion from Annex I of the Directive 98/8/EC resulted in the discontinuation of its application in mosquito control programs by the European, emerging the development and use of newsafer insecticides. Thus, relative research directed towards the discovery-development of novel molecules, capable to control the mosquito populations without exhibiting the disadvantages of synthetic pesticides. In this respect, the plant originated natural compounds constitute a large deposit of such molecules, inherently allowing the retrieval of various commercially successful molecules like pyrethrins. Todate, the search for novel, potent and safer pesticides from this deposit has already provided several candidates, either as pure compounds and/or their extracts. Specifically, various organic acids such as lactic and orthophosporic acids (Chakraborty et al., 2010), alkaloids (Talontsi et al., 2011) and plant proteins (Chowdhury et al., 2008) have been identified as efficient mosquito control agents. Furthermore, several plants were used as the maternal material to produce bioproducts which were applied against mosquitoes with hopeful results (Shaalan et al., 2005; Sukumar et al., 1991). On the other hand, the plant derived Essential Oils (EOs) constitute a special category of natural products that exhibit the major advantage -for the mosquito control endeavor- of exhibiting an insect oriented mode of action with low penetrability to the ecosystems that does not affect larger animals. In addition, the natural diversity of their constituents addresses effectively the problem of resistance development (Isman, 2000).

## **2. Literature review**

614 Integrated Pest Management and Pest Control – Current and Future Tactics

substitutes that also display the major disadvantage of resistance development (Alves et al.,

Recent research trend on mosquito chemical control mainly focuses on currently used compounds, aiming to enhance their potency and circumvent the problems connected with their application. In this respect, the so far developed pyrazole derivatives are quite efficient exhibiting however adverse environmental effects (Stevens et al., 2011), while the corresponding pyrroles display the desirable efficiency (Raghavendra et al., 2011) but adequate research on their environmental side effects is still underway. Amides, such as methazolamide and acetazolamide, were also evaluated as potent mosquito larvicides but were found to display significant bioaccumulation properties (Del Pilar Corena et al., 2006) which discourage their broad use. Finally, various novel pyrimido-quinolione molecules have been developed and assessed as highly toxic for other organisms (Rajanarendar et al., 2010). Ray (2010) recognized that the insecticide treated nets, in connection with the long lasting insecticidal nets, have resurrected the chemical control of malaria's mosquito vector. This may be rationalized considering that their targeted application resolves the problems connected with the environmental impacts of chemical control agents since limit their

Despite the numerous efforts and progress achieved, the efficacy of insecticidal nets in malaria prevention still constitutes a hot issue, since depends strongly upon a plethora of additional factors (Killeen & Smith, 2007). In particular, despite efforts (Pennetier et al., 2010) to overcome the recognized for longtime resistance development issues of insecticidal nets, todate these problems have not been resolved (Yadouleton et al., 2011). An additional drawback derives by the combined impact of herbicide application that promotes the cross-

The corresponding biological control has dictated the development of novel-alternative mosquito control tools, including the sterile males technique (Patersson et al., 1968), the genetically modified mosquitoes (Gu et al., 2011; Lavialle-Defaix et al., 2011), the entomopathogenic fungi (Van Breukelen et al., 2011; Kanzok & Jacobs-Lorena, 2006) and bacteria. Among the tools developed the bacterial pathogens application is considered as the most prominent intervention, displaying species selective insecticidal ability (Hayes et al., 2011) which is considered as an efficient means for mosquito control without harmful impacts for the environment (Caquet et al., 2011). Major thresholds limiting the wider application of this technique are related with the induced pathogen introduction among the natural mosquito populations (Hancock et al., 2011) and the threats connected with bioaccumulation and resistance development (Tilquin et al., 2008). In general, the biological control tools are still under development, presenting todate a low degree of maturity for

Temephos was considered as one of the most potent-safe insecticides. Its recent exclusion from Annex I of the Directive 98/8/EC resulted in the discontinuation of its application in mosquito control programs by the European, emerging the development and use of newsafer insecticides. Thus, relative research directed towards the discovery-development of novel molecules, capable to control the mosquito populations without exhibiting the disadvantages of synthetic pesticides. In this respect, the plant originated natural compounds constitute a large deposit of such molecules, inherently allowing the retrieval of

2010; Daaboub et al., 2008; Lima et al., 2011).

expansion, availability and environmental penetration.

large-scale interventions.

resistance to mosquito populations (Boyer et al., 2006; Riaz et al., 2009).

## **2.1 Umbelliferae (Apiaceae) family: A source of potent natural agrochemicals**

Many EOs originated from diverse plant families have been considered and studied as potential sources of natural agrochemicals. In this respect, previous research results on Umbeliferae (Apiaceae) family plant materials revealed the significant acaricidal activities of butylidenepthalides isolated from *Angelica acutiloba* Kitagawa var. *sugiyame* Hikino (Kwon & Ahn, 2002) and the similar activity of the EO of *Foeniculum vulgare,* attributed to the presence of *p*-anisaldehyde and (+,−)-fenchone in the EO (Lee, 2004). These EOs were practically inactive in fumigant toxicity tests against *Lycoriella mali* though they are known to contain the active monoterpenes *α*-pinene and *β*-pinene (Choi et al., 2006), which are common constituents of many Umbelliferae EOs. Methanolic extracts of *Angelica dahurica*, *Cnidium officinale*, and *Foeniculum vulgare* were also tested against the Coleoptera *Lasioderma sericorne*, *Sitophilus oryzae* and *Callosobruchus chinensis* exhibiting a moderate activity only the second extract (Kim et al., 2003a; Kim et al., 2003b). Other EOs of this family screened as inactive against coleoptera were originated from the species *Anethum graveolens* L., *Apium graveolens* Houtt., *Coriandrum sativum* L., *Cuminum cyminum* L. and *Petroselinum sativum* L. (Regnault-Roger & Hamraoui, 1994; Papachristos & Stamopoulos, 2002). On the contrary, the EOs of *Pimpinella anisum* L. and *Cuminum cyminum* L. displayed excellent ovicidal and insecticidal activities against the *Tribolium confusum* du Val and the *Ephestia kuehniella* Zeller (Tunc et al., 2000). In addition, the aqueous extract of *Pimpinella anisum* exhibited good repellent effect against the adults of sweet potato whitefly *Bemisia tabaci* (Ateyyat et al., 2007).

These rather controversial results are not connected with the impressive activities that Umbelliferae EOs were found to exhibit against the Diptera, with the EO of *Ammi visnaga*  displaying -among 19 EOs- the most potent ovicidal activity against *Mayetiola destructor*  (Lamiri et al., 2001). In addition, tests against *Droshophila melanogaster* of furanocoumarins and pthalides isolated from *Angelica acutiloba* Kitagawa var. *sugiyame* Hikino revealed the hypothesis that the insecticidal properties of the plant extracts are connected with the acetylcholinesterase inhibition (Miyazawa et al., 2004). Finally, alkylpthalides originated from *Cnidium officinale* Makino were tested as extremely effective against *Droshophila melanogaster* (Tsukamoto et al., 2005).

Essential Oils of Umbelliferae

*Apium graveolens* Seeds

*Bupleurum*

*Carum ptroselinum*

*Conium*

*Conopodium*

*Cryptotaenia canadensis*

*Eleoselinum*

*Ferula*

*Foeniculum*

*Foeniculum vulgare*

*Foeniculum*

*Foeniculum*

*Foeniculum vulgare*

*Ferula galbaniflua* (not

*Chaerophyllum*

*Apium graveolens* Seeds *Aedes aegypti,*

*Carum carvi* Fruits *Aedes aegypti, Culex*

*Carum carvi* Seeds *Aedes aegypti,*

(not

Fresh foliage, root, fruits

*Daucus carota* Roots *Aedes aegypti, Culex*

mentioned)

(not

*vulgare* Fruits *Aedes aegypti,*

Stems, inflorencenc es, leaves

(Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control 617

*Anopheles ditrus* Larvicidal Pitasawat et al.,

*quinquefasciatus* Larvicidal Lee, 2006

mentioned) *Culex pipiens* Larvicidal Khater and

*Anopheles ditrus* Larvicidal Pitasawat et al.,

*Culex pipiens* Larvicidal Eckenbach et al.,

*quinquefasciatus* Larvicidal Lee, 2006

mentioned) *Aedes aegypti* Larvicidal Orozco & Lentz,

repellent

*Anopheles ditrus* Larvicidal Pitasawat et al.,

*Culex pipiens* Larvicidal Manolakou et al.,

2007

2009

2009

2007

2009

1999

2009

2005

2005

2007

2009

Mehlhorn, 2006

Trabousli et al.,

Larvicidal Amer and

Shalaby, 2008

Repellent Tuetun et al.,

**Species Part used Mosquito species Bioactivity Reference**

*Athamanta densa* Aerial parts *Culex pipiens* Larvicidal (present study)

*fruticosum* Aerial parts *Culex pipiens* Larvicidal Evergetis et al.,

*heldreichii* Aerial parts *Culex pipiens* Larvicidal (present study)

*divaricatum* Aerial parts *Culex pipiens* Larvicidal (present study)

*capillifolium* Aerial parts *Culex pipiens* Larvicidal Evergetis et al.,

*Coriander sativum* Seeds *Ochlerotatus caspius* Larvicidal Knio et al., 2008

*asclepium* Aerial parts *Culex pipiens* Larvicidal Evergetis et al.,

*Ferula assa-foetida* Stems *Culex quinquefasciatus* Larvicidal Pavela, 2009

*lancerottensis* Stems *Culex quinquefasciatus* Larvicidal Pavela, 2008 *Ferulago nodosa* Aerial parts *Culex pipiens* Larvicidal (present study)

*vulgare* Fruits *Aedes aegypti* Repellent Kim et al., 2002

*vulgare* Flowers *Culex pipiens* Larvicidal,

*Anopheles stephensi, Aedes aegypti, Culex quinquefasciatus*

*Aedes, Anopheles, Armigeres, Culex, Mansonia*

## **2.2 Umbelliferae (Apiaceae) family: A strong focal point for mosquito control**

**Table 1** summarizes the test results against various mosquito species reported for all extracts and EOs derived from plants belonging to the Umbeliferae family. Same table also contains the test results of fourteen EOs, which appear herein for the first time. Results indicate that the organic phase of the *Cryoptaenia canadensis* extract is the most active against fourth instars of *Culex pipiens*, leading to the isolation –from the extract- of the acetylated very toxic (LC50 values lower than 10 mgl-1) molecules of falcarinol and falcarindiol (Eckenbach et al., 1999). The larvicidal properties of hexane soluble fraction of *Apium graveolens* seeds -a plant with pleasant aroma- and three isolated compounds (sedanolide, senkyunolide-N, senkyunolide-J) against *Aedes aegypti* mosquitoes highlighted sedanolide as very active (100% mortality at 50 mgl-1, Momin & Nair, 2001). As a consequence, a gel containing 5% of the *Apium graveolens* hexane extract was developed, providing full protection to volunteers from mosquito bites for two hours (Tuetun et al., 2009), while the ethanolic formulations from the same plant also provided protection against *Aedes aegypti*. Another formulation containing the aforementioned hexane extract and 5% vanillin showed strong repellent activities against different mosquito species (Tuetun et al., 2005, see also **Table 1** for details). The crude seed extract had no adverse effects on human volunteers skins when tested for several anti-mosquito properties (Choochote et al., 2004). This plant's EO exhibits potent larvicidal activity against two laboratory-reared mosquito species, the malaria vector *Anopheles dirus* and the vector of dengue *Aedes aegypti* (Pitasawat et al., 2007).

