Plants in the Genus Tephrosia: Valuable Resources for Botanical Insecticides

Simple Summary There is an increasing interest in botanical insecticides worldwide. Plants from the genus Tephrosia are rich in bioactive phytochemicals, particularly rotenoids which include rotenone, deguelin, rotenolone, and tephrosin. Rotenoids have strong insecticidal activities against a wider range of pests. However, there has been no treatise thus far focusing on Tephrosia as insecticidal plants. This article is intended to review phytochemicals produced by selected species, their insecticidal activities, and the current status on the use of Tephrosia as botanical insecticidal plants for insect pest control. Abstract Synthetic insecticides are effective in controlling insect pests but can also harm nontarget organisms and the environment. During the last 40 years, there has been an increasing interest in alternative insecticides, particularly those derived from plants, commonly known as botanical insecticides. However, commercially available botanical insecticides remain limited. Rotenone is one of the earliest identified compounds and was used as fish poison and pest management. Due to its link with Parkinson disease, the use of rotenone was banned in many developed countries. Rotenone used to be isolated from Derris spp. and Lonchocarpus spp., and it can also be isolated from Tephrosia species. In this article, we present basic botanical information on selected Tephrosia species and their major compounds related to insecticidal activities and highlight the current use of extracts derived from some species, Tephrosia vogelii in particular, for control of insect pests in stored grains and crop production. The crude extracts contain multiple bioactive compounds, mainly rotenone, deguelin, rotenolone, and tephrosin, which act in either additive or synergistic fashion, resulting in effective control of insect pests. There are about 400 species in the genus Tephrosia, and species and even strains or variants vary greatly in these active compounds. We argue that a systematic evaluation of bioactive compounds in different species are needed, and species or strains with high insecticidal activities should be selected for use in the sustainable control of insect pests.


Introduction
Application of synthetic insecticides is the most common way of controlling insect pests in crop production [1]. The use of insecticides is credited for protecting crops from insect damage and enhancing crop productivity. It was estimated that without the use of pesticides, global food production loss would be 35-45% [2]. However, since the publication of Silent Spring [3], the application of synthetic insecticides has become one of the most controversial topics and remains the forefront regulatory issue  50 species are native to equatorial Africa, of which 30 are found in Kenya; 70 are found in South Africa; 35 occur in India; and 30 are native of South America [21,22]. They are erect herbs or soft or woody shrubs with dense foliage grown to a height ranging from 0.5 to 4 m. They have the potential to restore soil fertility due to their ability to fix nitrogen [16,18]. Leaves are compound, leaflets are inverted lance-shaped or obovate, 7-15 cm long and 0.3-1 cm wide. Flowers are pea shape, white, purple or pinkish, 7 mm long, in a few-flowered, leaf-opposed, raceme-like clusters. Plants are self-pollinated and produce linear-long pods, 2.5-4.0 cm long and 3-4 mm wide. Seeds are ellipsoid, dark brown. Most species are diploid with a 2n chromosome number of 11. Species commonly associated with insecticidal activities include Tephrosia candida, (Roxb.) DC.; Tephrosia elata, Deflers; Tephrosia purpurea (L.) Pers.; Tephrosia villosa (L.) Pers.; Tephrosia virginiana (L.) Pers.; and T. vogelii Hook. f. (Table 1).

