Insects: an underrepresented resource for the discovery of biologically active natural products.

Nature has been the source of life-changing and -saving medications for centuries. Aspirin, penicillin and morphine are prime examples of Nature׳s gifts to medicine. These discoveries catalyzed the field of natural product drug discovery which has mostly focused on plants. However, insects have more than twice the number of species and entomotherapy has been in practice for as long as and often in conjunction with medicinal plants and is an important alternative to modern medicine in many parts of the world. Herein, an overview of current traditional medicinal applications of insects and characterization of isolated biologically active molecules starting from approximately 2010 is presented. Insect natural products reviewed were isolated from ants, bees, wasps, beetles, cockroaches, termites, flies, true bugs, moths and more. Biological activities of these natural products from insects include antimicrobial, antifungal, antiviral, anticancer, antioxidant, anti-inflammatory and immunomodulatory effects.

Introduction

The chemical and structural diversity of natural products are unparalleled by any synthetic based library and have inspired a wealth of life-changing and -saving medicines for centuries. In 1803, the remarkable pain-relieving natural product, morphine, was isolated from the Papaver somniferum plant and in the 1870׳s served as the template for codeine. Two decades later, salicin, the medicinally active natural product from the willow bark, Salix alba, was isolated and characterized by Henri Leroux2, 3. From this acetylsalicylic acid, an anti-inflammatory agent known commercially as aspirin was synthesized by the Bayer scientist Felix Hoffman. Aspirin has been a commercial success for the Bayer Company since its market debut in 1899. Thirty years later penicillin was isolated from the fungus Penicillium notatum by Alexander Fleming and proven to be a groundbreaking antibiotic by Howard Florey and Ernest Chain2, 4, 5. This discovery revolutionized medicine, earning Fleming, Chain and Florey the 1945 Nobel Prize in physiology or medicine for their efforts2, 5. Then in 1971, the most effective malarial treatment to date was discovered while scientists were reviewing ancient tomes on traditional Chinese medicine. It was within these texts that artemisinin was identified as a promising drug discovery lead. The head scientist, Youyou Tu, earned the Lasker prize in 2011 and the 2015 Nobel Prize in physiology and medicine for this impressive discovery4, 5.

The aforementioned examples and the fact that 33% of drugs approved from 1981--2010 and a staggering 68% of all approved antibacterial drugs are natural products or derivatives thereof prove that nature is an important source of new target molecules6, 7. However, the focus of natural product medicine has been overly focused on terrestrial plants, various algae and fungi. An underrepresented source of inspiration, entomotherapy, has been in practice for as long as and often in conjunction with medicinal plants. Entomotherapy is the use of insects as medicine and is an important alternative to modern therapy in many parts of the world including India, Mexico, Korea, China, Spain, Brazil, Argentina, Ecuador and various African countries today8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. Honey, a sweet bee byproduct, is valued as an antioxidant and antimicrobial agent, suitable for the battle against heart disease and skin disorders23, 24, 25, 26, 27. Propolis, a gap filling “glue” used in the construction of the beehive, has been evaluated extensively for its potential antioxidant, antimicrobial, anti-inflammatory, cardioprotective, immunomodulatory and antiangiogenic properties28, 29, 30. Venom, especially from bees, has been studied as an alternative medicine for inflammation and cancer therapy31, 32, 33, 34. Success has also been achieved in identifying biologically active isolates from insect bodies, excretions and secretions. Antimicrobial peptides, an innate component of insect immunity within the hemolymph (blood), have shown activity against fungi, parasites, viruses and most importantly antibiotic resistant bacteria35, 36, 37. Insect toxin isolates pederin and cantharidin from defense secretions of the rove and blister beetles, respectively, have peaked scientific interest as potential anticancer therapies38, 39.

Insects as natural product resources have also been featured in reviews published from 2010–201514, 38, 40, 41, 42, 43, 44. However, the scope of the aforementioned articles often includes other arthropods, covers a limited selection of insects, focuses heavily on insect byproducts and incorporates uses outside of pharmaceutical applications. This review focuses on the continued cultural use of insects as medicine worldwide, validation of their medicinal properties and identification, characterization and synthesis of insect natural products and derivatives.

Like plants and other invertebrates, insects are hosts for many types of microbial endophytes that include bacteria and fungi. These endophytic microorganisms may be involved or working together with their insect hosts in the production of a natural product reported. The potential involvement of endophytes should be taken into account when we isolate the natural products from insects.

Insects belong to the kingdom Anamalia, phylum Arthropoda, and class Insecta. Members of this class are further divided into 29 orders though 81% belong to one of the following four orders: Coleoptera, Diptera, Hymenoptera and Lepidoptera46, 47. This review article is split into sections based on the different orders and occasionally further subdivided by families. Sections are titled with scientific nomenclature with common names in parentheses.

Hymenoptera (bees, wasps, ants and sawflies)

Symphyta (sawflies, wood wasps, and horntails)

The family Symphyta is the smallest of the hymenoptera representing only 7% (8000--10,000 species) of the order. Like ants, they feed on leaves or burrow into wood earning them the position as pests by most foresters. In 2013, researchers out of China and Australia found medicinal value in these pests. Two small molecules with strong antimicrobial activity (Fig. 1) were isolated from the methanolic extract of Australian sawfly larvae. The novel macrocarpal (1) and known grandinol (2) were evaluated against Bacillus subtilis (B. subtilis) and exhibited inhibition zones of 8.0 and 7.0 mm, respectively.

Figure 1

Structures of two compounds with antimicrobial activity isolated from Australian sawfly larvae.

Additionally, the hemolymph of sawfly larvae has been found to be rich in phenolic compounds, such as novel flavonol oligoglycosides, flavonoid glycosides and naphthodianthrones, all with known potential health benefits that were not evaluated in the studies50, 51.

Formicidae (ants)

Ants are one of the most familiar members of the hymenoptera order. This is a social family with an impressive life span, the record being 29 years. In 2005, the number of valid described species was approximately 12,000. Ants and ant byproducts are widely used in traditional folk medicine across the globe. In southern India, mud from the interior portion of an anthill is applied topically for the treatment of scabies by the Paniyan tribe. In northern India, scabies, wounds and boils are treated through the topical application of a paste made from the crushed black ants, Bothroponera rufipes. Additionally, ground B. rufipes mixed with water is gurgled to relieve toothaches and daily consumption of 1–2 whole ants reportedly reduces blood pressure. Weaver ants (Oecophylla smaragdina) have been used in the treatment of severe cough, cold and flu in Myanmar, Africa, Australia and India. In Thai culture, they are used for detoxification of blood, arresting hemorrhage during miscarriages, restoration of uterus and removal of any aftermath from the uterine canal after childbirth, stimulating pulse and heartbeat, and dizziness. The leaf cutting ant, genus Atta, is used for the treatment of sore throat, stomachache, chest palpitations and heart disease in Latin America. Dinoponera are Neotropical ants used in the treatment of asthma in Latin America and earaches, rheumatism, and back pain in northeastern Brazil52, 53. In 2012, investigation into Dinoponera venom revealed antinociceptive activity supporting its use in pain treatment. Furthermore, it showed neuroprotective effects when administered as an intraperitoneal treatment as measured by a pentylenetetrazole induced seizure model in mice. This is an important proof of concept step in the search for new treatments for epilepsy.

Red ants of the ChangBai mountain (Tetramorium spp.), utilized in traditional Chinese medicine, were found to contain three novel coumarin compounds and two known amide alkaloids (3–7, Fig. 2). Each compound was evaluated for antibacterial activity against Gram-positive B. subtilis and Gram-negative Escherichia coli. Compound 6, known alkaloid N-(2-hydroxyethyl)-benzamide, showed strong activity against B. subtilis comparable to the positive control, penicillin G. The novel coumarins, compounds 3--5, were mildly active and compound 7, known alkaloid N-phenyl-3,4,5-trihydroxybenzamide, was inactive. None of the compounds exhibited activity against Gram-negative bacteria E. coli. Solenopsin A (8), are well-known antiangiogenic active alkaloids isolated from Solenopsis invicta and Solenopsis germinate fire ants. The key mechanism of action reportedly involves selective inhibition of protein kinase B (AKT), a molecular target of growing interest in cancer18, 44, 56, 57. In 2012, three new alkaloids (9--11) were isolated from the alate queen ants of S. richteri, S.invicta and hybrid (S. richteri × S. invicta) and two years later an additional 36 alkaloids58, 59. However, the biological activities of these alkaloids have yet to be evaluated.

Figure 2

Structures of coumarins and alkaloids isolated from ants.

