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Indian Journal of Traditional Knowledge

Vol. 16(4), October 2017, pp 583-587

Correlation between bioacumulated phytocompounds and bioactivities in


Nasutitermes macrocephalus (Silvestri, 1903) (Isoptera: Termitidae) and its nest
extracts against multi-resistant bacterial strains
Thiago P Chaves1,2, Iamara S Policarpo3, Henrique DM Coutinho4*, Rmulo RN Alves5,
Alexandre Vasconcellos3 & Ana Claudia D Medeiros2
1
Departamento de Cincias da Natureza, Universidade Federal do Piau, BR 135, km 03, Planalto Horizonte, 64900-000,
Bom Jesus - PI, Brazil; 2Laboratrio de Desenvolvimento e Ensaios de Medicamentos (LABDEM) Universidade Estadual da Paraba,
Rua das Baranas, 351 - Bairro Universitrio, 58429-500, Campina Grande PB, Brazil; 3Departamento de Sistemtica e Ecologia,
CCEN, Universidade Federal da Paraba, Laboratrio de Termitologia, PB, 58051-900, Joo Pessoa, Brazil; 4Laboratrio de
Microbiologia e Biologia Molecular, Universidade Regional do Cariri, Crato -CE, 63105-000, Brasil;
5
Departamento de Biologia, Universidade Estadual da Paraba, Laboratrio de Zoologia, PB, 58051-900, Joo Pessoa, Brazil
E-mail: hdmcoutinho@urca.br

Received 28 July 2016, revised 24 January 2017

Termites are among the species most commonly used in folk medicine in various locations worldwide. Of these,
Nasutitermes macrocephalus is often used in the treatment of various diseases that affect humans, however, with no
supporting scientific evidence. From this perspective, this study aimed to evaluate the antibacterial activity and modulating
effect of bacterial resistance of N. macrocephalus and its nest extract. The minimum inhibitory concentration (MIC) of
antimicrobial agents used in clinical practice is determined in the presence and absence of the ethanol extract of
N. macrocephalus and its nest in a subinhibitory concentration (125 g/mL) against multi-resistant strains of Escherichia
coli and Staphylococcus aureus. The extract showed no significant antimicrobial activity (MIC > 1000 g/mL). However,
there was a considerable reduction in MIC of antimicrobials when combined with the extract, especially erythromycin,
ampicillin and gentamicin. The results indicate that N. macrocephalus and its nest are a promising source of substances that
can be used in combating multi-drug-resistant bacteria. Additional studies are needed to identify the active components
responsible for such activity.

Keywords: Termites, Ethnozoology, Natural products, Bacterial resistance, Modulatory activity


IPC Int. Cl.8: A01N, A61K, C12N, C12M, C07, C08
Insects, a group of animals more numerous and by the local population2, including Nasutitermes
diverse than any other, have played an important role macrocephalus, which is employed in the treatment of
in various parts of the world as sources of medicinal various diseases and conditions, such as influenza,
resources and food, as aesthetic-decorative asthma, bronchitis, coughs, sinusitis, tonsillitis and
inspiration, and in the religious and magical practices hoarseness5. This species is commonly found in South
of different ethnic groups1. Whole insects and other America, mainly in tropical forests and ecosystems of
arthropods, as well as substances extracted from them, semi-arid regions6,7. N. macrocephalus builds arboreal
have been used worldwide as medicinal resources by nests, with a volume that may exceed 1000 liters8. In
human cultures2. For a variety of reasons, insects and tropical forests, the population of these nests may
their chemical defense systems represent a valuable vary from 150.000 to 1.500.000 individuals, with 15
source of novel chemistry that certainly merits further to 544 individuals/m2,9. It is a keen consumer of wood
investigation as a source of new medicinal at various stages of decomposition9. The Caatinga,
compounds as well as other applications3. Termites predominant ecosystem of the Brazilian semiarid
(Isoptera) are among the insect species most region, presents a large number of plants and animals
frequently used in traditional folk medicines around that have been used by the local population for
the world4. In Brazilian semi-arid region various medicinal purposes, thus suggesting a great potential
species of termites have been used in folk medicine from the perspective of bioprospecting2. Therefore,
____________ studies to analyze the pharmacological potential of
*Corresponding author animals and plants of the Caatinga may contribute to
584 INDIAN J TRADIT KNOWLE VOL 16, NO. 4. OCTOBER 2017

