Bioavailability of Antioxidant Compounds and Effect on Markers of Inflammation by Ingestion of a Hibiscus Sabdariffa Beverage With Glicemic and Insulinemic Response
Phase: NA
Conditions: Markers of Inflammation, Hyperglycemia, Postprandial, Hyperinsulinism
Sponsor: Instituto Tecnológico y de Estudios Superiores de Occidente

 
 
 
 
 
 
 
 
 
 
OFFICIAL TITLE 
BIOAVALABILITY OF ANTIOXIDANT COMPUNDS AND EFFECT ON MARKERS 
OF INFLAMMATION BY INGESTION OF A HIBISCUS SABDARIFFA BEVERAGE 
WITH GLICEMIC AND INSULINEMIC RESPONSE 
 
 
Study Protocol 
 
 
 
 
 
 
 
 
Date  January 14th, 2021 

 
 
 INTRODUCTION 
 
 
There are numerous plant foods that are a source of bioactive compounds, which 
can induce an anti-inflammatory effect on various pathways of inflammatory 
processes in the body as it may be useful in decreasing markers of inflammation 
expressed by COVID-19 infectious disease and conditions such as obesity and its 
comorbidities. Given the above, Hibiscus sabdariffa hibiscus possesses 
advantages as a potential adjuvant in the management of COVID-19, as studies on 
the phytochemical properties of H. sabdariffa show that it has several health 
benefits, and could be used as a potent material for the therapeutic treatment of 
various diseases. Due to its high content of bioactive compounds, these can exert 
antioxidant, anti-inflammatory and anticarcinogenic effects, as well as help control 
blood glucose levels, prevent cardiovascular disease and obesity. In addition, it is a 
traditional component of the Mexican diet, of common consumption, easy 
incorporation in the diet, versatility in preparation and national production.   
Therefore, to evaluate the bioavailability of bioactive compounds present in a 
beverage developed from the TECNM/ITESO collaboration, as well as the 
glycemic and insulinemic response produced by its consumption; besides 
establishing the effect on some inflammation markers that may be activated as a 
consequence of the SARS-COV-2 virus infection. This will help to increase 
knowledge about potential treatment/prevention schemes, avoid the development 
of severe manifestations of the disease, as well as boost the production and 
market of a national product. 
 
 
 

 
 BACKGROUND INFORMATION 
 
 GENERAL CHARACTERISTICS Hibiscus sabdariffa. 
 
 
Hibiscus sabdariffa L., also known as roselle, karkadé, rosa de jamaica, among 
other names, is an herbaceous medicinal plant, which in Mexico is better known as 
jamaica. It is an annual cultivated shrub that belongs to the Malvaceae family 
(Duarte-Valenzuela et al., 2016), and grows to a maximum height of 3 m, with tri- 
or pentalobed leaves about 15 cm long, alternating on the stem. Its flowers 
measure 8 to 10 cm in diameter, are red at the base and pale at the ends, while 
the calyx is deep red, fleshy and rich in malic acid (Secretaría de Agricultura y 
Desarrollo Rural, 2021). In Mexico, hibiscus calyxes are used in the preparation of 
a refreshing drink widely consumed by the population, which gives it both 
economic and cultural importance (Duarte-Valenzuela et al., 2016), however, 
according to national production is insufficient and at least 50% of the hibiscus 
consumed is imported.  
In Latin America it is used as a food and source of dietary fiber (Da-Costa-Rocha 
et al., 2014). Despite being native to Asia, it is also cultivated in many areas, 
including Central America and Africa. This species is usually cultivated for its fibers 
and calyxes, and includes three different genotypes: green, red which is the most 
commonly used type) and dark red (Andzi Barhé & Feuya Tchouya, 2016). 
Hibiscus calyxes have been recognized as a source of dietary fiber (DF) with 
antioxidant activity (Sayago-Ayerdi & Goñi, 2010) as well as anti-hypertensive, 
anti-hypercholesterolemic and anticarcinogenic effects (Lin et al., 2012). 
The calyx is commercially important in the food industry for the production of foods 
and beverages such as tea, juices, jams, jellies and syrups (Borrás-Linares, 
Fernández-Arroyo, et al., 2015). H. sabdariffa herbal tea is commonly used as the 
sole ingredient for infusions. In many countries, the leaves are also consumed as a 
green leafy vegetable (Zhen et al., 2016). In general, there are several reports 
available on the consumption of the dried or fresh calyxes, seeds and leaves of H. 
sabdariffa used in the preparation of herbal medicines, beverages and fermented 
drinks or even cooked or raw (Da-Costa-Rocha et al., 2014). Hibiscus (Hibiscus 
sabdariffa L.) is an important crop for the tropical areas of Mexico. The area 
planted in Mexico is 20,061.31 ha, from which 7,538 tons of dried calyxes are 
obtained (SIAP, 2019), an amount that supplies less than 50% of the national 
demand; the deficit is imported from countries in Africa and Asia. In Mexico, the 
main hibiscus flower producing states are: Guerrero, Michoacán, Oaxaca, Puebla 
and Nayarit (SIAP 2019). 

 
The state of Nayarit is among the top 5 producers nationwide (SIAP, 2019), with a 
production of 177.60 tons per hectare. Jala, is the municipality that occupies the 
second place in the whole country with a work in 60 hectares harvested, whose 
commercialization is propitiated mainly in the neighboring municipalities of 
Ahuacatlán, Ixtlán del Río, and cities such as Guadalajara and Monterrey. 
Although hibiscus production is of greater importance in the northern part of 
Nayarit, specifically in the municipalities of Huajicori, Rosamorada, Ruiz and 
Acaponeta, the incorporation of its production in the south and center such as La 
Yesca, Amatlán de Cañas, Ahuacatlán, Santiago Ixcuintla, Tepic and Jala, have 
made the latter the best positioned with its variety (SIAP, 2019; Cardona; 2020). 
 
FUNCTIONAL 
PROPERTIES 
OF 
COMPOUNDS 
PRESENT 
IN 
Hibiscus 
sabdariffa. 
 
H. sabdariffa is an important source of CF (Patel, 2014). Many studies have shown 
that H. sabdariffa calyxes contain bioactive compounds (BCs) that are generally 
believed to be the active components responsible for the therapeutic effects in 
such extracts, such as organic acids, anthocyanins, flavonoids, and phenolic acids 
(Sayago-Ayerdi et al., 2021; Ojulari et al., 2019), Table 2.1 presents some of the 
major FCs that make up the extracts of hibiscus calyxes.  
In H. sabdariffa calyxes, their CBs are minority food components, considered non-
nutrients, partially bioavailable in the body and have been shown to have various 
positive effects on consumer health. CF and carotenoids exert their main biological 
action through mechanisms of antioxidation and free radical sequestration, while 
the main effect of phytosterols is through the inhibition of intestinal cholesterol 
absorption (Sayago-Ayerdi & Goñi, 2010). 
On the other hand, the biological activities of phenolic compounds in health 
promotion and disease prevention depend on intake, absorption, transport to target 
organs and metabolism. Despite claims that the health-promoting benefits of 
consuming H. sabdariffa extracts are related to the polyphenol content, there have 
been studies on the pharmacokinetics of H. sabdariffa anthocyanins in humans 
(Frank et al., 2005). Anthocyanins are a group of phenolic compounds found in a 
wide variety of flowers and fruits that exhibit a reddish-purple color and have been 
used as natural alternatives for the replacement of synthetic coloring additives in 
the food industry. The calyx of H. sabdariffa has several anthocyanins Table 2.1., 
these manage to competitively inhibit the angiotensin converting enzyme (ACE) 
such is the example of the inhibition of this enzyme in the lung of a rabbit, in a 
study with H. sabdariffa being this enzyme the main catalyst for the conversion of 
angiotensin I to angiotensin II (vasoconstrictor) leading to an increase in blood 
pressure (BP) (Ojeda et al., 2010). This mechanism of action is supported by 

 
human clinical trials that linked the hypotensive activity of H. sabdariffa extract to 
ACE inhibition and serum sodium lowering (Beltrán-Debón et al., 2015). 
 
