Food and Nutrition Sciences, 2016, 7, 383-395

Published Online April 2016 in SciRes. http://www.scirp.org/journal/fns

http://dx.doi.org/10.4236/fns.2016.75040

Clinical, Hematological and Biochemical

Health Benefit Effects of Hibiscus sabdariffa
Lin Dried Calyces Beverage in Human
Ghislain Maffo Tazoho1, Inocent Gouado2*, Mathieu Ndomou2, Salomon Tchuandom Bonsi3,
Yvonne Mbaduet Wamba4, Esther Etengeneng Agbor1
1
Department of Biochemistry, Faculty of Science, University of Dschang, Dschang, Cameroon
2
Department of Biochemistry, Faculty of Science, University of Douala, Douala, Cameroon
3
Penka-Michel’s District Hospital Laboratory, Penka-Michel, Cameroon
4
Espace Priorité Santé du Cameroun Health Center, Dschang, Cameroon

Received 23 February 2016; accepted 26 April 2016; published 29 April 2016

Copyright © 2016 by authors and Scientific Research Publishing Inc.

This work is licensed under the Creative Commons Attribution-NonCommercial International License (CC
BY-NC).
http://creativecommons.org/licenses/by-nc/4.0/

Abstract
Background/Objective: This study was carried out to investigate the health benefit effects of Hi-
biscus sabdariffa Lin (H. sabdariffa L.) dried calyces beverage on some clinical, biochemical and
hematological parameters in humans. Methods: The dried calyces were harvested in the two re-
gions (Adamaoua and West) of Cameroon. The proximate, mineral composition and phytochemical
screening were evaluated. A standardized extraction procedure was set up and from the calyces;
we prepared a drink for 32 male volunteers’ subjects aged from 21 to 32 years, specially recruited
for the experiment. Each participant consumed 500 mL twice a day (in the morning and in the
evening) as supplement beverage during two weeks. The anthropometrics (age, height, weight,
body mass index (BMI)), clinical (systolic and diastolic blood pressure), hematological (RBC, Hb,
PCV, MCV, MCH, MCHC, WBC, Lymphocytes, MID cells, Granulocytes, platelet and MPV) and bio-
chemical (TC, HDL-C, LDL-C, TG, serum iron, blood glucose, creatinine, urea, ASAT and ALAT) pa-
rameters were determined in the blood on days 0 and at the end of each week. Results: Crude pro-
tein, lipid, fiber and ash content of calyx ranged respectively from 4.57 - 5.98, 10.10 - 11.33, 20.39 -
22.30 and 9.15% - 10.38% while the levels of minerals were ranged from 512.0 - 740.6, 77.8 -
177.7, 52.84 - 52.85, 1.10 - 2.10, 41.2 - 119.5, 3.25 - 8.20 and 0.56 - 17.5 mg/100g respectively for
Ca, Mg, K, Na, P, Fe and Zn. The phytochemical screening tests revealed the presence of alkaloids,
flavonoids, tannins, phenols and anthocyanins on methanol and aqueous extracts. A significant
increase of RBC, Hb, PCV, MPV, HDL-C, TG and creatinine and a significant decrease of WBC, MID
cells, LDL-C and TC (p < 0.05) were observed during the study period. Furthermore, there was no
significant change on BMI, MCV, MCH, MCHC, lymphocyte, granulocyte, platelet, serum iron, blood
*
Corresponding author.

How to cite this paper: Maffo Tazoho, G., Gouado, I., Ndomou, M., Tchuandom Bonsi, S., Mbaduet Wamba, Y. and Agbor,
E.E. (2016) Clinical, Hematological and Biochemical Health Benefit Effects of Hibiscus sabdariffa Lin Dried Calyces Beverage
in Human. Food and Nutrition Sciences, 7, 383-395. http://dx.doi.org/10.4236/fns.2016.75040
G. Maffo Tazoho et al.

glucose, ASAT, ALAT and urea levels. Conclusion: H. sabdariffa L. dried calyces from Cameroon are
rich sources of crude fibers and minerals. The H. Sabdariffa L. dried calyces drink can be safely
used for people suffering for anemia. It also revealed good cholesterol lowering potential. No he-
patoxicity and no kidney damage have been observed as far as serum enzymes were concerned.