The iron ant, Tetraponera rufonigra, is used in local tribes of northeastern India to treat foot and mouth disease in cattle. One to two ants are crushed into a powder and added to fodder which is then fed to the cattle up to 3 times a day. Studies into the venom of Tetraponera ants revealed a unique class of neurotoxic bioactive compounds termed tetraponerines. Tetraponerines are tricyclic alkaloids isolated for the first time in 1987 from venom of the New Guinean pseudomyrmecine ant. These molecules, designated T1–T8 (12–15, Fig. 3), are split evenly into two groups based on the grouping of cyclic rings, 5-6-5 or 6-6-560. In each group, further division exists based on the stereochemistry and length of the alkyl chain (pentyl or propyl). In 2001, additional investigation into Tetraponera from India, China and Africa revealed the presence and distribution of the tetraponerines are not equivalent across the species. An Indian ant (T. allaborans) possessed T2, T4 and T8, while the Chinese ant (T. binghami) contained T5, T6, T7 and T8. The remaining tested species, T. rufonigra (India), T. penzigi (Africa), T. clypeata (Africa) and T. sp. cf. emeryi (Africa) showed no evidence of tetraponerines. The natural application of tetraponerines as neurotoxic agents led to interest in their cytotoxic profiles. An evaluation of the anti-proliferative activity of T5–T8 against 4 different carcinoma cell lines highlighted the importance of C5 configuration on potency. Furthermore, T7 exhibited an impressive cytotoxic profile on the breast carcinoma cell line MCF-7. A structure-activity relationship (SAR) study was conducted to analyze the effect of varying alkyl chain lengths and 6-6-6 tricyclic rings on cytotoxicity against human colorectal (HT29) cancer cells. Results revealed the alkyl chain to have the greatest effect on cytotoxicity with a decrease in IC50 with increasing chain length, whereas the nature of the ring structure and stereochemistry had no effect.

Figure 3

Structures of tetraponerines isolated from venom of the New Guinean pseudomyrmecine ant.

Chinese black ants, Polyrhachis dives, are traditionally used to treat numerous ailments, including rheumatoid and osteoarthritis, inflammatory diseases, and central nervous system (CNS) associated disorders. Recently, several dopamine derivatives (Fig. 4) were isolated from an ethanol extract: (±)-polyrhadopamine A (16), (±)-polyrhadopamine B (17), polyrhadopamines C–E (18–20), trolline (21) and compounds 22–24. The compounds were evaluated for inhibition of Rho-associated protein kinase ROCK isoforms, ROCK1 and ROCK2, known targets for the potenital treatment of cardiovascular, neurological, oncological and renal diseases. Compounds (±)-polyrhadopamine A (16), (+)-polyrhadopamine B (17a) and trolline (21) inhibited both ROCK isoforms. Compounds 22–24 inhibited only the ROCK2 isoform. Potential for the treatment of CNS disorders was further evaluated utilizing a neural stem cell (NSC) proliferation assay. Only (–)-polyrhadopamine B (17b) exhibited a potent dose-dependent promotion of NSC proliferation. Additionally, the immunosuppressive and anti-inflammatory effects of the compounds were studied to verify activity against rheumatoid and osteoarthritis. One dopamine derivative, polyrhadopamine C (18), was found to have anti-proliferative effects against T-lymphocytes isolated from Kunming mice comparable to the control, cyclosporine A. Likewise, this same compound had comparable inhibitory effects on tumor necrosis factor (TNF)-α production in lipopolysaccharides (LPS)-induced RAW264.7cells as the control indomethacin. The combined immunosuppressive and anti-inflammatory activity is strong evidence that polyrhadopamine C (18) could be effective against rheumatoid arthritis. Last year, an additional thirteen nitrogen containing compounds were isolated from P. dives ants and evaluated for their anti-inflammatory, immunosuppressive, and renoprotective activities.

Figure 4

Structures of dopamine derivatives isolated from Chinese black ants.

The compounds 5-(3-indolylmethyl)-nicotinsaureamide (25, Fig. 5) and β-carboline-3-carboxamide (26) suppressed the proliferation of T-cells, key cellular targets for rheumatoid arthritis therapy64, 65. Additionally, compounds 25, 27, and 3-hydroxypyridine significantly reduced collagen IV, fibronectin, and interleukin-6 (IL-6) overproduction supporting the use of the ants as a tonic for kidney problems. Furthermore, compounds 25, 26, 29, and 30 led to moderate inhibition of TNF-α production, compound 29 inhibited cyclooxygenase-1, and compound 28 significantly inhibited janus kinase (JAK)-3 kinase all of which indicate anti-inflammatory activity.

Figure 5

Structures of additional compounds isolated from Chinese black ants.

Vespidae (paper, potter, and hover wasps; hornets; yellow jackets)

Wasps are another familiar group of the hymenoptera known for their wondrous paper and clay nests and the act of stinging as a defense mechanism. The Vespidae have approximately 5,000 species classified into six subfamilies: Vespinae, Polistinae, Stenogastrinae, Eumeninae, Masarinae and Euparagiinae. In Latin America and Korea, the Apoica, Brachygastra and Synoeca genera of the Polistinae (paper wasps) are valued traditional therapies for asthma and cough12, 17. Additionally, Apoica and Protonectarina are thought to cure mumps, hemorrhage, and menstruation problems. Polistes, the most diverse genus of the Polistinae, are used to treat colds and severe cough in northeastern India and parts of Latin America11, 12. Also, the buffer extract of Polistes mandarinus was cytotoxic against cervix cancer cell line, HeLa, at an equivalent level to the positive control drug mitomycin C. As eusocial insects wasps are known to produce antimicrobial substances to provide a collective immunity against microorganisms. Malaysian female wasp venom from various genera of the Stenogastrinae (hover wasps) subfamily were evaluated for antimicrobial activity. The venom was extracted with methanol and tested against Gram-positive bacteria, B. subtilis, Gram-negative bacteria, E. coli, and yeast, S. cerevisiae. Each extract produced inhibition zones approximately half the size of the positive controls, penicillin G and kanamycin, indicative of mild activity against the bacterial and yeast strains tested. The venom of the Brazilian social wasp, Synoeca cyanea (Polistinae), was found to inhibit bacterial growth of Gram-positive Enterococus faecalis and Gram-negative E. coli bacteria by 93% at 100 µg. The bite or sting of the Vespa orientalis (Vespinae) is used to treat coughs, colds and stomach pains in the Galo and Nyishi tribes of northeastern India. Crude venom of the V. orientalis wasp was found to have strong activity against B. subtilis though not as potent as the positive control tetracylcin. The venom was also shown to have mild activity against Staphylococcus aureus, E. coli and Klesiella pneumonia. In addition to antibacterial activity venom is known to possess neurotoxic effects and in many cases is pharmacologically active against tumor cells56, 70. For example polybioside (31, Fig. 6), an isolate of Polybia paulista (Polistinae) venom, was found to be neuroactive. Its neuroactive effect was evidenced through the expression of c-Fos protein, a known biochemical marker for stimulated neurons, in various areas of a rat brain after intracerebroventricular (ICV) application.

Figure 6

Structures of polybiosides isolated from the venom of Polybia paulista.

Apidae and Halictidae (bees)

Bees are the most popular insect of the hymenoptera albeit for the honeybees (Apis) and bumblebees (Bombus) which ironically represent a tiny fraction of the 20,000 species. Likewise, the Apis, numbering only 170 species, has been used extensively in medicine. In 2013, an Asiatic honeybee (Apis cerana) chymotrypsin inhibitor (AcCI) gene was identified to encode an 85 amino acid protein that includes a 20 amino acid signal sequence. The recombinant 65-amino acid mature peptide inhibited chymotrypsin with an IC50 of 24.71 nmol/L and showed anti-elastolytic activity via the inhibition of human elastase (IC50 = 38.50 nmol/L) and porcine elastase (IC50 = 70.21 nmol/L). This unique mode of action provides a novel approach for anti-inflammatory therapies.

