Proximal composition of bee pollen and its functional effect on stress

Abstract

Bee pollen is an agglomerate of microscopic grains rich in proteins and phenolic compounds. Other products rich in flavonoids and vitamins, as well as bee pollen, have been shown a positive effect on stress, which is a physical condition that can lead to several somatic disorders. In this work the compositional characteristics of bee pollen were measured, and its effect in volunteers’ adult men was evaluated by a cortisol salivary test, and by responding to Lip’s Stress Symptom Inventory. The volunteers were given bee pollen for a month. After that, the salivary cortisol was measured and applied the questionnaire. Bee pollen shown an adequate nutritional composition. Its administration had a 23% reduction in salivary cortisol. The values obtained in the questionnaire were consistent with the results of the cortisol dosage, which relieved the symptoms reported by the volunteers. A phytochemical screening was also performed on this material showing the presence of flavonoids, which may be an active compound responsible for the functional effect in the stress relieving of the participants.

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The Journal of Phytopharmacology 2022; 11(5):330-334
Online at: www.phytopharmajournal.com
Research Article
ISSN 2320-480X
JPHYTO 2022; 11(5): 330-334
September- October
Received: 03-07-2022
Accepted: 26-08-2022
©2022, All rights reserved
doi: 10.31254/phyto.2022.11503
Elizabete Lourenco da Costa
Anhembi Mocumbi University, Almeida
Lima St. 1134, São Paulo - SP, Brazil
José Marcelino Antonio
Catholic University of Santos,
Conselheiro Nebias Av. 300, Santos -
SP, Brazil
Odilon Antonio Pereira
Catholic University of Santos,
Conselheiro Nebias Av. 300, Santos -
SP, Brazil
Ellen Virgínia de Oliveira Nascimento
Catholic University of Santos,
Conselheiro Nebias Av. 300, Santos -
SP, Brazil
Juliana Tonini Mesquita
Adolfo Lutz Institute, Dr. Arnaldo Av.
355 - São Paulo – SP, Brazil
Correspondence:
Dr. Elizabete Lourenco da Costa
Anhembi Mocumbi University, Almeida
Lima St. 1134, São Paulo - SP, Brazil
Email: betelcosta@gmail.com
Proximal composition of bee pollen and its functional effect
on stress
Elizabete Lourenco da Costa1, José Marcelino Antonio2, Odilon Antonio Pereira2, Ellen Virgínia de
Oliveira Nascimento2, Juliana Tonini Mesquita3
ABSTRACT
Bee pollen is an agglomerate of microscopic grains rich in proteins and phenolic compounds. Other
products rich in flavonoids and vitamins, as well as bee pollen, have been shown a positive effect on
stress, which is a physical condition that can lead to several somatic disorders. In this work the
compositional characteristics of bee pollen were measured, and its effect in volunteers’ adult men was
evaluated by a cortisol salivary test, and by responding to Lip’s Stress Symptom Inventory. The
volunteers were given bee pollen for a month. After that, the salivary cortisol was measured and applied
the questionnaire. Bee pollen shown an adequate nutritional composition. Its administration had a 23%
reduction in salivary cortisol. The values obtained in the questionnaire were consistent with the results of
the cortisol dosage, which relieved the symptoms reported by the volunteers. A phytochemical screening
was also performed on this material showing the presence of flavonoids, which may be an active
compound responsible for the functional effect in the stress relieving of the participants.
Keywords: Bee Pollen; Stress; Proximal Composition; Functional Food.
INTRODUCTION
Bee pollen (BP) is an agglomerate of microscopic grains which are the male reproductive cells of the
plants. Such grains are collected by Apis mellifera and carried back to the hive, where they will serve as
a protein source to feed the worker larvae until the third day of life and will be part of the composition of
royal jelly [1]. As it is a product rich in essential nutrients such as amino acids, vitamins, and minerals, it
has been used in several countries as a diet supplement [2]. Besides its nutritional value, there is evidence
of its therapeutic effect on health. Some of its indications include an anticarcinogenic activity,
antioxidant, and antibacterial activity [3].
The therapeutic effect of several apicultural products in general is assigned to their phenolic compound
contents, which have an antioxidant activity. Other products rich in phenolic compounds have also been
indicated in the treatment of stress symptoms [4].
Stress is defined traditionally as an organism’s responses to physical, mental, or infectious aggressions
which may be capable of disrupting homeostasis, characterized by negative effects, mainly anxiety.
These processes can be associated with the hypothalamic pituitary adrenal axis (HPA), that stimulates
the adrenal gland to release cortisol in response to stress [5]. (Gerber et al., 2018).