**Table 1** summarizes the test results against various mosquito species reported for all extracts and EOs derived from plants belonging to the Umbeliferae family. Same table also contains the test results of fourteen EOs, which appear herein for the first time. Results indicate that the organic phase of the *Cryoptaenia canadensis* extract is the most active against fourth instars of *Culex pipiens*, leading to the isolation –from the extract- of the acetylated very toxic (LC50 values lower than 10 mgl-1) molecules of falcarinol and falcarindiol (Eckenbach et al., 1999). The larvicidal properties of hexane soluble fraction of *Apium graveolens* seeds -a plant with pleasant aroma- and three isolated compounds (sedanolide, senkyunolide-N, senkyunolide-J) against *Aedes aegypti* mosquitoes highlighted sedanolide as very active (100% mortality at 50 mgl-1, Momin & Nair, 2001). As a consequence, a gel containing 5% of the *Apium graveolens* hexane extract was developed, providing full protection to volunteers from mosquito bites for two hours (Tuetun et al., 2009), while the ethanolic formulations from the same plant also provided protection against *Aedes aegypti*. Another formulation containing the aforementioned hexane extract and 5% vanillin showed strong repellent activities against different mosquito species (Tuetun et al., 2005, see also **Table 1** for details). The crude seed extract had no adverse effects on human volunteers skins when tested for several anti-mosquito properties (Choochote et al., 2004). This plant's EO exhibits potent larvicidal activity against two laboratory-reared mosquito species, the malaria vector *Anopheles dirus* and the vector of dengue *Aedes aegypti* (Pitasawat et al., 2007).

**2.2 Umbelliferae (Apiaceae) family: A strong focal point for mosquito control**

**Species Part used Mosquito species Bioactivity Reference**  *Ammi visnaga* Seeds *Culex quinquefasciatus* Larvicidal Pavela, 2008

> *Anopheles stephensi, Aedes aegypti, Culex quinquefasciatus*

*archangelica* Fruits *Culex quinquefasciatus* Larvicidal Pavela, 2009 *Angelica sylvestris* Aerial parts *Culex pipiens* Larvicidal (present study) *Apium graveolens* Seeds *Aedes aegypti* Larvicidal Momin & Nair,

> *Aedes aegypti, Aedes gardnerii, Aedes lineatopennis, Anopheles barbirostris, Armigeres subalbatus, Culex tritaeniorhynchus, Culex gelicus, Culex vishnui* group, *Mansonia uniformis*

Larvicidal Amer and

Larvicidal, effects on growth and development

Larvicidal, adulticidal, repellent

Mehlhorn, 2006

Promsiri et al.,

Choochote, 2004

2006

2001

2005

Repellent Tuetun et al.,

*Anethum graveolens*

*Anethum graveolens*

*Angelica*

(not mentioned)

Leaves,

*Apium graveolens* Seeds *Aedes aegypti*

*Apium graveolens* Seeds

twigs *Aedes aegypti*

Essential Oils of Umbelliferae

(Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control 619

Another EO found to possess potent larvicidal, oviposition-deterrent, vapor toxicity and repellent activities against *Aedes aegypti* was isolated from ajowan (*Tachyspermum ammi*, Pandey et al. 2009). *Anethum graveolens* extract exhibited larval toxicity with LC50 values from 27 to 20 mgl-1 (for 24 and 48 hours exposures respectively), while on growth survival and prolongation tests of the various instar larvae of *Aedes aegypti,* the second instar larvae was determined as the more susceptible. The lowest concentration of crude extracts of *Anethum graveolens* used (caused more than 50% larval mortality) was not toxic to guppy

Among all EOs tested for mosquito control, the most potent was derived from *Foeniculum vulgare*, which caused the highest mortality against *Aedes albopictus* (Conti et al., 2010) and moderate against *Anopheles dirus* and *Aedes aegypti* (Pitasawat et al., 2007). Main component of this EO is methyl chavicol (more than 43%), while its methanolic extract (*trans-*anethole chemotype) was moderately active against *Aedes aegypti*, the yellow fever mosquito (Orozco & Lentz, 2005). The hexane fraction from its fruit-derived parts showed 99% repellency against *Aedes aegypti*, while the other fractions (chloroform, ethyl acetate and water: 37, 37 and 17% respectively) were practically inactive (Kim et al., 2002). Repellency and toxicity were also studied against *Culex pipiens* (Trabousli et al., 2005), indicating that the EO of *Foeniculum vulgare* was the most effective, while the repellency assays revealed protection

*Pimpinella anisum* L. EO proved to possess equally potent larvicidal and ovicidal activities against *Anopheles stephensi*, *Aedes aegypti, Culex quinquefasciatus* and only larvicidal against *Ochlerotatus caspius* (Prajapati et al., 2005; Knio et al., 2008). Similar larvicidal activity results were also observed when the EOs of *Coriander sativum* and *Petroselinum crispum* were tested against *Ochlerotatus caspius* (Knio et al., 2008). The larvicidal tests of EOs of genus *Carum* were performed for *Carum carvi* against *Anopheles dirus* and *Aedes aegypti* and for *Carum petroselinum*  against *Culex pipiens* (Lee, 2006; Pitasawat et al., 2007; Khater & Shalaby, 2008). The results were directly similar to those of the EO of *Daucus carota* (against *Anopheles dirus* and *Aedes aegypti*) proving their inability to cause 100% mortality at the lowest concentration (Lee, 2006). Among the methanolic extracts of 118 Euroasiatic plants, tested for their larvicidal effects against *Culex quinquefasciatus,* the species *Ammi visnaga* and *Seseli pallasii* were determined as two of the most toxic materials tested, with LC50 values lower than 10 mgl-1 (Pavela, 2008, 2009). On the other hand, the extracts of *Angelica archangelica* and *Imperatoria ostruthium* exhibited LC50 values lower than 70 mgl-1, while *Seseli tortuosum* and *Ferula lancerottensis* displayed moderate larvicidal activity (LC50 values around 430 mgl-1). The only inactive Apiaceae plant tested was *Ferula assa-foetida* (LC50 value higher of 1000 mgl-1), with the EO of *Ferula galbaniflua* exhibiting the weakest activity against *Culex quinquefasciatus* and *Anopheles stephensi* (mortality level less than 14% of dead larvae after 48 hours, Amer & Mehlhorn, 2006a). The same authors also reported that *Anopheles stephensi* was the most resistant to dill (*Anethum graveolens*), while the *Culex quinquefasciatus* the more sensitive. Dill was also evaluated for persistency to larvicidal effects under different conditions for 1 month after the preparation of its solutions. In all cases (open, closed, in light or in dark) the EO was active only when was used

fish (*Poecilia reticulata*) at concentrations of 12.5 mgl-1 (Promsiri et al., 2006).

time for almost one hour when applied at concentration of 3%.

immediately after preparation (Amer & Mehlhorn, 2006b).

Table 1.  Reported phytochemicals derived from plants belonging to Apiaceae family against

Aerial parts *Culex pipiens* Larvicidal (present study)

Larvicidal, adulticidal, ovicidal, ovipositiondeterrent, repellent

Larvicidal, ovipositiondeterrent, vapor toxicity, repellent

*Anopheles stephensi, Aedes aegypti, Culex quinquefasciatus*

*ostruthium* Roots *Culex quinquefasciatus* Larvicidal Pavela, 2009

2009

2009

Prajapati et al.,

2005

al.,2009

Pandey et al.,

2009

**Species Part used Mosquito species Bioactivity Reference**

*Foeniculum*

*Heracleum*

*Imperatoria*

*Laserpitium*

*Oenanthe*

*Petroselimum*

*Peucedanum*

*Peucedanum*

*Pimpinella*

*Pimpinella tragium* ssp tragium

*Smyrnium*

*Thamnosciadium*

*Trachyspermum*

various mosquito species.

*ammi* Seeds *Anopheles stephensi*

*Pimpinella anisum* Seeds

Another EO found to possess potent larvicidal, oviposition-deterrent, vapor toxicity and repellent activities against *Aedes aegypti* was isolated from ajowan (*Tachyspermum ammi*, Pandey et al. 2009). *Anethum graveolens* extract exhibited larval toxicity with LC50 values from 27 to 20 mgl-1 (for 24 and 48 hours exposures respectively), while on growth survival and prolongation tests of the various instar larvae of *Aedes aegypti,* the second instar larvae was determined as the more susceptible. The lowest concentration of crude extracts of *Anethum graveolens* used (caused more than 50% larval mortality) was not toxic to guppy fish (*Poecilia reticulata*) at concentrations of 12.5 mgl-1 (Promsiri et al., 2006).

Among all EOs tested for mosquito control, the most potent was derived from *Foeniculum vulgare*, which caused the highest mortality against *Aedes albopictus* (Conti et al., 2010) and moderate against *Anopheles dirus* and *Aedes aegypti* (Pitasawat et al., 2007). Main component of this EO is methyl chavicol (more than 43%), while its methanolic extract (*trans-*anethole chemotype) was moderately active against *Aedes aegypti*, the yellow fever mosquito (Orozco & Lentz, 2005). The hexane fraction from its fruit-derived parts showed 99% repellency against *Aedes aegypti*, while the other fractions (chloroform, ethyl acetate and water: 37, 37 and 17% respectively) were practically inactive (Kim et al., 2002). Repellency and toxicity were also studied against *Culex pipiens* (Trabousli et al., 2005), indicating that the EO of *Foeniculum vulgare* was the most effective, while the repellency assays revealed protection time for almost one hour when applied at concentration of 3%.

*Pimpinella anisum* L. EO proved to possess equally potent larvicidal and ovicidal activities against *Anopheles stephensi*, *Aedes aegypti, Culex quinquefasciatus* and only larvicidal against *Ochlerotatus caspius* (Prajapati et al., 2005; Knio et al., 2008). Similar larvicidal activity results were also observed when the EOs of *Coriander sativum* and *Petroselinum crispum* were tested against *Ochlerotatus caspius* (Knio et al., 2008). The larvicidal tests of EOs of genus *Carum* were performed for *Carum carvi* against *Anopheles dirus* and *Aedes aegypti* and for *Carum petroselinum*  against *Culex pipiens* (Lee, 2006; Pitasawat et al., 2007; Khater & Shalaby, 2008). The results were directly similar to those of the EO of *Daucus carota* (against *Anopheles dirus* and *Aedes aegypti*) proving their inability to cause 100% mortality at the lowest concentration (Lee, 2006).

Among the methanolic extracts of 118 Euroasiatic plants, tested for their larvicidal effects against *Culex quinquefasciatus,* the species *Ammi visnaga* and *Seseli pallasii* were determined as two of the most toxic materials tested, with LC50 values lower than 10 mgl-1 (Pavela, 2008, 2009). On the other hand, the extracts of *Angelica archangelica* and *Imperatoria ostruthium* exhibited LC50 values lower than 70 mgl-1, while *Seseli tortuosum* and *Ferula lancerottensis* displayed moderate larvicidal activity (LC50 values around 430 mgl-1). The only inactive Apiaceae plant tested was *Ferula assa-foetida* (LC50 value higher of 1000 mgl-1), with the EO of *Ferula galbaniflua* exhibiting the weakest activity against *Culex quinquefasciatus* and *Anopheles stephensi* (mortality level less than 14% of dead larvae after 48 hours, Amer & Mehlhorn, 2006a). The same authors also reported that *Anopheles stephensi* was the most resistant to dill (*Anethum graveolens*), while the *Culex quinquefasciatus* the more sensitive. Dill was also evaluated for persistency to larvicidal effects under different conditions for 1 month after the preparation of its solutions. In all cases (open, closed, in light or in dark) the EO was active only when was used immediately after preparation (Amer & Mehlhorn, 2006b).