The venom of A. cerana was evaluated for its anti-diabetic and anti-dandruff activities. Researchers assessed its antidiabetic properties by measuring blood sugar, cholesterol and triglyceride content in test subjects before and 30 min after bee stinging. Prior to being stung the blood sugar levels ranged from 91–120 mg/dL, cholesterol was 189–235 mg/dL and triglycerides were 110–184 mg/dL. After bee stinging values fell to 80–94, 178–199 and 84–138 mg/dL for blood sugar, cholesterol and triglyceride levels, respectively. Anti-dandruff activity was investigated by testing the inhibitory effect of the venom on Malassezia furfur a known dandruff causing organism. Ketoconazole, a common active ingredient in anti-dandruff shampoos, was used as a positive control. Interestingly, at a concentration of 5 mg/mL the venom was more than twice as effective as ketoconazole. Last year, Indian Apis dorsata bee venom (ADBV) was found to possess anti-inflammatory, anti-nociceptive, and anti-arthritic properties. These properties were evinced through the venom׳s ability to reduce joint inflammation, paw edema and arthritic index along with the reduction of pain perception parameters such as dorsal flexion, motility, and stair climb score. Furthermore, it significantly decreased the major pro-inflammatory mediators like TNF-α and IL-6 in rats induced with arthritis. Crude bee venom extracts from A. cerana, A. dorsata and Apis florea were tested against six Gram-negative bacteria and fungal strains to investigate antimicrobial properties with ampicillin as a control. The venoms were as effective as ampicillin against E. coli, Salmonella typhimurium and Xanthomonas subtilis and were more active than ampicillin against Klebsiella pneumonia.

Portuguese propolis from the beehive of Apis mellifera was recently assessed for total phenolic and flavonoid content, anti-inflammatory and antimicrobial activity. The propolis samples were collected from three regions in Portugal including: Bragança, Coimbra and Beja. The Bragança propolis extract had the strongest antioxidant profile with a total phenolic and flavonoid content of 277.17 and 142.32 mg/g, respectively. Anti-inflammatory activity was assessed by inhibition of hyaluronidase, an enzyme that degrades hyaluronic acid leading to bone loss, pain and inflammation. All extracts inhibited hyaluronidase activity by 50%–75% at a concentration of 25 mg/mL and not surprisingly, the Bragança extract was the most effective. Antimicrobial activity was evaluated against S. aureus (Gram-positive), P. aeruginosa (Gram-negative), E. coli (Gram-negative) and C. albicans (yeast). All extracts showed activity against each strain with the weakest activity being against C. albicans and the strongest activity against S. aureus. Propolis generated by the Brazilian stingless bee, Melipona orbignyi, was recently evaluated for its chemical composition and antimicrobial, antioxidant and cytotoxic activities. The 80% ethanol extract was determined to contain aromatic acids, phenolic compounds, alcohols, terpenes and sugars in its chemical profile. Antioxidant activity was confirmed by its ability to scavenge free radicals, inhibit hemolysis and lipid peroxidation in human erythrocytes incubated with an oxidizing agent. The extract was active against S. aureus bacterium (Gram-positive) and the fungus C. albicans but not the E. coli (Gram-negative) bacterium. The cytotoxic activity of M. orbignyi ethanol extract was evaluated against the human K562 erythroleukemia cell line which showed a decrease in viability in contact with the extract which was attributed to cell necrosis. A phytochemical screening of the ethanolic extract of Brazilian red propolis indicated the presence of phlobaphene tannins, catechins, chalcones, aurones, flavonones, flavonols, xanthones, pentacyclic triterpenoids and guttiferones. Given the strong phenolic content, it is not surprising the extract exhibited strong antioxidant activity that was in fact far superior to the control Trolox, as measured by 2,2′-diphenyl-1-picrylhydrazyl (DPPH) sequestering. Additionally, the extract exhibited intense cytotoxicity against tumor cell lines SF-295 (human glioblastoma), OVCAR-8 (ovary) and HCT-116 (colon). Geopropolis from the Brazilian stingless bee, Scaptotrigona postica, was evaluated for chemical composition and effectiveness against antiherpes simplex virus-1 (HSV-1). The chemical composition was found to consist mostly of pyrrolizidine alkaloids and C-glycosyl flavones. Treatment with the extract reduced the presence of HSV-1 by 98% which was attributed to inhibition of viral replication and virus entry into cells. Propolis from the Brazilian stingless bee, Tetragonisca fiebrigi, was evaluated for antimicrobial, antioxidant and anti-inflammatory activity. The ethanol extract was analyzed for chemical composition utilizing gas-chromatography mass spectrometry (GC--MS). The major components of the extract, determined by GC--MS, were phenolic compounds, aromatic acids, alcohols, terpenes, and sugars. Antimicrobial activity was evaluated against various Gram-positive and Gram-negative bacteria and fungi. The extract was active against all strains tested but was most effective against Gram-positive bacteria. Antioxidant activity was also noted through scavenging free radicals, inhibiting hemolysis and lipid peroxidation in human erythrocytes incubated with an oxidizing agent. Furthermore, anti-inflammatory potential of the extract was confirmed via inhibition of the hyaluronidase enzyme and cytotoxic activity was concentration-dependent against K562 (leukemia) cells, with a predominance of death by necrosis.

The Indian propolis from the stingless bee, Trigona sp., was evaluated for its chemical composition and antimicrobial activity. The chemical composition of the ethanol extract contained 24 compounds belonging to the following classes: alkanes, thiophilic acids, aromatic acids, aliphatic acids, sugars, esters, and terpines. The antimicrobial activity was tested against 6 standard bacterial strains (E. coli, S. typhimurium, S. aureus, etc.) several multi drug resistant strains (K. pneumoniae and A. baumannii, etc.) and a standard yeast strain (C. glabrata). The extract proved potent against all strains tested with minimum inhibitory concentration (MIC) values ranging from 1.21–9.75 µg/mL. Although in general, MICs for Gram-positive bacteria were lower than that for Gram-negative bacteria. Methanol extracts of Nigerian insect propolis were recently evaluated for activity against the malarial parasite Plasmodium berghei. Mice were infected with P. berghei and evaluated for parasitaemia levels over several days. Survivorship of mice treated with the positive control, chloroquine, was not significantly higher than those treated with the extract. Thus, the extract presents a promising lead for new anti-malarial drugs.

The honey from A. mellifera is used in traditional Mexican medicine for the treatment of cough, bronchitis, asthma, rheumatism and diarrhea. A daily spoonful of honey is believed to cure coughs, fevers and stomach pains in the Galo and Nyishi tribes of northeastern India. Additionally, the honeycomb is rubbed on the skin to treat irritation. In northeastern India honeybee (A. cerana, A. florae, and A. mellifera) honey is thought to cure coughs, fevers, stomach pains and the comb, skin irritations. In parts of India honey from the honeybee, Apis indica, is also used to treat colds, headache, asthma and sore throat, indigestion and urinary disorders16, 80. Honey is also an important staple in Argentinian homeopathy. It is taken orally to treat dyspepsia, constipation, colds, influenza and asthma. It is also applied topically to relieve ocular illness, insect bites, bruises, arthritis and muscular. Likewise, in Mexican traditional medicine honey is used as a topical anti-septic agent to treat cutaneous injuries and infections and as an anti-inflammatory agent against muscle fatigue and sprains.

Honey made from the Brazilian stingless bee, Melipona marginata, was evaluated for potential anti-inflammatory activity of honey extract on skin inflammation. High performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC--UV--ESI-MS) identified 11 phenolic compounds such as kaempferol and caffeic acid. Topical treatment using M. marginata honey extract confirmed its anti-inflammatory activity by reducing edema (54%), leucocyte migration, and production of ROS (55%) in the skin of mice after 12-O-tetradecanoylphorbol-13-acetate (TPA) application. Honey made from the Brazilian bee Melipona subnitida was evaluated for its chemical profile and antioxidant activity. The chemical profile consisted of gallic acid, vanillic acid, 3, 4-dihydroxybenzoic acid, cinnamic acid, isorhamnetin, naringenin, quercetin, trans-trans and cis-trans abscisic acid. Three different methods were employed to verify antioxidant activity of pure honey and various extracts including inhibition of cooxidation in β-carotene/linoleic acid mixturesand the scavenging of DPPH and 2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The pure honey and all extracts prevented the bleaching of β-carotene in the carotene/linoleic acid mixtures and scavenged free radicals (DPPH and ABTS) with varying degrees of efficiency. Six honeys from the Republic of Mauritius were analyzed for antimicrobial and antioxidant potential. Antimicrobial properties were evaluated against five strains and antioxidant capacity was determined using eight assays: trolox equivalent antioxidant capacity (TEAC), ferric reducing antioxidant power (FRAP), iron (II) chelating activity, hydroxyl radical (OH·) scavenging, DPPH free radical scavenging, hypochlorous acid (HOCl), nitric oxide (NO·) and ABTS diammonium salt radical (ABTS·+) scavenging. All honeys had similar antioxidant profiles and were found to have higher antibacterial activity than antifungal, supporting its reported effects in wound healing. Four Malaysian honey samples, acacia (A. mellifera), pineapple (A. mellifera), borneo (A. cerana) and tualang (A. dorsata) were evaluated for chemical composition and antioxidant activities. Total phenolic, flavonoid and protein content were measured. Tualang had the highest phenolic, flavonoid and protein content and strongest antioxidant activity as measured by the FRAP assay and DPPH radical scavenging activity. The seasonal effect on antimicrobial activity of honey from the Brazilian stingless bee, Melipona compressipes manaosensis, was recently evaluated. Honey harvested in November (rainy season) was only effective against E. coli and S. sonnei. However, honey harvested in March (dry season) was effective against S. aureus, E. coli, P. vulgaris, S. sonnei, and Klebsiella sp. Wild honey produced by four species of bees, A. cerana, A. andreniformis, A. koschevnikovi and A. nuluensis, in Malaysia were evaluated for antioxidant and acetylcholinesterase inhibitor potential. Antioxidant properties were assessed by radical scavenging activity as determined from DDPH assay, ferric reducing/antioxidant power (FRAP) assay, and ABTS decolorization assay. The 80% methanol extract of the wild A. cerana honey performed the best as measured by DPPH assay (84%), FRAP assay (37 µmol/L Fe (II)/1 g dry sample) and ABTS decolorization assay (8 mg AEAC/1 g dry sample).