Table 
2.1. 
Flavonoid, 
anthocyanin, 
organic 
acids, 
phenolic 
acids 
and 
hydroxycinnamic acids content of calyxes of H. sabdariffa. 
Bioactives 
Content 
Organic acids 
-Hydroxycitric acid, hibiscus acid, hibiscus 
acid dimethylester, 3-deoxy-D-
lixoheptulosaric acid (DHA), hydroxyethyl 
ester of hibiscus acid, 
trimethylhydroxycitric acid 
4000±1000 
(mg/100 g) 
 
Fenolic acids 
- Ellagic acid, ferulic acid, P-coumaric 
acid, 3-O-caffeoylquinic acid, chlorogenic 
acid, neochlorogenic acid, methyl 
digallate, methyl chlorogenate, 
coumaroylquinic acid, dihydroferulic acid 
4-o-glucuronide, 5-O-caffeoylshikimic acid 
ethyl chlorogenate, gallic acid, 
protocatechuic acid glycoside, pyrogallol, 
3,4-dihydroxybenzoic acid, 3, 4-
dihydroxyphenylacetic acid, 3-
hydroxybenzoic acid, 4-hydroxybenzoic 
acid, methyl gallate, 3,4-
dihydroxyphenylpropionic acid, 4-
hydroxyphenylacetic acid, syringic acid, 3-
methoxy-4-hydroxyphenylacetic acid, 5-(3 
', 4' -dihydroxyphenyl)-γ-valerolactone, 4-
hydroxyphenylpropionic acid, 3-
hydroxyphenylacetic acid, 3-methoxy-4-
hydroxyphenylpropionic acid, 3-
hydroxyphenylpropionic acid. 
2400±300-
1000±400 
(mg/100 g) 
 
Hydroxycinamic 
acids 
-3-caffeoylquinic acid, coumaroylquinic 
acid, 5-caffeoylquinic acid, 4-
caffeoylquinic acid, methyl chlorogenate, 
2-O-trans-caffeoyl-hydroxycitric acid, 
caffeic acid, isomer of caffeoylquinic acid, 
coumaroylquinic acid, caffeoyl-
methylquinic acid, coumaric acid, 
caffeoylshikimic acid, feruloylquinic acid, 
ferulic acid 
 

 
Flavonoids 
-Quercetin, Luteolin, kaempferol, 
Myricetin, Epigallocatechin, Glycosides, 
Hibiscitrin (hibiscetin-3-glucoside), 
Gossypetin, Kaempherol-3-sambubioside 
(Leucoside), Myricetin-3-sambubioside, 
Quercetin-3-sambubioside Quercetin-3-O-
glucoside, (Isoquercitrin) Quercetin-3-O-
galactoside, Kaempherol-3-O-ruthinoside, 
Methyl-epigallocatechin Kaempferol-3-
glucoside, Kaempherol 3-O-(6′′-O-acetyl) 
glucoside, Syringetin-3-O-glucoside 
Kaempherol-3-p-coumaroylglucoside. . 
419±2 - 
2260±70 
(mg/100 g) 
Antocianidins  
- Delphinidin, hibiscentin cyanidin, 
hibiscin, delphinidin-3-glucoside, cyanidin-
3-glucoside, delphinidin-3-sambubioside 
cyanidin-3-sambubioside. 
115 ± 259  
(mg/100 g) 
Source (Borrás-Linares, Herranz-López, et al., 2015; di Lorenzo et al., 2021; Jabeur et al., 2019; 
Pimentel-Moral et al., 2018; Widowati et al., 2017) 
 
Therefore, the biological properties of the antioxidants found in hibiscus depend on 
their bioaccessibility and bioavailability. 
 
This is why in vivo studies of FC bioavailability are becoming more common and 
necessary, as this allows us to show beyond the detailed characterization of the 
compounds present in the various extracts. In the case of hibiscus, there are 
studies that have shown, both in hypercholesterolemic animals and in animals fed 
a high-fat diet, how the intake of the compounds present influences the decrease in 
markers of oxidative stress and in expression of satiety hormones (Moyano et al., 
2016; Sayago-Ayerdi et al., 2014), On the other hand, it has been until the 
characterization of hibiscus calyx extracts from improved varieties and the in vitro 
predigestion of these calyxes, that it has been possible to establish a wide 
percentage of compounds present in Hibiscus capable of being bioaccessible 
(Sayago-Ayerdi et al., 2021). In this analysis, 49 compounds were obtained, of 
which five are organic acids such as Hibiscus, which is found in high concentration 
in the characterization but, after in vitro digestion, less than half remains, indicating 
that it may be bioavailable; Something similar happens with hydroxycitric acid, but 
this bioaccessibility is even more marked in the case of flavonoids such as 
quercertin-3-sambubioside or kaempferol-3-glucosides, which are not even 

 
detected after in vitro digestion. Likewise, anthocyanins such as delphinidin-3-
sambubioside contained in hibiscus are almost totally bioaccessible after digestion 
(Sayago-Ayerdi et al., 2021). This indicates the potential bioaccessibility of the 
phenolic compounds present in hibiscus calyxes that are the product of decoction. 
 