Essential Oils of Umbelliferae

**3.2 Essential oils isolation**

*Athamanta densa* Boiss. &

*Chaerophyllum heldreichii*

*Laserpitium pseudomeum*  Orph., Heldr. & Sart. Ex

*Pimpinella tragium* ssp

*Pimpinella rigidula* (Boiss. &

*Scaligeria cretica* (Miller)

*Seseli parnassicum* Boiss. &

*Smyrnium rotundifolium*

*Thamnosciadium junceum*

Table 2. Collection data.

\*=Greek Endemic.

*Peucedanum neumayeri* (Vis.)

Boiss.*\**

(Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control 621

The freshly collected plant materials (steams, leaves and flowers) were washed thoroughly, chopped off finely and subjected to steam distillation in a Clevenger-type apparatus, using the Microwave Accelerated Reaction System (MARS 5) at 1400 W for 40 min with 3 L of H2O in order to obtain their EOs. The resulting oils were dried over anhydrous sodium sulphate

Orph. *\** AD Flowering 15.06.2005 Mt. Parnassos, Sterea

*Ferulago nodosa* (L.) Boiss. FN Flowering 02.05.2005 Antikyra, Sterea

Reichenb PN Flowering 28.08.2004 Mt. Smolikas, Hepiros,

*Peucedanum officinale* L. PO Flowering 15.07.2004 Mt. Oiti, Sterea Hellas,

tragium Vill. \* PT Flowering 15.07.2004 Mt. Oiti, Sterea Hellas,

*Pimpinella peregrina* L. PP Flowering 14.05.2005 Iraklio, Is. Crete, olive

Orph.) H. Wolf *\** PR Flowering 17.08.2004 Molai, Peloponnisos,

Boiss. SC Flowering 22.05.2005 Vouliagmeni, Sterea

Heldr. \* SP Flowering 15.07.2004 Mt. Oiti, Sterea Hellas,

(Sibth. & Sm.) Hartvig \* TJ Flowering 25.07.2004 Mt. Parnnassos, Sterea

Miller SR Flowering 02.05.2005 Distomo, Sterea

**Stage Date Location**

LP Flowering 15.07.2004 Mt. Oiti, Sterea Hellas,

Mt. Parnon, Peloponnisos, forest streams

Hellas, vertical cliffs

Mt. Parnon, Peloponnisos, forest clearings

Hellas, olive groves

rocky slopes

forest clearings

rocky slopes

rocky slopes

groves

roadside

Hellas, seaside

forest clearings

Hellas, roadside

Hellas, alpic ravine

and stored at 4 oC. The EO yield of each plant is included in **Table 3**.

*Angelica sylvestris* L. AS Flowering 05.09.2004

Orph. Ex Boiss. \* CH Flowering 25.07.2004

**Species Abbreviation Vegetative**

Finally, an interesting result was obtained during the study of several EOs using coupled gas chromatography-electroantennographic detection (GC-EAD), on the hypothesis that compounds can be detected by the antennae of the yellow fever mosquito, *Aedes aegypti*. Thus, cumin aldehyde and cumin alcohol the *Cuminum cynimum* EO components were identified as such molecules. It must be noted that for both components, their EO (cumin oil) was also EAD-active (Campbell et al., 2011)

## **2.3 Greek Umbelliferae (Apiaceae) plants extract activities against Culex pipiens mosquitoes**

The larvicidal activity of the EO obtained from the stem of Greek *Foeniculum vulgare* was determined against *Culex pipiens* larvae, while methyl chavicol was determined as its main component (more than 32%). Although the LC50 value of methyl chavicol was more than 80 mgl-1, the respective EO was determined as 2.1-fold more toxic (Manolakou et al., 2009). *Culex pipiens* larvae were also used to test the mosquito control properties of EO from various naturally growing plants throughout Greece, belonging to the following six different Apiaceae family taxa: *Heracleum sphondylium, Seseli montanum, Conopodium capillifolium, Bupleurum fruticosum, Oenanthe pimpinelloides, Eleoselinum asclepium.* All EOs tested displayed good larvicidal activities with LC50 values ranging from 40.26-96.96 mgl-1 (Evergetis et al., 2009)

As a continuation of our ongoing efforts to exploit the use of natural products for the development of environmentally friendly means for the mosquito population control, our interest was stimulated on the investigation of Umbeliferae (Apiaceae) plants EOs. In this context, we report herein the chemical composition and larvicidal activity results for 14 EOs originated from different taxon obtained during Greek Umbelliferae biodiversity studies (**Table 1**).

## **3. Materials and methods**

## **3.1 Plant material**

Fourteen different taxa of the Umbeliferae (Apiaceae) family, Apioideae subfamily belonging to seven tribes and twelve different genera have been collected during the present study. Representatives of the Apieae Tribe are *Pimpinella peregrina* L., and 5 Greek endemics, namely *Athamanta densa* Boiss. & Orph., *Pimpinella tragium* ssp *tragium* Vill., *Pimpinella rigidula* (Boiss. & Orph.) H. Wolf, *Seseli parnassicum* Boiss. & Heldr. and *Thamnosciadium junceum* (Sibth. & Sm.) Hartvig.; of Smyrnieae tribe *Scaligeria cretica* (Miller) Boiss. and *Smyrnium rotundifolium* Miller; of Angeliceae tribe *Angelica sylvestris* L.; of Scandiceae tribe the Greek endemic *Chaerophyllum heldreichii* Orph. Ex Boiss.; of Peucedaneae tribe *Ferulago nodosa* (L.) Boiss., *Peucedanum neumayeri* (Vis.) Reichenb, *Peucedanum officinale* L., and of Laserpitieae tribe the Greek endemic *Laserpitium pseudomeum* Orph., Heldr. & Sart. Ex Boiss (Pimenov & Leonov, 1993; Tutin et al., 1968).

Full collection details are provided in **Table 2.** A voucher specimen of each plant is deposited in the herbarium of the Agricultural University of Athens, Athens, Greece.

## **3.2 Essential oils isolation**

620 Integrated Pest Management and Pest Control – Current and Future Tactics

Finally, an interesting result was obtained during the study of several EOs using coupled gas chromatography-electroantennographic detection (GC-EAD), on the hypothesis that compounds can be detected by the antennae of the yellow fever mosquito, *Aedes aegypti*. Thus, cumin aldehyde and cumin alcohol the *Cuminum cynimum* EO components were identified as such molecules. It must be noted that for both components, their EO (cumin oil)

**2.3 Greek Umbelliferae (Apiaceae) plants extract activities against Culex pipiens**

The larvicidal activity of the EO obtained from the stem of Greek *Foeniculum vulgare* was determined against *Culex pipiens* larvae, while methyl chavicol was determined as its main component (more than 32%). Although the LC50 value of methyl chavicol was more than 80 mgl-1, the respective EO was determined as 2.1-fold more toxic (Manolakou et al., 2009). *Culex pipiens* larvae were also used to test the mosquito control properties of EO from various naturally growing plants throughout Greece, belonging to the following six different Apiaceae family taxa: *Heracleum sphondylium, Seseli montanum, Conopodium capillifolium, Bupleurum fruticosum, Oenanthe pimpinelloides, Eleoselinum asclepium.* All EOs tested displayed good larvicidal activities with LC50 values ranging from 40.26-96.96 mgl-1

As a continuation of our ongoing efforts to exploit the use of natural products for the development of environmentally friendly means for the mosquito population control, our interest was stimulated on the investigation of Umbeliferae (Apiaceae) plants EOs. In this context, we report herein the chemical composition and larvicidal activity results for 14 EOs originated from different taxon obtained during Greek Umbelliferae biodiversity

Fourteen different taxa of the Umbeliferae (Apiaceae) family, Apioideae subfamily belonging to seven tribes and twelve different genera have been collected during the present study. Representatives of the Apieae Tribe are *Pimpinella peregrina* L., and 5 Greek endemics, namely *Athamanta densa* Boiss. & Orph., *Pimpinella tragium* ssp *tragium* Vill., *Pimpinella rigidula* (Boiss. & Orph.) H. Wolf, *Seseli parnassicum* Boiss. & Heldr. and *Thamnosciadium junceum* (Sibth. & Sm.) Hartvig.; of Smyrnieae tribe *Scaligeria cretica* (Miller) Boiss. and *Smyrnium rotundifolium* Miller; of Angeliceae tribe *Angelica sylvestris* L.; of Scandiceae tribe the Greek endemic *Chaerophyllum heldreichii* Orph. Ex Boiss.; of Peucedaneae tribe *Ferulago nodosa* (L.) Boiss., *Peucedanum neumayeri* (Vis.) Reichenb, *Peucedanum officinale* L., and of Laserpitieae tribe the Greek endemic *Laserpitium pseudomeum* Orph., Heldr. & Sart. Ex Boiss

Full collection details are provided in **Table 2.** A voucher specimen of each plant is

deposited in the herbarium of the Agricultural University of Athens, Athens, Greece.

was also EAD-active (Campbell et al., 2011)

**mosquitoes**

(Evergetis et al., 2009)

studies (**Table 1**).

**3.1 Plant material**

**3. Materials and methods**

(Pimenov & Leonov, 1993; Tutin et al., 1968).

The freshly collected plant materials (steams, leaves and flowers) were washed thoroughly, chopped off finely and subjected to steam distillation in a Clevenger-type apparatus, using the Microwave Accelerated Reaction System (MARS 5) at 1400 W for 40 min with 3 L of H2O in order to obtain their EOs. The resulting oils were dried over anhydrous sodium sulphate and stored at 4 oC. The EO yield of each plant is included in **Table 3**.

\*=Greek Endemic.

Table 2. Collection data.

Essential Oils of Umbelliferae

**3.5 Larvicidal bioassays**

**3.6 Data analysis**

**4. Results and discussion 4.1 Phytochemical analysis**

*Thamnosciadium* Hartvig genera.

indices) are presented in **Table 4**.

humidity and photoperiod of 14:10 h (L:D).

LC90 values, and slopes were calculated (SPSS 11.0).

(Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control 623

diameter x 5 cm depth) filled with 150 ml of tap water. Egg rafts are removed daily and placed in cylindrical enamel pans (with diameter of 35 cm and 10 cm deep), in order to hatch. Larvae are reared under the same conditions of temperature and light and are fed daily with baby fish food (TetraMin, Baby Fish Food) at a concentration of 0.25 gl-1 of water until pupation. Pupae

Stock solutions of EOs tested were prepared in ethanol and maintained in a freezer as 1% mgl-1solutions. They were dissolved in double distilled water to produce solutions of the tested materials in concentrations ranging from 5 to 150 mgl-1. Prior to biological

The larval mortality bioassays were carried out according to the test method for larval susceptibility, proposed by the World Health Organization (WHO, 1981). Twenty 3rd to 4th instar larvae of the species *Culex pipiens* biotype *molestus* were collected from the colony, placed in a glass beaker with 250 ml of aqueous suspension of the tested material at various concentrations and an emulsifier was added in the final test solution (less than 0.05%). Four replicates were made per each concentration and a control treatment with tap water and emulsifier was also included. Beakers with larvae were placed at 25±2 oC, 80±2% relative

Larvicidal effect was recorded 48 h after treatment. Data obtained from each dose–larvicidal bioassay (total mortality, mgl-1 concentration in water) were subjected to probit analysis in which probit–transformed mortality was regressed against log10–transformed dose; LC50,

Fourteen distinct Umbeliferae taxa (twelve genera) are studied herein, one of which is endemic to Greece (*Thamnosciadium* Hartvig). It must be noted that there are no literature reports and studies on the EOs and their chemical compostitions for the material obtained from the plants *Athamanta densa* Boiss. & Orph. (AD), *Chaerophyllum heldreichii* Orph. Ex Boiss. (CH), *Laserpitium pseudomeum* Orph., Heldr. & Sart. Ex Boiss. (LP), *Peucedanum neumayeri* (Vis.) Reichenb (PN), *Pimpinella tragium* ssp tragium Vill. (PT), *Pimpinella rigidula*  (Boiss. & Orph.) H. Wolf (PR), *Scaligeria cretica* (Miller) Boiss. (SC), *Seseli parnassicum* Boiss. & Heldr. (SP) and *Smyrnium rotundifolium* Miller (SR). In addition, the discussion section on the related *taxa* EOs compositions includes ten (out of twelve) genera studied herein, since there are also no previous reports on the composition of EOs obtained from *Conium* L. and

In total seventy phytochemicals, representing 76.64 to 99.83 % of the respective EOs samples have been identified as their constituents using combined GC and GC/MS analyses and in certain occasions verified by NMR studies. The detailed qualitative and quantitative analytical data of the main constituents of steam volatiles (and their respective retention

are then collected and introduced into the adult rearing cages.

determinations the toxicity of each EO was evaluated (data not shown).