Coleoptera (beetles)

The coleopterans are the largest insect order with 350,000-386,000 species in 176 families occupying the land, freshwater and sea46, 88, 89. Approximately 62% of beetles belong to the following six mega families, each of which have at least 20,000 members: Staphylinidae, Carabidae, Cerambycidae Curculionidae, Chysomelidae, and Scarabaeidae. Although diverse and abundant, with the exception of the Scarabaeidae, mention of medicinal use for these mega families in the literature within the last 5 years is scarce. For example, the Chrysomelidae have approximately 38,000 species and the Curculionidae have nearly 60,000 species, yet the only mention of medicinal use was found in Latin American folk medicine46, 88. Adult Chrysomelidae, commonly known as leaf beetles, are taken for epilepsy and their caterpillars are further utilized for earache, stroke, swelling, wounds, seborrheic dermatitis, inflammation and thrombosis treatment. Snout beetles (Curculionidae) are leveraged for the relief of fever, headache and boils.

The Tenebrionidae, though not a mega family, have an extensive collection of 19,000 species. One such species is Blaps japanensis, a Chinese medicinal insect, which has been used for the treatment of fever, cough, rheumatism, cancer, and inflammatory disorders. As such, four new dimeric compounds, blapsols A–D (32--35, Fig. 7) and five known dopamine dimers (24, 36--39) isolated from Blaps japanensis were evaluated for their activity against cyclooxygenase (COX) enzymes: COX-1 and COX-290. Cyclooxygenases are responsible for catalyzing the conversion of arachidonic acid to prostaglandins, important mediators of pain, fever, and inflammation. The COX-1 isoform is expressed constitutively in tissues and is responsible for maintaining a homeostatic concentration of prostaglandins. The second, COX-2, is induced by stimuli including mitogens, hormones, cytokines, and growth factors. Selective inhibition of COX-2 is therefore desirable as disruption of COX-1 activity leads to adverse side effects like ulcers of the upper gastrointestinal tract.

Figure 7

Structures of dopamine dimers isolated from Blaps japanensis, a Chinese medicinal insect.

All compounds inhibited COX-2 and only compound 32 inhibited COX-1, thus supporting the use of these insects to treat pathogenic inflammation. The genotoxic and cytotoxic effects of darkling beetles, Ulomoides dermestoides (Tenebrionidae), used in traditional Argentinian medicine to treat asthma, Parkinson׳s disease, diabetes, arthritis, HIV and cancer, were recently evaluated. The dichloromethane extract was cytotoxic against human lung carcinoma epithelial cells (A549) and normal mononuclear cells. Additionally, the extract induced significant DNA damage in both tumor and healthy cells. However, if targeted directly to tumor cells, there is still potential for anticancer treatment despite the deleterious side effects.

The Scarabaeidae make up about 8% of all coleopterans and have been mentioned frequently in the literature for their medicinal value. The large chafer beetle Holotrichia parallela (Scarabaeidae), traditionally used as drug in China and East Asia for the treatment of gout, tetanus, erysipelas and superficial infections, was recently evaluated for antioxidant activity. Both water and ethanolic extracts were subjected to various in vitro assays including linoleic acid peroxidation inhibition, metal-chelating activity and DPPH radical scavenging activity. The ethanol extract had potent metal-chelating activity and inhibition of lipid peroxidation which was attributed to the presence of Catechin. The water extract, rich in proteins, proved to be an excellent antioxidant in the scavenging of DPPH radicals and metal-chelating activity. Holotrichia diomphalia (Scarabaeidae) larvae is a traditional crude drug in China for the treatment of chronic liver cirrhosis, contusion, edema, furuncle, and apoplexy. A chemical evaluation of the larval extract found four known compounds including the flavonoid tricin as well as cholesterol, palmitinic acid and eicosane. Additionally, ethanol and petroleum ether extracts of H. diomphalia larvae showed antifungal activities in vitro against Pyricularia oryzae by morphologically deforming hyphal growth from conidia. Minimum morphological deformation concentration (MMDC) values from ethanol and petroleum ether extracts were 7.8 and 125 µg/mL, respectively.

The extracts of rhinoceros beetle larvae, Allomyrina dichotoma (Scarabaeidae), were found to play a vital role in reactive oxygen species (ROS) scavenging in response to oxidative stress. Antioxidant properties of various extracts, including methanol, water, chloroform, ethyl acetate and hexane, were measured through DPPH radical scavenging, superoxide anion radical scavenging and singlet oxygen quenching assays. The methanol extract had the most effective DPPH radical scavenging activity and was 1.7 times more efficient than the standard, ascorbic acid, at singlet oxygen quenching. Also, the extracts protected the biological systems in E. coli and lactate dehydrogenase against detrimental effects of 1O2 of type II photosensitization in vitro. Last year, A. dichotoma larvae were shown to have hepatoprotective and anticancer activities. Oral administration of powdered larvae lessened diethylnitrosamine (DEN)-induced hepatotoxicity in mice and an ethyl acetate extract showed cytotoxicity against various cancer cells through induction of apoptosis, necrosis, and autophagy.

In 2012, the antioxidant activity of Protaetia brevitarsis (Scarabaeidae) at various growth stages, larvae, pupae and imago, was evaluated. The larvae and imago extracts had equivalent singlet oxygen quenching ability to the control, ascorbic acid, as well as comparable DPPH and ABTS radical scavenging activity. Two years later the larvae was shown to also have liver protective and antineoplastic activity. Oral administration of the powdered larvae reduced signs of acute and chronic liver injuries in DEN-induced hepatotoxic mice. Also, a hexane extract proved highly effective against a variety of cancer cell lines with minimal effect on healthy cells. The mechanism of action was determined to be apoptosis via the perturbation of mitochondrial membrane potential.

In northeastern India, the dung beetle, Catharsius sp. (Scarabaeidae), is made into a paste and taken orally for the relief of diarrhea. Catharsius molossus is also a valued Chinese medicinal insect with multiple reported uses including arresting convulsions, removing blood stasis, relaxing the bowels, and counteracting toxins. Recently, five small molecules including three novel N-acetyldopamine dimers, termed molossusamide A--C (40--42, Fig. 8), and two known compounds (38 and 43) were isolated and evaluated for biological activities using cytotoxicity, anti-influenza, enterovirus 71 (EV71) inhibition and anti-inflammation (COX--1/2). None of the compounds exhibited cytotoxicity against cancer cells or anti-viral (influenza and EV71) effects. Compound 38 inhibited the COX isozymes and was several times more effective against COX-2.

Figure 8

Structures of actyldopamine dimers isolated from Catharsius molossus and antioxidant lipids from Bruchidius dorsalis.

New antioxidant lipids, designated dorsamin-A763 (44), -A737 (45), -A765 (46), -A739 (47), and -A767 (48) were isolated via an activity-directed fractionation of the chloroform extract of Bruchidius dorsalis (Bruchidae) larvae. Compounds 44--48 exhibited ABTS radical scavenging activity comparable with or stronger than that of the control Trolox. The lucanid beetle, Serrognathus platymelus castanicolor (Lucanidae), was evaluated for antioxidant properties at three life stages: larvae, pupae and adult. The pupal methanol extract methanol extracts (PME) had the highest DPPH and ABTS radical scavenging activity and a 1O2 quenching ability comparable to the control ascorbic acid.