Keywords
Hibiscus sabdariffa Lin, Dried Calyces, Chemical Composition, Health, General Well-Being, Human

1. Introduction
The important role of diets in diseases prevention and health promotion has been clearly mentioned and the
health benefits of plant foods are not only attributable to their macro- and/or micronutrients content but also to
the presence of phytochemical compounds [1]. These plant foods which might provide therapeutic benefits are
generally called “functional foods”. The International Life Sciences Institute [2] defined functional foods as
“foods that, by virtue of the presence of physiologically active compounds provide a health beyond basic nutri-
tion”. A plethoric of public health guidelines are aimed to recommend generous intakes of plant foods including
fruits, vegetable, whole grain cereals and diet low in saturated fatty acids. In many African culture, vegetables
form an important part of healthy traditional diet to improve nutrition, boost food security, foster rural develop-
ment, support sustainable land care an offer health protecting properties [3]. Hibiscus sabdariffa Lin is one of
such plant foods of nutritional importance. The Hibiscus calyces have been found to be rich in vitamins, carbo-
hydrate, protein, antioxidant compounds and minerals [4] [5]. These calyces are used to make beverages gener-
ally called hibiscus tea [6].
In Cameroon, the Hibiscus calyces beverage, a non-alcoholic local beverage, is generally obtained by boiling
calyces in water (often with natural additives such as: pineapple, Cymbopogon citratus) follow by a filtration
and a sugar adding. This beverage, easy to make is a very cheaper drink comparatively to soft drink or others
fruits juice sold in local market. However, the nutritive value and functional properties of Hibiscus calyces cul-
tivated in Cameroon is not well documented. The consumption of H. sabdariffa L. drink tea may have some
positive effects on preventing chronic diseases and promoting health or general well-being in consumers. A pre-
vious study with this drink tea has shown positive effects on some biochemical parameters such as a significant
increase of hemoglobin and significant decrease of total cholesterol level [7]. However, there is a need to inves-
tigate more such as the evaluation of the effect of its consumption on hematopoietic system and other biochemi-
cal parameters.
Then, this study was aimed to determine the proximate and mineral composition of Hibiscus calyces culti-
vated in Cameroon and evaluate the effect of their consumption as supplemented-beverage on some clinical, bi-
ochemical and hematological parameters in human subjects.

2. Material and Methods

2.1. Study Areas
The study was conducted in two agro ecological areas, precisely in the high Guinean savanna zone (Adamaoua
region of Cameroon) and in the Western high plateaus zone (West region of Cameroon). The Guinean savanna
zone records a monomodal rainfall pattern. The mean annual rainfall is approximately 1500 mm with approx-
imately 150 days of rainfall. The temperature of this zone is of moderate type with a monthly mean ranged be-
tween 20 to 26˚C. The Western high plateaus zone is characterized by two seasons with unequal length. The dry
season starts in mid-November and end in mid-March while the raining season starts in mid-March and end in
mid-November. The temperature is moderately low with a mean of 19˚C while the rains are abundant with an
annually mean of 2000 mm.

2.2. Plant Material

H. sabdariffa L. dried calyces were collected close to the farmers from Adamaoua and West regions of Camer-

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oon in 2012 during the harvest period. Dried calyces collected in each area were placed in polyethylene bags and
transported to the laboratory. A part of dried calyx was crushed into fine powder for proximate and mineral
composition analyses.

2.3. Hibiscus sabdariffa L. Beverage Preparation

The H. sabdariffa L. beverage was prepared using the modified method of Maffo et al. [7]. For preparation of 20
litres of tea, 430 g of dried calyces were previously washed using tap water and boiled in a pot with 12 litres of
water for 30 minutes using a gas cooker. After cooling, the mixture was filtered and the red filtrate was obtained.
Concomitantly, 1.5 kg of sugar powder was dissolved in 8 litres of water and boiled for 10 minutes. That solu-
tion was added to the red filtrate obtained as described above, and homogenized to obtain the drink. That drink
was then kept fridge at 4˚C using polyethylene bottles of 1.5 litres.

2.4. Aqueous Extract Preparation

A small quantity of the drink was set apart and filtered through Whatman filter paper No 1 and the extract was
obtained by complete evaporation of water in an oven at 45˚C. The solid extract was used for phytochemical
screening.

2.5. Methanol Extract Preparation

To prepare the methanol extract, the calyx was crushed into fine powder and 100 g of it was infused in 400 mL
of methanol. The content was mixed thoroughly and left for 48 hours with two times daily shaking to improve
the extraction. Thereafter, the homogenate obtained was then filtered with Whatman filter paper No 1, and the
extract was concentrated in a hot air oven at 63˚C. The solid extract was used for phytochemical screening.