Tephrosia vogelii
Among the 400 species, T. vogelii is the most intensively studied one. It is a herb or small tree, native to tropical Africa and can attain a height of 2 to 3 m in a growing season of 5 to 7 months. Flower color may be white, purple, or red. It produces various compounds, such as flavonoid, steroid, and rotenoids (Table 1). Rotenoids mainly include four compounds ( Figure 1): (a) Rotenone, it has a molecular formula C 23 H 22 O 6 , crystal with a melting point of 165 • C. Aphids are killed by 3 mg/kg of rotenone in solution and oral LD 50 value in rats was about 132 mg/kg body weight [23]. (b) Deguelin, a derivative of rotenone with an empirical formula of C 23 H 22 O 6 , the same as rotenone, crystal, and melting point is 171 • C [24]. Its LD 50 value to humans ranged from 10 to 100 g [25], but such values to insects are unknown. (c) Rotenolone, crystal with a chemical formula of C 23 H 22 O 7 . (d) Tephrosin, a nearly colorless rotenoid with a formula of C 23 H 22 O 7 and melting point of 198 • C, and it is thought to be the oxidation product of deguelin [26]. The lethality values of both rotenolone and tephrosin are unknown. The content of rotenoids in T. vogelii leaves was higher than that in petal, stems, and roots, accounting for 80% to 90% of the total rotenoids [27].
Insects 2020, 11, x 3 of 18 Africa; 35 occur in India; and 30 are native of South America [21,22]. They are erect herbs or soft or woody shrubs with dense foliage grown to a height ranging from 0.5 to 4 m. They have the potential to restore soil fertility due to their ability to fix nitrogen [16,18]. Leaves

Tephrosia vogelii
Among the 400 species, T. vogelii is the most intensively studied one. It is a herb or small tree, native to tropical Africa and can attain a height of 2 to 3 m in a growing season of 5 to 7 months. Flower color may be white, purple, or red. It produces various compounds, such as flavonoid, steroid, and rotenoids (Table 1). Rotenoids mainly include four compounds ( Figure 1): (a) Rotenone, it has a molecular formula C23H22O6, crystal with a melting point of 165 °C. Aphids are killed by 3 mg/kg of rotenone in solution and oral LD50 value in rats was about 132 mg/kg body weight [23]. (b) Deguelin, a derivative of rotenone with an empirical formula of C23H22O6, the same as rotenone, crystal, and melting point is 171 °C [24]. Its LD50 value to humans ranged from 10 to 100 g [25], but such values to insects are unknown. (c) Rotenolone, crystal with a chemical formula of C23H22O7. (d) Tephrosin, a nearly colorless rotenoid with a formula of C23H22O7 and melting point of 198 °C, and it is thought to be the oxidation product of deguelin [26]. The lethality values of both rotenolone and tephrosin are unknown. The content of rotenoids in T. vogelii leaves was higher than that in petal, stems, and roots, accounting for 80% to 90% of the total rotenoids [27].    [69,70] Rotenoids belong to advanced members of the isoflavonoid group [71]. Their biosynthesis is based on shikimic/chorismic acid pathway, covering a group of plant natural products that is based on the rotoxen skeleton [71]. Rotenone was reported to be the most toxic of the rotenoids followed by deguelin [28]. Together, the four main compounds contribute greater than 95% of the toxicity [72]. Rotenone is a contact and ingestion compound [7]. Its mode of action involves the inhibition of the electron transport at the mitochondrial level, blocking phosphorylation of ADP (adenosine di-phosphate) to ATP (adenosine triphosphate), thereby inhibiting insect metabolism [28]. It is a selective, nonsystemic insecticide with contact and stomach action and secondary acaricidal activity [73]. Rotenone has been used for centuries as a selective fish poison [28] and also as an insecticide for controlling a wide range of arthropod pests including cucumber beetle, flea beetles, harlequin bug, leafhoppers, scales, spittlebugs, squash bugs, thrips, and some fruit worms [29]. Dried leaves of T. vogelii were used to protect stored legume seeds from damage by the bruchids [30].