BIOAVAILABILITY OF BIOACTIVE COMPOUNDS BY CLINICAL TRIALS 
 
 
In vitro models provide a first and valuable insight into the transformations of 
bioactive compounds (BCs) during their gastric processing before reaching the 
target tissues. Human and animal models have been used to study the 
bioavailability and metabolism of BCs by in vivo assays. Each model has its 
advantages and disadvantages, some are the established ethical aspects, the 
Declaration of Helsinki (Osuna et al., 2016) must be taken into account, in addition 
to the possible ethical requirements of each institution and country. For human 
models, the informed consent of the volunteers is required (Fernández-Ochoa et 
al., 2022). The first step metabolism consists of the bioactive compound crossing 
the intestinal barrier before reaching the systemic circulation, at this point it is 
crucial to take into account whether it is an oral intake, whether the CB is free from 
the food matrix, encapsulated or in emulsion because it has to be able to cross the 
epithelial barrier. The second step is bioavailability, many researches focus on 
assessing the bioavailability of CBs, this means the proportion of an administered 
CB that is able to reach the systemic circulation and is a function of the opposite 
processes of absorption and clearance and can be calculated by considering the 
area under the curve (ABC) of the initial concentration of the CB between the 
amount found in the ABC at the intravenous level. The bioavailability of CBs, 
mainly phenolic CF compounds, is highly variable and it has been reported, for 
example, that flavonoids are very poorly absorbed glycosides that require the 
presence of β-glucosidases enzymes found in the small intestine before being 
absorbed as aglycones (Ávila-Román et al., 2021). 
The main objective of bioavailability studies is to determine the formation of active 
metabolites from CB after absorption. It is important to evaluate whether 
metabolism has an effect on their biological activities, rather than the initial CB. 
Once absorbed in the liver, CBs are subjected to undergo different bioconversion 
reactions, from which they go on to produce metabolites according to two phases, 
phase I and II. Within these phases, conjugation reactions such as methylation, 
sulfation and glucuronidation take place, which facilitate their elimination via the 
biliary and urinary tract. Methylation reactions are catalyzed by catechol-
methyltransferase with S-adenosyl-L-methionine as the methyl donor (Lee et al. 
2002). Sulfation is catalyzed by sulfotransferases where 3'-phosphoadenosine-5'-

 
phosphosulfate is the sulfate group donor (Falany, 1997). CF themselves and their 
derived metabolites are transported to the liver by portal circulation and can be 
transferred back to the intestine through the enterohepatic cycle (Figure 2.1.) 
(Ávila-Román et al., 2021). 
The bioavailability of BCs, mainly FCs, can be evaluated by assessing their 
metabolites in plasma and urine samples after ingestion of them in pure form, 
injected or as nutraceuticals. It can be assessed indirectly by the antioxidant 
capacity of plasma after consumption. They can also be evaluated by a single dose 
by acute assays, where in these assays the concentration of phenols increases in 
plasma samples transiently, and the capacity of the organism to take up these 
compounds from the food matrix is reflected. However, it is highly recommended to 
evaluate the entire diet of volunteers to whom such assay will be conducted and 
the use of repeated doses (Pandareesh et al., 2015). 
The bioavailability and metabolism of phenolic compounds is a key point that 
needs to be investigated for a better understanding of the biological mechanisms of 
action, enabling the development of better applications based on these 
compounds. In recent years there has been an increase in this type of studies, 
which have succeeded in identifying new bioavailable metabolites in different 
biological matrices. Phase II metabolism, as well as colon metabolism, is the most 
prominent of these advances. All these advances have been made possible by the 
continuous progress of analytical and metabolomic approaches. 
 
. IMMUNOMODULATORY RESPONSE OF BIOACTIVE COMPOUNDS  
 
 
Inflammation is the response of an organism's immune system to damage caused 
to its cells and vascularized tissues by bacterial pathogens and by any other 
biological, chemical, physical or mechanical aggressor (García Barreno, 2008). 
Inflammation constitutes a non-specific response of the immune system to 
causative agents of diverse nature, which determine physiological changes that 
include increased blood flow and vascular permeability with the release of 
mediators derived from arachidonic acid, modified amino acids and cytokines, 
which amplify the process, while anti-inflammatory molecules (IL10, IL4, TGFβ, 
IL13, GC) are regulators of these events. Cytokines are a diverse group of proteins 
that include monokines, lymphokines, interleukins, interferons, colony-stimulating 
factors, and chemokines. They are small non-antibody proteins that are 
synthesized "as needed" during the immune response, usually produced as 
precursor forms by activated cells that mediate the inflammatory process 
(Brzustewicz & Bryl, 2015). 

 
Proinflammatory cytokines are referred to as those that promote inflammation, with 
IL-1, IL-6 and TNF-α being prototypical. They act as endogenous pyrogens, induce 
the synthesis of secondary mediators and proinflammatory cytokines by 
macrophages and mesenchymal cells, stimulate the production of acute phase 
proteins and attract inflammatory cells. The net effect of an inflammatory response 
is determined by the balance between pro- and anti-inflammatory cytokines (IL-4, 
IL-10 or IL-13). Activated macrophages produce, in response to pathogens and 
other damaging stimuli, tumor necrosis factor (TNF), a cytokine with a relative 
molecular mass of 17 kDa that behaves as a necessary and sufficient mediator of 
local and widespread inflammation. TNF amplifies and prolongs the inflammatory 
response by activating other cells which, in response, release cytokines such as 
interlucin 1 (IL-1) and high mobility group protein 1 and amphoterin (HMGB1), and 
other mediators such as eicosanoids, nitric oxide and reactive oxygen species, 
which enhance inflammation and cause tissue injury (Garcia Barreno, 2008). 
Although it is true that the inflammatory process is essential for the survival of 
organisms, given the enormous number of existing environmental pathogens, this 
reaction capacity can often be exacerbated, generating morbidity and mortality due 
to the generation of a series of diseases. Therefore, it is important to maintain a 
balance between inflammation and anti-inflammation, where food is a key factor 
that can contribute to this (Caballero-Gutiérrez & Gonzáles, 2016). Phytochemicals 
present in food, in their original form or when metabolized, can form active 
metabolites and behave as intracellular messengers activating or inhibiting the 
expression of genes involved in inflammatory processes. One of the diets that 
meets this characteristic is the Mediterranean diet, rich in vegetables, legumes, 
fresh and dried fruits, cereals, low in saturated fats, constant in wine, olive oil and 
fish, which promotes the reduction of cardiovascular events, cancer and chronic 
diseases. The families of the main phytochemicals with anti-inflammatory 
properties 
are 
carotenoids, 
phenolic 
compounds, 
alkaloids, 
nitrogenous 
compounds and organosulfur compounds (Caballero-Gutiérrez & Gonzáles, 2016). 
In 2019, the World Health Organization announced the existence of the infectious 
disease called COVID-19, caused by the SARS-CoV-2 virus, after an outbreak in 
Wuhan, China; subsequently, COVID-19 pandemic was declared on March 11, 
2020 (López Pérez et al., 2020). SARS-CoV-2 is the causative virus of COVID-19 
disease, which causes a predominantly respiratory syndrome and manifests with a 
broad spectrum ranging from moderate to severe clinical complications and even 
death. The pathogenesis of SARS-CoV-2 is complicated and multifactorial and 
involves both viral and host factors. Currently, nutritional deficiencies have been 
identified as being related to increased host susceptibility, which places the host in 
a situation where the pathogen can cause greater damage and progress to more 
severe levels of disease. Deficiencies of some nutrients are related to the 
exacerbated response of tissue-resident innate immune cells, which activates 
phagocytic function and signals triggering the inflammatory cascade. Likewise, 
malnutrition at the cellular level negatively affects several aspects of adaptive 