The Meloidae are a smaller group within the Coleopterans numbering only 2500 species. But thanks to their defensive secretions, the meloidae have a long history of medicinal applications. Therapeutic uses date back to 1264 in which they were reportedly used for wart removal in China and cancer treatment during the middle ages in Europe. Presently in parts of Spain, oil from the red-striped oil beetle, Berberomeloe majalis (Meloidae), is applied externally for wound and wart treatment and as an analgesic. Palembus dermestoides (Meloidae) is used for the treatment of sexual impotence, ophthalmological problems, rheumatism, weakness in folk Latin American medicine. Additionally, Pseudomeloe andensis is used for the treatment of warts in folk Latin American medicine. In current traditional Korean medicine the Meloidae are used externally for boils, fungal infection and facial paralysis due to stroke. Internally they are levered for gonorrhea, lymphangitis, neoplasm, rabies, syphilis and edema. In Mexico they are macerated with alcohol and rubbed on the head to combat baldness.

Scientific explorations into these beetles have revealed their bioactivity is attributable to terpene-related compounds. The monoterpene, cantharidin (CTD, 49, Fig. 9), was the first to be isolated and characterized (c.1914) from blister beetles colloquially known as the ‘Spanish fly’39, 46, 88. The demethylated CTD analogue, (R)-(+)-palasonin (50), was discovered in the dried bodies of the South African Hycleus oculatus, H. tinctus, and H. lunata beetles and is the only CTD analogue found in plant species as well39, 103, 104. Imide analogues of CTD and palasonin, cantharimide (51) and palasonimide (52) respectively, in which the oxygen atom in the anhydride core of the molecules is replaced by a nitrogen atom were also found in the H. lunata beetle. Within the last decade cantharimide, 5-hydroxy cantharimide (53) and norcantharidin (NCTD, 54) were isolated from the native Korean and Chinese blister beetle, Mylabris phalerate PALLAS.

Figure 9

Structures of monoterpene cantharidin (CTD), norcantharidin (NCTD), and their derivatives isolated from African, Korean, and Chinese Beetles.

The earliest isolated compound, CTD, is the highest concentrated terpene in blister beetles and has been explored extensively to further understand its pharmacological and toxicological properties against cancer. Cantharidin is often reported as inducing apoptosis and cell cycle arrest through a variety of underlying pathways39, 105. The primary mechanism is the inhibition of protein phosphatase 1 (PP1) and protein phosphatase 2 A (PP2A). These two phosphatases are known to be involved in various cellular processes including DNA damage, cell cycle arrest, and apoptosis. Researchers from North-Eastern Hill University reported CTD treatment caused plasma membrane disintegration, the appearance of membrane vacuoles and blebbing on murine Ehrlich ascites carcinoma cells which are hallmarks of apoptosis. Further evaluation revealed CTD acted via an intrinsic pathway involving disruption of the mitochondrial membrane, rerelease of cytochrome c and downstream activation of caspases directly involved in apoptosis. Similar mechanisms for apoptosis were reported for in vitro tests on human skin and gastric cancer cells in addition to cell cycle arrest at the G2/M phase109, 110. Cantharidin has been reported to effect human lung cancer cells via the intrinsic pathway by increasing reactive oxygen species (ROS) and calcium concentration leading to disruption of the mitochondrial membrane or inhibition of the phosphatidylinositol 3-kinase/Akt signaling pathway111, 112. This leads to the selective attenuation of one of the gelatinases, matrix metalloproteinase (MMP)-2, which can degrade components of extracellular matrix (ECM) and inhibits migration and invasion of human lung cancer cells. Also an extrinsic pathway whereby the death receptor is activated by an extracellular response followed by caspase 8 and caspase 3 activation leading to apoptosis has been postulated. Recently, it was discovered CTD also suppresses human umbilical vascular endothelial cell proliferation, migration, and tube formation, abrogates vascular endothelial growth factor (VEGF)-induced activation of signal transducer and activator of transcription 3 (STAT3) and suppressed the phosphorylation of JAK1, AKT and Extracellular signal–regulated kinase (ERK). All of which are markers of angiogenesis which is required for the large scale growth of tumor cells. Suppression of angiogenesis further supports the promise of cantharidin as a potential anticancer drug.

Cantharidin has also been found to induce a special type of cell suicide in erythrocytes-termed eryptosis. The erythrocytes of healthy subjects exposed to cantharidin were found to exhibit cell membrane scrambling and cell shrinkage, both hallmarks of eryptosis. Eryptosis eliminates infected or defective erythrocytes thus counteracting parasitemia in malaria and contributes to the pathophysiology of several clinical disorders including metabolic syndrome and diabetes, malignancy, cardiac and renal insufficiency, hemolytic uremic syndrome, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson׳s disease. Cantharidin was recently evaluated for its potential immunomodulatory effects through regulation of dendritic cells (DC). Cantharidin modulates the differentiation and maturation of DCs with alteration of the expression of surface markers and cytokines secretion, causing deviation of standard DC differentiation toward a state of less maturation. Thus, CTD could treat or prevent disorders involving unwanted immune responses, such as allergies, transplant rejection, or autoimmune diseases.

Though CTD has a variety of therapeutic uses, it has a number of toxic side effects, such as kidney and gastrointestinal damage and disruption of reproductive functions107, 117, 118. Its closely related naturally occurring analogue, norcantharidin (NCTD), has similar therapeutic uses but with lower toxicity risk117, 118. As a chemo-preventive agent NCTD has inhibited tumor progression in non-small cell lung cancer (NSCLC), prostate cancer and leukemia, in vitro, through cell-cycle arrest and apoptosis119, 120, 121. Just last year NCTD was also linked to the suppression of the canonical wingless type (Wnt) signaling pathway which is known to cause a variety of human cancers including NSCLC. Additional therapeutic benefits, cited in a recent review of NTCD, include the modulation of cancer stem cell self-renewal pathways, overcoming multidrug resistance and as radiation sensitizer.

The presence of nitrogen in cantharimide and 5-substituted cantharimide improves their solubility and cytotoxicity against human hepatocellular carcinoma cell lines. Given the therapeutic potential of CTD, NCTD and cantharimide, thousands of related analogues have been designed and synthesized with the aim of improving their anticancer potency and decreasing their toxicity. In 2014, a novel CTD analogue, N-benzylcantharidinamide (55, Fig. 10), was found to suppress the metastatic potential of Hep3B hepatocellular carcinoma cells through inhibition of MMP-9 expression. Cancer cells produce proteolytic enzymes like MMPs to invade surrounding tissue such as extracellular connective tissues and the basement membrane. An additional twenty-three N-substituted NCTD analogues were synthesized and evaluated for cytotoxicity against five human cancer cell lines that same year. Lipophilicity of the derivatives was altered through the N-substituents which included alkyl, alkyloxy, terpenyl or terpenyloxy groups. The lipophilicity of a molecule is an important factor in drug development as it affects solubility and permeability through membranes. Eleven of the molecules showed significant inhibition against all five cancer cell lines tested with a general trend of increased cytotoxicity with increasing side chain length. Thus, the lipophilicity of the N-position of norcantharimide is an important parameter affecting the biological activity of the compound. In fact, two compounds N-farnesyloxy-norcantharimide (56) and N-farnesyl-norcantharimide (57) showed a 2--5-fold increase in cytotoxicity as compared to NCTD against liver carcinoma cells. Even more impressive neither had significant adverse effects on normal murine embryonic liver cells. Not surprisingly, microscopy of the treated cells showed typical morphological features of apoptosis, such as cell shrinkage, chromatin condensation and DNA fragmentation. The biological activity of norcantharimide analogues with terminal phosphate esters was evaluated against a panel of nine human cancer cell lines: human colon carcinoma cell lines (HT29 and SW480); human breast carcinoma cell line (MCF-7); human ovarian carcinoma cell line (A2780); human lung carcinoma cell line (H460); human skin carcinoma cell line (A431); human prostate carcinoma cell line (DU145); human neuroblastoma cell line (BE2-C); and human glioblastoma cell line (SJ-G2). Multiple analogues were at least equipotent with NCTD and a number displayed about a 5-fold increase in cell death with the most potent being bis-trichloroethyl (58) and diphenyl (59) analogues.

Figure 10

Structures of synthetic cantharimide and norcantharimide derivatives.

In fact, the anti-proliferative activity of these analogues generally correlated with the relative ease of phosphate ester hydrolysis. A series of synthetic NCTD analogues were evaluated against a panel of nine human cancer cell lines including colon carcinoma (HT29 and SW480), breast carcinoma (MCF-7), ovarian carcinoma (A2780), lung carcinoma (H460), skin carcinoma (A431), prostate carcinoma (DU145), neuroblastoma (BE2-C) and glioblastoma (SJ-G2). The diastereomeric analogue, Novo-6 (60, Fig. 11), displayed a high level of anticancer activity against all tumor cells but with selectivity towards colon and glioblastoma cancer cell. Incorporation of an isopropyl tail at the hydroxyl position (61) increased the selectivity towards colon and glioblastoma but still completely void of any activity against the remaining tumor cell lines as per the parent molecule. Furthermore the introduction of a terminal phosphate moiety (62) at the same position produced a different trend in cytotoxicity with strong activity on neuroblastoma cells, suggesting an alternate mode of action. In recent years, a series of 5-benzoyloxymethylnorcantharidin derivatives with various substituents at the C1 position were synthesized in an effort to find PP2B-selective inhibitors an attribute of the well-known immunosuppressant drug cyclosporine A. The lead compound NCA-01 (63) and its derivatives (64--68) evinced potent inhibition of PP2B, approximately 70% or more, with negligible inhibition of PP1 and PP2A.