\*=Greek Endemic.

Table 3. Essential oils yields.

## **3.3 Gas Chromatography-Mass Spectrometry (GC-MS) analyses**

Gas Chromatography (GC). All GC analyses were carried out on a Agilent Technologies 7890A gas chromatograph, fitted with a HP 5MS 30m x 0.25mm x 0.25μm film thickness capillary column and FID. The column temperature was programmed from 60 to 280 °C at a initial rate of 3 °C/min. The injector and detector temperatures were programmed at 230 and 300 °C, respectively. Helium was used as the carrier gas at a flow rate 1 ml/min.

Gas Chromatography-Mass Spectrometry (GC-MS). The GCMS analyses were performed on the same instrument using the Agilent 5957C, VL MS Detector with Triple-Axis Detector system operating in EI mode (equipped with a HP 5MS 30m x 0.25mm x 0.25μm film thickness capillary column), using He (1 ml/min) as the carrier gas. The initial temperature of the column was 60 °C. The column was heated gradually to 280 °C with a 3 °C/min rate. The identification of the compounds was based on comparison of their retention indices (RI) (Van den Dool & Kratz, 1963), obtained using various n-alkanes (C9-C24). Also, their EImass spectra were compared with the NIST/NBS and Wiley library spectra and the literature (Adams, 1995; Massada, 1976). Additionally, the identity of the indicated phytochemicals was confirmed by comparison with available authentic samples.

#### **3.4 Mosquito rearing**

A colony of the species *Culex pipiens* biotype *molestus* is maintained for more than 25 years in the laboratory of Entomology of the Benaki Phytopathological Institute, Kifissia, Greece. Adult mosquitoes are kept in wooden framed cages (33x33x33 cm) with a 32x32 mesh at 25±2 oC, 80±2% relative humidity and photoperiod of 14:10 (L:D) h. Cotton wicks saturated with 10% sucrose solution are used as food source. Females lay eggs in round, plastic containers (10 cm diameter x 5 cm depth) filled with 150 ml of tap water. Egg rafts are removed daily and placed in cylindrical enamel pans (with diameter of 35 cm and 10 cm deep), in order to hatch. Larvae are reared under the same conditions of temperature and light and are fed daily with baby fish food (TetraMin, Baby Fish Food) at a concentration of 0.25 gl-1 of water until pupation. Pupae are then collected and introduced into the adult rearing cages.

## **3.5 Larvicidal bioassays**

622 Integrated Pest Management and Pest Control – Current and Future Tactics

*Angelica sylvestris* L. Aerial 920 0,5 *Athamanta densa* Boiss. & Orph. *\** Aerial 450 0,5

Boiss. \* Aerial 530 0,7 *Ferulago nodosa* (L.) Boiss. Aerial 400 0,7

& Sart. Ex Boiss. *\** Aerial 270 0,9 *Peucedanum neumayeri* (Vis.) Reichenb Aerial 600 0,5 *Peucedanum officinale* L. Aerial *180 0,8 Pimpinella tragium* ssp tragium Vill. \* Aerial 650 1,5 *Pimpinella peregrina* L. Aerial 500 0,4

H. Wolf *\** Aerial 235 0,7 *Scaligeria cretica* (Miller) Boiss. Aerial 200 0,5 *Seseli parnassicum* Boiss. & Heldr. \* Aerial *200 0,5 Smyrnium rotundifolium* Miller Aerial 530 0,9

Sm.) Hartvig \* Aerial 600 2,0

Gas Chromatography (GC). All GC analyses were carried out on a Agilent Technologies 7890A gas chromatograph, fitted with a HP 5MS 30m x 0.25mm x 0.25μm film thickness capillary column and FID. The column temperature was programmed from 60 to 280 °C at a initial rate of 3 °C/min. The injector and detector temperatures were programmed at 230

Gas Chromatography-Mass Spectrometry (GC-MS). The GCMS analyses were performed on the same instrument using the Agilent 5957C, VL MS Detector with Triple-Axis Detector system operating in EI mode (equipped with a HP 5MS 30m x 0.25mm x 0.25μm film thickness capillary column), using He (1 ml/min) as the carrier gas. The initial temperature of the column was 60 °C. The column was heated gradually to 280 °C with a 3 °C/min rate. The identification of the compounds was based on comparison of their retention indices (RI) (Van den Dool & Kratz, 1963), obtained using various n-alkanes (C9-C24). Also, their EImass spectra were compared with the NIST/NBS and Wiley library spectra and the literature (Adams, 1995; Massada, 1976). Additionally, the identity of the indicated

A colony of the species *Culex pipiens* biotype *molestus* is maintained for more than 25 years in the laboratory of Entomology of the Benaki Phytopathological Institute, Kifissia, Greece. Adult mosquitoes are kept in wooden framed cages (33x33x33 cm) with a 32x32 mesh at 25±2 oC, 80±2% relative humidity and photoperiod of 14:10 (L:D) h. Cotton wicks saturated with 10% sucrose solution are used as food source. Females lay eggs in round, plastic containers (10 cm

and 300 °C, respectively. Helium was used as the carrier gas at a flow rate 1 ml/min.

phytochemicals was confirmed by comparison with available authentic samples.

**3.3 Gas Chromatography-Mass Spectrometry (GC-MS) analyses**

**aerial parts (g) Volume of oil (mL)**

**Species Part distilled Weight of**

*Chaerophyllum heldreichii* Orph. Ex

*Pimpinella rigidula* (Boiss. & Orph.)

*Thamnosciadium junceum* (Sibth. &

Table 3. Essential oils yields.

**3.4 Mosquito rearing**

\*=Greek Endemic.

*Laserpitium pseudomeum* Orph., Heldr.

Stock solutions of EOs tested were prepared in ethanol and maintained in a freezer as 1% mgl-1solutions. They were dissolved in double distilled water to produce solutions of the tested materials in concentrations ranging from 5 to 150 mgl-1. Prior to biological determinations the toxicity of each EO was evaluated (data not shown).

The larval mortality bioassays were carried out according to the test method for larval susceptibility, proposed by the World Health Organization (WHO, 1981). Twenty 3rd to 4th instar larvae of the species *Culex pipiens* biotype *molestus* were collected from the colony, placed in a glass beaker with 250 ml of aqueous suspension of the tested material at various concentrations and an emulsifier was added in the final test solution (less than 0.05%). Four replicates were made per each concentration and a control treatment with tap water and emulsifier was also included. Beakers with larvae were placed at 25±2 oC, 80±2% relative humidity and photoperiod of 14:10 h (L:D).

## **3.6 Data analysis**

Larvicidal effect was recorded 48 h after treatment. Data obtained from each dose–larvicidal bioassay (total mortality, mgl-1 concentration in water) were subjected to probit analysis in which probit–transformed mortality was regressed against log10–transformed dose; LC50, LC90 values, and slopes were calculated (SPSS 11.0).

## **4. Results and discussion**

## **4.1 Phytochemical analysis**

Fourteen distinct Umbeliferae taxa (twelve genera) are studied herein, one of which is endemic to Greece (*Thamnosciadium* Hartvig). It must be noted that there are no literature reports and studies on the EOs and their chemical compostitions for the material obtained from the plants *Athamanta densa* Boiss. & Orph. (AD), *Chaerophyllum heldreichii* Orph. Ex Boiss. (CH), *Laserpitium pseudomeum* Orph., Heldr. & Sart. Ex Boiss. (LP), *Peucedanum neumayeri* (Vis.) Reichenb (PN), *Pimpinella tragium* ssp tragium Vill. (PT), *Pimpinella rigidula*  (Boiss. & Orph.) H. Wolf (PR), *Scaligeria cretica* (Miller) Boiss. (SC), *Seseli parnassicum* Boiss. & Heldr. (SP) and *Smyrnium rotundifolium* Miller (SR). In addition, the discussion section on the related *taxa* EOs compositions includes ten (out of twelve) genera studied herein, since there are also no previous reports on the composition of EOs obtained from *Conium* L. and *Thamnosciadium* Hartvig genera.

In total seventy phytochemicals, representing 76.64 to 99.83 % of the respective EOs samples have been identified as their constituents using combined GC and GC/MS analyses and in certain occasions verified by NMR studies. The detailed qualitative and quantitative analytical data of the main constituents of steam volatiles (and their respective retention indices) are presented in **Table 4**.

Essential Oils of Umbelliferae

myrcene,

(Baser et al., 2002).

2001) and *P. cervariifolium* (Bazgir et al., 2005).

and roots (Bernard & Clair, 1997), with

(Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control 625

The determined chemical composition of the EO from the aerial part of *Angelica sylvestris* L. (AS) is consistent with the literature reports for EOs obtained from its seeds (Bernard, 2001)

components. Same compounds were reported as the prevailing phytochemicals in the EOs of *A. archangelica* L. *sensu lato* (Bernard, 2001; Nykanen et al., 1991; Bernard & Clair, 1997; Chalcat & Garry, 1997; Nivinskiene et al., 2005), while the EO of *A. glauca* is reported to

et al., 2004; Kaul et al., 1996). Other *Angelica* L. taxa, such as *A. sinensis* (Dung et al., 1996; Kim et al., 2006), *A. gigas* (Kim et al., 2006), *A. acutiloba* (Kim et al., 2006), *A. heterocarpa*  (Bernard, 2001; Bernard & Clair, 1997) and *A. tenuissima* (Ka et al., 2005) display a

In addition to *α*-pinene, which is the main constituent as previously reported by Demetzos et al. (2000), the studied EO of *Ferulago nodosa* (L) Boiss. (FN) was found to contain thirteen new components for the *taxon's* EO. More specifically, the molecules of *trans*-2-hexenal,

terpinene, bornyl acetate, *β*-elemene, *β*-caryophylene, germacrene D and bicyclogermacrene were also determined as constituents of this EO. With the exception of *trans*-2-hexenal all the abovementioned compounds have been assayed in the EOs of the following *Ferulago* W.D.J. Koch taxa; *F. asparagifolia* (Baser et al., 2001), *F. phialocarpa* (Masoudi et al., 2004b), *F. macrocolea* (Rustaiyan et al., 2005), *F. galbaniflua* (Rustaiyan et al., 2002a), and *F. thirkeana*