Tephrosia candida
Tephrosia candida, commonly known as white hoarypea, is a perennial shrub, native to India. It grows up to 3.5 m. A distinct morphological characteristic of T. candida is that it produces greater biomass than T. vogelii. T. candida produces flavonoids including candidol, candidone, ovalichalcone, dehydrorotenone, candidin, and prongachin; and rotenoids, such as tephrosin and deguelin; and sterol (Table 1) [74]. Dehydrorotenone produced in stem and leaves is a stomach or contact poison and toxic to insects [20,75]. Tephrosin isolated from roots can interfere with insect growth and development [20,49]. Deguelin occurs mainly in roots and has antifeedant and growth inhibition activities, which are used for control of insects and nematodes [20,49]. A study conducted in Malawi showed that extracts derived from T. candida for control of aphid in common bean were less effective than those extracted from T. vogelii, which was explained by the fact that active compounds in T. candida were lower than T. vogelii [31].
Tephrosia candida, however, could be used as a cover crop for repellency of larval and adult diaprepes root weevil (Coleoptera: Curculionidae). This weevil, Diaprepes abbreviates (L.), is a major threat to the sustained profitability of citrus production in Florida and the Caribbean region. Adults were deterred from feeding and produced proportionally fewer eggs when caged with foliage of T. candida compared to foliage of T. vogelii, suggesting that the leaves of T. candida might contain antifeedants with activity toward adult diaprepes root weevil [76]. Additionally, feeding damage was observed by larvae on roots of T. candida and T. vogelii, but weight gain and survival of larvae fed with T. candida were greatly reduced compared to those fed with T. vogelii. These results implied that some compounds in root of T. candida are toxic to larvae. The authors suggested that phytochemicals responsible for the antifeedant and toxic properties of T. candida toward D. abbreviates are not shared by T. vogelii, and T. candida could be used as a cover crop in citrus field for control of diaprepes root weevil [76]. In fact, T. candida has been widely used in mixed cropping regimes and as a fallow crop in tropical production systems in Vietnam [77] and India [78] where it is valued for its contribution to soil fertility and simultaneously for repelling insect pest [49,50,79].

Tephrosia purpurea
Tephrosia purpurea is a highly branched suberect herbaceous perennial, about 1.5 m in height with spreading branches [80]. The plant grows abundantly in the upper Gangetic plains, and western Himalayas. More than 44 phytochemicals have been structurally identified from this species, which include rotenoids, flavanols, glycosides, isoflavones, sterols, and chalcones (Table 1) [81]. T. purpurea is known for its strong insecticidal efficacy. Sahayaraj [57] evaluated the potential of T. purpurea essential oil from stem and roots. Hexadecanoic acid was found to be the most abundant compound present. Essential oil showed strong repellent activity for males compared with females of banana stem weevil (Odoiporus longicollis), a serious pest of banana. The insecticidal efficacy was attributed to the presence of compounds like rotenone and hexadecanoic acid. T. purpurea leaf extract was able to control first to fourth instars larvae and pupae of A. aegypti [82]. Furthermore, the whole plant extract of T. purpurea was tested for its larvicidal activity against the larvae of Culex quinquefasciatus. The extract showed 100% mortality in very small doses suggesting its beneficial use in controlling the mosquito reproduction [58]. Winter season is more suitable for collection of plant materials due to the presence of high content of rotenone in this season [83].

Tephrosia villosa
Tephrosia villosa is a multibranched, perennial herb, up to 90 cm high, densely clothed with white, silky hair, found in India. Roots and seedpods produce flavonoids, including tephcalostaan, villosin, and tephrinone (Table 1). Whole plants contain rotenoids, dehydrorotenone, prenylated flavonone. The ethanol extract of roots, leaves, fruit, and twigs of T. villosa showed significant activity against southern house mosquito (Culex quinquefasciatus) larvae [65]. A defensin (TvD1) isolated from T. villosa showed inhibitory activities to mealworm (Tenebrio molitor) [84]. Plant defensin, a small, cationic, cysteine-rich broad-spectrum antimicrobial peptide, has four or five disulfide bridges and has been shown to be a component of the innate immunity system in plants. Over expression of defensin gene (TvD1) in tobacco exhibited strong activity against first and second instar larvae of taro caterpillar (Spodoptera litura), an important polyphagous insect attaching 44 families of economically important plants [66].