According to hypotheses of Meyerhof and Schmidt [6]. the stress can promote a cumulative effect,
influenced by individual differences and environment, but these symptoms can possibly make the body
more susceptible to illness, by suppressing the immune system [7].
The loss of nutrients such as vitamin C, zinc and mineral salts can take place under stressful situations,
worsening the organism’s symptoms, and on other hand, lifestyle, specific nutrients, like antioxidants,
omega-3 fatty acids, and vitamins, can modulate the cognitive function and mental health [8]. In this
sense, BP contains a high nutritional value due its composition of essential amino acids, fatty acids,
vitamins, fibres and flavonoids, which may be an alternative in the stress treatment.
The objective of this work is to research if the intake of BP has an influence on the stress level of Fire
Service Professionals, as individuals who are constantly encountering risk and to characterize the bee
pollen composition.

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331
MATERIALS AND METHODS
Bee pollen was purchased from local commerce in the Pedro de
Toledo municipality (SP, Brazil), in the rainforest region.
To access the centesimal composition the sample was submitted to the
following analysis: dry matter, protein and ash contents were
determined according to the official AOAC methods [9]. Dry matter
was obtained by desiccation at 105ºC until constant weight, whereas
the ash content was given by incineration at 550ºC, crude protein was
determined by the Kjeldhal procedure, using 6.25 as converting factor,
and the Bligh and Dyer [10]. method was used for lipids quantification.
The determination of the carbohydrates was carried out by difference.
The following pharmacological groups were researched in the
phytochemical characterization of BP: alkaloids, anthraquinone
glycosides, cardioactive glycosides, flavonoids, tannins and saponin
glycosides, which followed the procedures used by Jacome et al [11].
The polyphenol content was obtained by adapting Augusto’s Method
[12], where an aliquot of bee pollen sample previously left in contact
with water for 24 hours and kept out of the light, was added to a
reagent solution of Folin-Ciocalteu (1:10, v/v) (Merck, Lot:
HC894012) for 5 minutes, and then added to a solution of 2 mL of
sodium carbonate (4%, p/v), followed by two-hours out of the light.
After that period, the sample was read by a spectrophotometer
UV/VIS (Genesis 2), =740 nm. For the calculation of the results, a
standard curve was made with gallic acid and was performed (Sigma
Aldrich, Lot: 117K0057) in a concentration ranging from 10 to
60mg/L (R2=0,9999, y=0,0121x+0,0028). The polyphenol content
was expressed in mg GAE/100 g.
The subjects consisted of 52 males from local Fire Service, volunteers
in the municipality of Santos, SP, Brazil. Aged between 20 and 49
years old, who were randomly assigned to a double-blind study. The
volunteers received an Informed Consent Form, which was read by
them, containing the aims of the research, responsible researches,
mode of consumption, the length of the experiment and the possible
adverse effects.
The subjects were randomly assigned into two groups, the
experimental group (EG) and the control group (CG). The
experimental group received flasks containing commercial dehydrated
bee pollen, while the control group received flasks containing oat
flakes as a placebo. A daily intake of five capsules was recommended
to both groups, three in the morning and two in the afternoon,
totalling 2.5 g per day for 30 days.
The bee pollen was encapsulated in the Pharmaceutical Laboratory of
the Catholic University of Santos.
The ethical aspects were respected in accordance with Resolution
196/96 of the National Health Council referring to the
recommendations on research involving humans.
Samples of the participants’ saliva were collected in the morning
period, before starting the treatment and at the end of the experiment
for salivary cortisol dosage. The participants were asked to continue
to fast and to rest for one hour prior to the collection.
A cotton cylinder previously moistened with saliva was used and
stored in a plastic device (sarstedt salivetteTM). The samples were
refrigerated and immediately delivered for cortisol analysis by the
immunoassay technique using the DSL-10-67100 kit.
The samples were centrifuged for 2 min/1000 rpm. The aliquots of
25µL were transferred to the cavities of microtiter plates coated with
IgG and 100 µL of enzyme conjugate diluent solution (1:50, v/v) was
added to each plate. After being gently rotated, 100 µL of antiserum-
cortisol was added, and then the plate was incubated for 45 minutes at
25℃ on an orbital shaker at 300 rpm.
After washing the cavities with a specific solution, a chromogen
solution of tetramethylbenzidine was added and after a 30 min orbital
agitation (300 rpm), 100 µL of stop solution was added and then the
sample was read by a spectrophotometer Lab systems Multiclan MS at
450 nm.