Figure 11

Structures of synthetic cantharidin and norcantharidin derivatives.

In a study last year, a series sulfonamide NCTD derivatives were synthesized and evaluated for their cytotoxic effects on human lung (A549), liver (HepG2), cervical (HeLa) and colon (HCT-8) carcinoma cell lines together with a genetically normal human cell line (WI-38). One compound in particular, 69, exhibited potent cytotoxic effects on all carcinoma cell lines, whereas it was less toxic to normal cells than its parent compound, NCTD. The mechanism of action was determined to be inhibition of PP1 and microtubule formation in tumor cells and suggests that compound 69 is a promising anticancer drug candidate.

Blattodea (cockroaches and termites)

Blattodea is a smaller insect order of approximately 7600 species comprised of cockroaches (4600) and termites (3000). The giant cockroach, Rhyparobia maderae (Blaberidae), is used as an asthma treatment in Latin American folk medicine. Additionally, the cockroach Eurycotis manni (Blattidae) is prescribed for headache relief. The American cockroach, Periplaneta americana (Blattidae), is used for a variety of conditions including: heartburn, asthma, stomach ache, intestinal colic, earache, alcoholism, epilepsy, vomit, boil, hemorrhage, bronchitis, diarrhea, gonorrhea, panaris, cancer, stroke, burns and menstrual cramps. In parts of India, it is cooked in a soup to treat postpartum, uterine problems and urinary obstructions. Additionally, the whole body is consumed to treat gangrene, dyspnea, asthma and tuberculosis and applied topically for tetanus and earache relief43, 80. In China, the ethanol extract of the dried whole body of P. americana have been used as traditional Chinese medicine for the treatment of blood stasis syndrome, acne and abdominal mass for hundreds of years. Given the varied therapeutic uses for the pest, the ethanol extract of the insect was screened for bioactive compounds resulting in the discovery of four new isocoumarins periplatins A--D (70--73, Fig. 12) along with three known ones (74--76). The compounds were tested against various cancer cell lines and compounds 72--74 showed significant cytotoxic activities against human liver (HepG2) and breast cancer (MCF-7) cells. In a small scale clinical study, sixty septic patients were treated with P. americana extract which successfully improved the condition and prognosis in patients with sepsis.

Figure 12

Structures of isocoumarins periplatins isolated from Periplaneta americana.

Blatta orientalis (Blattidae) is used topically for the treatment of tetanus and ear pain in Mexican traditional medicine. Recently, the anti-asthmatic and anti-anaphylactic activities of B. orientalis mother tincture was evaluated. The results indicated nonselective anti-cholinergic and anti-histaminic activities were responsible for its anti-asthmatic activity. Furthermore, mast cell stabilization, lowering elevating IgE antibody and reducing oeosinophil led to both passive and active anaphylactic activity.

The Chinese cockroach, Eupolyphaga sinensis (Corydiidae), is a key ingredient in a number of traditional Chinese medicinal products including Shu-Mai Tang, Dahuang Zhechong pill, Shensong Yangxin capsule, Shen-Yuan-Dan, Yi Guan Jin and Yaotongning131, 132, 133, 134, 135, 136. Shu-Mai Tang is prescribed for the treatment of ischemic heart disease and was found to have a therapeutic effect against inflammation-induced myocardial fibrosis leading to the attenuation of left ventricular remodeling and improved cardiac function. The Dahuang Zhechong pill is clinically used for the treatment of hepatic diseases, gynecopathy, and atherosclerosis in China. Further studies into its role in atherosclerosis treatment revealed its ability to significantly inhibit proliferation of vascular smooth muscle cells through depressing PDGF expression in the cells, retarding the cell cycle and promoting cellular apoptosis. Shenong Yangxin capsule is prescribed for the treatment of arrhythmias in China and was recently found to have potential use in diabetic cardiopathy. The Chinese herb modified Yi Guan Jian is an effective regimen that is usually used in outpatients with chronic liver diseases such as fibrosis and cirrhosis. Shen-Yuan-Dan is used for the treatment of ischemic heart disease, proven to relieve angina and reduces cardiac dysfunction Traditional Chinese medicine Yaotongning Capsule is a common remedy to treat rheumatism in China and possesses diverse biological activities including anti-inflammation.

Another traditional use of Eupolyphaga sinensis is the treatment of cancer. Researchers extracted the adult insect with petroleum ether, ethyl acetate and n-butanol and evaluated the in vitro activity of the extracts in ten cancer cell lines. All extracts had significant antiproliferative activity against all cell lines. An LC-MS evaluation of the ethyl acetate extract was analyzed further to isolate the potential bioactive compounds. Four compounds including two new isocoumarins (77--78, Fig. 13), a new alkaloid deemed eupolyphagin (79), and a known N-acetyldopamine dimer were isolated but were poorly active against the panel of tumor cells. That same year the effect of Eupolyphaga sinensis ethanol extracts on tumor reduction was evaluated using hepatocarcinoma H22-bearing mice. The extract exhibited significant inhibition of tumor growth, promoted Th1 type cytokine production (IFN-γ and TNF-α) and induced apoptosis of hepatocarcinoma via increasing BAX/BCL-2 ratio and activation of caspase-3. In 2014, its effect against hepatocellular carcinoma was further attributed to targeting the protein kinase C (PKC), AKT, mitogen-activated protein kinase (MAPK) signaling and related metastasis signaling which are known targets for potential cancer therapies. That same year the 70% ethanol extract of Eupolyphaga sinensis was found to reduce tumor progression in human and murine chronic myeloid cells. The mechanism was believed to involve G2/M cell cycle arrest and inhibition of epidermal growth factor receptor (EGFR) phosphorylation in the EGFR signaling pathway of human and murine chronic myeloid tumor cells. This extract was also found to inhibit NSCLC progression through the inhibition of angiogenesis. The proposed underlying mechanism was interruption of the auto-phosphorylation of kinase insert domain receptor (KDR) and subsequently AKT and ERK1/2.

Figure 13

Structures of isocoumarins and an alkaloid isolated from Eupolyphaga sinensis.

Polyphaga plancyi, a close relative of Eupolyphaga sinensis that is widely distributed in China, has also been used in Chinese traditional medicine to treat diseases such as amenorrhea and fracture and its extracts were shown to have anticancer and anti-inflammatory activities. A recent study of P. plancyi Bolivar isolated five new compounds (80--84, Fig. 14). Compounds 81 and 83 inhibit JAK3 kinase with IC50 values of 12.6 and 5.0 μmol/L, respectively. In addition, compound 83 inhibit DDR1 kinase with an IC50 value of 4.87 μmol/L. Both JAK3 and DDR1 are potential drug targets for cancer.

Figure 14

Structures of new compounds isolated from Polyphaga plancyi Bolivar.

The winged termite, Reticulitermes (Rhinotermitidae), is roasted with sugar and consumed to treat eye diseases and diabetes in certain regions within India. In the Kerala state in southern India, the Kurichchan tribe consume a decoction of termites and Vitex negundo (a five-leaved Chinese chastetree shrub) twice daily to cure ulcers. The tribes of Irular and Mudugar dry and powder Odontotermes formosanus (Termitidae) which is then added as needed to milk for general health improvement and relief of body pain. These termites are also fried in oil, powdered and administered orally for rheumatic diseases. The termites of Microcerotermes exignus (Termitidae) and Nasutitermes macrocephalus (Termitidae) are used for asthma, bronchitis and flu in Latin American folk medicine. In Zambia, Hodotermes mossambicus (Hodotermitidae) is used to alleviate childhood malnutrition. In Somalia, Macrotermes bellicosus (Termitidae) is used to suture wounds. In Nigeria, Macrotermes nigeriensis is leveraged for wounds and used to alleviate sickness in pregnant women. In Brazil, various genera of the family Termitidae are used for asthma, cough, flu, sore throat, sinusitis, tonsillitis and hoarseness. In India, Odontotermes formosanus of the Termitidae family is thought to cure ulcers, improve health, relieve body pain, rheumatism and anemia, and enhance lactation in women.