Tephrosia virginiana
Tephrosia virginiana, commonly known as devil's shoestring as its roots are very long and stingy, such that they can be used for twine, occurs only in North America, ranging from Texas in the southwest to Florida in the east, north to Ontario, and west to Nebraska. Roots of this species were used as piscicide by Native Americans [85]. The roots contain rotenone, tephrosin, and toxicarol (Table 1) [69]. Root extracts are toxic to aphids, houseflies, potato beetle, fleas, and lice infecting dogs and poultry. Rotenone content in roots extracts harvested at the full-bloom stage was the highest compared to those harvested at dominant, emergence, and mature seed stages, and the extracts were highly toxic to houseflies (Musca domestica L.) [70]. Detailed information on phytochemicals of this species, however, have not been reported thus far.

The Use of Tephrosia Plants for Managing Insect Pests
The discussed Tephrosia species are rich in bioactive compounds and show insecticidal activities against different insect pests. Based on the current information, the use of Tephrosia plants for insect pest management can be summarized as the follows (Figure 2).

The Use of Tephrosia Plants for Managing Insect Pests
The discussed Tephrosia species are rich in bioactive compounds and show insecticidal activities against different insect pests. Based on the current information, the use of Tephrosia plants for insect pest management can be summarized as the follows (Figure 2).

Commercial Formulation of Rotenone
Commercially, rotenone is generally extracted from the roots of cube plants (Lonchocarpu utilis) and barbasco (Lonchocarpu urucu) which was referred to as Cube resin as well as from derris plants (Derris elliptica). Rotenone is also extracted from Tephrosia spp and Dalbergia paniculata [28]. Although rotenone content in cube and derris plants is higher, about 5% in dried derri roots, the cultivation of these plants is difficult because of the liana type of growth and the labor involved in harvesting the small fibrous roots. On the other hand, most Tephrosia species have large biomass. A yield of over 14 metric tons of dried leaves and stems of T. vogelii could be obtained per hectare in the U.S. [17]. Regarding the content of rotenone in plant organs, leaflets contain 80% to 90% of rotenone [17]. Additionally, breeding effort by the U.S. Department of Agriculture (USDA) in the 1970s showed that some breeding lines could have rotenone content of more than 4.5% in leaflets [17]. Thus, extraction of rotenone from T. vogelii plants could be much easier and more convenient than from cube or derri plants.
Most commercial products of rotenone come from Central and South America [28]. Rotenone almost insoluble in water, and very soluble in many organic solvents, such as ethanol, acetone, chloroform, and ether [86]. Rotenone is unstable in light and air, and not environmentally persistent [87]. It degrades rapidly under natural conditions [24]. Rotenone powders lose much of their toxicity within weeks. Thus, its storage must be protected from air, light, and alkali, and the storage temperature should not exceed 25 °C. Solutions of rotenone in organic solvent, when exposed to light and air, become successively yellow, orange, and finally deep red due to oxidation [88]. Rotenone controls aster beetles, aphids, cabbage worms, cucumber beetles, Japanese beetles, and other insects [29]. Insects poisoned by rotenone experience a drop in oxygen consumption, respiratory depression,

