Phytochem Rev DOI 10.1007/s11101-015-9404-6

Chemical constituents and biological activities of South American Rhamnaceae J. Alarco´n • C. L. Cespedes

Received: 20 January 2015 / Accepted: 19 March 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Secondary metabolites have diverse functions into plants. These functions are products of the coevolution which result in synthesis of constitutive and/or induced chemical defense generated for protection against to different phytopathogenic attack. Many phytochemical studies are biodirected with the aim of finding biopesticides of botanical origins. Some taxa of Americas such as Rhamnaceae family are toxic to insects, fungi and several bacteria strains, and these effects has been associated with the presence of alkaloids, phenolics and terpenes. The natural compounds that have been isolated represent a valuable resource for future studies of plant chemical defense and the role of these substances in chemical ecology. Here are show recent advances in the phytochemistry and biological activities of selected members of Rhamnaceae from Latin-America.

J. Alarco´n (&) Laboratorio de Sı´ntesis y Biotransformacio´n de Productos Naturales, Grupo de Investigacio´n Quı´mica y Biotecnologı´a de Productos Naturales Bioactivos, Departamento de Ciencias Ba´sicas, Facultad de Ciencias, Universidad del Bı´o-Bı´o, Chillan, Chile e-mail: [email protected] C. L. Cespedes Laboratorio de Fitoquı´mica Ecolo´gica y Bioquı´mica, Grupo de Investigacio´n Quı´mica y Biotecnologı´a de Productos Naturales Bioactivos, Departamento de Ciencias Ba´sicas, Facultad de Ciencias, Universidad del Bı´o-Bı´o, Chillan, Chile

Keywords Rhamnaceae  Cyclopeptidealkaloids  Condalia  Talguenea  Discaria  Triterpenes  Insecticidal activity

Introduction Looking for new biologically active molecules useful as therapeutic agents, active against several diseases or as useful agents to control pest insect, fungi, bacteria, or other organisms, remains a global research priority. The essential strategy in order to identify these new molecule entities remains focused on the study of secondary metabolites present in endemic plants. The geographical and climatic features of Chile have allowed the development of unique flora being important natural resources of the country. Nonetheless, the percentage of species that have been studied from the standpoint of biological activity and potential agrochemicals is quite limited. The effects of synthetic pesticides on humans and biodiversity are widely documented. At present, environmentally-friendly pesticides are strongly preferred to synthetics. It is noteworthy that the crop protection role of pesticides, and other techniques, i.e. biopesticides, plant extracts, prevention methods, organic methods and plant resistance to certain pests are under an integrated pest management (IPM). The risks and benefits of these must be assessed on a sound scientific basis. Therefore, it is important do a

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significant reduction in the use of synthetic pesticides under an IPM program (Rosner and Markowitz 2013; Rhodes et al. 2013). Biopesticides are derived from natural materials such as animals, plants, bacteria, and certain minerals. They are usually biodegradable in short periods of time. Plants, the most common source of biopesticides, produce a great variety of secondary metabolites that lack apparent function in physiological or biochemical processes; these compounds (or allelochemicals) are important in mediating interactions between plants and their biotic environment (Berenbaum 1989, 2002; Kessler and Baldwin 2002). There is increased interest for application of secondary metabolites in IPM, this has prompted the search for new sources of biologically active natural products, with new modes of action (Conner et al. 2000; Eisner et al. 2000; Meinwald 2001), characteristics that which enhance their value as practical pesticides (Akhtar et al. 2008; Isman 2006). Here, we review recent results on the bioactivity of extracts, fractions, mixtures and pure compounds from selected members of the Rhamnaceae family from South America. Many of these substances provide defense mechanisms against bacterial, fungal and herbivore predator attacks in these plants. Endemic Chilean plants of Rhamnaceae are an interesting resource to study, because there is very little background on it. Rhamnaceae are a cosmopolitan family (more commonly in the subtropical and tropical regions) of flowering plants, mostly trees, shrubs, and some vines, and one herb, commonly called the Buckthorn Family, consisting of *50 genera and *900 species. The earliest fossil evidence of Rhamnaceae is from the Eocene. They are characterized by flowers with petalopposed stamens (obhaplostemony) and tendency toward xeromorphism. Obhaplostemony is a relatively rare feature in angiosperms, and this has resulted in Rhamnaceae being associated with other families such as Vitaceae and Cornaceae exhibiting this arrangement (Richardson et al. 2000). The xeromorphic adaptations exhibited by some members of the family include reduced or absent leaves, crowding of leaves, shortening of branch axes, presence of thorns or spines, and low, shrubby habit. In addition to ornamental shrubs Ceanothus and Colletia (Richardson et al. 2000), there are few plants of economic value in Rhamnaceae, the most notable being the jujube (Ziziphus jujuba), a fruit tree for this plant have been