ensuring that local natural resources are properly diluted with methanol at 1000 g/mL. To the 5 mL of
valued, not only ecologically, but also economically each test solution was added the same volume of 2 %
and socially. In this context, pharmacological and (w/v) AlCl3 solution in methanol. This mixture
biochemical investigations to identify the true efficacy remained undisturbed for 10 min before the UV
of medicines produced from termites are essential, spectrophotometric reading at 415 nm wavelength.
since its use is widespread in traditional medicine for The total flavonoids were determined by the
the treatment of various diseases and conditions in the calibration curve using quercetin (Sigma-Aldrich) as
Brazilian semi-arid region2. Therefore, this study aimed standard at concentrations between 2 and 30 g/mL.
at evaluating, through experimental models in vitro, the
Determination of condensed tannins
extract of N. macrocephalus and its nest as an
The content of condensed tannins was verified
antibacterial agent and as a modifier of resistance
through the method of vanillin-HCl described by
against pathogenic microorganisms.
Makkar & Becker12, where 0.25 mL of the sample
Methodology were added to 1.5 mL of vanillin solution in methanol
Zoological material (4 % w/v) and subsequently, 0.75 mL of concentrated
The collection of N. macrocephalus along with the HCl (37 %). After HCl addition, the tube content was
nest was performed on the farm Farinha, municipality shaken in water bath at 30 C for 3-4 sec before
of Pocinhos, semiarid region of Paraba state, (707S, reading on a spectrophotometer at 500 nm
3607W). The species was identified by Prof. wavelength. Catechin was used as standard.
Alexandre Vasconcellos from the Department of
Determination of saponins
Systematics and Ecology at the Federal University of
The quantification of total saponins followed the
Paraba (UFPB). A sample was deposited to the
method described by Makkar et al.13. 250 L of 8 %
Isoptera collection at the Center of Exact and Natural
vanillin solution in ethanol was added to 250 L
Sciences UFPB under the number CICB 68.
extract solution in 80 % methanol; then 2.5 mL of
Extract preparation 72 % sulfuric acid were added. The tubes were
Twenty gram of termites with nest was subjected to incubated at 60 C in a water bath for 10 min and
extraction by maceration with 100 mL of absolute transferred to an ice bath, staying for 4 min. The
ethanol for 5 days at room temperature (253 C) absorbance reading at 544 nm was performed against
with occasional stirring. After filtration, the extracts a blank consisting of the vanillin solution, 80 %
were concentrated on a rotary evaporator at 40 C. methanol and sulfuric acid. The calibration curve was
Before microbiological testing the extract was diluted obtained from a disogenin solution at concentrations
into 10 % Dimethylsulphoxide (DMSO) solution. between 100 and 500 g/mL.
Phytochemical tests Drugs
Determination of total polyphenols All the drugs tested were obtained from Sigma
The total polyphenol content of plant extracts was Chemical Corp., St. Louis, MO, USA, and dissolved
measured using spectrophotometry in the visible region by in sterile water before use.
the method of Folin-Ciocalteu described by Chandra and Minimal inhibitory concentration (MIC) and determination &
Mejia10 with minor modifications. The extracts (25 mg) were modulation activity
dissolved in distilled water to obtain a final concentration We used the clinical isolates of Staphylococcus
200 g/mL. From each solution, a 1 mL aliquot was added aureus UEPB01, resistant to ampicillin, ciprofloxacin,
to 1 mL of 1 mol/L Folin-Ciocalteu reagent. This mixture cephalexin, erythromycin, penicillin and amoxicillin
remained undisturbed for 2 min before the addition of 2 mL and Escherichia coli UEPB01, resistant to amoxicillin,
of 20 % (w/v) Na2CO3 solution and left undisturbed for 10 cephalothin, levofloxacin, chloramphenicol,
min. Thereafter the reading was performed tetracycline and gentamicin. The tested bacterial strains
Spectrophotometer Shimadzu, model UV-mini 1240, at 757 were incubated at 37 C for 24 h in MuellerHinton
nm. The calibration curve was obtained with gallic acid at agar and maintained on agar slants in tubes on the same
concentrations between 1 and 40 g/mL. medium. The minimum inhibitory concentration (MIC)
Determination of total flavonoids was determined by microdilution method in 96-well
The total flavonoids were determined by the plates14 using Mueller-Hinton broth. Colonies of
method described by Meda et al.11. The extracts were microorganisms were suspended in 0.9 % saline
CHAVES et al.: NASUTITERMES MACROCEPHALUS AND PHYTOCOMPOUNDS 585