Commercial Formulation of Rotenone
Commercially, rotenone is generally extracted from the roots of cube plants (Lonchocarpu utilis) and barbasco (Lonchocarpu urucu) which was referred to as Cube resin as well as from derris plants (Derris elliptica). Rotenone is also extracted from Tephrosia spp and Dalbergia paniculata [28]. Although rotenone content in cube and derris plants is higher, about 5% in dried derri roots, the cultivation of these plants is difficult because of the liana type of growth and the labor involved in harvesting the small fibrous roots. On the other hand, most Tephrosia species have large biomass. A yield of over 14 metric tons of dried leaves and stems of T. vogelii could be obtained per hectare in the U.S. [17]. Regarding the content of rotenone in plant organs, leaflets contain 80% to 90% of rotenone [17]. Additionally, breeding effort by the U.S. Department of Agriculture (USDA) in the 1970s showed that some breeding lines could have rotenone content of more than 4.5% in leaflets [17]. Thus, extraction of rotenone from T. vogelii plants could be much easier and more convenient than from cube or derri plants.
Most commercial products of rotenone come from Central and South America [28]. Rotenone almost insoluble in water, and very soluble in many organic solvents, such as ethanol, acetone, chloroform, and ether [86]. Rotenone is unstable in light and air, and not environmentally persistent [87]. It degrades rapidly under natural conditions [24]. Rotenone powders lose much of their toxicity within weeks. Thus, its storage must be protected from air, light, and alkali, and the storage temperature should not exceed 25 • C. Solutions of rotenone in organic solvent, when exposed to light and air, become successively yellow, orange, and finally deep red due to oxidation [88]. Rotenone controls aster beetles, aphids, cabbage worms, cucumber beetles, Japanese beetles, and other insects [29]. Insects poisoned by rotenone experience a drop in oxygen consumption, respiratory depression, and ataxia, which lead to convulsions, paralysis, and death by respiratory arrest [73]. Due to its sensitive to air, light, and temperature, rotenone should be applied during cloudy sky or evening with appropriate dosage to maximizing insect control efficacy and reduce application frequency [10].
Rotenone was a registered pesticide in the U.S. under the Federal Insecticide Fungicide Rodenticide Act in 1947. Its formulations include crystalline preparations (about 95%), dust (0.75%), and emulsifiable solutions (about 50%). Rotenone is also formulated with other pesticides, such as pyrethrins, carbaryl, lindane, piperonyl butoxide, and others in products to control insects, mites, ticks, lice, spiders, and undesirable fish [28]. However, due to its potentially adverse impacts on aquatic ecosystems [89] and more recently the link to Parkinson's disease in human beings [90,91], the U.S. has banned all uses of rotenone except as a piscicide since 2012, and the European Union (EU) began a phase out of rotenone in 2008 [92]. The Codex Alimentarius Commission in 2009 proposed to remove rotenone from the list of approved substances for plant protection, which was supported by Argentina, Japan, and Kenya but opposed by Australia, Brazil, Iran, Mexico, the Philippines, Thailand, U.S., and the International Federation of Organic Agriculture Movements (IFOAM) [93]. However, rotenone is still being used in China for controlling insect pests during vegetable production, such as head cabbage with the maximum residue limit set at 0.5 mg/kg [94].
Globally, the demand for natural pesticides is growing [7,8] due to the increasing interest in organically produced safe food. Policy changes in the U.S., EU, and some other countries about the safety data and maximum residue limits for synthetic pesticides as well as rotenone may have changed the commercial scope for botanical pesticides [95]. However, some countries, such as Brazil, China, and India have led the way in policy changes that could enabled more commercialization and use of botanical pesticides [8]. Nevertheless, the use of plant extracts for insect control has been a tradition, which remains strong across the African continent and some other regions [95].