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made the complete genome sequence which could reveal the molecular mechanisms underlying some specific properties of the jujube usefulness for improve the resources from Rhamnaceae (Liu et al. 2014). Chilean Rhamnaceae are spiny shrubs found in the central zone of Chile, where there are 30 species distributed in 7 genera (Marticorena and Quezada 1985). Following the work done in the genus Colletia, the number of species has been reduced to 18 in the same 7 genera. In this regard, it is important to note that genus Discaria has a high degree of polymorphism. Tortosa (1983) met in D. chacaye based on morphological characters to different polymorphs of D. serratifolia. In Gondwanic Discaria, phenotypically intermediate individuals exist between D. chacaye (G. Don) Tortosa and D. articulate (Phil.) Miers, and between D. chacaye and related Ochetophila trinervis (Gillies ex Hook & Arn.) Poepp. Ex Miers (Medan et al. 2012). Such genera are: Colletia (6), Condalia (1), Discaria (5), Retanilla (2), Rhamnus (1), Talguenea (1), and Trevoa (1) (Marticorena and Quezada 1985; Tortosa 1992; Marticorena 1990). The leaves, aerial, seeds, and root parts of plants of this family are traditionally use in traditional medicine for inflammation, fever, insomnia, relieve pain, convulsions and viral treatments (Kundu et al. 1989; Lee et al. 2003). In Rhamnaceae probably Zizyphus genus (endemic to Indo-Pakistan region) is one of more worldwide studied species (Mahajan and Chopda 2009). Zizyphus nummularia is very important in ethnomedicinal system of Indo–Pakistan subcontinent and its bark is known to have antibacterial and analgesic activities (Shah et al. 1990, Trevisan et al. 2009). Discaria genus is a small shrub found in Southern lands of Argentina and Chile. Its root bark is used in traditional folk medicine for the treatment of diabetes, stomach disorders, and as a fever-lowering agent (Giacomelli et al. 2001). Infusions of this plant have been used for long time in folk-medicine for the treatment of many diseases (Montes and Wilkomirsky 1981). Particularly, the Chilean folklore medicine uses ‘‘trevu’’ (Trevoa trinervis) for inflammation treatments caused by wounds and burns. Different fractions of this plant possess anti-inflammatory and antipyretic activities (Delporte et al. 1997). At 700 s decade some studies were focused mainly on the isolation and characterization of alkaloids with benzylisoquinoline and cyclopeptides skeleton (Torres and Sa´nchez 1971; Torres et al. 1979; Rivera et al. 1984; Pacheco et al. 1973).

Phytochem Rev

Based on previously published information about members of this family and our observation that these plants appeared to be highly resistant to both insect and pathogen attack in the field, we undertook examination of literature of members of this family. Our goal is to correlate phytochemical composition and biological activity to identify biopesticides of botanical origin for insect control studies.

4, also six alkaloid with aporphine skeleton: boldine 9, norboldine 10, isocorydine 13, norisocorydine 14, roemerine 2 and aporphine 15 (Figs. 1, 2). Recently studies reported the presence of three known alkaloids: coclaurine 7, N-methylcoclaurine 4, and R(-)-armepavine 3 isolated and identified from aerial part of Talguenea quinquenervia (Alarcon et al. 2011).