 
immunity, including cytokine production, as well as antibody- and cell-mediated 
immunity of the immune system (Kunnumakkara et al., 2021). 
The mechanism of SARS-CoV-2 infection begins when the virus enters the human 
body and binds to the angiotensin-converting enzyme 2 (ACE2) protein receptor. 
This group of proteins is widely expressed in the respiratory system, mainly in type 
II alveoli, oral mucosa, nasal epithelium and other organs. In addition to the 
respiratory tract, it can (adhere, infiltrate, bind, infect) other organs such as the 
cornea, heart, kidney, among others.  Similarly, the virus binds to the 
transmembrane serine type II protease (TMPRSS2). A wide expression of this 
TMPRSS2 protein is reported in the nasal cavity, lungs, colon, gall bladder, kidney, 
prostate, pancreas, heart, among others and that is why it is susceptible to the 
virus (Sungnak et al., 2020). The cytokine cascade is positively correlated with the 
severity of infection, with the magnitude and severity of damage and with the risk of 
death. On the other hand, elevated levels of same cytokines such as IL-2R, IL-6 
and IL-10 have been identified in subjects with COVID-19. In addition, in severe 
and advanced cases, a gradual reduction in absolute CD4+T, CD8+T, and B-cell 
counts has been found. A retrospective analysis in patients with COVID-19 and 
pneumonia demonstrated increased expression in serum samples of IL-6, 
interleukins, IL-10, IL-2, and interferon, as well as a decrease in CD3+ and CD4+, 
NK, and CD8+ (Kunnumakkara et al., 2021). 
Certain benefits are more noticeable in cultures such as Asian cultures where the 
consumption of spices and various foods rich in phenolic compounds (PC) is very 
common in the diet. There are studies that attribute to these compounds the 
inhibition of infection by preventing the entry of the virus into the organism, such as 
the case of luteolin and quercertin in cultures of Vero E6 cells with an EC50 of 
10µM and 83µM, respectively, where it was found that luteolin presented a high 
affinity for the protein present in SARS-CoV-S (Zhou et al., 2020). Likewise, 
another assay with resveratrol, which is found in grape skins, showed that it can 
modulate the severity of COVID-19 through regulating ACE2 expression and 
function (Horne & Vohl, 2020). The search for various CF-rich plants is a good 
alternative as an adjuvant to COVID-19 infection. However, it has the disadvantage 
that the extracts are not fully characterized to be able to identify the potential that 
these foods possess. The biological activity of FCs depends on their bioavailability 
and metabolic rate, as well as their digestive accessibility, which is determined by 
their release from the food matrix and their transepithelial passage efficiency. Many 
phenolic compounds can also be dose-dependent and can be recovered in urine in 
various metabolites (Mateos et al., 2016). This is why hibiscus extracts have ample 
percentage of bioaccessible compounds after digestion and this indicates that 
there may be a correlation between immune response and severity of progression 
with COVID-19 disease. There are numerous fruits, species and plants, as well as 
isolated bioactive compounds, which are the target of various pathways of 

 
inflammatory processes, and which induce an anti-inflammatory effect in several 
chronic diseases, such as obesity, dyslipidemias and diabetes mellitus. 
 
HYPOGLYCEMIC EFFECT OF HIBISCUS 
 
One of the physiological mechanisms benefited by the consumption of hibiscus is 
the hypoglycemic effect that it is capable of producing by the action of its nutritional 
and functional components, positioning hibiscus as a beverage capable of reducing 
the incidence and maintaining glycemic control in individuals with type 2 diabetes 
mellitus (DM2), since it has been stated in various studies that low consumption of 
fiber and antioxidants, accompanied by high consumption of sugars, are the main 
causes that lead to its development (Mayasari et al., 2018), together with physical 
inactivity, excess body weight and genetic inheritance (Uyaguari-Matute et al., 
2021). 
Of course, Hibiscus sabdariffa L. has been catalogued as a functional food with a 
great hypoglycemic potential, being this granted by its abundant content of 
bioactive compounds (Villanueva-Carvajal et al., 2013), mainly organic acids, 
anthocyanins, flavonoids and polyphenols (Bule et al., 2020). 
Among the wide variety of studies focused on the therapeutic properties of hibiscus 
are in vivo assays. Janson et al., (2021) focused on the glycemic response of 
obese rats when fed a high-fat diet accompanied by an aqueous extract of hibiscus 
in two proportions (250 and 500 mg/kg body weight) for 8 weeks, the result of 
which was a decrease in fasting blood glucose values, serum insulin and the value 
of the homeostatic model for evaluating insulin resistance (HOMA-IR). They also 
observed that postprandial glucose levels after 2 h were significantly reduced, in 
addition to showing a lower area under the curve than that of the control group. 
Thus, they claim that hibiscus is able to improve insulin sensitivity, which leads to 
increased glucose uptake in muscle and adipose tissue, thus reducing insulin 
resistance. A study with similar conditions (experimental model and high-fat diet) 
showed similarly favorable results, since the dose used of Hibiscus sabdariffa 
(HS), and the mixture of HS with agave fructans, decreased glucose concentration 
due to the increase in glucagon-like peptide type 1 (GLP-1) values after 
supplementation (Moyano et al., 2016). 
Among the clinical studies conducted, those where the population is represented 
by diabetic individuals stand out, where hibiscus tea was administered (4 g. of 
hibiscus distributed in 4 intakes) was administered to elderly women with 
hypertension and diabetes (following their pharmacological treatment without 
modifications) which showed as a result the reduction of fasting blood glucose 
levels, which is attributable to the reduction of cortisol, it should be noted that this 

 
hormone plays an important role in the homeostatic control of blood glucose by 
activating gluconeogenesis (Yusni & Meutia, 2020). On the other hand, Sarbini et 
al., (2019) presented contradictory results to the aforementioned, since the values 
of blood glucose, fasting insulin, and HOMA-IR, showed no significant differences 
between the placebo group and the HS group (administration for 8 weeks of 50 mg 
of pure extract in 2 capsules), although this is attributed to the consumption of 
antihypertensive drugs and failure in the effective dose. For this reason, it is 
preferable to perform clinical studies in apparently healthy people, in order to have 
a clearer answer about the hypoglycemic effect of hibiscus, without the intervention 
of pharmacological factors involved. 
Similarly, Mayasari et al., (2018) focused their research on prediabetic women 
reporting the decrease in fasting blood glucose levels, after administration of 
Roselle-Stevia tea bags (5 g and 125 mg, respectively) twice daily for 14 days. 
Therefore, they assure that the amount provided is the effective dose for the 
reduction of fasting and two-hour postprandial glucose values in prediabetic 
individuals.  
Likewise, several authors suggest that the antiglycemic effect provided by the 
existing compounds in hibiscus, may follow several mechanisms including 
inhibition of α-amylase and α-glucosidase enzymes, decrease of advanced 
glycation end products in plasma and reduction of oxidative stress caused by 
hyperglycemia (Bule et al., 2020), it is also able to increase insulin secretion and 
improve insulin resistance in diabetic individuals (Sarbini et al., 2019). 
The aforementioned biological activity is dependent on the bioaccessibility and 
bioavailability of bioactive compounds, given that a considerable amount of them 
are bound to dietary fiber, as mentioned by Sáyago-Ayerdi & Goñi (2010) in their 
research when they named this interaction as "antioxidant dietary fiber", generating 
a favorable added value for human health when released during gastrointestinal 
digestion (Moyano et al., 2016). Villanueva-Carvajal et al., (2013) stated that the 
breakdown of the food matrix (calyxes of Hibiscus sabdariffa) facilitates the 
exposure of the functional groups of phenolic compounds allowing to exert the 
antioxidant effect, which they hypothesize may be generated by the high 
concentration of organic acids, such as hydroxycitric acid and hybisic acid, the 
above was confirmed after a simulation of gastrointestinal digestion in vitro. 
One of the mechanisms by which hibiscus induces a hypoglycemic effect is 
through the inhibition of the aforementioned enzymes, which are related to 
carbohydrate digestion. As background we have the study conducted by 
Gondokesumo et al, (2017) where they point out that at a concentration of 37.5 µM 
of a methanolic extract of Hibiscus sabdariffa is able to inhibit the action of the 
enzyme α-glucosidase (IC50: 15.81 ± 0.78 µg/mL) while the mean inhibitory 
activity of the enzyme α-amylase was 18.09 ± 0.28 µg/mL at a concentration of 
34.29 µM, this effect is attributed to the high concentrations of flavonoids in 