solution and the suspension adjusted by Table 1 Concentration of secondary metabolites (mg/g)
spectrophotometric method at 625 nm to a final determined for the extract of N. microcephalus and this nest
concentration of 5 x 106 CFU/mL. Were performed Metabolites Total Total Condensed Total
serial dilutions of the extract in the range of 1000 to polyphenols flavonoids tannins saponins
3.9 g/mL and antibiotics in the range of 2500 to Concentration 27. 56 11.75 12.46 50.19
2.4 g/mL. DMSO 10 % was included as negative 4.31a 1.03b 2.14c 19.12d
control. The plates were incubated at 37 1 C for antibacterial properties necessary in order to address
24 h. Bacterial growth was indicated by addition of 20 this situation16. From this perspective, various animal
L of 0.01 % resazurin aqueous with incubation at 37 species have been methodically tested by researchers
C 1 C for 2 h, and MIC values were identified as as a source of drugs for modern medical science, and
the lowest concentration in which no bacterial growth the current number of animal sources for the
is visible. Evaluation of extracts as modulators of production of basic medicines is extensive17.
antibiotic resistance was performed according to Arthropods have attracted attention because of their
Coutinho et al.15. The MIC of the antibiotic was potential as a source of medically relevant substances,
determined in presence and absence of sub-inhibitory especially the class Insecta3. Among insects, termites
concentrations (MIC/8). Plates were incubated as are some of the most commonly used in traditional
described above and each assay was performed in folk medicine in the world15,18. In work of Figueiredo
triplicate. et al.19, a compilation of data was performed where
Statistical analysis of microbiological tests the species N. macrocephalus was the most frequently
The test results were expressed as geometric recorded and was found to be widely used as a
means. The two-way analysis of variance followed by therapeutic tool in the treatment of asthma,
Bonferroni post-test was applied using the GraphPad hoarseness, and sinusitis, among other ailments.
Prism 5.0 software. The results of this study corroborate the works of
Coutinho et al.15,17 and Chaves et al.20 in which, even
Results and discussion
without seeing direct antimicrobial activity of
Through chemical testing it was possible to
N. corniger extracts, they found a reduction in the
determine the presence and concentration of substances
MIC of specific antibiotics to be associated with these
originating from the plant secondary metabolism. The
extracts. The authors indicated the species N. corniger
concentration of these compounds is present in
and possibly other species of termites as a source of
Table 1 and expressed in equivalent milligrams of
natural products to modify activity of antibiotics
standards used. In the microbiological testing, there
against multi-resistant bacteria.
was no significant antimicrobial activity of the
extract of N. macrocephalus against S. aureus The synergism observed in this study between the
UEPB01 and E. coli UEPB01 (MIC > 1000 g/mL). N. macrocephalus extract and antibiotics tested may
However, the extract when combined with be due to the presence of secondary metabolites
antimicrobials in subinhibitory concentration present in the extract that are synthesized by plants in
(125 g/mL) afforded a significant reduction in the response to microbial infections. The chemical
MIC of the antibiotic tested, except for levofloxacin analysis carried out in this work revealed the presence
against E. coli (Figs. 1&2). of polyphenols, flavonoids, tannins and saponins in
The synergistic effect of the extract tested with the extract. According Cowan21, these metabolites
antibiotics against S. aureus UEPB01 was demonstrated have several activities, including the antimicrobial
especially when combined with erythromycin, causing a activity and a previous study with N. corniger
marked reduction in MIC from 2500 to 996 g/mL. This revealed almost the same concentration of these
reduction was also observed in the MIC of ampicillin, metabolites20 with similar biological results. All these
992 to 78 g/mL (Fig. 1). Against E. coli UEPB01 this metabolites can affect the lipid bilayer of the bacteria,
effect was observed in association with gentamicin disrupting the cell membrane and enhancing the
extract, reducing the MIC of 156 g/mL to 39 g/mL influx of antibiotics and consequently their antibiotic
(Fig. 2). effect. N. macrocephalus is a xylophage species, and
In recent years, there has been observed an increase the presence of these secondary metabolites are from
of bacterial resistance to conventional antibiotics, the support plant used as a food source; these
making the development of new drugs with compounds may be found in the digestive system of
586 INDIAN J TRADIT KNOWLE VOL 16, NO. 4. OCTOBER 2017