Crude Extracts for Insect Control in Field Crop Production
Water, dilute liquid soap, or organic solvent-assisted extractions of bioactive compounds from plants continue although sophisticated procedures for extraction have been advanced. Crude or unrefined plant extracts are directly used for control of insect pests. Such practices remain in low input farming, particularly in Africa and even grow elsewhere like organic farming [96]. Insecticidal activities of the extracts could be due to the action of a single compound, additive or synergistic effects of several compounds. The combined effects have been referred to as phytocomplex [97]. A study conducted in Tanzania showed that extracts derived from T. vogelii significantly controlled aphid (Aphis fabae Scopoli), foliage beetle (Ootheca mutabilis (Schonherr) and O. benigseni Weise), and flower beetle (Epicauta albovittata Gestro and Epicauta limbatipennis Pic) in the common bean production field, and the application resulted in significantly higher yield than the control treatments [98]. Similarly, studies performed in Malawi and Tanzania showed that pest abundance (aphid, bean foliage beetle, and flower beetle) was lower when synthetic pesticides were used, while application of extracts derived from T. vogelii had relatively higher number of pests but lower than control treatments. More importantly, beneficial arthropod numbers were higher in plants treated with T. vogelii extract than those treated with synthetic insecticides, suggesting that T. vogelii extract had little effect on beneficial arthropods [96]. Such effects could be attributed to lower persistence of plant extracts and different modes of action [96]. To make the insecticide, dry powdered leaves were mixed with water containing 1% liquid soap at 10% w/v ratio for 24 h. Diluted solutions containing 1-2% of the extract were sprayed in the early evening to reduce exposure to sunlight and lessen effects on beneficial insects. Such practices have largely been used in southern and eastern Africa to control field pests rather than storage pests [99]. Previous studies indicate that T. vogelii is very effective in controlling a number of hard-to-kill field insects, including cucumber beetle, leafhoppers, squash bugs, flea beetles, harlequin bug, spittlebugs, thrips, scales, and some fruit worms [99]. Anjarwalla et al. [100] also reported the efficacy of extracts derived from T. vogelii in controlling bruchids in beans and cowpeas. Chemical analysis of T. vogelii indicated the presence of the rotenoids, including deguelin, tephrosin, and rotenone (with deguelin being the most abundant) [18,39]. These results demonstrated that the use of T. vigelii extracts to control insect pests can be as effective as synthetic insecticides in terms of crop yields, while conserving the non-target arthropods. Due to the locality of plant materials and convenience in extraction and application, the crude extracts can be more easily integrated in to agro-ecologically sustainable crop production systems [96].

Tephrosia as Cover Plant for Biocontrol of Insects and Soil Nitrogen Enrichment
Another role of Tephrosia plants is their repellency of insect pest. As mentioned above, adults of diaprepes root weevil were deterred from feeding of foliage on T. candida and produced fewer eggs, and T. candida could be used as a cover crop in citrus field for control of diaprepes root weevil [76]. Additionally, acetone extracts from leaves of T. vogelii had obvious antifeedant, inhibitory effect on growth and development and ovicidal activity, as well as acute stomach action [101]. The rotenoid compounds from T. elata showed significant antifeedant activity against Maruca testulalis, Spodoptera. exempta, and Eldana sacchariana [54]. Essential oil derived from T. purpurea showed stronger repellent activity against male banana stem weevils than the female ones [57]. The potentials of these species for repellency of insect pests deserve further investigation.
Tephrosia species are also widely used as cover crops by planting with rubber, oil palm, citrus, coffee, tea, coconut, and also annual crops [102]. Some species are reported to be green manure plants in agriculture and sometimes as a windbreak, contour hedge or shade plant because of the dense foliage and good root anchorage [103]. T. noctiflora is one of the Tephrosia species first being used as a green manure [103]. T. candida proved to be one of the most satisfactory green manures because it can flourish in poor soil for several years and has a dense foliage [103]. T. candida fallows alone can raise maize grain yield by 300% [104]. In South Rwanda, T. vogelii and Cajanus cajan were intercropped with Sorghum bicolor during the long rainy season. After sorghum was harvested, green manures were cut into small pieces and incorporated together with the sorghum residues. After four years of such intercropping, results showed that only T. vogelii led to a significant increase in yields. The yield of subsequently cropped beans increased by 25% compared to the control [105].