2.6. Phytochemical Screening

The secondary metabolites such as alkaloids, flavonoids, tannins, phenols, saponins, sterols, triterpenoids and
anthocyanins were investigated in the aqueous and methanol extracts using the methods described by Harbone
[8].

2.7. Proximate Composition

Crude protein, lipid, ash and dry matter were determined according to AOAC method [9]. Briefly, samples were
dried at 105˚C overnight and dry matter was calculated. Total nitrogen (micro-kjeldahl) was determined and
protein was calculated as N × 6.25. Ash content was determined by incinerating samples at 500˚C to constant
weight. Total lipids were determined by extracting a known weight of sample with Ether exhaustively using a
soxhlet apparatus. Crude fiber content was determined using Sharer and Kurschner method [10]. Each sample
was analysed in triplicate.

2.8. Mineral Composition

Minerals were determined in sample extracts, prepared by dry-ashing [11]. The amount of zinc and iron were
determined according to the analytical method of atomic absorption spectroscopy [12]. The phosphorus was de-
termined by the ammonium molybdate/ammonium vanadate method of Chapman and Pratt [13]. Calcium and
magnesium were determined by the titration method of Chapman and Pratt [14]. Sodium and potassium were
determined according to AOAC [9] using flame photometer.

2.9. Study Subjects

Thirty two male subjects were recruited for the study (aged 21 to 32 years with a mean of 25.38 ± 3.35 years).
Written informed consent was obtained from each subject. Table 4 describes the anthropometric characteristics
of subjects during the study period. Participants were excluded if they were taking any medications, smoker and
if they were diabetics. None of the subject had reported of taking H. sabdariffa L. beverage one month prior to
the study and they were advised not to consume H. sabdariffa L. beverage from another source during the ex-

385
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perimental period. The consumption of the beverage covered two weeks period during which each subject con-
sumed 1 litre a day (500 mL in the morning and 500 mL in the afternoon). No change happened in the normal
food diet routine of the subjects; only H. sabdariffa L. beverage was added as a supplement. This protocol was
approved by the National Ethics Committee of Cameroon according to the authorization N°107/CNE/SE/2012.

2.10. Blood Sample Collection and Preparation

The collection of fasting blood sample were performed on days 0, 7 and 14 between 6.00 and 8.00 AM in the
health centre “Espace priorité santé du Cameroun” at Dschang. Blood samples were collected in tubes with and
without EDTA as anticoagulant. Serum was obtained after centrifugation of the blood collected in the tube
without anticoagulant, and stored in Eppendorf tubes at −18˚C for further analyses. Blood collected in the tubes
with anticoagulant was transported the same day at Penka-Michel’s district hospital laboratory for haematologi-
cal parameters investigation. The blood glucose level was also investigated the same day using serum obtained
from the blood collected in tube without anticoagulant.

2.11. Biochemical and Haematological Analyses

Haematological parameters [red blood cell (RBC), packed cell volume (PCV), white blood cell (WBC), haemo-
globin (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hae-
moglobin concentration (MCHC),lymphocytes, MID cells, granulocytes, platelet, mean platelet volume (MPV)]
were measured using an automated haematological analyser, PE 600 (23 parameters). Triglycerides (TG) were
measured using TECO DIAGNOSTICS kit (Lakeview Ave, Anaheim) while high density lipoprotein cholesterol
(HDL-C) was measured using IMNESCO (Neustadt/Wied-Germany) kit. The concentration of low density lipo-
protein cholesterol (LDL) was determined using the Friedewald formula [15]. Alanine aminotransferase (ALAT),
aspartate aminotransferase (ASAT) and creatinine were measured using Hospitex Diagnostics kit (Osman Noro,
Sesto Florentino), while serum iron, urea, glycaemia and total cholesterol (TC) were measured using CHRONO-
LAB kit (Barcelona, Spain).

2.12. Statistical Analyses

Results were reported as means ± SD and statistical analyses were done using Graph pad prism version 5.00
software. The following statistical tests were performed: one way ANOVA and Newman-Keuls. A p value of
less than .05 was considered significant.