The EO of *Peucedanum officinale* L. (PO) is dominated by bornyl acetate, which was previously found only in *P. scoparium* (Masoudi et al., 2004a). It is also characterized by the presence of 2,3,4-trimethyl benzaldehyde, which has not been previously reported as constitutent of *Peucedanum* L. EOs. In addition, the EO tested was found to contain five molecules, namely *α*-pinene, sabinene, myrcene, limonene and *β*-selinene, never reported in a EO of *P. officinale* (Jaimand et al., 2006). These five phytochemichals are abundant in the general profile of *Peucedanum* L. EOs, as reported for *P. scoparium* (Masoudi et al., 2004a), *P. zenkeri* (Menut et al., 1995), *P.vertcillare* (Fraternale et al., 2000), *P. petiolare* (Rustaiyan et al.,

The EOs of *Pimpinella* L. have been thoroughly studied, mainly because the application of their several taxa as culinary herbs and/or spices. Though the EOs of fourteen (14) *taxa* were studied, only one (Tabanca et al., 2005) refers to PP (*Pimpinella peregrina* L.) and none to PT (*Pimpinella tragium* ssp tragium VIII) and PR (*Pimpinella rigidulla* Boiss. & Orph. H. Wolf). The main constituent of EO of PO is *α*-bergamontenene, reported so far only for *P. anagodendron* (Velasco-Negueruela et al. 2005) and *P. anisum* (Santos et al., 1998). Two additional components determined herein, *β*-bisabolene and *β*-sesquiphellandrene, have not been reported in previous studies for PP but are well documented for *P. anagodendron*  (Velasco-Negueruela et al. 2005), *P. junoniae* (Velasco-Negueruela et al. 2003), *P. anisum*  (Santos et al., 1998), *P. anisetum* (Baser et al., 1999; Tepe et al., 2006) and *P. tragioides* (Askari & Sefidcon, 2007)*.* New entries, for this genera EO components list, are isoledene, aristolene, calarene and *β*-selinene which were also assayed in the EO of PP. On the contrary, the EO of PR is characterized by the complete absence of monoterpenes, advocating previous record of *β*-selinene and introducing *α*-amorphene, *α*-selinene and *trans*-isomyristicin as components of the *Pimpinella* L. EOs. Finally, the EO of PT has only two differences as

contain *β*-phellandrene as major component and only small portions of

completely different, both qualitative and quantitative, EO composition profile.

aComparison of mass spectra with MS libraries and retention times

bComparison of experimental RI with reported RI

cComparison with authentic compounds

RI: Retention indices calculated against C8 to C24 n-alkanes on the HP 5MS column.

Table 4. Chemical constituents of the essential oils tested.

aComparison of mass spectra with MS libraries and retention times

Table 4. Chemical constituents of the essential oils tested.

RI: Retention indices calculated against C8 to C24 n-alkanes on the HP 5MS column.

bComparison of experimental RI with reported RI cComparison with authentic compounds

The determined chemical composition of the EO from the aerial part of *Angelica sylvestris* L. (AS) is consistent with the literature reports for EOs obtained from its seeds (Bernard, 2001) and roots (Bernard & Clair, 1997), with -pinene and -phellandrene being the major components. Same compounds were reported as the prevailing phytochemicals in the EOs of *A. archangelica* L. *sensu lato* (Bernard, 2001; Nykanen et al., 1991; Bernard & Clair, 1997; Chalcat & Garry, 1997; Nivinskiene et al., 2005), while the EO of *A. glauca* is reported to contain *β*-phellandrene as major component and only small portions of -pinene (Aghinotri et al., 2004; Kaul et al., 1996). Other *Angelica* L. taxa, such as *A. sinensis* (Dung et al., 1996; Kim et al., 2006), *A. gigas* (Kim et al., 2006), *A. acutiloba* (Kim et al., 2006), *A. heterocarpa*  (Bernard, 2001; Bernard & Clair, 1997) and *A. tenuissima* (Ka et al., 2005) display a completely different, both qualitative and quantitative, EO composition profile.

In addition to *α*-pinene, which is the main constituent as previously reported by Demetzos et al. (2000), the studied EO of *Ferulago nodosa* (L) Boiss. (FN) was found to contain thirteen new components for the *taxon's* EO. More specifically, the molecules of *trans*-2-hexenal, myrcene, -phellandrene, -terpinene, *β*-phellandrene, *cis*-ocimene, *trans*-ocimene, *γ*terpinene, bornyl acetate, *β*-elemene, *β*-caryophylene, germacrene D and bicyclogermacrene were also determined as constituents of this EO. With the exception of *trans*-2-hexenal all the abovementioned compounds have been assayed in the EOs of the following *Ferulago* W.D.J. Koch taxa; *F. asparagifolia* (Baser et al., 2001), *F. phialocarpa* (Masoudi et al., 2004b), *F. macrocolea* (Rustaiyan et al., 2005), *F. galbaniflua* (Rustaiyan et al., 2002a), and *F. thirkeana*  (Baser et al., 2002).

The EO of *Peucedanum officinale* L. (PO) is dominated by bornyl acetate, which was previously found only in *P. scoparium* (Masoudi et al., 2004a). It is also characterized by the presence of 2,3,4-trimethyl benzaldehyde, which has not been previously reported as constitutent of *Peucedanum* L. EOs. In addition, the EO tested was found to contain five molecules, namely *α*-pinene, sabinene, myrcene, limonene and *β*-selinene, never reported in a EO of *P. officinale* (Jaimand et al., 2006). These five phytochemichals are abundant in the general profile of *Peucedanum* L. EOs, as reported for *P. scoparium* (Masoudi et al., 2004a), *P. zenkeri* (Menut et al., 1995), *P.vertcillare* (Fraternale et al., 2000), *P. petiolare* (Rustaiyan et al., 2001) and *P. cervariifolium* (Bazgir et al., 2005).

The EOs of *Pimpinella* L. have been thoroughly studied, mainly because the application of their several taxa as culinary herbs and/or spices. Though the EOs of fourteen (14) *taxa* were studied, only one (Tabanca et al., 2005) refers to PP (*Pimpinella peregrina* L.) and none to PT (*Pimpinella tragium* ssp tragium VIII) and PR (*Pimpinella rigidulla* Boiss. & Orph. H. Wolf). The main constituent of EO of PO is *α*-bergamontenene, reported so far only for *P. anagodendron* (Velasco-Negueruela et al. 2005) and *P. anisum* (Santos et al., 1998). Two additional components determined herein, *β*-bisabolene and *β*-sesquiphellandrene, have not been reported in previous studies for PP but are well documented for *P. anagodendron*  (Velasco-Negueruela et al. 2005), *P. junoniae* (Velasco-Negueruela et al. 2003), *P. anisum*  (Santos et al., 1998), *P. anisetum* (Baser et al., 1999; Tepe et al., 2006) and *P. tragioides* (Askari & Sefidcon, 2007)*.* New entries, for this genera EO components list, are isoledene, aristolene, calarene and *β*-selinene which were also assayed in the EO of PP. On the contrary, the EO of PR is characterized by the complete absence of monoterpenes, advocating previous record of *β*-selinene and introducing *α*-amorphene, *α*-selinene and *trans*-isomyristicin as components of the *Pimpinella* L. EOs. Finally, the EO of PT has only two differences as

Essential Oils of Umbelliferae

**4.2 Larvicidal assays**

mgl-1 respectively).

overlap.

confidence limits (CL)

biotype *molestus*.

*Angelica sylvestris* >150 *Pimpinella peregrina* >150

(Apiaceae) Family Taxa as Emerging Potent Agents for Mosquito Control 627

Bulatovic et al. 2006) and in *S. buchtormence*. These compounds were also present in the EOs of *S. resinosum* and *S. tortuosum*, obtained from the fruits and not the herbal part of the plants (Dogan et al. 2006). The *L. khorassanicum* and *L. staurophyllum* EOs were assayed to contain mostly aliphatic terpenes, while the corresponding cyclic terpenes were present in smaller amounts compared to EOs of Seseli L. (Sedghat et al. 2003; Sefidkon et al. 1997).

Finally, the investigated EO of *Athamanta densa* Boiss. & Orph., contains as major constituents myristicin and various unidentified alkaloids, which account for almost 24 % of its weight. The literature reports of EOs of *Athamanta* L. indicate that they mainly contain either myristicin, such as the EOs of *A. sicula* (Camarda & Di Stefano, 2003), *A. turbith sensu lato* (Tomic et al., 2009), *A. macedonica* (Verykokidou et al., 1995) and *A. haynaldi* (Zivanovic

The investigated EOs were evaluated ─for the first time─ in respect to their larvicidal activities against 3rd- 4th instar larvae of *Culex pipiens*. The relative results expressed as the respective LC50 and LC90 values are included in **Table 5**. Among the EOs tested only two were rather inactive (AS and PP, displaying LC50 values above 150 mgl-1), while the EOs of SC and SP were moderately active displaying LC50 values above 100 mgl-1 (111.99 and 122.54

**Essential Oils tested LC50 (95% CL)a LC90 (95% CL)a Slope (±SE)**  *Athamanta densa* 10.15 (9.49-10.73) 15.75 (14.52-17.76) 6.72±0.80 *Pimpinella tragium* ssp tragium 40.13 (32.43-45.95) 71.10 (61.51-91.00) 5.15±0.52b *Pimpinella rigidula* 40.31 (34.75-43.64) 60.41 (55.66-70.57) 7.29±1.44b *Thamnosciadium junceum* 44.17 (41.52-46.62) 64.42 (59.94-71.28) 7.82±0.86b *Peucedanum neumayeri* 47.40 (40.25-54.15) 81.47 (68.63-113.57) 5.44±0.53b *Chaerophyllum heldreichii* 53.61 (50.29-56.55) 75.96 (71.53-82.15) 8.46±0.87 *Laserpitium pseudomeum* 56.73 (53.50-59.60) 79.59 (75.18-85.71) 8.46±0.86 *Ferulago nodosa* 67.39 (64.17-70.41) 95.59 (89.90-103.94) 8.43±0.84 *Smyrnium rotundifolium* 80.32 (76.88-84.16) 105.30 (98.33-116.61) 10.89±1.29 *Peucedanum officinale* 86.46 (82.27-90.30) 125.05 (117.23-136.95) 7.99±0.84 *Scaligeria cretica* 111.99 (107.86-115.47) 133.83 (128.35-143.21) 6.58±0.73b *Seseli parnassicum* 122.54 (115.54-141.06) 167.15 (143.83-268.76) 6.30±0.68

a LC values are expressed in mgl-1 and they are considered significantly different when 95% CL fail to

b Since goodness–of–fit test is significant (P<0.05), a heterogeneity factor is used in the calculation of

Table 5. LC50 and LC90 values for the tested essential oils against larvae of *Culex pipiens*

et al., 1994), or apiole as in *A. sicula* (Camarda & Di Stefano, 2008).

compared to the genus EO components, an unidentified component and *β*-elemenone. In general, its composition is in accordance with the phytochemical profiles reported for the EOs of *P. aromatica* (Baser et al., 1996), *P. serbica* (Ivanic et al., 1983), *P. flabellifolia* (Tepe et al., 2006), *P. aurea* (Tabanca et al., 2005; Assadian et al., 2005), *P. acuminata* (Melkani et al., 2006), *P. barbata* (Fakhari & Sonboli, 2006), *P. rupicola* (Velasco-Negueruela et al. 2005), *P. corymbosa*  and *P. puberula* (Tabanca et al., 2005).