 
hibiscus. Another previous study (Alegbe et al., 2019) points out that the aqueous 
extract of hibiscus administered to diabetic mice at 100, 200 and 400 mg/kg for 14 
days inhibited α-amylase and α-glucosidase enzymes considering an IC50 of 
490.79 and 475.31 µg / ml, respectively; this fact was reflected in the decrease of 
blood glucose values. They also performed an isolation of protocatechinic and 
gallic acids with ethyl acetate, proving that these compounds exert an inhibitory 
activity of the enzymes in question, besides being able to reduce blood glucose 
values and oxidative stress. Another relevant aspect is the capacity that these 
compounds showed for the regeneration of the islets of Langerhans of the 
pancreas, which coincides with that published by Adeyemi & Adewole (2019) who 
report that the methanolic extract of Hibiscus sabdariffa L. which they fed to rats 
with type I diabetes induced β-cell proliferation, as well as pancreatic islet volume, 
area and diameter, which translated into increased insulin secretion and enhanced 
glucose homeostatic control. Added to this study, the hibiscus extract reflected an 
improvement in the maintenance of body weight of the rats, even after the 
treatment was withdrawn, contrary to the control modulated with insulin protamine 
zinc, which continued with a reduction in weight when withdrawn. 
 
DIGESTION AND ABSORPTION OF CARBOHYDRATES.  
 
The hypoglycemic effect that hibiscus is capable of producing is related to the 
digestion and absorption of carbohydrates. These two mechanisms regulate the 
decomposition of food into small particles capable of crossing the intestinal lumen 
and reaching the portal system for subsequent distribution in the human body, 
contributing to a myriad of energy-demanding reactions. In this sense, nutrients are 
obtained from food through a series of processes in the gastrointestinal tract, 
where carbohydrates stand out as the major source of energy (Nadia et al., 2021). 
Carbohydrates are hydrolyzed by enzymes to produce monosaccharides or 
disaccharides, which are absorbed in the small intestinal epithelium through 
passive mechanisms, as in the case of fructose, and active mechanisms, for the 
absorption of glucose and galactose (de Koning et al., 2020). 
Carbohydrates can be ingested in the form of complex structures, such as 
polysaccharides containing very long carbohydrate chains (starch), as well as 
simpler structures, such as disaccharides (lactose, sucrose, maltose and 
trehalose) and monosaccharides (glucose, galactose and fructose) (McQuilken, 
2021). 
The digestion of this macronutrient is achieved in two stages, the first corresponds 
to the intraluminal phase, which is mediated by salivary and pancreatic amylase, 
and the second, by the mucosal phase where an interaction with the apical surface 

 
of the enterocyte occurs (de Koning et al., 2020). Regarding the luminal phase, this 
begins in the mouth where carbohydrates begin to be assimilated by the reaction 
carried out by the enzyme α-amylase, or also known as ptyalin, which is produced 
by the salivary glands, in order to hydrolyze the α (1,4) bonds of linear glucose 
polymers, obtaining as a product smaller oligomers, such as maltose, maltotriose 
and α-limit dextrins (Sanders, 2016). It should be emphasized that this enzyme 
leaves intact α-bonds (1,6), α-terminal bonds (1,4) and also those that are close to 
branch points. In addition, α-amylase is usually inactivated by the modification of 
gastric juices on the pH of the stomach, so that carbohydrate digestion is 
incomplete, continuing until it reaches the small intestine. 
As the pH of the chyme is neutralized in the small intestine by the action of 
bicarbonate, pancreatic α-amylase begins to be released to continue the chemical 
digestion of carbohydrates.  
The products of the previous reactions carried out by the salivary and pancreatic α-
amylase enzymes are again hydrolyzed on the apical surface of the enterocytes, 
although this time the enzyme complexes responsible are glucoamylase, 
saccharase-isomaltase and glucosidases. Glucoamylase goes on to hydrolyze the 
α (1,4) bonds at the non-reducing end of maltotriose and α-limiting dextrins, 
resulting in glucose and isomaltose monomers. The latter is not able to be 
hydrolyzed by this enzyme due to its inability to break α (1,6) bonds, giving rise to 
the saccharase-isomaltase enzyme, which is divided into 2 subunits, one of which 
is saccharase-maltase, responsible for catalyzing the digestion of sucrose and 
maltose, obtaining as product the monosaccharides that compose it; and the 
second subunit isomaltase-maltase, in charge of digesting isomaltose and maltose. 
And finally, there are the enzymes α and β Glucosidase, the first catalyzes the 
hydrolysis of terminal α-linked D-glucose residues (1,4) next to the non-reducing 
ends, thus releasing β-glucose. It should be noted that this enzyme is capable of 
hydrolyzing α (1,6) linkages, thus marking the end of the digestion of the 
macronutrient of interest. On the other hand, the enzyme β-Glucosidase is 
responsible for catalyzing the hydrolysis of the β (1,4) linkage of lactose, to obtain 
glucose and galactose, as well as breaking down glycolipids, obtaining a 
monosaccharide, such as glucose or galactose, and ceramide (Sanders, 2016). 
 
Regulation of plasma glucose levels. 
 
