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
diverse than any other, have played an important role
in various parts of the world as sources of medicinal
resources and food, as aesthetic-decorative
inspiration, and in the religious and magical practices
of different ethnic groups1. Whole insects and other
arthropods, as well as substances extracted from them,
have been used worldwide as medicinal resources by
human cultures2. For a variety of reasons, insects and
their chemical defense systems represent a valuable
source of novel chemistry that certainly merits further
investigation as a source of new medicinal
compounds as well as other applications3. Termites
(Isoptera) are among the insect species most
frequently used in traditional folk medicines around
the world4. In Brazilian semi-arid region various
species of termites have been used in folk medicine
____________
*Corresponding author
by the local population2, including Nasutitermes
macrocephalus, which is employed in the treatment of
various diseases and conditions, such as influenza,
asthma, bronchitis, coughs, sinusitis, tonsillitis and
hoarseness5. This species is commonly found in South
America, mainly in tropical forests and ecosystems of
semi-arid regions6,7. N. macrocephalus builds arboreal
nests, with a volume that may exceed 1000 liters8. In
tropical forests, the population of these nests may
vary from 150.000 to 1.500.000 individuals, with 15
to 544 individuals/m2,9. It is a keen consumer of wood
at various stages of decomposition9. The Caatinga,
predominant ecosystem of the Brazilian semiarid
region, presents a large number of plants and animals
that have been used by the local population for
medicinal purposes, thus suggesting a great potential
from the perspective of bioprospecting2. Therefore,
studies to analyze the pharmacological potential of
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
valued, not only ecologically, but also economically
and socially. In this context, pharmacological and
biochemical investigations to identify the true efficacy
of medicines produced from termites are essential,
since its use is widespread in traditional medicine for
the treatment of various diseases and conditions in the
Brazilian semi-arid region2. Therefore, this study aimed
at evaluating, through experimental models in vitro, the
extract of N. macrocephalus and its nest as an
antibacterial agent and as a modifier of resistance
against pathogenic microorganisms.
Methodology
Zoological material
The collection of N. macrocephalus along with the
nest was performed on the farm Farinha, municipality
of Pocinhos, semiarid region of Paraba state, (707S,
3607W). The species was identified by Prof.
Alexandre Vasconcellos from the Department of
Systematics and Ecology at the Federal University of
Paraba (UFPB). A sample was deposited to the
Isoptera collection at the Center of Exact and Natural
Sciences UFPB under the number CICB 68.
Extract preparation
Twenty gram of termites with nest was subjected to
extraction by maceration with 100 mL of absolute
ethanol for 5 days at room temperature (253 C)
with occasional stirring. After filtration, the extracts
were concentrated on a rotary evaporator at 40 C.
Before microbiological testing the extract was diluted
into 10 % Dimethylsulphoxide (DMSO) solution.
Phytochemical tests
Determination of total polyphenols
The total polyphenol content of plant extracts was
measured using spectrophotometry in the visible region by
the method of Folin-Ciocalteu described by Chandra and
Mejia10 with minor modifications. The extracts (25 mg) were
dissolved in distilled water to obtain a final concentration
200 g/mL. From each solution, a 1 mL aliquot was added
to 1 mL of 1 mol/L Folin-Ciocalteu reagent. This mixture
remained undisturbed for 2 min before the addition of 2 mL
of 20 % (w/v) Na2CO3 solution and left undisturbed for 10
min. Thereafter the reading was performed
Spectrophotometer Shimadzu, model UV-mini 1240, at 757
nm. The calibration curve was obtained with gallic acid at
concentrations between 1 and 40 g/mL.
Determination of total flavonoids
The total flavonoids were determined by the
method described by Meda et al.11. The extracts were
diluted with methanol at 1000 g/mL. To the 5 mL of
each test solution was added the same volume of 2 %
(w/v) AlCl3 solution in methanol. This mixture
remained undisturbed for 10 min before the UV
spectrophotometric reading at 415 nm wavelength.
The total flavonoids were determined by the
calibration curve using quercetin (Sigma-Aldrich) as
standard at concentrations between 2 and 30 g/mL.