Major components of the EO of *Scaligeia cretica* (Miller) Boiss (SC) are *α*-pinene, *β*-farnesene and germacrene D, which have also been detected in previous studies on the EOs of *Scaligeria* DC. In this respect, the EO of *S. lazica* contains *β*-farnesene as major and *α*-pinene, germacrene D as minor components (Baser et al., 1993). On the contrary, the EO of *S. tripartite* contains *β*-farnesene and germacrene D as minor compounds, while *α*-pinene is absent (Tabanca et al., 2007). Compounds assayed herein and never reported before in the EOs of *Scaligeria* DC are *α*-terpineol, *β*-elemene and *β*-humulene.

The EOs of *Laserpitium pseudomeum* Orph. Heldr. & Sart Ex Poiss. (LP) contains *α*-pinene, *β*-pinene, sabinene and *β*-phellandrene as major components, all well known constitutents of the EOs of *Laserpitium* L. Previous literature reports indicated that the EOs of *L. latifolium* contains *α*-pinene and *β*-pinene as major components (Borg-Karlson et al., 1994), the *L. petrophilum α*-pinene and sabinene (Baser et al., 1997), while the molecule of *β*phellandrene is present in traces in both EOs. On the contrary, the phytochemical profile of the EO of *L. siler* is completely different containing mainly limonene and perillaldehyde (Chizzola et al., 1999)

The EO composition of *Smyrnium* L. has also been scarcely investigated, since only three *taxa's* EOs, namely *S. perfoliatum* (Molleken et al., 1998a; Tirillini et al., 1996; Tirillini & Tosi, 1992), *S. cordifolium* (Amiri et al., 2006) and *S. olusatrum* (Molleken et al., 1998b), have been studied todate. The studied of EO of *Smyrnium rotundifolium* Miller (SR) contains 7 major components, with the molecule of *α*-selinene reported for the first time as EO component of *Smyrnium* L.. Other compounds present in large quantitites are furanodiene (reported as major constitutent in *S. olusatrum*), myrcene, furanoeremophil-1-one, 1*β*acetoxyfuranoeudesm-4(15)-ene, 1*β*-acetoxyfurano eudesm-3-ene (detected in *S. olusatrum*  and *S. perfoliatum,* Molleken et al., 1998) and germacrone (present in *S. cordifolium*)*.*

The phytochemical profile of *Chaerophyllum* L. EOs was studied previously for *C. macropodum* (Baser et al., 2006), *C. crinitum* (Baser et al., 2006; Nematollahi et al., 2005), *C. macrospermum* (Sefidcon & Abdoli, 2005; Rustaiyan et al., 2002b, Mamedova, 1994), *C. bulbosum sensu lato* (Mamedova & Akhmedova, 1991; Kokkalou & Stefanou, 1989), *C. aksekiense* (Baser et al., 2000b), *C. coloratum* (Vajs et al., 1995), *C. azoricum* (Pedro et al., 1999) and *C. prescotii* (Letchamo et al., 2005). The more significant differentiation among the literature results and the assayed herein EO of *Chaerophyllum heldreichi* Orph. Ex Boiss (CH) comprises the identification for first time of *α*-terpineol as main component of EO of *Chaerophyllum* L..

The EO of *Seseli parnassicum* Boiss. & Heldr. (SP) was found to contain three new compound entries, *β*-humulene, *β*-selinene and *β*-sesquiphellandrene, as compared with the EO of the Seseli L. *taxa* (also including the synonymous *Lomatopodium* Fisch. et C.A. Mey *taxa*). The remaining components are in accordance with the EO content of same *taxa* plants, such as *S. montanum* (Evergetis et al., 2009), *S. campestre* and *S. peucedanoides* (Baser et al. 2000a; Bulatovic et al. 2006) and in *S. buchtormence*. These compounds were also present in the EOs of *S. resinosum* and *S. tortuosum*, obtained from the fruits and not the herbal part of the plants (Dogan et al. 2006). The *L. khorassanicum* and *L. staurophyllum* EOs were assayed to contain mostly aliphatic terpenes, while the corresponding cyclic terpenes were present in smaller amounts compared to EOs of Seseli L. (Sedghat et al. 2003; Sefidkon et al. 1997).

Finally, the investigated EO of *Athamanta densa* Boiss. & Orph., contains as major constituents myristicin and various unidentified alkaloids, which account for almost 24 % of its weight. The literature reports of EOs of *Athamanta* L. indicate that they mainly contain either myristicin, such as the EOs of *A. sicula* (Camarda & Di Stefano, 2003), *A. turbith sensu lato* (Tomic et al., 2009), *A. macedonica* (Verykokidou et al., 1995) and *A. haynaldi* (Zivanovic et al., 1994), or apiole as in *A. sicula* (Camarda & Di Stefano, 2008).

## **4.2 Larvicidal assays**

626 Integrated Pest Management and Pest Control – Current and Future Tactics

compared to the genus EO components, an unidentified component and *β*-elemenone. In general, its composition is in accordance with the phytochemical profiles reported for the EOs of *P. aromatica* (Baser et al., 1996), *P. serbica* (Ivanic et al., 1983), *P. flabellifolia* (Tepe et al., 2006), *P. aurea* (Tabanca et al., 2005; Assadian et al., 2005), *P. acuminata* (Melkani et al., 2006), *P. barbata* (Fakhari & Sonboli, 2006), *P. rupicola* (Velasco-Negueruela et al. 2005), *P. corymbosa*

Major components of the EO of *Scaligeia cretica* (Miller) Boiss (SC) are *α*-pinene, *β*-farnesene and germacrene D, which have also been detected in previous studies on the EOs of *Scaligeria* DC. In this respect, the EO of *S. lazica* contains *β*-farnesene as major and *α*-pinene, germacrene D as minor components (Baser et al., 1993). On the contrary, the EO of *S. tripartite* contains *β*-farnesene and germacrene D as minor compounds, while *α*-pinene is absent (Tabanca et al., 2007). Compounds assayed herein and never reported before in the

The EOs of *Laserpitium pseudomeum* Orph. Heldr. & Sart Ex Poiss. (LP) contains *α*-pinene, *β*-pinene, sabinene and *β*-phellandrene as major components, all well known constitutents of the EOs of *Laserpitium* L. Previous literature reports indicated that the EOs of *L. latifolium* contains *α*-pinene and *β*-pinene as major components (Borg-Karlson et al., 1994), the *L. petrophilum α*-pinene and sabinene (Baser et al., 1997), while the molecule of *β*phellandrene is present in traces in both EOs. On the contrary, the phytochemical profile of the EO of *L. siler* is completely different containing mainly limonene and perillaldehyde

The EO composition of *Smyrnium* L. has also been scarcely investigated, since only three *taxa's* EOs, namely *S. perfoliatum* (Molleken et al., 1998a; Tirillini et al., 1996; Tirillini & Tosi, 1992), *S. cordifolium* (Amiri et al., 2006) and *S. olusatrum* (Molleken et al., 1998b), have been studied todate. The studied of EO of *Smyrnium rotundifolium* Miller (SR) contains 7 major components, with the molecule of *α*-selinene reported for the first time as EO component of *Smyrnium* L.. Other compounds present in large quantitites are furanodiene (reported as major constitutent in *S. olusatrum*), myrcene, furanoeremophil-1-one, 1*β*acetoxyfuranoeudesm-4(15)-ene, 1*β*-acetoxyfurano eudesm-3-ene (detected in *S. olusatrum*

The phytochemical profile of *Chaerophyllum* L. EOs was studied previously for *C. macropodum* (Baser et al., 2006), *C. crinitum* (Baser et al., 2006; Nematollahi et al., 2005), *C. macrospermum* (Sefidcon & Abdoli, 2005; Rustaiyan et al., 2002b, Mamedova, 1994), *C. bulbosum sensu lato* (Mamedova & Akhmedova, 1991; Kokkalou & Stefanou, 1989), *C. aksekiense* (Baser et al., 2000b), *C. coloratum* (Vajs et al., 1995), *C. azoricum* (Pedro et al., 1999) and *C. prescotii* (Letchamo et al., 2005). The more significant differentiation among the literature results and the assayed herein EO of *Chaerophyllum heldreichi* Orph. Ex Boiss (CH) comprises the identification for first time of *α*-terpineol as main component of EO of

The EO of *Seseli parnassicum* Boiss. & Heldr. (SP) was found to contain three new compound entries, *β*-humulene, *β*-selinene and *β*-sesquiphellandrene, as compared with the EO of the Seseli L. *taxa* (also including the synonymous *Lomatopodium* Fisch. et C.A. Mey *taxa*). The remaining components are in accordance with the EO content of same *taxa* plants, such as *S. montanum* (Evergetis et al., 2009), *S. campestre* and *S. peucedanoides* (Baser et al. 2000a;

and *S. perfoliatum,* Molleken et al., 1998) and germacrone (present in *S. cordifolium*)*.*

EOs of *Scaligeria* DC are *α*-terpineol, *β*-elemene and *β*-humulene.

and *P. puberula* (Tabanca et al., 2005).

(Chizzola et al., 1999)

*Chaerophyllum* L..

The investigated EOs were evaluated ─for the first time─ in respect to their larvicidal activities against 3rd- 4th instar larvae of *Culex pipiens*. The relative results expressed as the respective LC50 and LC90 values are included in **Table 5**. Among the EOs tested only two were rather inactive (AS and PP, displaying LC50 values above 150 mgl-1), while the EOs of SC and SP were moderately active displaying LC50 values above 100 mgl-1 (111.99 and 122.54 mgl-1 respectively).

a LC values are expressed in mgl-1 and they are considered significantly different when 95% CL fail to overlap.

b Since goodness–of–fit test is significant (P<0.05), a heterogeneity factor is used in the calculation of confidence limits (CL)

Table 5. LC50 and LC90 values for the tested essential oils against larvae of *Culex pipiens* biotype *molestus*.

Essential Oils of Umbelliferae

43-44.

380-381.

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65, 721-724.

287.

21-32.

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**27**

*1México 2Portugal*

*Flourensia cernua* **DC: A Plant from**

**Mexican Semiarid Regions with a Broad**

**Spectrum of Action for Disease Control**

Mexico has an extensive variety of plants, it is the world´s fourth richest country in this aspect. Some 25,000 species are registered, and it is thought that there are approximately 30, 000 not described. Particularly the regions of the north of Mexico, with their semiarid climate, have a great number and variety of wild plants grown under extreme climatic conditions. Wild species which have compounds with flavonoid structures, sesquiterpenoids, acetylenes, *p*-acetophenones, benzofurans, and benzopyrans grow in these regions. The polyphenolic compounds include tannins and flavonoids which have therapeutic uses due to their anti-inflammatory, antifungal, antibacterial, antioxidant, and

*Flourensia cernua* is an endemic species which grows in semiarid zones of Mexico and contains polyphenolic, lactone, benzofuran, and benzopyran compounds which give it a

In this work, *F. cernua* is reviewed in terms of its geographical distribution in Mexico, traditional uses, bioactive compounds identified for controlling fungi, bacteria, and insects,

It is commonly known in different ways, as it is found in the United States of America as well as in Mexico. The names given in the United States of America are: tarbush, hojase, American-tarbush, black-brush, varnish-brush, and hojasen (Correl and Johnston, 1970; Vines, 1960). In Mexico, it is known as hojasen, tarbush, black-brush (Arredondo, 1981).

**1. Introduction**

healing properties.

potential use for disease control.

as well as cytotoxic activity.