Diptera (flies and mosquitoes)

Dipterans, commonly called true flies, are a large and diverse species of 120,000--150,000 colonized on all continents and aquatic environments. Though often associated with forensic science, these insects have varied therapeutic uses as well. For example, adult horseflies (Tabanidae) are used in traditional Korean medicine to treat amenorrhea, abdominal blood stasis and indigestion. Houseflies, Musca domestica (Muscidae), are boiled with water and lemon to relieve vomiting and diarrhea in traditional Mexican medicine. They are also popular in Latin American medicine for boils, baldness, eyesores, external sebaceus lamps, stye, ophthalmological problems, dermatosis, cysties and erysipelas. In China, the use of housefly larvae to cure osteomyelitis, decubital necrosis, lip boil, ecthyma and malnutrition stagnation dates back to the Ming/Qing Dynasty.

Chitosan from housefly and blowfly, Chrysomya megacephala (Calliphoridae), larvae have been investigated for antioxidant activity in recent years146, 147. Chitosan is a partially de-acetylated polymer produced from the alkaline de-acetylation of chitin and is reportedly used as a wound healing, antimicrobial and hemostatic agent. The chitosan extracts indeed had potent antioxidant properties comparable to ascorbic acid as measured by DPPH radical and superoxide anion scavenging activity. Antifungal activity was tested against a panel of fungi including R. stolonifer, R. solani, T. cucumeris, S. sclerotiorum, C. lunata, S. rolfsii and F. fulva. The strongest activity was against R. stolonifer with an IC50 of 0.16 mg/mL.

Blowfly excreta and secretion extracted from Sarconesiopsis magellanica and Lucilia sericata (Calliphoridae) larvae were evaluated for antibacterial activity against a panel of six bacteria split evenly between Gram positive and Gram negative bacterium. Both species were determined effective against the selected strains however the S. magellanica extract required a lower concentration to ensure therapeutic efficacy. Native and inoculated extracts of housefly larva were evaluated for antibacterial and anti-tumor activity in vitro against various Gram negative and Gram positive bacteria, fungi and human colon cancer cells. The native extract included whole bodies of the larvae unmodified and the inoculated included larvae which were exposed to Shigella dysenteriae 51302. Neither extract had activity against the fungi, both were strongest against Gram positive bacteria and each exhibited dose-dependent activity against the cancer cell line.

Hemiptera (true bugs, aphids, cicadas and scale insects)

Hemiptera is the fifth largest insect order with 90,000 species described to date46, 150. Hemipterans are often cited in the literature as a healthy alternative food source and only more recently been explored for bioactive substances. The cochineal insect, Dactylopius coccus (Dactylopiidae), whole body of which is used for nasal congestion and ear pain in holistic Mexican medicine. A major isolate of D. coccus, carminic acid, was determined to be as powerful a free radical scavenging agent as ascorbic acid and stronger than trolox. Furthermore, it can protect against enzymatically induced β-carotene bleaching as well as quercetin. The shell of the lac insect Kerria lacca (Kerriidae) is utilized for the treatment of diarrhea and indigestion in parts of India. Shellac, a natural polymer resin secreted from K. lacca, is valued as a treatment for measles, macula and scabies in traditional Chinese medicine. Recently, several novel sesquiterpene acids were isolated from this shellac including four α-cedrene types (Shellolic acid A–C, Laccishellolic acid, 85--88, Fig. 15), two R-curcumene types (Shellolic acid D–E, 89--90) and Shellolic acid F (91). The anti-tumor and antibacterial activities were evaluated revealing that none had cytotoxic activity and only shellolic acid A had antimicrobial activity against B. subtilis.

Figure 15

Structures of sesquiterpene acids isolated from Kerria lacca.

The stinkbug Encosternum delegorguei (Tessaratomidae), an edible treat in Zimbabwe and South Africa, was recently proven to contain significant amounts of protein, fat and minerals validating its use as a nutritional food source153, 154. In addition to being a healthy appetizer, the stinkbug has acclaimed traditional medicinal values, such as decreasing hypertension and curing asthma and heart diseases. Likewise, the Chinese stinkbug, Aspongopus chinensis (Pentatomidae), is a nutrient-rich edible insect which has been employed to relieve pain, and treat nephropathy and kidney disease in China. In recent years, much research has gone into identifying bioactive small molecules from this insect. Researchers evaluated petroleum ether, ethyl acetate and n-butanol A. chinensis extracts for cytotoxicity against a panel of human cancer cell lines including colorectal (HCT-116), lung (H-125), prostate (LNCaP), melanoma (MDA), pancreatic (PANC-1), breast (MCF-7), ovarian (OVC-5), leukemia (CEM), glioma (U251N) and murine normal cells (CFU-GM) as a control. Each exhibited significant activity against the cell lines and the ethyl acetate extract was analyzed further for active compounds. The compounds isolated from the ethyl acetate extract, however, all exhibited no or poor activity against the cancer cells. In 2014, a novel trimer of N-acetyldopamine (NADA) termed (±)-aspongamide A (92, Fig. 16) was isolated from A. chinensis along with a racemic mixture of NADA dimeric enantiomers(38). The compounds were tested as potential therapeutic agents to treat chronic kidney disease (CKD). Aspongamide A (92) promotes a significant decrease in expression and phosphorylation of Smad3 a key role in the TGF-B/Smad signaling pathway which regulates CKD pathogenesis. The dimeric NADA derivatives (±) 38 were observed to cause an increase in collagen I and α-smooth muscle actin (α-SMA) expression, indicating benefit for restoring skin after wounds as collagen I and α-SMA are implicated in the process of fibrosis and wound healing.

Figure 16

Structures of N-acetyldopamine trimers and dimers isolated from Aspongopus chinensis.

That same year an additional 29 small molecules were isolated from the insect eight of which were novel bioactive compounds including four norepinephrine derivatives (aspongopusamides A--D, 93--96), three sesquiterpenoids (aspongnoids A--C, 97--99, Fig. 17) and one lactam (asponglactam A, 100). A subset of the insect-derived substances was evaluated as renal protection agents in high-glucose-induced mesangial cells and COX-2 inhibition. Aspongopusamide A (103) caused a significant decrease in collagen IV, fibronectin, and IL-6 production in high-glucose-induced mesangial cells and inhibited COX-2 (IC50 = 6.50 µmol/L). Aspongopusamide B also provided protective effect on diabetic nephropathy and aspongopusamide C (95) inhibited COX-2 with an IC50 of 117 µmol/L. Last year, an additional 9 novel small molecules (101--109) were isolated from A. chinensis. With the exception of (±) aspongadenine A (103) and (±) aspongester A (109), all had the ability to promote the proliferation of neural stem cells. The most active compound was (+)-asponguanine B (100a).

Figure 17

Structures of sesquiterpenoids and other small molecules isolated from Aspongopus chinensis.

Orthoptera (grasshoppers and crickets)

There are some 22,500 species of orthopterans described to date. Orthopterans are sometimes singled out as pests though that determination is based on select members. One notable family includes the acridids, plague locusts that at times enter gregarious stages forming swarms numbering in the millions. In Mexico, people eat the grasshopper, Schistocerca piceifrons (Acrididae), to relieve hiccups and the cricket, Gryllus spp., for stuttering. The mole cricket, Gryllotalpa Africana (Gryllotalpidae), is used in traditional Korean medicine for retention of urine, urolithiasis (stones in bladder), edema, lymphangitis and furuncles. The house cricket, Acheta domesticus (Gryllidae), is used for the treatment of scabies, asthma, eczema, lithiasis, earache, oliguresis, rheumatism, urine retention, urinary incontinence and ophthalmological problems in Latin America. The crickets, Paragryllus temulentus (Gryllidae) and Gryllus assimilis (Gryllidae), were additionally cited for rheumatism and warts, respectively. The desert locus Schistocerca gregaria (Acrididae) was found to be rich in sterols with therapeutic uses including cardiovascular protective and immune regulatory effects, anti-inflammatory and anticancer benefits. The known anticancer agents pancratistatin (110, Fig. 18), narciclasine (111) and an additional derivative, ungeremine (112), were isolated from the Texas grasshopper, Brachystola magna (Romaleidae). The compounds were further tested against murine lymphocytic leukemia cells and a variety of human tumor cell lines and as expected all were active against the cell lines with narciclasine being the most potent on each cell line. The chitosan from two species of grasshoppers, Calliptamus barbarous (Acrididae) and Oedaleus decorus (Acrididae), were found to have potent antimicrobial activity against several strains of fish, clinical and food-borne pathogens. Additionally, the chitosan was proven to have antioxidant activity as measured by DPPH radical scavenging activity and ferric reducing power. The nutritional value of the short-horned grasshopper, Chondacris rosea (Acrididae), and mole cricket, Brachytrupes orientalis (Gryllidae), were recently investigated. These food staples of Arunachal Pradesh, India were found to possess a substantial amount of protein, minerals and energy.