Chemical constituents

Cyclopeptide alkaloids

The plants belonging to Rhamnaceae family have been noted to produce a variety of characteristic secondary metabolites covering from triterpenes, cyclopeptides alkaloids, benzylisoquinoline alkaloids (BIAs), and flavonoids. Several Chilean members are characterized by the presence of alkaloid derivatives of 1-bencyl tetrahydroisoquinolines, cyclopeptides alkaloids, sapogenines and triterpenes.

Cyclopeptides from terrestrial plants can be divided into two types: one is the normal cyclopeptides, which usually consist of less than 14 amino acid residues with no disulfide bond. Generally, these peptides are the second metabolites of plant and about 455 cyclopeptides have been discovered in higher plants belonging to 26 families, 65 genera, and 120 species (Tan and Zhou 2006; Trevisan et al. 2009). In particular, plants of Caryophyllaceae and Rhamnaceae families commonly contain cyclopeptides. For example, from D. crenata and R. ephedra was isolated crenatine A 16 (Silva et al. 1974; Bhakuni et al. 1974), together with integerresine 17 (Bhakuni et al. 1974). Additionally from root of Condalia buxifolia from Brazil and Paraguay were isolated condaline A together with other three known cyclopeptide alkaloids, additionally, antibacterial activities of these alkaloids were reported (Morel et al. 2002).

Benzylisoquinoline and aporphine alkaloids BIAs are structurally a diverse group of specialized metabolites with a long history of investigation. Although the ecophysiological functions of most BIAs are unknown, the medicinal properties of many of these compounds have been exploited for centuries (Hagel and Facchini 2013), including narcotic analgesics (codeine and morphine), antimicrobial agent’s (sanguinarine and berberine) and the antitussive, anticancer drug noscapine. Magnocurarine 1 was isolated from Colletia histrix (Torres and Sa´nchez 1971) Discaria serratifolia is endemic plant of Central-Region Chile; it shows a great degree of polymorphism and has been described eight varieties. From D. serratifolia var. Montana was isolated 1,2-dimethoxy-11-hydroxyapomorphine 11 (Rivera et al. 1984). Previously Torres et al. (1979) from D. serratifolia var. discolor reported R(-)-Omethylarmepavine 5, R(-)-N-demethylcolettine 6, R(-)-armepavine 3, and R(-)-N-methylcoclaurine 4. Pacheco et al. (1973) from D. crenata isolated R(-)- armepavine 3, and R(-)-N-methylcoclaurine 4. From aerial parts of D. chacaye was isolated 1,2,11trimethoxynoraporphine 12 (Correa et al. 1987). From aerial parts and roots of Retanilla ephedra (Silva et al.1974) were isolated four alkaloids with 1-bencylisoquinoline skeleton: coclaurine 7, R(-)armepavine 3, nor-armepavine 8, N-methylcoclaurine

Terpenoids Rhamnaceae species are known to contain triterpenoids metabolites, especially pentacyclic triterpenoids. Studies in Chilean Rhamnaceae has reported until now 18 triterpenes, of which four compounds (18, 19, 20 and 21) belong to lupane group (Fig. 3), one compound (22) to ursane group (Fig. 4), three compounds (23, 24 and 25) to oleanane type group (Fig. 4), three (28, 29 and 30) (Fig. 5) to ceanothane group (Fig. 6) and four (31, 32, 33, 34, and 35) to dammarane group (Fig. 7), and two known sterol 28, and 29 (Fig. 5). The ceanothane type triterpenoids, with a contracted A-ring, have been isolated from species such as Ziziphus (Ganapaty et al. 2006; Guo et al. 2009; Jagadeesh et al. 2000; Lee et al. 2003; Suksamrarn et al. 2006), Ceanothus (Li et al. 1997), Paliurus (Lee et al. 1991, 1997), Colubrina (Roitman and Jurd 1978), Hovenia

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Phytochem Rev HO

Fig. 1 Benzylisoquinolinic and aporphinic alkaloids isolated from Chilean Rhamnaceae

O +

N

HO

CH3 CH3

N

O

CH3

HO 1

Others metabolites

O O HN O

R

O

N H

Fig. 2 Cyclopeptidic Rhamnaceae

N H

N

N

16

alkaloids

isolated

17

from

Chilean

(Yoshikawa et al. 1998) and Alphitonia (Guise et al. 1962). To the best of our knowledge, to date, only twenty of the ceanothane type triterpenes have been reported and noticeable all of them were obtained from members of this family. Thus, the ceanothane type triterpenoids could be a chemical marker for the Rhamnaceae with diverse uses such as in chemosystematics or chemotaxonomy.