Th0is experiment consisted in the application of a validated
questionnaire, the “Inventory of Stress Symptoms for adults,”
developed by LIPP [13]. in the professional Fire Service on the first
and the last days of the experiment.
This questionnaire is composed of 37 items of a somatic approach and
19 of a psychological approach, applied and interpreted by a
psychologist, in accordance with the Federal Council of Psychology,
for the classification of a stress phase in which the volunteer was
present for two trials of the study, before consumption bee pollen and
after 28 days, taking the capsules of bee pollen or placebo (oat), daily.
The descriptive statistics and the graphs were performed using
Microsoft Excel for Windows. The student’s t-test was used to
determine the differences between the mean values at 5% significance
level using STATISTICA for Windows, version 5.0 [14].
RESULTS
Table 1 presents the centesimal composition of the pollen, the
presence/absence of phytochemical groups and the total content of
phenolic compounds.
Table 1: Proximal composition and phytochemical screaming of bee
pollen
Chemical composition (%)
Phytochemical groups
Moisture
17.5±0.48
Alkaloids and saponin
glycosides
-
Ash
2.3±0.33
Anthraquinone and
cardioactive glycosides
-
Protein
19.2±0.12
Flavonoids
+
Lipids
6.3±0.24
Tannin
-
Carbohydrates
54.7
Total polyphenols
84.3mg/g
The centesimal composition of the bee pollen has been shown in
accordance with the requirements in Normative Instruction no. 3 of
January 19, 2001 determining the identity and quality parameters of
fresh and dehydrated bee pollen in Brazil [15].
The results were negative for the phytochemical groups with potential
anti-nutritional activity such as tannins, alkaloids, saponin glycosides,
anthraquinone glycosides, cardioactive glycosides, and positive for
flavonoids due to their antioxidant activity. The total phenolic content
was 843 mg/100 g, including flavonoids.
Cortisol is a hormone produced in stressful situations and since the
volunteers are constantly exposed to high-risk activities, we opted for
salivary cortisol research as it is a less invasive method of collection.
The mean salivary cortisol observed in the experimental group before
the BP administration was 19.8±13.9 nmol/L with a minimum of 0
nmol/L and a maximum of 41.4 nmol/L values. For the control group,
before the administration of the placebo, a mean of 16.8±11.3 nmol/L
of salivary cortisol was observed; 2.8 nmol/L and 41.4 nmol/L were
the respective minimum and maximum values.
After the experimental period, the group that consumed the Bee
Pollen showed a significant reduction of salivary cortisol to 12.7±7.3
nmol/L (p= 0.0136), while the control group increased to 19.2± 12.8
nmol/L (Figure 1).

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Figure 1: Salivary cortisol in nmol/L of male individuals before (t0) and after (ft) the consumption of bee pollen (Exp) or placebo (Co) for 30 days (different
letters indicate the statistical differences in the significance level of 5% of probability by student’s t-test.
The salivary cortisol from the experimental group that consumed the
Bee Pollen was also statistically lower than the group that received the
placebo after 30 days (p= 0.454).
According to Lipp [13]. the analysis of LSSI is divided into four
phases: the alert phase (AP) is a positive diagnosis of stress where a
human being adapts themselves to an adrenaline producing action; in
the resistance phase (RP) the organism prevents the total energy waste
and as the individual continues to produce cortisol, they are
vulnerable to infections. In the exhaustion phase (EP) the tension
exceeds manageable limits. The increased cortisol has a negative
effect and diseases may start to arise. The exhaustion phase (EP) is
pathological, which can lead to a big imbalance resulting in
depression, ulcers, hypertension, psoriasis among others somatic or
auto-immune diseases.
The results obtained with the LSSI application showed agreement
with the salivary cortisol measurements.
Before the consumption of the BP, 31% of the individuals from the
experimental group were in the resistance phase, 34% (n=9) were in
the alert phase and 31% (n=8) had no symptoms. After 30 days of
consumption nobody was in the quasi-exhaustion phase, 4% (n=1)
were in the resistance phase, 8% (n=2) were in the alert phase while
88% (n=23) moved to a phase without symptoms (Figure 2).
For the control group, 12% of the individuals were in the quasi-
exhaustion phase before receiving the placebo, 23% (n=6) were in the
resistance phase, 15% (n=4) were in the alert phase and 50% (n=13)
had no symptoms. 30 days after consuming the placebo, nobody was
in the exhaustion phase, while 19% (n=5) were in the resistance
phase, 19% (n=5) were in the alert phase and 62% (n=16) were
classified as symptom free (Figure 2).