As a consequence of food consumption, blood glucose levels rise in response to 
carbohydrate consumption, which is influenced by the type of carbohydrate, the 
elimination of glucose from the bloodstream and the interaction with other food 
components (Sanders, 2016), so control systems are required to detect these 
changes in order to maintain blood glucose within normal values. Some of these 

 
processes is the secretion of insulin and glucagon, these mechanisms are 
synchronized which allows maintaining stable blood glucose levels during the 
interprandial period (Pinés Corrales et al., 2020). 
Insulin is a polypeptide of 51 amino acids distributed in 2 chains, which are linked 
by different disulfide bridges (5.8KDa) (Gómez Ayala, 2008). It is synthesized by 
the β cells of the Islets of Langerhans in the pancreas. Insulin action is mediated by 
intracellular signaling cascades, mainly by the entry of glucose into the β-cells of 
the pancreas through GLUT4 transporters (Sanders, 2016), which leads to 
increased metabolism causing elevation of the ATP/ADP complex, and 
consequently inhibition of the K+-ATP channel causing depolarization of the cell 
leading to an influx of Ca2+ and insulin secretion. Once released into the 
bloodstream, insulin binds to its receptor, which is composed of an extracellular α-
subunit, which receives insulin, and a transmembrane β-subunit, which translates 
the signal through tyrosine kinase. As a consequence of the binding of insulin to its 
receptor, the signaling cascade is activated, managing to carry out its function by 
counteracting the action of hormones that cause hyperglycemia, and the regulation 
of glucose metabolism (Bonilla-Ocampo, 2014). In the presence of hypoglycemia, 
there are several response mechanisms: the first is the decrease in insulin 
secretion, the second physiological defense is the secretion of glucagon and the 
third is the increase in epinephrine secretion (Nares, M., Gonzalez et al., 2018). 
Glucagon is a polypeptide made up of 29 amino acids, which is synthesized and 
secreted by the α cells of the Islets of Langerhans in the pancreas. Its release 
responds to plasma glucose levels lower than physiological concentrations by 
stimulating glycogenolysis, i.e. the hydrolysis of glycogen into glucose. Therefore, it 
is evident that glucagon and insulin have antagonistic effects. 
There are extrinsic and intrinsic factors that modify the postprandial glycemic 
response, and therefore the glycemic index of foods, summarized in the physical 
form of the food (potato), the preparation and cooking used (raw and cooked 
carrot), the maturity of the fruits (banana), the production and manufacturing 
process of the products, the macronutrient mix (joint consumption of proteins and 
fats), the acidity of the food, as well as the consumption of fiber (Dávila, 2016). 
The concept of the glycemic index has been expanded over the years, giving rise 
to mathematical products such as the glycemic load (GL), which is defined as the 
quantity and quality of carbohydrates consumed and estimates the total glycemic 
effect of the diet, for its obtaining it is necessary to multiply the amount of CHO 
contained in the food ingested by the GI value (GL = GI x available carbohydrates). 
The increase to the glycemic load is associated with an unfavorable physiological 
response, mainly for people living with diabetes, since blood glucose and insulin 
values rise sharply. Foods with GL ≤10 are considered low GL and foods with GL ≥ 
20 are considered high GL. 

 
 
OBJECTIVES 
 
 
 
General objective  
 
To evaluate the in vivo bioavailability of bioactive compounds from a H. sabdariffa 
beverage and their immunomodulatory potential against moderate inflammation 
caused by SARS-CoV2 as well as enzymatic inhibition of α-amylase and α-
glucosidase enzymes in vitro.  
 
Specific objectives  
 
1. To perform HPLC-DAD-MS characterization of a Hibiscus sabdariffa hibiscus-
based beverage. 
 
2. To perform an acute single-blind clinical trial, administering a hibiscus drink to 
normal-weight adults. 
 
3. To evaluate the bioavailability of organic acids and phenolic compounds in urine 
and plasma samples present in the H. sabdariffa beverage. 
 
4. To evaluate the immunomodulatory potential of the metabolites identified as 
bioavailable in plasma samples.   
 
5. To evaluate the enzymatic inhibition of α-amylase and α-glucosidase exerted by 
the digested, placebo and treatment beverages. 
 
6. To evaluate the in vivo GR and IR of the placebo beverage and treatment 
beverage. 
  

 
. GENERAL METHODOLOGY 
 
 
First Stage  
 
 
Obtaining hibiscus-based beverage and placebo. 
 
The hibiscus-based beverages (H.sabdariffa) and placebo were obtained from the 
reformulated company NUTRIYÉ SAPI de CV, located in the city of Guadalajara, 
Jalisco, Mexico.  
 
 
Participants. 
 
Twelve normo-weight volunteer participants without any medical condition were 
included. The invitation to participate was made through ITESO's communications 
office, as well as through announcements in social networks. They were selected 
according to the following criteria: 
Within the inclusion criteria to be considered are those mentioned in Table 4.2. 
  
 
Table 4.2. Non-inclusion and inclusion criteria for volunteer selection. 
Inclusion criteria  
Non-inclusion criteria 
Normolipidemia 
Total cholesterol 
(plasma) < 200 
mg/L-1 
Hypertension 
Hyperthyroidism 
Triglycerides < 150 
mg/L-1 
Diabetes 
In diet 
 
Fasting glucose 
(plasma) < 100 
mg/L-1 
Smokers 
Pregnant or lactating 
women 
Normoglycemia 
18.5 – 24.9 
Cardiovascular 
disease 
In medical treatment / 
supplements 
BMI [kg/m2] 
18 – 40  
Secondary 
Hepatic insufficiency 

 
hyperlipidemia 
Abdominal 
circumference 
[cm]. 
Men<90 Women < 
80 
Renal 
insufficiency 
Infection / 
inflammatory disease 
(6 weeks prior to 
study) 
Fat percentage 
[%] [cm 
Men<19 Women < 
33 
 
 
Systolic pressure 
[mmHg]. 
< 140 
 
 
Diastolic pressure 
[mmHg]. 
< 90 
 
 
Alcohol  
Last consumption 
one week and a half 
 
 
 
 
The study adhered to the ethical principles for research on human subjects of the 
Declaration of Helsinki and was submitted to the ITESO Research Ethics 
Committee. 
 
 
Acute Trial Clinical Intervention  
 
An acute single-blind trial was conducted. Participants consumed a hibiscus-based 
drink (H. sabdariffa), or a 60ml placebo drink, and then received a Western-type 
diet characterized by excessive consumption of red meat, simple carbohydrates, 
ultra-processed products, colorants, saturated fats, refined sugars and very little 
intake of fiber and vitamins. After a washout period of more than 6 days, the 
participants underwent a second intervention with a different product from the first 
one (beverage to be evaluated or placebo, as appropriate), and the samples were 
repeated according to the following scheme 
 
 

 
 
The protocol was previously explained to the volunteers and all those who give 
their written informed consent will be candidates to participate (Annex 1). On the 
day of the intervention, participants will arrive at 7 a.m., Mexico Central Time, with 
an overnight fast of 10-12h. They were requested a rest period of 30 min prior to 
blood pressure taking at the time of admission, in addition to the fact that they had 
to be seated, upright and with their feet on the floor during the process; the 
measurement was performed using a manometer on the left arm, elevated at the 
level of the heart (Gómez-León Mandujano et al., 2016). 
Two types of sampling were performed: 
(a) Urine. 
 