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2

On the other hand, from Discaria crenata was isolated rutin, after of an acid hydrolysis affords quercetin, glucose and rhamnose (Pacheco et al. 1973). Biological activities Studies on the biological activity of compounds isolated from Chilean Rhamnaceae are quite scarce. Alkaloids and pentacyclic triterpenes (PTs) as mentioned above constitute the chemical basis of this family. In particular PTs possess several interesting biological activity such as anti-HIV, anti-tumor, antidiabetic, anti-inflammatory, antibacterial, antiviral, antiparasitic, hepatoprotective, wound healing, antioxidant, antipruritic, antiangiogenic biological

Phytochem Rev

COOH O

HO

HO

18

HO 20

19

CH2OH

21

Fig. 3 Triterpenes with lupane skeleton from Chilean Rhamnaceae

COOH

HO

COOH

HO 22

COOH O

AcO 23

24

25

Fig. 4 Triterpenes with oleanane and ursane skeleton

GluO HO

27

26

Fig. 5 Sterols isolated from Chilean Rhamnaceae

COOH

OHC

COOH

OHC HO

28

COOH

HOOC HO

29

30

Fig. 6 Triterpenes with ceanothane skeleton from Chilean Rhamnaceae

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Phytochem Rev Fig. 7 Triterpenes with dammarane skeleton with Chilean Rhamnaceae

HO O

O H3CO

OH

HO O

O O

O

31

CH2OH

HO

HO 32

33

OH

OH O

O O

O O

CH2OH

HO

H

HO 34

activities antiallergic and immunomodulatory activities (Zhang et al. 2011). The BIAs constitute a group of natural products of diverse structure that are widely present in many plants and mammalian species. Around an amount near to 2500 compounds of 1-BIAs structures have been identified and shown a wide range of biological activities including anticancer, antimalarial, anti-HIV, antiplatelet and vaso-relaxant. Several natural and synthetic benzylisoquinoline derivatives have also displayed affinities for dopamine and serotonin receptors, which are important neurotransmitters in the central nervous system (CNS) (Hawkins and Smolke 2008).

35

studies of the latter activity showed that the 3-hydroxyl, 17-carboxyl acid, and 19-isopropyl (or isopropylidene) moieties are essentially pharmacophores (Lee et al. 1998). The study revealed that Rhamnaceae family is rich in ceanothic acid, a ring A-homologue of betulinic acid. Assays for determining the antineoplastic effect of different fractions obtained from T. trinervis were performed by Delporte et al. (1997) against the following cell lines, P-388 (lymphoid neoplasm from DBA/2 mouse) A-549 (human lung carcinoma), HT29 (human colon carcinoma) and Mel-28 (human malignant melanoma). The results show that fractions used do not have any effect on the cell lines tested.

Anti-inflammatory activity Insecticidal activity From de standpoint of biological activity of South American Rhamnaceae, there are few reports. Infusion of T. trinervis is used by Chilean folklore medicine for treatment of inflammation cause by wound and burns (Mun˜oz et al. 1981). Other studies have showed that different extract of this plant do not have toxic effect on mice at dose level assayed (Delporte et al. 1997). Antineoplastic affect The lupane-type triterpene, betulinic acid, is widely distributed in plants. This pentacyclic compounds has proved to be selectively cytotoxic to melanoma (Pisha et al. 1995) and are active against the HIV virus (Fujioka et al. 1994). Structure–activity relationship