**2. Common names**

Diana Jasso de Rodríguez1, F. Daniel Hernández-Castillo1,

Susana Solís-Gaona1, Raúl Rodríguez- García1

*1Universidad Autónoma Agraria Antonio Narro (UAAAN), Buenavista, Saltillo, Coahuila*

and Rosa M. Rodríguez-Jasso2

*2Centre of Biological Engineering, University of Minho,Campus Gualtar*

## *Flourensia cernua* **DC: A Plant from Mexican Semiarid Regions with a Broad Spectrum of Action for Disease Control**

Diana Jasso de Rodríguez1, F. Daniel Hernández-Castillo1, Susana Solís-Gaona1, Raúl Rodríguez- García1 and Rosa M. Rodríguez-Jasso2 *1Universidad Autónoma Agraria Antonio Narro (UAAAN), Buenavista, Saltillo, Coahuila 2Centre of Biological Engineering, University of Minho,Campus Gualtar 1México 2Portugal*

## **1. Introduction**

638 Integrated Pest Management and Pest Control – Current and Future Tactics

Yadouleton, A., Martin, T., Padonou, G., Chandre, F., Asidi, A., Djogbenou, L., Dabiré, R.,

in Northern Benin. *Parasites & Vectors*, 4, 60, doi: 10.1186/1756-3305-4-60. Zivanovic, P., Djokovic, D., Vajs, V., Slavkovska, V., Todorovic, B., & Milosavljevic, S. (1994).

*Pharmazie*, 49, 463-464.

Aïkpon, R., Boko, M., Glitho, I. & Akogbeto, M. (2011). Cotton pest management practices and the selection of pyrethroid resistance in *Anopheles gambiae* population

Essential oils flowers and fruits of *Athamanta haynaldii* Bort. Et Uchtr. (Apiaceae).

Mexico has an extensive variety of plants, it is the world´s fourth richest country in this aspect. Some 25,000 species are registered, and it is thought that there are approximately 30, 000 not described. Particularly the regions of the north of Mexico, with their semiarid climate, have a great number and variety of wild plants grown under extreme climatic conditions. Wild species which have compounds with flavonoid structures, sesquiterpenoids, acetylenes, *p*-acetophenones, benzofurans, and benzopyrans grow in these regions. The polyphenolic compounds include tannins and flavonoids which have therapeutic uses due to their anti-inflammatory, antifungal, antibacterial, antioxidant, and healing properties.

*Flourensia cernua* is an endemic species which grows in semiarid zones of Mexico and contains polyphenolic, lactone, benzofuran, and benzopyran compounds which give it a potential use for disease control.

In this work, *F. cernua* is reviewed in terms of its geographical distribution in Mexico, traditional uses, bioactive compounds identified for controlling fungi, bacteria, and insects, as well as cytotoxic activity.

## **2. Common names**

It is commonly known in different ways, as it is found in the United States of America as well as in Mexico. The names given in the United States of America are: tarbush, hojase, American-tarbush, black-brush, varnish-brush, and hojasen (Correl and Johnston, 1970; Vines, 1960). In Mexico, it is known as hojasen, tarbush, black-brush (Arredondo, 1981).

*Flourensia cernua* DC: A Plant from

**5. Phytochemical analysis**

et al., 1968).

**7. Active chemicals**

**6. Properties and documented actions**

Mexican Semiarid Regions with a Broad Spectrum of Action for Disease Control 641

Fig. 2. *Flourensia cernua* plant at su in a wilderness site in Northeast Mexico.

ecographical distribution and a possible chemical adaptation to the environment.

flowers for indigestion and gastrointestinal problems (Arredondo, 1981).

1973). Also there are benzopyrans (Fig.3e) and benzofurans (Fig.3f).

The *Flourensia genus* is important due to the great amount of secondary metabolites it possesses; these are widely used for biological and ecological applications. Nine species of *Flourensia* have been reported, being *Flourensia cernua* the one with the highest number of chemicals (Aregullín and Rodríguez, 1983) with economical potential. The authors correlated the presence of benzofurans and benzopyrans with biological activity. The fact that these secondary metabolites are not present in other species, led to a correlation between the

Several medicinal properties have been reported for the tea obtained from the leaves or

The green fruits are innocuous for cattle. However, dry fruits are toxic and when consumed at approximately 1% of the animal weight they cause death during the first 24 hours (Sperry

Fractionation of a CH2Cl2-MeOH (1:1) extract of the aerial parts of *Flourensia cernua* led to the isolation of three phytotoxic compounds, namely dehydroflourensic acid (Fig.3a), flourensadiol (Fig.3b), and methyl orsellinate (Fig.3c) and seven hitherto unknown γlactones were obtained (Fig.3d), these being tetracosane-4-olide, pentacosane-4-olide, hexacosane-4-olide, heptacosane-4-olide, octacosane-4-olide, nonacosane-4-olide and triacontane-4-olide. Besides, a previously known flavonoid, ermanin (Dominguez et al.,

## **3. Geographical distribution**

In Mexico, *F. cernua* is found in the Chihuahuan and Sonoran deserts, as well as in the states of Coahuila, Chihuahua, Durango, Hidalgo, Nuevo León, San Luis Potosí, Sonora, and Zacatecas (Valdés, 1988; Martínez, 1993) (Fig. 1). In the United States of America, it is found West of Texas and South of New Mexico and Arizona (Vines, 1960).

It is found in altitudes ranging from 1000 to 2000 masl. In studies carried out in Mexico, it is observed that the prevailing altitude for this species is 1900 masl and slopes from 1 to 6 per cent (Arredondo, 1981) although it is also found in altitudes from 300 to 400 Northeast of Coahuila.

## **4. Associated species**

The main shrub species to which *Flourensia cernua* (Fig.2) is related to are: *Larrea tridentata*, *Yucca filifera*, *Atriplex canescens*, *Castela texana*, *Acacia fernesiana*, *Prosopis juliflora*, *Agave lechuguilla*, *Parthenium incanum*, *Fouquieria splendens*, and *Acacia constricta* (Comisión Técnico Consultiva para la Determinación de los Coeficientes de agostadero, 1979).

Fig. 1. Geographical distribution of *Flourensia cernua* DC in Mexico.

Fig. 2. *Flourensia cernua* plant at su in a wilderness site in Northeast Mexico.

## **5. Phytochemical analysis**

640 Integrated Pest Management and Pest Control – Current and Future Tactics

In Mexico, *F. cernua* is found in the Chihuahuan and Sonoran deserts, as well as in the states of Coahuila, Chihuahua, Durango, Hidalgo, Nuevo León, San Luis Potosí, Sonora, and Zacatecas (Valdés, 1988; Martínez, 1993) (Fig. 1). In the United States of America, it is found

It is found in altitudes ranging from 1000 to 2000 masl. In studies carried out in Mexico, it is observed that the prevailing altitude for this species is 1900 masl and slopes from 1 to 6 per cent (Arredondo, 1981) although it is also found in altitudes from 300 to 400 Northeast of

The main shrub species to which *Flourensia cernua* (Fig.2) is related to are: *Larrea tridentata*, *Yucca filifera*, *Atriplex canescens*, *Castela texana*, *Acacia fernesiana*, *Prosopis juliflora*, *Agave lechuguilla*, *Parthenium incanum*, *Fouquieria splendens*, and *Acacia constricta* (Comisión Técnico

West of Texas and South of New Mexico and Arizona (Vines, 1960).

Consultiva para la Determinación de los Coeficientes de agostadero, 1979).

Fig. 1. Geographical distribution of *Flourensia cernua* DC in Mexico.

**3. Geographical distribution**

Coahuila.

**4. Associated species**

The *Flourensia genus* is important due to the great amount of secondary metabolites it possesses; these are widely used for biological and ecological applications. Nine species of *Flourensia* have been reported, being *Flourensia cernua* the one with the highest number of chemicals (Aregullín and Rodríguez, 1983) with economical potential. The authors correlated the presence of benzofurans and benzopyrans with biological activity. The fact that these secondary metabolites are not present in other species, led to a correlation between the ecographical distribution and a possible chemical adaptation to the environment.

## **6. Properties and documented actions**

Several medicinal properties have been reported for the tea obtained from the leaves or flowers for indigestion and gastrointestinal problems (Arredondo, 1981).

The green fruits are innocuous for cattle. However, dry fruits are toxic and when consumed at approximately 1% of the animal weight they cause death during the first 24 hours (Sperry et al., 1968).

## **7. Active chemicals**

Fractionation of a CH2Cl2-MeOH (1:1) extract of the aerial parts of *Flourensia cernua* led to the isolation of three phytotoxic compounds, namely dehydroflourensic acid (Fig.3a), flourensadiol (Fig.3b), and methyl orsellinate (Fig.3c) and seven hitherto unknown γlactones were obtained (Fig.3d), these being tetracosane-4-olide, pentacosane-4-olide, hexacosane-4-olide, heptacosane-4-olide, octacosane-4-olide, nonacosane-4-olide and triacontane-4-olide. Besides, a previously known flavonoid, ermanin (Dominguez et al., 1973). Also there are benzopyrans (Fig.3e) and benzofurans (Fig.3f).

*Flourensia cernua* DC: A Plant from

*Flourensia* spp. extracts concentration.

inhibition level was 98.6%.

Mexican Semiarid Regions with a Broad Spectrum of Action for Disease Control 643

however, inhibition was slightly lower than *F. microphylla* at low concentration. The highest

Fig. 4. Mycelial growth inhibition (percentage of *Rhrizoctonia solani*) as a function of

for inhibiting mycelial growth of *C. gloesporoides* and *P. digitatum*, (Fig. 5).

offering an alternative production of antimicrobial agents.

In studies carried out by our research group, Guerrero-Rodriguez et al. (2007) proved the *F. cernua* effect on mycelial inhibition of *Alternaria alternata*, *Colletotrichum gloesporoides*, and *Penicillum digitatum*, where methanol:chloroform (1:1) solvents and sequential extractions with hexane, diethyl ether, and ethanol were used. *A. alternata* reported the highest mycelial inhibition, took place with hexane fractions (91.9%) and methanol: chloroform (88.4%) at 4000 mg L-1. 2000 mg L-1 ethanol extract caused the lowest production of *C. gloesporoides* conidium. The four extracts reduced conidium production for *P. digitatum*, however they didn´t present statistical differences. In general, the ethanolic extract was the most efficient

Studies carried out in the UAAAN by Castillo et al. (2010) on plant extract of *Larrea tridentata*, *Flourensia cernua*, *Agave lechuguilla*, *Opuntia* sp, and *Yucca* sp; obtained with alternative organic solvents (lanolin and cocoa butter) and water were tested against *Rhizoctonia solani* pathogens. The obtained results were as follows: The *L. tridentata* and *F. cernua* extracts by means of lanolin and cocoa butter at a 2000 and 1000 ppm total tannins inhibited the growth of *R. solani* 100%. Lanolin and cocoa butter solvents allowed a high recovery of polyphenolic molecules of strong antifungal activity against *R. solani* thus

Fig. 3. Chemical of six active compounds from *Flourensia cernua*.

## **8. Plant fungicide, bactericide, and insecticide activity**

## **8.1 Antifungal activity**

Fungicide activity *in vitro* of leaves extracts at solution concentration of 1,000 mg L-1 on *Rhizoctonia solani*, *Pythium* sp. and *Fusarium oxysporum* was reported (Saeedi-Ghomi & Maldonado, 1982). The leaf fractions of hexane, diethyl ether and ethanol were active against *Colletotrichum fragariae* Brooks, *C. gloesporioides* Penz and Sacc. The essential oils from the hexane fraction were active at 1 µg doses, whereas the diethyl ether and ethanol fractions were active at 10 µg doses. The ethanol fraction was active against *C. accutatum* Simmons only at 400 µg. (Tellez et al., 2001).