For determination of phenolic compounds and organic acids in urine samples, a 
basal sample (T=0) was taken in a collection bottle. Once the hibiscus-based drink 
or placebo was ingested, samples were collected at the following times: 3h, 6h, 
12h and 24h. 
 
b) Blood 
 
For the studies of inflammation levels (IL-6 and TNF-a), and for the bioavailability 
of phenolic compounds and organic acids, blood samples were taken with the help 
of collecting tubes by venous puncture. Eight samples were taken, the first on 
arrival of the patients at basal time (T=B), then at 30 min (T=2), 1h (T=3), 1:30h 
(T=4), 2h (T=5), 3h (T=6), 4h (T=7) and 6h (T=8) after having ingested the 
hibiscus-based drink or the placebo. 
In both interventions, patients received a Western diet (breakfast and lunch) 
characterized by excessive consumption of red meat, simple carbohydrates, ultra-
processed products, dyes, saturated fats, refined sugars and very little intake of 
fiber and vitamins, adjusted to their physical characteristics, and supervised by a 
team of specialist nutritionists. The participants remained at the trial site without 
any type of physical exercise during the course of the study and in a relaxed 
environment with selected recreational activities; they also had a water fountain 
within their reach from which they could drink indefinitely and a team of medical 
services that was located at the trial site until its completion. 
 
Sample handling. 

 
 
For biochemical tests, plasma samples were separated by centrifugation at 3000 
rpm for 10 minutes at 4 °C and divided into aliquots and stored at -20 °C until 
further analysis. On the other hand, urine samples were divided into aliquots and 
frozen at -20 °C until further analysis. 
 
Extraction of phenolic metabolites and organic acids from biological samples. 
 
A liquid-liquid extraction and protein precipitation with acetonitrile and methanol 
was used to isolate metabolites from plasma following the procedure described by 
(Day et al., 2001; Gomez-Juaristi et al., 2018). with some modifications. A thawed 
plasma sample of 400 μl was acidified with 10 μl of 50% (v/v) aqueous formic acid. 
After shaking the aqueous mixture, it was added dropwise to 900 μl of cold 
acetonitrile containing 10% (w/v) ascorbic acid and shaken three times for 20 s 
before centrifugation at 14000 rpm for 10 min at 4 °C. The supernatant was 
decanted and the pellet was again extracted with 900 μl of methanol. After 
centrifugation (14000 rpm, 10 min 4 °C), the two supernatants were combined and 
dried under a stream of nitrogen at 30 °C. The dried samples were resuspended in 
150 μl of aqueous formic acid (0.1% v/v) containing 10% acetonitrile acidified with 
0.1% formic acid and centrifuged at 4°C for 15 min at 14000 rpm. The final 
supernatant was collected, filtered (0.45 μm pore size, cellulose acetate membrane 
filters, millipore) and 30 μL was analyzed by HPLC-DAD-MS. Urine samples were 
diluted with an equivalent volume of HPLC-grade water and centrifuged at 14000 
rpm (10 min, 4 °C). The supernatants were filtered (cellulose acetate membrane 
filters with a pore size of 0.45 μm, millipore) and a 5 uL aliquot was directly injected 
into the HPLC-DAD-MS apparatus. 
 
Characterization and quantification of phenolic compounds and organic acids from 
H. sabdariffa hibiscus-based beverage and biological samples by HPLC-DAD-MS. 
 
The phenolic fraction and organic acids of the H. sabdariffa hibiscus-based 
beverage and the placebo beverage provided by Nutriyé were characterized. 
Where an aliquot of each beverage was filtered through a 0.45 μm millipore pore 
size filter and separately injected under gradient conditions into an HPLC high-
performance liquid chromatograph coupled to an Agilent 1260 series mass 
spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with a UV-
Vis diode array detector (DAD). 

 
Metabolite identification in plasma and urine samples was carried out in the same 
system performed by the following methodology according to Mateos et al., (2016), 
using 30 µl of plasma and 5 µl of urine were injected separately under isocratic 
conditions in a high-performance liquid chromatograph (HPLC-DAD-MS) coupled 
to a mass spectrometer (Agilent 1260). Water (Phase A) and acetonitrile (Phase B) 
acidified with 0.1% formic acid at a flow rate of 0.4 ml/min were used as mobile 
phase. The phase was initially programmed with 95% of solvent A and then 5% of 
B. The elution program was increased to 10% of solvent B in 10 min and 60% of 
solvent B in 5 min, which was maintained for 2 min. The initial conditions (5% of 
solvent B) were recovered 1 min and maintained for 2 min. The conditions for the 
mass chromatograph were as follows. Identification of CF and organic acids was 
carried out using an Agilent 1260 series HPLC system (Agilent Technologies, 
Santa Clara, CA, USA) equipped with a UV-Vis diode array detector (DAD). 
Samples were injected onto a Poroshell 120 EC-C18 column (4.6 mm × 150 mm, 
particle size 2.7 μm) (Agilent Technologies). Detection will be performed in a 
specific nm range. For MS analysis, an Agilent Quadrupole LC / MS 6120 
equipped with an electrospray ionization interface in negative ionization mode will 
be used with specific conditions of: drying gas flow; nebulizer pressure; gas drying 
temperature; and capillary voltage. Data analysis was performed using OpenLab 
CDS ChemStation Edition software (Agilent Technologies, Santa Clara, CA, USA). 
Characterization was based on retention time and mass spectrometry data. 
Compounds were first detected using a single MS scan in the specific range (m/z), 
followed by a targeted search based on peaks showing major signals in the UV-Vis 
chromatograms. Due to the lack of standards for phase II metabolites, tentative 
quantification of calibration curves corresponding to phenolic compounds and 
precursor organic acids was performed. The urine results will be normalized with 
the volume excreted in each interval studied. 
 
Enzyme inhibition. 
 
In vitro gastrointestinal digestion of the beverages.  
 
Samples will be subjected to an in vitro digestion model adapted from the 
methodology by Blancas-Benitez et al., (2018). First, the samples will be subjected 
to an enzymatic hydrolysis process with pepsin at a temperature of 37°C for 1 h 
(step 1); the phenolic compounds (FC) released in the supernatant at this stage 
are considered as the FC present in the gastric fraction (GasF). Subsequently, the 
samples are hydrolyzed with pancreatin 5 mg/ml, P-1750, Sigma Aldrich) and α-
amylase (120 mg/ml, A-6255, Sigma Aldrich) (step 2). The CF released at this 
stage of digestion is considered the CF present in the intestinal fraction (IntF).  

 
 
 α-amylase activity. 
 
The products obtained from the various stages of sample digestion will be used to 
determine the α-amylase enzyme activity (U/mL) using the kinetic method 
proposed in the specifications of the amylase activity assay kit (Sigma-Aldrich 
#MAK009), which consists of taking an aliquot of 50 µL of the various samples and 
depositing them in a 96-well plate, then mixed with 100 µL of master solution, 
previously prepared with 50 µL of amylase assay buffer and 50 µL of α-amylase 
substrate mixture, then incubated at 25°C and at minute 3, the absorbance at 405 
nm is measured to obtain the initial time (T-initial) using the Synergy HT microplate 
reader (Biotek Instruments, Inc. , USA), readings will be taken every 5 min until the 
values of the samples approach that obtained at the highest concentration of the 
standard (20 nM/well) giving rise to the T-final. A calibration curve will be 
performed with Nitrophenol at concentrations of 4, 8, 12, 16 and 20 nM/well. One 
enzyme unit is the amount of amylase that cleaves ethylidene-pNP-G7 to generate 
1 mM p-Nitrophenol per min. The enzyme activity will be expressed as percentage 
inhibition (I%) and determined with the equation (Eq 6.1): 
𝐼% = 
(𝐵 × 𝐹𝑎𝑐𝑡𝑜𝑟 𝑑𝑒 𝑑𝑖𝑙𝑢𝑐𝑖ó𝑛)
𝑇𝑖𝑒𝑚𝑝𝑜 𝑑𝑒 𝑟𝑒𝑎𝑐𝑐𝑖ó𝑛 × 𝑉   
Ec 4.1 
 
Where:  
B refers to the amount of Nitrophenol generated, reaction time is the T-final minus 
T-initial in minutes and V denotes the volume of sample added to the well (µL). 
 