Fig. 2 Graph demonstrating the modulatory activity of the


Fig. 1 Graph demonstrating the modulatory activity of the ethanol ethanol extract of N. macrocephalus on resistance of E. coli
extract of N. macrocephalus on resistance of S. aureus UEPB01 to UEPB01 to antibiotics. *** statistically significant with p
antibiotics. *** statistically significant with p value < 0.001. value < 0.001.; ns value not statistically significant with p
the animal droppings or used to build a nest22. > 0,05.
Evidence of antimicrobial products isolated from these natural products. To the moderns and occidental-
these animals has been reported, like the peptides based societies, this study demonstrate that natural
such as spinigerin and termicin isolated from products from these termite can enhance the antibiotic
Pseudocanthotermes spiniger, which showed activity of drugs to be used against multi-drug resistant
antifungal and antibacterial activity23. Studies bacteria, a huge concern for all countries and
addressing the molecular biology of the termites populations worldwide.
Nasutitermes demonstrated their potential as
Conclusion
producers of antimicrobial peptides24. The results indicate that the N. macrocephalus
In a broader perspective, natural product obtained extract and its nest have modulatory activity for
from various kinds of animals has been demonstrated bacterial resistance against strains resistant to multiple
to have potential to accentuate the action of drugs, highlighting the possibility of its use in
antibiotics against multiresistant bacteria. Substances antimicrobial therapy.
extracted from the skin of Rhinella jimi25, Ameiva
ameiva and Tropidurus hispidus26, the body fat of References
Tupinambis merianae27, Gallus gallus domesticus28 1 Morris B, Insects and human life, (Berg, New York), 2004.
and Spilotes pullatus29 showed promising results 2 Alves RRN, Fauna used in popular medicine in Northeast
regarding the modification of bacterial resistance. Brazil, J Ethnobiol Ethnomed, 5 (2009) 1-11.
3 Dossey AT, Insects and their chemical weaponry: New
Among the compounds from natural sources, extracts potential for drug discovery, Nat Prod Rep, 27 (2010)
are complex mixtures which make microbial 1737-57.
adaptability difficult, and for this reason, their use as 4 Wilsanand V, Utilization of termite Odontotermes
an antimicrobial agent lowers the possibility of formosanus by tribes of South India in medicine and food,
Nat Prod Rad, 4 (205) 121-5.
microorganisms acquiring resistance to its action15.
5 Vasconcellos A & Moura FMS, Wood litter consumption by
When combined with antibiotics, extracts can have three species of Nasutitermes termites in an area of the
direct action against various bacterial species, either Atlantic Coastal Forest in northeastern Brazil, J Insect Sci,
increasing or modulating the activity of a particular 10 (2010) 1-9.
antibiotic, thereby reversing the natural resistance of 6 Alves RRN, Barbosa JAA, Santos SLDX, et al., Animal-based
bacteria to specific antibiotics30. Remedies as Complementary Medicines in the Semi-arid
Region of Northeastern Brazil, ECAM, 2009 (2009a) 1-15.
Traditional significance of study to the society/researchers 7 Alves RRN, Neto NAL, Brooks SE, et al.,
Commercialization of animal-derived remedies as
This study has a great significance for two different complementary medicine in the semi-arid region of
kinds of societies: to the traditional population groups, Northeastern Brazil, J Ethnopharmacol, 124 (2009b) 600-8.
this study contributes with an accurate demonstration 8 Martius C, Diversity and ecology of termites in Amazonian
that some drugs have not be used in association with forest, Pedobiology, 38 (1994) 40728.
CHAVES et al.: NASUTITERMES MACROCEPHALUS AND PHYTOCOMPOUNDS 587