Tephrosia for Controlling Insect Pests in Stored Grains
Tephrosia plants have long been used for protecting grains from weevils [106]. Dried leaves have the potential to protect stored legume seeds from damage by bruchids in Southern Africa [107]. The insecticidal and repellent properties of T. vogelii were tested against Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae) in stored maize grain. T. vogelii caused 85.0-93.7% insect mortality in 21 days. The mean lethal exposure times (LT 50 ) to achieve 50% mortality varied from five to six days (7.5-10.0% w/w) to seven to eight days (2.5-5.0% w/w) [108].
Dry plant materials used for stored grains are generally recommended about 5% (v/v). However, due to the differences in the content of active compounds among varieties or even strains or variants, the application rates may vary. For example, the quantity of T. vogelii chemotype 1 (a strain or variant with higher insecticidal activity) used for control of adult bruchids could be much lower than that of chemotype 2 (a strain or variant with little insecticidal activity) [39]. The exposure of adult bruchids to chemotype 1 admixed with cowpeas led rapidly to higher levels of mortality than with chemotype 2. Interestingly, the exposure of bruchids to deguelin, the most abundant compound in the crude extract, supposedly less toxic than rotenone, caused a significantly higher mortality, while tephrosin was significantly less toxic than deguelin. The LC 50 for tephrosin was calculated at approximately 200 mg/kg, whereas the LC 50 values for rotenone, deguelin, sarcolobine, and toxicarol were below 10 mg/kg. The effect of obovatin 5-methyl ether against bruchids did not differ significantly from the control or the extract derived from chemotype 2 plants [39].

Concerns over the Use of Tephrosia Species as Botanical Insecticides
This review shows that Tephrosia plants are promising genetic resources for developing botanical insecticides. However, duo to the variable contents in active compounds among species or variants, along with incomplete understanding of their insecticidal activities, some concerns over the use of Tephrosia plants herein should be raised.
Not all species have anticipated insecticidal activities. As early as in the 1930s, Wilbaux [109] and Roark [16] suggested that only some species of Tephrosia might be the sources of rotenone. Irvine and Freyre [110] screened 16 Tephrosia species, 14 were contained some rotenoids, five contained rotenoids in their leaves, and T. vogelii had the highest leaf rotenoid content, ranging from 0.65% to 4.25%. Additionally, distinct chemotypes or variants occur in T. vogelii. Chemical analysis of plant material across Malawi identified two distinct chemotypes, one containing rotenoids for their biological activity against insects [111,112] and the other was characterized by flavones, flavanones, and flavonols [18]. Subsequent bioassays revealed that insecticidal activities of chemotype 1 were due to the presence of rotenoids, including deguelin, dehydrodeguelin, rotenone, and tephrosin; while the flavonoids in chemotype 2 were inactive [39] and had little active against insects. It was reported that about 25% of the plants of T. vogelii grown in Malawi belonged to the chemotype 2. Thus, the use of wrong species or chemotypes, such as chemotype 2 could result in ineffective in pest management as mentioned by Stevenson and Belmain [95].
Insecticidal activities may not completely rely on rotenone. In the same study [112], chemotype 1 contained deguelin as the major rotenoid along with tephrosin, and rotenone as a minor component. As mentioned above, extracts from chemotype 1 plants showed insecticidal activities, meaning deguelin plays a critical role in control of insect pests. This finding is important. Currently, it is rotenone that has been banned in several countries [92], but there has been no documentation about any regulations of deguelin. Interestingly, it has been known that plants containing high deguelin have been used as an anthelmintic agent in certain regions of China [113] and as a traditional Thai medicine to treat hepatitis and hepatic dysfunction [114]. Recently, deguelin has been shown to have potent anticancer activity against multiple cancer types and can impede carcinogenesis by enhancing cell apoptosis and preventing malignant transformation and tumor proliferation [115]. Tephrosia species or chemotypes with high deguelin contents but low levels of rotenone could be ideal plant materials for developing botanical insecticides. These results may imply that future chemical evaluation of Tephrosia species or chemotypes should focus more on deguelin. A study conducted in West Java, Indonesia showed that acetone extracts derived from leaf samples collected from seven geographical locations varied in LC 50 (ranging from 0.137% to 0.371%) against cabbage head caterpillar (Crocidolomia pavonana). More importantly, compounds other than rotenone are also responsible for the insecticidal activity [116]. Early studies also showed that the leaflets of T. vogelii contained more deguelin than rotenone, the reverse was general found in the petioles, stems, and roots [17]. One variety contained deguelin but no rotenone [27]. Varietal or strain variation in active compounds should be isolated individually and labelled as different chemotypes or specific strain. They should be produced in isolation for seed production or simply propagated by vegetative means to maintain the genetic identity for further evaluation and potentially released as new cultivars for commercial production.
A major challenge in production of T. vogelii is poor seed production [17], which has affected breeding effort on developing new cultivars and large-scale production of selected desirable chemotypes or strains [17]. With the advance of plant tissue culture or microprogation [117], this problem can be easily resolved. Micropropagation through shoot culture using existing meristems can produce a large quantity of plantlets without somaclonal variation [118]. Thus, the availability of genetic identical plantlets can be used for commercial production. Due to their rapid growth characteristic, a large quantity of biomass can be used for extracting bioactive compounds for pest management.
Precautions should be taken when handling Tephrosa extracts as some species or variants contain high rotenone, and rotenone has been linked with Parkinson's disease. Additionally, the extracts may have other potential hazardous effects to nontarget organisms even though they are much safer than commercial rotenone insecticide. The precautions include safe procedures for extraction, appropriate labeling of the extracted products, methods for storage and transportation, and safe methods for field applications at right time and correct doses.