Determination of condensed tannins
The content of condensed tannins was verified
through the method of vanillin-HCl described by
Makkar & Becker12, where 0.25 mL of the sample
were added to 1.5 mL of vanillin solution in methanol
(4 % w/v) and subsequently, 0.75 mL of concentrated
HCl (37 %). After HCl addition, the tube content was
shaken in water bath at 30 C for 3-4 sec before
reading on a spectrophotometer at 500 nm
wavelength. Catechin was used as standard.
Determination of saponins
The quantification of total saponins followed the
method described by Makkar et al.13. 250 L of 8 %
vanillin solution in ethanol was added to 250 L
extract solution in 80 % methanol; then 2.5 mL of
72 % sulfuric acid were added. The tubes were
incubated at 60 C in a water bath for 10 min and
transferred to an ice bath, staying for 4 min. The
absorbance reading at 544 nm was performed against
a blank consisting of the vanillin solution, 80 %
methanol and sulfuric acid. The calibration curve was
obtained from a disogenin solution at concentrations
between 100 and 500 g/mL.
Drugs
All the drugs tested were obtained from Sigma
Chemical Corp., St. Louis, MO, USA, and dissolved
in sterile water before use.
Minimal inhibitory concentration (MIC) and determination &
modulation activity
We used the clinical isolates of Staphylococcus
aureus UEPB01, resistant to ampicillin, ciprofloxacin,
cephalexin, erythromycin, penicillin and amoxicillin
and Escherichia coli UEPB01, resistant to amoxicillin,
cephalothin, levofloxacin, chloramphenicol,
tetracycline and gentamicin. The tested bacterial strains
were incubated at 37 C for 24 h in MuellerHinton
agar and maintained on agar slants in tubes on the same
medium. The minimum inhibitory concentration (MIC)
was determined by microdilution method in 96-well
plates14 using Mueller-Hinton broth. Colonies of
microorganisms were suspended in 0.9 % saline
 CHAVES et al.: NASUTITERMES MACROCEPHALUS AND PHYTOCOMPOUNDS
solution and the suspension adjusted by
spectrophotometric method at 625 nm to a final
concentration of 5 x 106 CFU/mL. Were performed
serial dilutions of the extract in the range of 1000 to
3.9 g/mL and antibiotics in the range of 2500 to
2.4 g/mL. DMSO 10 % was included as negative
control. The plates were incubated at 37 1 C for
24 h. Bacterial growth was indicated by addition of 20
L of 0.01 % resazurin aqueous with incubation at 37
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
is visible. Evaluation of extracts as modulators of
antibiotic resistance was performed according to
Coutinho et al.15. The MIC of the antibiotic was
determined in presence and absence of sub-inhibitory
concentrations (MIC/8). Plates were incubated as
described above and each assay was performed in
triplicate.
Statistical analysis of microbiological tests
The test results were expressed as geometric
means. The two-way analysis of variance followed by
Bonferroni post-test was applied using the GraphPad
Prism 5.0 software.
Results and discussion
Through chemical testing it was possible to
determine the presence and concentration of substances
originating from the plant secondary metabolism. The
concentration of these compounds is present in
Table 1 and expressed in equivalent milligrams of
standards used. In the microbiological testing, there
was no significant antimicrobial activity of the
extract of N. macrocephalus against S. aureus
UEPB01 and E. coli UEPB01 (MIC > 1000 g/mL).
However, the extract when combined with
antimicrobials in subinhibitory concentration
(125 g/mL) afforded a significant reduction in the
MIC of the antibiotic tested, except for levofloxacin
against E. coli (Figs. 1&2).
The synergistic effect of the extract tested with
antibiotics against S. aureus UEPB01 was demonstrated
especially when combined with erythromycin, causing a
marked reduction in MIC from 2500 to 996 g/mL. This
reduction was also observed in the MIC of ampicillin,
992 to 78 g/mL (Fig. 1). Against E. coli UEPB01 this
effect was observed in association with gentamicin
extract, reducing the MIC of 156 g/mL to 39 g/mL
(Fig. 2).
In recent years, there has been observed an increase
of bacterial resistance to conventional antibiotics,
making the development of new drugs with
585
Table 1 Concentration of secondary metabolites (mg/g)
determined for the extract of N. microcephalus and this nest
Metabolites Total Total Condensed Total
polyphenols flavonoids tannins saponins
Concentration 27. 56 11.75 12.46 50.19
4.31a 1.03b 2.14c 19.12d
antibacterial properties necessary in order to address
this situation16. From this perspective, various animal
species have been methodically tested by researchers
the current number of animal sources for the
production of basic medicines is extensive17.