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Extracts obtained from a common shrub that occurs as part of vegetative species growing on arid lands of North-Central Chile and Argentina known as ‘‘piquilin’’ Condalia microphylla (Rhamnaceae) showed insect growth inhibitory activity against the fall armyworm Spodoptera frugiperda, yellow meal worm Tenebrio molitor and fruit fly Drosophila melanogaster larvae in artificial diet feeding assays (Cespedes et al. 2013). The effects of these extracts on mortality, antifeedancy and growth inhibition were examined (Tables 1 and 2). The phytochemical profile of the most active extract was examined with conventional chromatographic and spectroscopic procedures. This n-hexane extract showed a high

Phytochem Rev Table 1 Results obtained in tests with not choice measured in mortality percentage of S. frugiperda, T. molitor and D. melanogaster larvae, after application of the extracts from C. microphylla at different concentrations in larvae’s diet Samples

S. frugiperda

T. molitor

D. melanogaster

LD50 S. frugiperda molitor

LD50 T. melanogaster

LD50 D. melanogaster

0.0

0.0

0.0

n.d

n.d

n.d

10

0.0

0.0

0.0

25

0.0

0.0

0.0

50

0.0

0.0

0.0

9.4

14.2

7.65

100

0.0

0.0

0.0

10 25

70 ± 0.6b 55 ± 0.7b

45 ± 0.6b 70 ± 0.7b

61.0 ± 0.6b 90.0 ± 0.6b

50

100 ± 0.8c

100

100 ± 1.0

c

10

80 ± 0.2

b

3.89

5.2

3.23

25

90 ± 0.9

c

50

100 ± 1.0c

100

100 ± 1.0

c

b

9.7

20.4

17.9

10.8

–

–

Cone. (ppm)

Control

Aqueous

Ethyl Acetate n-hexane

Methanol

Gedunin

83.4 ± 0.8c 100 ± 1.0

c

100 ± 1.0c 100 ± 1.0c

73.5 ± 0.3

d

94.5 ± 0.6d

84.7 ± 0.5

b

100 ± 0.6c

95 ± 0.6b 100 ± 1.0 b

100 ± 1.0c

10

70 ± 0.5

25

50 ± 0.7b

55 ± 0.7b

65 ± 3.4b

50

100 ± 1.0c

80 ± 0.5b

100 ± 4.5c

100

100 ± 1.0c

100 ± 1.0c

100 ± 4.5c

10

37 ± 0.2

40 ± 0.7

100 ± 0.6b c

a

25

45.5 ± 0.3

50

73.50.6b

d

30 ± 1.5

22 ± 0.8

a

69 ± 0.6a

59 ± 0.9

d

100 ± 1.0c

89 ± 0.4c

100 ± 1.0c

Each value corresponds to the average of the five different experiments ±SE. The values followed by the same letter are not significantly different. The significance level P \ 95 %. The time for S. frugiperda was after 21 days, for T. molitor was 25 days, and for D. melanogaster 72 h a

This value correspond to LD95 (Cespedes et al. 2005)

percentage of hentriacontane and triacontane. The observed mortality strongly correlates with the contents of these long-chain n-alkanes compounds, the LD50 for n-hexane, ethyl acetate and methanol extracts against S. frugiperda, were 3.89, 9.4, and 9.7 ppm; against T. molitor 5.2, 14.2, and 20.4 ppm, and against D. melanogaster 3.23, 7.65 and 17.9 ppm, respectively (Cespedes et al. 2013). Many secondary metabolites from plants are synthesized as well as constitutive and/or induced chemical defense generated for protection against to different phytopathogenic attack. T. quinquenervia (Rhamnaceae) ‘‘Talguen’’ is a notorious weed and possess several medicinal applications. Some taxa from Americas such as Rhamnaceae members are toxic to insects, fungi and several bacteria strains, and these effects has been associated with the presence of

alkaloids, phenolics and terpenes. The methanol extract from the roots of T. quinquenervia afforded 9 triterpenes which belong to lupane, oleanane, ursane, sterol and ceanothane groups, these compounds showed insecticidal effects (Tables 3, 4) (Quiroz et al. 2015). Our results indicate that these compounds are involved in interference of sclerotization and moulting (Quiroz et al. 2015). These triterpenes showed to have selective effects on the pre-emergence metabolism of the insect and selectivity towards Lepidoptera and Diptera, but not to coleoptera. The results were fully comparable to know natural insect growth inhibitors such as gedunin and Cedrela extracts and have had a possible role as natural insecticidal agents. To the best our knowledge, this was the first report about the isolation of ceanothane triterpenes type from this plant