Gamboa et al. (2003) used an extraction method by soxhlet to obtain methanolic extracts which were evaluated on soil pathogene *Rhizoctonia solani* and on phytopathogene algae *Phytophthora infestans*, 20,000 µl L-1 were the required dose for 86% pathogene inhibition.

Mata et al. (2003) reported that the fractionation of an extract of the aerial parts of *F. cernua* led to the isolation of three phytotoxic compounds namely: Flourensadiol, methyl orsellinate, and dehydroflourensic acid.

In a study carried out at our lab the inhibitory effect of ethanolic extracts was evaluated for three *Flourensia* species: *F. cernua*, *F. microphylla*, and *F. retinophylla* on three pathogens: *Alternia* sp., *Rhizoctonia solani* (Fig.4), and *Fusarium oxysporum*, which attack commercial cultivars (Jasso de Rodriguez et al., 2007). The variance analysis on the pathogen mycelial development showed highly significant differences (p<0.01) on extract, dose, and extract interactions x dose. *F. microphylla* inhibited *Alternaria* sp 42.5% at a 10 µl L-1, reaching 76.8% at a 100 µl L-1. *F. cernua* and *F. retinophylla* showed a similar effect for high concentrations,

Fungicide activity *in vitro* of leaves extracts at solution concentration of 1,000 mg L-1 on *Rhizoctonia solani*, *Pythium* sp. and *Fusarium oxysporum* was reported (Saeedi-Ghomi & Maldonado, 1982). The leaf fractions of hexane, diethyl ether and ethanol were active against *Colletotrichum fragariae* Brooks, *C. gloesporioides* Penz and Sacc. The essential oils from the hexane fraction were active at 1 µg doses, whereas the diethyl ether and ethanol fractions were active at 10 µg doses. The ethanol fraction was active against *C. accutatum*

Gamboa et al. (2003) used an extraction method by soxhlet to obtain methanolic extracts which were evaluated on soil pathogene *Rhizoctonia solani* and on phytopathogene algae *Phytophthora infestans*, 20,000 µl L-1 were the required dose for 86% pathogene inhibition.

Mata et al. (2003) reported that the fractionation of an extract of the aerial parts of *F. cernua* led to the isolation of three phytotoxic compounds namely: Flourensadiol, methyl

In a study carried out at our lab the inhibitory effect of ethanolic extracts was evaluated for three *Flourensia* species: *F. cernua*, *F. microphylla*, and *F. retinophylla* on three pathogens: *Alternia* sp., *Rhizoctonia solani* (Fig.4), and *Fusarium oxysporum*, which attack commercial cultivars (Jasso de Rodriguez et al., 2007). The variance analysis on the pathogen mycelial development showed highly significant differences (p<0.01) on extract, dose, and extract interactions x dose. *F. microphylla* inhibited *Alternaria* sp 42.5% at a 10 µl L-1, reaching 76.8% at a 100 µl L-1. *F. cernua* and *F. retinophylla* showed a similar effect for high concentrations,

Fig. 3. Chemical of six active compounds from *Flourensia cernua*.

**8. Plant fungicide, bactericide, and insecticide activity**

**8.1 Antifungal activity**

Simmons only at 400 µg. (Tellez et al., 2001).

orsellinate, and dehydroflourensic acid.

however, inhibition was slightly lower than *F. microphylla* at low concentration. The highest inhibition level was 98.6%.

Fig. 4. Mycelial growth inhibition (percentage of *Rhrizoctonia solani*) as a function of *Flourensia* spp. extracts concentration.

In studies carried out by our research group, Guerrero-Rodriguez et al. (2007) proved the *F. cernua* effect on mycelial inhibition of *Alternaria alternata*, *Colletotrichum gloesporoides*, and *Penicillum digitatum*, where methanol:chloroform (1:1) solvents and sequential extractions with hexane, diethyl ether, and ethanol were used. *A. alternata* reported the highest mycelial inhibition, took place with hexane fractions (91.9%) and methanol: chloroform (88.4%) at 4000 mg L-1. 2000 mg L-1 ethanol extract caused the lowest production of *C. gloesporoides* conidium. The four extracts reduced conidium production for *P. digitatum*, however they didn´t present statistical differences. In general, the ethanolic extract was the most efficient for inhibiting mycelial growth of *C. gloesporoides* and *P. digitatum*, (Fig. 5).

Studies carried out in the UAAAN by Castillo et al. (2010) on plant extract of *Larrea tridentata*, *Flourensia cernua*, *Agave lechuguilla*, *Opuntia* sp, and *Yucca* sp; obtained with alternative organic solvents (lanolin and cocoa butter) and water were tested against *Rhizoctonia solani* pathogens. The obtained results were as follows: The *L. tridentata* and *F. cernua* extracts by means of lanolin and cocoa butter at a 2000 and 1000 ppm total tannins inhibited the growth of *R. solani* 100%. Lanolin and cocoa butter solvents allowed a high recovery of polyphenolic molecules of strong antifungal activity against *R. solani* thus offering an alternative production of antimicrobial agents.

*Flourensia cernua* DC: A Plant from

1983; Towers et al., 1975).

Extracts and

concentrations (μl L-1)

Mexican Semiarid Regions with a Broad Spectrum of Action for Disease Control 645

conditions: One where the inoculated media was kept in darkness and the other one where the inoculated media was UV irradiated (280-400 nm) for 15 min previous to incubation in darkness. Bioactivity was greatly increased by the UV irradiation (Aregullin & Rodriguez,

Molina-Salinas et al. (2006), evaluated the crude extracts effect of methanol, acetone, and hexane of the aerial parts of *Artemisa ludoviciana* Nutt., *Chenopodium ambrosioides* L., *Murrubium vulgare* L., *Mentha spicata* L., and *Flourensia cernua* DC to inhibit the growth or death of *Mycobacterium tuberculosis* strains H37Rv and CIBIN:UMF:15:99. Results showed that from the evaluated plants, *F. cernua* was the only active plant which inhibited and killed *Mycobacterium tuberculosis* strains H37Rv and CIBIN:UMF:15:99. The hexane extract showed a minimal inhibitory concentration (MIC) 50 and 25 µg L-1 against sensitive and resistant strains, respectively; acetone extract was active against and only CIBIN:UMF:15:99 (MIC=100 µg L-1 ). It may be concluded that hexane extract of *F. cernua* leaves could be an important source of bactericidal compounds against multidrug-resistant *M. tuberculosis*.

In a study carried out by our research group, the antibacterial activity of *F. cernua* obtained with hexane, ether, ethanol, and metanol-chloroform mixture, at different dose, was evaluated on *Pseudomonas cichorii* (Pc), *Xanthomonas axonopodis* pv. phaseoli (Xap), and *Pectobacterium caratovora* subsp. atroseptic (Pca) (Peralta, 2006). All the extracts showed activity on Xap and Pc, however, none of them showed any inhibition effect when evaluated on Pca. *F. cernua* hexane extract at a 4000 μl ml-1 (P≤0.05) concentration showed the highest inhibition on Xap

Hexane 500 1.89 a 37.23 a 0 a 1000 26.41 B ab 51.55 C ab 0 A a 2000 44.34 b 74.77 b 0 a 4000 83.96 c 82.51 c 21.73 B Diethyl ether 500 0 a 2.41 a 11.01 c 1000 12.95 A ab 0 B a 5.29 A b 2000 17.27 b 2.08 a 0 a 4000 47.48 c 63.44 b 3.96 Ab Ethanol 500 18.7 a 0 a 4.58 a 1000 20.87 A ab 0 A a 5.6 B ab 2000 43.04 b 1.47 a 13.49 c 4000 52.17 c 14.73 b 9.92 B Meth-Chlor 500 0 a 0 a 5.93 c 1000 6.77 A b 0 A a 3.56 A a 2000 0 a 17.46 b 3.86 a 4000 0 a 0 a 4.91 b

Table 1. Interactions of *F. cernua* extracts x concentrations of the CFU ml-1 inhibition

precentage, of three bacteria at 24 h of incubation. Meth-Chlor= Methanol-Chloroform; Pc = *Pseudomonas cichorii*; Xcp = *Xanthomonas axenopodis* pv. Phaseoli; Pca = *Pectobacterium caratovora* subsp. Atroseptica; A: Extract, \*\* = p< 0.05; B: Concentration \*\* = p< 0.05.

Inhibition of CFU ml-1 Pc A B Xap A B Pca A B

(82.51%) and Pc (83.96%). The other extracts showed a lower activity (Table 1).

Fig. 5. Percentage of mycelial inhibition of four extracts from *Flourensia cernua* DC at four concentrations on three postharvest pathogens: A) *Alternaria alternata*, B) *Colletotrichum gloeosporioides*, and C) *Penicillium digitatum*. yP ≤0.01 (LSD). Con=Control; Hex=Hexane; Eth=Ether; Et=EtOH, and Me:CH=MeOH-CHCl3.

#### **8.2 Antibacterial activity**

The mixtures of benzofurans and benzopyrans of *F. cernua*, were tested against Gram positive and Gram negative bacteria, fungi and *Saccharomyces* under two experimental

Fig. 5. Percentage of mycelial inhibition of four extracts from *Flourensia cernua* DC at four concentrations on three postharvest pathogens: A) *Alternaria alternata*, B) *Colletotrichum gloeosporioides*, and C) *Penicillium digitatum*. yP ≤0.01 (LSD). Con=Control; Hex=Hexane;

The mixtures of benzofurans and benzopyrans of *F. cernua*, were tested against Gram positive and Gram negative bacteria, fungi and *Saccharomyces* under two experimental

Eth=Ether; Et=EtOH, and Me:CH=MeOH-CHCl3.

**8.2 Antibacterial activity**

conditions: One where the inoculated media was kept in darkness and the other one where the inoculated media was UV irradiated (280-400 nm) for 15 min previous to incubation in darkness. Bioactivity was greatly increased by the UV irradiation (Aregullin & Rodriguez, 1983; Towers et al., 1975).

Molina-Salinas et al. (2006), evaluated the crude extracts effect of methanol, acetone, and hexane of the aerial parts of *Artemisa ludoviciana* Nutt., *Chenopodium ambrosioides* L., *Murrubium vulgare* L., *Mentha spicata* L., and *Flourensia cernua* DC to inhibit the growth or death of *Mycobacterium tuberculosis* strains H37Rv and CIBIN:UMF:15:99. Results showed that from the evaluated plants, *F. cernua* was the only active plant which inhibited and killed *Mycobacterium tuberculosis* strains H37Rv and CIBIN:UMF:15:99. The hexane extract showed a minimal inhibitory concentration (MIC) 50 and 25 µg L-1 against sensitive and resistant strains, respectively; acetone extract was active against and only CIBIN:UMF:15:99 (MIC=100 µg L-1 ). It may be concluded that hexane extract of *F. cernua* leaves could be an important source of bactericidal compounds against multidrug-resistant *M. tuberculosis*.

In a study carried out by our research group, the antibacterial activity of *F. cernua* obtained with hexane, ether, ethanol, and metanol-chloroform mixture, at different dose, was evaluated on *Pseudomonas cichorii* (Pc), *Xanthomonas axonopodis* pv. phaseoli (Xap), and *Pectobacterium caratovora* subsp. atroseptic (Pca) (Peralta, 2006). All the extracts showed activity on Xap and Pc, however, none of them showed any inhibition effect when evaluated on Pca. *F. cernua* hexane extract at a 4000 μl ml-1 (P≤0.05) concentration showed the highest inhibition on Xap (82.51%) and Pc (83.96%). The other extracts showed a lower activity (Table 1).