Conclusions and Future Outlook
Several species of Tephrosia have long been recognized as insecticidal plants. Studies of these plants over the last 60 years have resulted in some important findings. (a) Species and varieties differ significantly in rotenoid contents. The USDA program conducted from the 1960s to 1970s clearly showed that rotenoid content varied from 0.65% to 4.25%, and hybridization and selection could further increase rotenoid content [17]. This finding suggests that rotenoid content can be improved through breeding. With the advance of molecular marker tools and omics technologies, we believe that rotenoid contents can be significantly enhanced by modern breeding technologies. (b) Chemotypes or variants occur within varieties of T. vogelii, individual plants may differ significantly in rotenone, deguelin, rotenolone, and tephrosin contents . (c) Deguelin could be an important compound against insect pests. Current data indicate that deguelin could be equal to or more important than rotenone in pest management. Deguelin has been widely used as pharmaceutical compounds [115], its use as an insecticide may be environmentally more benign than rotenone. Thus, the potential of deguelin deserves future investigation. (d) Progress made on the use of T. vogelii in control of insect pests in Africa is encouraging. The use of crude extracts can control insects under the threshold level, protect other arthropods, and result in high crop yield. Such work demonstrates the effectiveness of a phytocomplex for sustainable pest control in contrast to some negative effects with the use of synthetic insecticides.
The progress made in T. vogelii calls for action to explore the rich genetic resources of Tephrosia. (a) Bioactive compounds including rotenone, deguelin, rotenolone, and tephrosin in different organs of each species should be analyzed. Based on the results, species with high levels of active compounds and insecticidal activities should be further screened for individual rotenoid contents among variants or chemotypes. (b) Chemotypes or variants with high contents of the compounds should be propagated vegetatively though in vitro shoot culture to maintain their genetic identity and fidelity. (c) These chemotypes or variants should be used as parents for hybridization to produce segregated populations for selecting progenies with even higher bioactive compounds. The developed breeding lines should be evaluated in different regions for consistence in performance, and new cultivars with high levels of a compound or a group of compounds should be released. (d) Crude extracts should be extracted from the developed cultivars, and they should be evaluated and used for control of insect pests. (e) Attention should be given to deguelin, it should be evaluated for insect control and effects on nontarget organisms and the environment. It is anticipated that deguelin could be an important compound for management of insect pests in the near future.