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Phytochem Rev Table 2 Activity of extracts from C. microphylla on pupation and emergences parameters of fall armyworm (after 21 days of incubation)a Treatment

Control

Cone (ppm)

Mean time pupation (days)a

Pupation SP(%)e

Mean weight pupae (mg)c

Mean emergence (days)d

Emergence (%)f

Male (%)

Female (%)

0

22

88.2

309 ± 15.5a

33

77.5

35

42.5

2

22

60.6

190.5 ± 11.4a

31

8.3

8.3*

–

22.5

22.8

180.9 ± 9.78b

33

8.3

8.3*

–

b

–

10 n-hexane

Ethyl Acetate Methanol

Gedunin

25

24

16.8

50

n.d

0

122.7 ± 8.79

–

0.0

–

n.d

–

0.0

–

2

21.5

–

68.3

227.6 ± 11.4a

33

16.7

8.3

8.3

10

24

25.7

150.8 ± 7.54a

36

8.3

8.3

25

25

12.5

148.8 ± 7.44a

–

0.0

–

–

50

25

6.3

n.d

–

0.0

–

–

a

2

22

52.3

205.3 ± 10.3

32

16.6

8.3

8.3

10 25

25 25

26.3 6.3

119.9 ± 5.5b 109 ± 4.4b

35 –

8.3 –

8.3 –

–

50

25

0

n.d

–

–

–

–

10

22.5

49.8

111.5 ± 5.6b

34

16.6

–

8.3

25

24

24.2

67.1 ± 3.4c

35

15.6

4.2

10.4

55.1 ± 2.75c

36

4.17

11.1*

–

50

21.5

d

4.17

a

The values for growth bioassay were from weight, values taken at 22 ± 1 days before pupation, the criteria followed was to account larvae that formed pupae, the larvae that not formed pupae were counted as died larvae b

Values taken after pupation. The values for aqueous extract were omitted because are irrelevant and this extract not showed any effect at all assayed concentrations

c Means followed by the same letter within a column after ±SE values are not significantly different in a Student–Newman–Keuls (SNK) test at P \ 0.05 (treatments are compared by concentration to control), 95 % confidence limits d

Percentage with respect to control

e

SP: survival pupation = number of surviving pupae 100/total larvae for pupation

f

% = Number of adults emerged 100/total number of pupae. The asterisks indicates deformities

species. These types of compounds exhibited activity under a concentration dependent manner with a LD50 values between 41 and 17 lg/mL. The insecticidal activity of extracts obtained from aerial parts of T. quinquenervia (Gill. et Hook) were evaluated using bioassay against larvae of Drosophila melanogaster. All extracts tested had insecticidal activity, the most active being those that contain alkaloids. From the most active fraction, it was possible to identify the known alkaloids coclaurine, armepavine and Nmethylcoclaurine (Alarcon et al. 2011, 2015).

Conclusions Plants are able to produce a countless number of molecules, secondary metabolites, as a result of the interaction of these sets with its environment. These

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secondary metabolites have been shown to have different biological activities, many of which are currently take advantage of different purposes. The information has been compiled about Rhamnaceae family producing two important groups of secondary metabolites such as alkaloids and pentacyclic triterpenes, these compounds are responsible for the activities reported. In the particular way and based on our results obtained from C. microphylla, and T. quinquenervia and no published result, we suggest that the insect growth inhibition caused by n-hexane and ethyl acetate extracts could be due to synergistic effect by components into those extracts. These plant extracts may be considered to be efficient insect growth regulators (IGRs), as well as having similar activity to phytoecdysteroids, as was evidenced by their significant inhibition of the molting processes. These