Arthropods have attracted attention because of their
potential as a source of medically relevant substances,
especially the class Insecta3. Among insects, termites
are some of the most commonly used in traditional
folk medicine in the world15,18. In work of Figueiredo
et al.19, a compilation of data was performed where
the species N. macrocephalus was the most frequently
recorded and was found to be widely used as a
therapeutic tool in the treatment of asthma,
hoarseness, and sinusitis, among other ailments.
The results of this study corroborate the works of
Coutinho et al.15,17 and Chaves et al.20 in which, even
without seeing direct antimicrobial activity of
N. corniger extracts, they found a reduction in the
MIC of specific antibiotics to be associated with these
extracts. The authors indicated the species N. corniger
and possibly other species of termites as a source of
natural products to modify activity of antibiotics
against multi-resistant bacteria.
The synergism observed in this study between the
N. macrocephalus extract and antibiotics tested may
be due to the presence of secondary metabolites
present in the extract that are synthesized by plants in
response to microbial infections. The chemical
analysis carried out in this work revealed the presence
of polyphenols, flavonoids, tannins and saponins in
the extract. According Cowan21, these metabolites
have several activities, including the antimicrobial
activity and a previous study with N. corniger
revealed almost the same concentration of these
metabolites20 with similar biological results. All these
metabolites can affect the lipid bilayer of the bacteria,
disrupting the cell membrane and enhancing the
influx of antibiotics and consequently their antibiotic
effect. N. macrocephalus is a xylophage species, and
the presence of these secondary metabolites are from
the support plant used as a food source; these
compounds may be found in the digestive system of
586 INDIAN J TRADIT KNOWLE VOL 16, NO. 4. OCTOBER 2017
Fig. 1 Graph demonstrating the modulatory activity of the ethanol
extract of N. macrocephalus on resistance of S. aureus UEPB01 to
antibiotics. *** statistically significant with p value < 0.001.
the animal droppings or used to build a nest22.
Evidence of antimicrobial products isolated from
these animals has been reported, like the peptides
such as spinigerin and termicin isolated from
Pseudocanthotermes spiniger, which showed
antifungal and antibacterial activity23. Studies
addressing the molecular biology of the termites
Nasutitermes demonstrated their potential as
producers of antimicrobial peptides24.
In a broader perspective, natural product obtained
from various kinds of animals has been demonstrated
to have potential to accentuate the action of
antibiotics against multiresistant bacteria. Substances
extracted from the skin of Rhinella jimi25, Ameiva
ameiva and Tropidurus hispidus26, the body fat of
Tupinambis merianae27, Gallus gallus domesticus28
and Spilotes pullatus29 showed promising results
regarding the modification of bacterial resistance.
Among the compounds from natural sources, extracts
are complex mixtures which make microbial
adaptability difficult, and for this reason, their use as
an antimicrobial agent lowers the possibility of
microorganisms acquiring resistance to its action15.
When combined with antibiotics, extracts can have
direct action against various bacterial species, either
increasing or modulating the activity of a particular
antibiotic, thereby reversing the natural resistance of
bacteria to specific antibiotics30.
Traditional significance of study to the society/researchers
This study has a great significance for two different
kinds of societies: to the traditional population groups,
this study contributes with an accurate demonstration
that some drugs have not be used in association with
Fig. 2 Graph demonstrating the modulatory activity of the
ethanol extract of N. macrocephalus on resistance of E. coli
UEPB01 to antibiotics. *** statistically significant with p
value < 0.001.; ns value not statistically significant with p
> 0,05.
these natural products. To the moderns and occidental-
based societies, this study demonstrate that natural
products from these termite can enhance the antibiotic
activity of drugs to be used against multi-drug resistant
bacteria, a huge concern for all countries and
populations worldwide.
Conclusion
The results indicate that the N. macrocephalus
extract and its nest have modulatory activity for
bacterial resistance against strains resistant to multiple
drugs, highlighting the possibility of its use in
antimicrobial therapy.
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