Figure 18

Structures of small molecules isolated from Brachystola magna.

Lepidoptera (butterflies and moths)

Lepidopterans, which include butterflies and moths, are one of the largest insect orders with anywhere from 155,208 to 174,312 members to date. Moths especially have been leveraged at all life stages for medicinal purposes. In traditional Indian medicine, dog bites are treated by consummation and/or direct application of freshly laid eggs of Diacrisia obliqua (Arctiidae) to the affected area. Dried full grown larvae of Stomphosistis thraustica (Gracillaridae) are taken with herbs to treat common fever and to increase milk flow in lactating women. In Latin American folk medicine, the moths, Oiketicus kirbyi (Psychidae), are given for the treatment of asthma, earache and haemorrhage.

A novel small molecule, designated papilistatin (113, Fig. 19), was isolated from the wings of Byasa polyeuctes termessa, a Taiwan butterfly (Papilionidae). Papilistatin showed strong cytotoxicity against colon and pancreatic cancer cells and selective Gram-positive antibacterial activity. The cocoon of mulberry white caterpillar, Rondotia menciana (Bombycidae), was recently evaluated for the presence of flavonoids. Researchers discovered flavonol galactosides quercetin 3-O-galactosyl-galactosideand kaempferol 3-O-galactosyl-galactoside, the first reported flavonoids conjugated with galactose in animal species. The medicinal activity of these novel compounds was not evaluated however. The cocoon crude extract of Bombyx mori, a close relative of the R. menciana, was found to have significant effect on hypercholesterolemia and atherosclerosis, as evinced through lipid-lowering capability and decreasing the extent of atherosclerotic lesions in rabbits, induced with hyperlipidemia and atherosclerosis. Three years later, a detailed chemical profile of an aqueous extract of its cocoon revealed various flavonoids including quercetin 7-O-β-D-glucoside, kaempferol 7-O-β-D-glucopyranoside, coumaric acid glucoside, 2-hydroxy-nonadecanoic acid and 9,12-dihydroxy stearic acid. Not surprisingly, this flavonoid rich extract exhibited free radical scavenging activity and cardioprotection via reduction of apoptotic factors, oxidative stress, cardiac enzyme activity and interleukin-6 (pro-inflammatory marker).

Figure 19

Structures of small molecules isolated from lepidopterans and their excreta.

Even B. mori excreta is known to contain bioactive compounds useful in the treatment of diabetes, cancer, high cholesterol and high blood pressure, and is also commonly prescribed in traditional Chinese medicine to treat infectious diseases, headaches and abdominal pain162, 169. For example, the excreta possess ecdysterones (114), which are known to have analgesic effects in man. In 2014, a novel hydroxyl fatty acid termed bombyxmoric acid methyl ester was also discovered in B. mori droppings; however, it׳s medicinal properties were not evaluated. B. mori are not the only moths valued for the medicinal qualities of their feces. Another Chinese tradition is the consumption of insect tea which is prepared from the feces of moths, such as Aglossa dimidiata, Hydrillodes morosa and Nodaria niphona and mentioned in the “Compendium of Materia Medica” as a potential treatment for otitis media. Additional efforts in the past two years have been made to validate new therapeutic properties of insect tea. In a study on human tongue carcinoma cells, insect tea significantly induced apoptosis in cancer cells by upregulating BAX, CASP3, CASP9 and down regulating BCL2. This was likewise the case for human liver cancer cells evaluated last year. Anti-inflammatory activity was also evidenced through the down regulation of genes encoding nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), inducible nitric oxide synthase (iNOS), and COX-2. Furthermore, insect tea was found to be as effective at preventing gastric ulcers as the gastric ulcer drug, ranitidine and buccal mucosa cancer preventative effects in in vivo murine models173, 174.

Conclusions

Summary of small molecules isolated from insects and their biological activities is listed in Table 1. It is clear that Mother Nature has more to offer the world of medicine than just plants. Insects have a long and rich history in traditional medicine across the globe. Not to mention over one hundred small molecule isolates with bioactivity discovered within the past several years. Despite these discoveries, insect-derived products have yet to establish the recognition and market success of their plant derived counterparts. Undoubtedly, this is partly due to negative cultural attitudes towards insects, particularly in the West. However, public perception of insects should not deter scientific pioneers from advancing these largely untapped resources. Indeed, if given the proper attention, insect-derived substances hold great promise for the future of natural product drug discovery.

Table 1

Summary of small molecules isolated from insects and their biological activities.

Family	Genus	Common name	Molecule	Biological activity	Ref.
Hymenoptera (bees, wasps, ants and sawflies)
Symphyta	Pergidae	Sawfly	Macrocarpal (1); grandinol (2)	Antimicrobial	50
Formicidae	Tetramorium	Red ant	Compounds 3--7	Antimicrobial	55
Formicidae	Solenopsis	Fire ant	Solenopsin A (8)	Antiangiogenic	18, 44, 56, 57
Formicidae	Solenopsis	Fire ant	Compounds 9--11	--	58, 59
Formicidae	Tetraponera	Iron ant	Compounds 12--15	Antiproliferative	60, 62
Formicidae	Polyrhachis	Black ant	Polyrhadopamines A--E (16--20); Troline (21); compounds 22--30	Antiinflammatory; antiproliferative; renoprotective	63--65
Vespidae	Polybia	Wasp	Polybioside (31)	Neuroactive	70
Coleoptera (beetles)
Tenebrionidae	Blaps	Stink beetle	Blapsols A--D (32--35); compounds 24, 36--39	Antiinflammatory	90
Scarabaeidae	Catharsius	Dung beetle	Molossusamides A--C (40--42); compounds 38 and 43	Antiinflammatory	100
Bruchidae	Bruchidius	Seed beetle	Compounds 44--48	Antioxidant	101
Meloidae	Various	Blister beetle	Cantharidin (49)	Antiproliferative; immunomodulatory	103, 105, 107, 109--113,116, 117
Meloidae	Hycleus	Blister beetle	(R)-(+)-palasonin (50)	--	103, 104
Meloidae	Hycleus	Blister beetle	Cantharimide (51); palasonimide (52)	--	104
Meloidae	Mylabris	Blister beetle	5-Hydroxy cantharimide (53); norcantharidin (NCTD, 54)	--	39
Synthetic	--	--	N-benzylcantharidinamide (55)	Antiproliferative	122
Synthetic	--	--	N-farnesyloxy-norcantharimide (56); N-farnesyl-norcantharimide (57)	Antiproliferative	123
Synthetic	--	--	Compounds 58--59	Antiproliferative	125
Synthetic	--	--	Compounds 60--69	Antiproliferative	126, 127
Blattodea (cockroaches and termites)
Blattidae	Periplaneta	American cockroach	Periplatins A--D (70--73); compounds 74--76	Antiproliferative	128, 129
Corydiidae	Eupolyphaga	Chinese cockroach	Compounds 77--78; eupolyphagin (79)	Antiproliferative	137
Corydiidae	Polyphaga	Chinese cockroach	Plancyamides A (80) and B (82), plancypyrazine A (81); plancyols A (83) and B (84)	Antiproliferative	142
Hemiptera (true bugs, aphids, cicadas and scale insects)
Kerriidae	Kerria	Lac insect	Shellolic acid A (85)	Antimicrobial	152
Kerriidae	Kerria	Lac insect	Shellolic acids B--C (86--87), Laccishellolic acid (88); shellolic acids D–F (89--91)	--	152
Pentatomidae	Aspongopus	Chinese stinkbug	Aspongamide A (92)	Active against chronic kidney disease	156
Pentatomidae	Aspongopus	Chinese stinkbug	Aspongopusamides A--D (93--96)	Antiinflammatory	157
Pentatomidae	Aspongopus	Chinese stinkbug	Aspongnoids A--C (97--99); asponglactam A (100)	--	157
Pentatomidae	Aspongopus	Chinese stinkbug	Compounds 101--109	Promote neutral stem cell proliferation	158
Orthoptera (grasshoppers and crickets)
Romaleidae	Brachystola	Texas grasshopper	Pancratistatin (110); narciclasine (111); ungeremine (112)	Antiproliferative	160
Lepidoptera (butterflies and moths)
Papilionidae	Byasa	Taiwan butterfly	Papilistatin (113)	Antiproliferative	161
Bombycidae	Bombyx	Silk moth	Compound 114	Analgesic effects	162

--Not applicable.