Phytochem Rev

Table 3 Results obtained in tests with not choice measured in mortality percentage of D. melanogaster and C. pomonella larvae, after application of the extracts and fraction from T. quinquenervia at different concentrations in larvae’s diet Sample

CONC (ppm)

Aqueous

Control

Ethyl acetate

Hexane

Methanol

20

21

25

26

27

Gedunin

a

D. melanogastera,b

C. pomonellaa,b

0

0

91

10

80 ± 2.0

90 ± 1.0

50

80 ± 2.0

100 ± 0.0

100

87 ± 1.5

100 ± 0.0

10

17 ± 0.6

93 ± 0.6

50

27 ± 0.6

100 ± 0.0

100

87 ± 1.5

100 ± 0.0

10

30 ± 0.0

83 ± 1.5

50

37 ± 1.5

97 ± 0.5

100

77 ± 1.2

100 ± 0.0

10

17 ± 0.6

100 ± 0.0

50

25 ± 1.5

100 ± 0.0

100

40 ± 1.0

100 ± 0.0

10

57 ± 2.5

67 ± 1.5

50 100

93 ± 0.6 100 ± 0.0

100 ± 0.0 100 ± 0.0

10

83 ± 1.5

70 ± 1.0

50

83 ± 1.5

100 ± 0.0

100

100 ± 0.0

100 ± 0.0

10

13.3 ± 1.2

50 ± 1.7

50

100 ± 0.0

83 ± 2.1

100

100 ± 0.0

100 ± 0.0

10

20 ± 0.0

73 ± 0.6

50

100 ± 0.0

100 ± 0.0

100

100 ± 0.0

100 ± 0.0

10

43 ± 0.6

–

50

80 ± 1.7

–

100

100 ± 0.0

–

20

87 ± 3.9

50 100

100 ± 0.0

LD50 D. melanogaster

88

LD50 C. pomonella 7.19

12.3

94

9.66

111

8.38

17.7

34.9

41.4

24.8

21.8

38

20.4

17.3

39.7

–

11.9

–

Mean of three replicates

b

Means followed by the same letter within a column after ±SE values are not significantly different in a Student–Newman–Keuls (SNK) test at P \ 0.05 (treatments are compared by concentration to control), 95 % confidence limits

extracts possess potent insecticidal and growth inhibitory activities (Cespedes et al. 2013). Preceding experimental observations suggest that acute toxicity and growth inhibition of our extracts may be due to inhibition of a proteinase, ETH and other polyphenol oxidases (PPO) that could be bind to these type of compounds (n-alkanes) (Cespedes et al. 2013) or move throughout cellular membranes and could produce white substance accumulation

(deposits) in vacuoles in similar form to reported by Delgado et al. (2011) and Halse et al. (1993). This target has been demonstrated for other compounds of natural origin (Cespedes et al. 2005; Karban and Baxter 2001; Kessler and Baldwin 2002). The sites and mode of action of these extracts and their isolated components are being investigated and probably correspond to a combination of antifeedant action, as well as, neurodegenerative effects, midgut phenol

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Phytochem Rev Table 4 Activities of compounds 20, 21, 25, and 26 isolated from roots of T. quinquenervia on growth bioassay, pupation, and emergence parameters of D. melanogaster (after 14 days of incubations) Treatment

Control

Mortalitya,b (%)

Length gained (%)

% Pupation

100

100

Mean time pupation (days)

IG (%)

Emergence (%)

100

100

100

8.0

25

73.3 ± 2.5

77.1

35.1

27

5.0

26.7

87.5

26

73.3 ± 3.0

5.7

42.5

27

6.5

26.7

89.0

20

77.0 ± 1.5

18.8

44.2

23

5.5

23.3

33.3

21

87.7 ± 1.2

66.1

13.0

13.3

6.0

13.3

0

a

0

Weight gained (%)