9 Vasconcellos A, Biomass and abundance of termites in three 20 Chaves TP, Clementino ELC, Felismino DC, et al., Antibiotic
remnant areas of Atlantic Forest in northeastern Brazil, Rev resistance modulation by natural products obtained from
Bras Entomol, 54 (2010) 45561. Nasutitermes corniger (Motschulsky, 1855) and its nest, Saudi J
10 Chandra S & Mejia EG, Polyphenolic Compounds, Biol Sci, 22 (2015) 4048.
Antioxidant Capacity, and Quinone Reductase Activity of 21 Cowan MM, Plant products as antimicrobial agents, Clin
an Aqueous Extract of Ardisia compressa in Comparison Microbiol Rev, 12 (1999) 56482.
to Mate (Ilex paraguariensis) and Green (Camellia 22 Lima JT & Costa-Leonardo AM, Recursos alimentares
sinensis) Teas, J Agric Food Chem, 52 (2004) 35839. explorados pelos cupins (Insecta: Isoptera), Biota Neotrop, 7
11 Meda A, Lamien CE, Romito M, et al., Determination of the (2007) 243-50.
total phenolic, flavonoid and proline contents in Burkina 23 Lamberty M, Zachary D, Lanot R, et al., Constitutive
Fasan honey, as well as their radical scavenging activity, expression of a Cysteine-rich antifungal and a linear
Food Chem, 91 (2005) 5717. antibacterial peptide in a termite insect, J Biol Chem, 276
12 Makkar HPS & Becker K, Vanillin-HCl method for (2001) 408592.
condensed tannins: Effect of organic solvents used for 24 Bulmer MS & Crozier RH, Variation in positive selection in
extraction of tannins, J Chem Ecol, 4 (1993) 613-21. termite GNBPs and Relish, Mol Biol Evol, 23 (2006) 31726.
13 Makkar HPS, Siddhuraju P & Becker K, Plant Secondary 25 Brito SV, Ferreira FS, Siqueira-Jnior JP, et al.,
Metabolities, (Humana Press,Totowa, New Jersey), 2007. Phototoxic and modulatory effects of natural products
14 CLSI - Clinical and Laboratory Standards Institute. from the skin of Rhinella jimi (Stevaux, 2002), Braz J
Performance Standards for Antimicrobial Susceptibility Pharmacog, 22 (2012) 82-7.
Testing. Twenty-Second Informational Supplement, ninth 26 Santos IJM, Coutinho HDM, Matias EFF, et al.,
ed. Document M100-S22. NIH, Pensilvnia, USA, 2012. Antimicrobial activity of natural products from the skins
of the semiarid living lizards Ameiva ameiva (Linnaeus,
15 Coutinho HDM, Vasconcellos A, Lima MA, et al., Termite
1758) and Tropidurus hispidus (Spix, 1825), J Arid
usage associated with antibiotic therapy: enhancement of
Environ, 76 (2012) 138-41.
aminoglycoside antibiotic activity by natural products of
27 Ferreira FS, Brito SV, Costa JGM, et al., Is the body fat of the
Nasutitermes corniger (Motschulsky 1855), BMC CAM, 9
lizard Tupinambis merianae effective against bacterial
(2009) 1-4.
infections?, J Ethnopharmacol, 126 (2009) 233-7.
16 Andersson DI & Hughes D, Antibiotic resistance and its cost: 28 Aquino PEA, Leite JLA, Guedes TTAM, et al.,
is it possible to reverse resistance?, Nat Rev, 8 (2010) 26071. Modulao da atividade antibacteriana do tecido adiposo
17 Coutinho HDM, Vasconcellos A, Freire-Pessa HL, et al., da Gallus gallus domesticus (Linnaeus 1758), Comunicat
Natural products from the termite Nasutitermes corniger Sci, 5 (2014) 380-5.
lowers aminoglycoside minimum inhibitory concentrations, 29 Paiva AO, Sales DL, Dias DQ, et al., Antimicrobial activity
Pharmacog Mag, 6 (2010) 1-4. and chemical composition of fixed oil extracted from the
18 Alves RRN & Alves HN, The faunal drugstore: Animal-based body fat of the snake Spilotes pullatus, Pharma Biol, 52
remedies used in traditional medicines in Latin America, J (2014) 740-4.
Ethnobiol Ethnomed, 17 (2011) 143. 30 Souza DO, Tintino SR, Figueredo FG, et al., Atividade
19 Figueirdo RECR, Vasconcellos A, Policarpo IS, et al., Edible antibacteriana e moduladora de Cecropia pachystachya Trcul
and medicinal termites: a global overview, J Ethnobiol sobre a ao de aminoglicosdeos, Rev Cubana Plantas Med, 19
Ethnomed, 11 (2015) 29. (2014) 121-32.