Figure 2: Classification of stress phases in volunteers according to LSSI in the experimental group (a) and control group (b) before and after 30 days of using BP,
and placebo respectively. RP - resistance phase; AP - alert phase; QE - quasi-exhaustion phase; NS - no symptoms.

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DISCUSSION
The bee pollen composition may vary according to the processing
technique employed or the flora characteristic of the production
location. In relation to the moisture content, bee pollen collected in
the original form has to contain a maximum moisture reading of 30%
when it comes to dehydrated bee pollen at a temperature higher to
42ºC, the moisture content should not be higher than 4%.
The sample used showed adequacy established by the Brazilian
Legislation, in the case of dehydrated bee pollen, commercialized by a
traditional company in this branch; however, this is commonly sold by
small producers where the standardization of the final product cannot
always be achieved.
Melo, Este vinho and Almeida-Mouradian [16]. analysed different bee
pollen samples collected in several regions of Brazil observing the
presence of many microbial groups, such as molds and yeasts,
coliforms and total bacteria count in a magnitude of up to 104 CFU g-
1. If the humidity of a product like this is above the values
recommended by the current legislation, this contamination can draw
attention to the risk of the development of mycotoxigenic molds.
The total phenolic content found in the bee pollen used was 84,3 mg
GAE/g and this value was close to the interval observed by Altiner et
al [17]. in samples collected in the Turkey. These authors also
demonstrated that hydrolysis, simulating gastric process, rose the
antioxidant capacity, more than obtained by solvent extract.
As far as the benefits of polyphenols are concerned, Sakkara and
Shimon [18]. reviewed a potential anti-stress activity in experimental
animals and human trials where products rich in these compounds has
emerged as essential for prevent stress-related health problems.
The cortisol is a hormone essential to life because it is responsible for
adequate responses to stressful conditions such as serious diseases,
severe traumas and surgery. However, it is harmful to organisms when
continuously released, being responsible for catabolism and
neurophysiological changes [5].
As a strategy to develop a treatment without side effects, functional
foods with anti-stress action have been investigated. Moreover, a milk
protein hydrolysate (LactiumTM) has already been patented as it
demonstrated an anxiolytic effect in animals and human being
volunteers [19].
According to Noah et al [20]. the administration of high levels of
vitamin B6 and magnesium may be expected to have a favourable
impact on stress related with mood and anxiety. The recommended
daily intake of pyridoxine is 1.3 mg for adults and the deficiency of
this vitamin tends to selectively reduce the cerebral production of
serotonin and gamma-aminobutyric acid (GABA) that are crucial for
depression control, pain perception and anxiety.
Mean values of pyridoxine of 0.64 mg/100 g for pollen in natura and
0.55 mg/100 g for dehydrated pollen were found in the bee pollen
produced in the state of São Paulo/Brasil [21].
The results obtained in this study show that the bee pollen reduced the
salivary cortisol concentration after thirty days of intake in adults
submitted to stressful situations. This data is in agreement with the
evaluation obtained with the inventory application.
In addition to an increase in individuals in the classification of
“without symptoms”, the symptoms reported by the volunteers during
the period that they consumed BP were: an improvement in tightness
in the mandible, insomnia, professional performance, sense of
humour, sensation of well-being, greater physical and mental
disposition, facility for thinking, relaxation, tranquillity, motivation,
increase in libido, absence of dizziness and an increase in emotional
control.
CONCLUSION
The outcomes of this study suggest that the consumption of BP results
in a positive effect in the reduction of stress in males who are
constantly submitted to tense situations. Further studies are necessary
that will elucidate the mechanism of action, which may be attributed
to the presence of specific amino acids, flavonoids or vitamins.
Acknowledgements
The authors would like to thank the Fire Service from the
municipality of Santos, to Hexalab Consult Oria me Analyses
Quamina’s Ltda. for the polyphenol analyses and to the pharmacist
Carla Lanza for performing the phytochemical tests.
Conflict of Interest
None declared.
Financial Support
None declared.
ORCID ID
Elizabete Lourenço da Costa: https://orcid.org/0000-0002-7594-5098
José Marcelino Antonio: https://orcid.org/0000-0001-6288-6108
Odilom Pereira: https://orcid.org/0000-0002-1714-2959
Juliana Mesquita: https://orcid.org/0000-0002-1734-3400
Elen Virginia Nascimento: https://orcid.org/0000-0001-8077-1590
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HOW TO CITE THIS ARTICLE
Costa EL, Antonio JM, Pereira OA, Nascimento EVO, Mesquita JT. Proximal
composition of bee pollen and its functional effect on stress. J Phytopharmacol 2022;
11(5):330-334. doi: 10.31254/phyto.2022.11503