Mean of three replicates

b

Means followed by the same letter within a column after ±SE values are not significantly different in a Student–Newman–Keuls (SNK) test at P \ 0.05 (treatments are compared by concentration to control), 95 % confidence limits

oxidase, proteinase, ETH, tyrosinase or other PPOs and cuticle synthesis inhibition, as well as molting disruption and/or sclerotization toxicity, as has been found for other natural compounds (Kubo et al. 2003; Cespedes et al. 2000, 2001, 2004, 2005, 2006; Torres et al. 2003) and extracts (Feng et al. 1995). Thus, the effect of n-hexane, ethyl acetate and methanol extracts on reducing insect growth, increasing or shortening development time, modifying the apolysis during molting and producing a high mortality on T. molitor, S. frugiperda and D. melanogaster were more powerful than gedunin, and MeOH-Ced extract from Cedrela salvadorensis, used as positive control in several reports (Cespedes et al. 2000, 2004, 2005, 2006, 2013; Torres et al. 2003). Although chemically distinct, the level of insecticidal activity of metabolites and mixtures derived from Condalia species is comparable to that of the known insect growth regulator, gedunin and may be due to a synergistic effect shown by the ecdysone-like activity of the n-hexane extract in the test system used. On the other hand, our results showed that Chilean Rhamnaceae is rich in triterpenes, particularly pentacyclic triterpenes. These compounds possess a big variety of pharmacological properties; different studies have demonstrated the insecticidal activity or insect’s antifeedant effect of these (Gonza´lez-Coloma et al. 2011). This information helps to understand the defensive role in plants. Terpenes of ceanothane and oleanane type are clustered under constitutive or inducible from chemical defense of plant species under study. New biological activity studies of the isolated from these extracts are in progress. These plant isolates are IGRs, with activity similar to that of phytoecdysteroids, as was evidenced by their

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significant inhibition of molting processes. Our findings show that acute toxicity and insect growth inhibition observed may be due to the inhibition of acetylcholinesterase or butyrylcholinesterase enzymes. The activities showed by these plants, their metabolites and mixtures could to help to explain its resistance to pathogen attack. In summary, the insecticidal activity of n-hexane, ethyl acetate extracts, fractions and compounds from aerial parts of selected members of the Rhamnaceae could be due to synergistic effects shown by the components of the mixtures in the test system used in this investigation. Furthermore, the great inanition (extreme weakness from lack of nourishment) observed in insect growth regulatory and insecticidal assays may be due to inhibition of cholinesterases as well. The activity of this kind of plants and their metabolites, fractions and extracts is comparable to that of the insect growth regulator azadirachtin, gedunin, and toosendanin which suggests potential for further development as biopesticides that prevent the emergence of resistance in insect pest control and avoid the use of POPs under an IPM, which today definitively characterizing the impacts on crop and benefic insects, such as bees and honey-bees is experimentally challenging. Knowledge of insect regulator behavior, physiology and toxicology has expanded drastically on recent years; indeed, scientific research evaluating risks of pesticides to benefic and insect pest and other pollinators has improved markedly (Brady 2014), EPA recommending a modeling approach for summing up all exposure risks, taking into consideration life cycle differences in sensitivity as well as life cycle differences in exposure

Phytochem Rev

risk, this incentive the research of new sources of biopesticides friendly with the environment such the Rhamnaceae botanical family. Improvements in pesticide technology have a long history of unintended consequences for beneficial insects in general. Manny beneficial insects (honey bees for example) are uniquely vulnerable to nontarget impacts of agricultural pesticide use largely because, as the world’s premier managed pollinator species, they are deliberately transported by farmers into areas where crops are grown (often with the help of chemical pesticides) to provide pollination services. Thus, the search of new biopesticides has in effect become one biotechnological innovation that may not actually have unintended consequences for benefic insect and other organisms. Acknowledgments To Fondecyt program grant # 1130463. The authors wish to thank to internal grant from Direccio´n de Investigacio´n DIUBB # 083009-2R, # 122509 and # 132209 GI/ C, Universidad del Bio Bio, Chilla´n, Chile.

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