α-glucosidase activity. 
 
The inhibition of α-glucosidase of the beverages will be evaluated using the 
products of the different phases of the in vitro digestion and will follow the 
methodology described in the specifications of the α-glucosidase kit (Sigma-Aldrich 
#MAK123) where first 220 µL of deionized water will be placed in a well of a 
reading plate and in another well 20 µL of deionized water will be placed and 200 
µL of calibrator will be added to one of them. From the samples previously 
prepared in 50 mM phosphate buffer (pH 7. 0) 20 µL will be taken and placed in 
separate wells and 200 µL of master solution will be transferred to each of them, 
the solution will be previously prepared with 200 µL of ρ-nitrophenyl-α-D-
glucopyranoside (NPG) buffer and 8 µL of NPG substrate, then incubated at 37° C 
and absorbance will be measured at 405 nm using Synergy HT microplate reader 

 
(Biotek Instruments, Inc., USA), the initial measurement (T-initial) will be read and 
after 20 minutes the final absorbance (T-final) will be taken. 
The enzyme activity will be expressed in units per liter (unt/L) and calculated with 
the equation (Eq 6.2) where one enzyme unit is the amount of α-glucosidase that 
catalyzes the hydrolysis of 1 mM ρ-nitrophenyl-α-D-glucopyranoside per minute at 
pH 7.0. 
 
unt/L) = 
(𝐴𝑏𝑠 𝑓𝑖𝑛𝑎𝑙 − 𝐴𝑏𝑠 𝑖𝑛𝑖𝑐𝑖𝑎𝑙)
𝐴𝑏𝑠 𝑑𝑒𝑙 𝑐𝑎𝑙𝑖𝑏𝑟𝑎𝑑𝑜𝑟 −𝐴𝑏𝑠 𝑑𝑒𝑙 𝑎𝑔𝑢𝑎× 250 unt/L  
 
Ec 4.2 
 
Where:  
Abs of the calibrator is the value for the calibrator at 20 minutes, Abs of the water 
represents the value of the water at 20 minutes and unt are units. 
 
 
Evaluation of glycemic response. 
 
To obtain the biological sample, a lancet will be used to puncture the thumb of the 
volunteer's finger and the sample will be collected on highly sensitive test strips for 
capillary glucose measurement of the OneTouch Ultra® System. 
The portable system used for pre- and postprandial glucose measurement allows 
the monitoring of capillary glycemic response under experimental conditions for 
healthy subjects. With the use of this equipment, measurements will be obtained 
from t0 (preprandial), and at 15, 30, 45, 60, 90 and 120 min (postprandial). The 
results will be expressed in mg/dL. 
 
Insulin analysis. 
 
Participants will be required to hold a hand warmer (~40 °C) for 5 min before each 
capillary blood sampling, to stimulate peripheral blood flow. 
Blood samples (approx. 250 µL, 4-5 drops) will be drawn through finger pricks 
(OneTouch Ultra® lancets). Measurements over time will be similar to the glycemic 
response assessment technique. Samples will be placed in BD Microtainer SST 
tubes and kept at room temperature for 30 min. Samples will then be centrifuged 

 
(5000 rpm, 25°C) for 10 min to obtain serum and kept in a freezer at -20 degrees 
Celsius until further analysis (Hossain et al., 2021). 
Serum insulin concentrations will be analyzed using a two-site solid-phase enzyme 
immunoassay kit (Human Insulin ELISA Kit, Millipore, EZHI-14K), where the direct 
sandwich technique is used with two antibody solutions targeting insulin epitopes 
(Figure 6. 2) in such a way that the plate will be coated with a monoclonal antibody 
(capture) and then the sample will be added, so that the analyte of interest (insulin) 
will bind with the capture antibody and subsequently a horseradish peroxidase-
labeled detection secondary antibody will be added, this antibody will bind to the 
antigens that will be in turn bound to the capture antibody, consequently will be 
added the substrate 3,3',5,5'- tetramethylbenzidine in buffer (TMB), forming a 
bluish coloration dependent on the insulin concentration, the reaction will be 
terminated by the addition of stop solution (HCl) obtaining as colored product 
(yellow) after acidification, which is quantifiable at 450 nm in a microplate reader 
(BioTek®, Synergy HT, Winooski, Vermont, USA). The results will be expressed in 
μU/L. 
Statistical Analysis. 
 
Statistical analysis: first stage 
 
The normality of the data distribution will be evaluated by means of the Shapiro-
Wilk test; if so, a Student's t-test will be used, or, on the contrary, a nonparametric 
Mann-Whitney test will be used to evaluate the statistical differences between the 
variables. The level of statistical significance will be a value of (p<0.05). Statistical 
analysis will be performed with the STATISTICA program, version 10 
(Statsoft.1984-2011 Inc., Tulsa, OK, USA). 
 
Statistical analysis: second stage 
 
Each sample will be considered as an experimental unit. Statistical analysis will be 
performed with the STATISTICA program, version 10 (Statsoft. 1984-2011 Inc., 
Tulsa, OK, USA). Differences in the response variables in the samples will be 
evaluated through a repeated-means ANOVA analysis. The level of statistical 
significance will be a value of (p< 0.05). 
 
 Calculation of the area under the curve. 

 
 
The glycemic and insulinemic values obtained experimentally will be recorded in a 
database. The area under the curve (AUC) will be calculated according to the 
equation proposed by Wolover and Jenkins (1986) (Eq.4.7.): 
    𝐴𝑈𝐶= ((𝐴+ 𝐵+ 
𝐷
2) ∗15) + ((
𝐷
2 +
𝐸
2) ∗30) + ((
𝐸2∗30
2
∗(𝐸+ 𝐹))  Ec 4.7. 
Where A, B, C, D, E, F; represent the increase in glucose and insulin above 
baseline and 15 and 30 represent the time interval between blood samples. Any 
area below the fasting level will be discarded.   
 
 

 
EXPECTED RESULTS 
 
 
This research aims to identify which metabolites are bioavailable in plasma and 
urine samples after ingestion of a hibiscus drink and to identify inflammation 
markers in the same normal-weight volunteers, as a potential adjuvant in the 
management of inflammation caused by COVID-19 disease, and generate a 
hypoglycemic effect by reducing the levels of glucose and serum insulin, 
consequent to the generation of possible modifications on the physiological 
mechanisms, including the inhibition of the activity of the α-amylase and α-
glucosidase enzymes by the action of the bioactive compounds, as well as by the 
interaction with the rest of the ingredients that compose the beverage of interest. 
 
 

 
 
 
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