3. Results and Discussion

3.1. Results
3.1.1. Proximate and Mineral Composition of H. sabdariffa L. Dried Calyces
Table 1 shows the proximate composition of H. sabdariffa L. dried calyces cultivated in West and Adamaoua
regions of Cameroon. The dry matter, total protein, total lipid, total fiber and ash content of calyces harvested in
the West region were respectively 93.09 ± 0.06, 5.98 ± 0.11, 10.10 ± 0.23, 22.30 ± 0.15 and 10.38 ± 0.20%
while the values 94.72 ± 0.05, 4.57 ± 0.10, 11.33 ± 0.15, 20.39 ± 0.19 and 9.15 ± 0.19% were observed respec-
tively for the dry matter, total protein, total lipid total fiber and ash content of calyces harvested in the
Adamaoua region of Cameroon. The dry matter and crude lipid of calyces from Adamaoua region were signifi-
cantly higher (p < 0.05) than those of West region. However, the crude protein, crude fiber and ash content of
calyces harvested in the West region were significantly higher (p < 0.05) than those of Adamaoua region of

Table 1. Proximate composition of Hibiscus sabdariffa L. dried calyces (g/100g).

Sample Dry matter Crude protein Crude lipid Crude fibre Ash
b a b a
CW 93.09 ± 0.06 5.98 ± 0.11 10.10 ± 0.23 22.30 ± 0.15 10.38 ± 0.20a

CA 94.72 ± 0.05a 4.57 ± 0.10b 11.33 ± 0.15a 20.39 ± 0.19b 9.15 ± 0.19b

Values are means ± SD. Means in the same colon followed by different letters differ significantly at 5% probability. SD = standard deviation, CW =
calyx from west region, CA = calyx from Adamaoua region.

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Cameroon. Table 2 shows the values of some mineral analysed in H. sabdariffa L. dried calyces harvested in
two agro ecological areas in Cameroon. The mineral analysed were calcium, magnesium, potassium, sodium,
phosphorus, iron and zinc. The values of these mineral ranged from 512.0 - 740.6, 77.8 - 177.7, 52.84 - 52.85,
1.1 - 2.1, 41.2 - 119.5, 3.25 - 8.20 and 0.56 - 17.5 mg/100g respectively for Ca, Mg, K, Na, P, Fe and Zn. The
observations show that Mg, Na, P, Fe and Zn levels of dried calyces cultivated in West region of Cameroon
were higher than those cultivated in the Adamaoua region.

3.1.2. Phytochemical Composition of Aqueous and Methanol Extracts of Hibiscus sabdariffa L.

Dried Calyces
Several classes of secondary metabolites such as alkaloids, flavonoids, tannins, phenols and anthocyanins were
identified both in the aqueous and methanol extracts of H. sabdariffa L. dried calyx (Table 3).

3.1.3. Anthropometric Data of the Study Population

The people recruited for this study were aged between 21 to 32 years old. The mean age of the study population
was 25.38 ± 3.35 years while their mean height was 1.76 ± 0.06 m (Table 4). Besides, the subjects mean weight
were 69.23 ± 6.87, 68.38 ± 7.25 and 68.75 ± 7.22 kg respectively for day 0: (D0), D7 and D14 while the mean

Table 2. Mineral composition of Hibiscus sabdariffa L. dried calyces (mg/100g).

Sample Ca Mg K Na P Fe Zn

CA 740.6 77.8 52.85 1.10 41.2 3.25 0.56

CW 512.0 177.7 52.84 2.10 119.5 8.20 17.5

CA = calyx from Adamaoua region, CW = calyx from west region, Ca = calcium, Mg = magnesium, K = potassium, Na = sodium, P = phosphorus,
Fe = iron and Zn = zinc.

Table 3. Phytochemical screening of aqueous and methanol extracts of Hibiscus calyces.

Class of compound

Sample Alcaloids flavonoids tannins saponins sterols triterpenoids phenols anthocyanines

MECA + + + - - - + +

AECA + + + - - - + +

MECW + + + - - - + +

EACW + + + - - - + +

MECA = methanolic extract of calyx from Adamaoua region, AECA = aqueous extract of calyx from Adamaoua region, MECW = methanolic extract
of calyx from west region, EACA = aqueous extract of calyx from west region. + present, - absent.

Table 4. Characteristic data of the study population (n = 32).

Day 0 Day 7 Day 14

Range Means ± SD Range Means ± SD Range Means ± SD

a a
Age (year) 21 - 32 25.38 ± 3.35 21 - 32 25.38 ± 3.35 21 - 32 25.38 ± 3.35a

Height (m) 1.60 - 1.82 1.76 ± 0.06a 1.60 - 1.82 1.76 ± 0.06a 1.60 - 1.82 1.76 ± 0.06a

Weight (kg) 55.20 - 85.10 69.23 ± 6.87a 55.60 - 84.40 68.38 ± 7.25a 55.10 - 85 68.75 ± 7.22a

BMI (kg∙m−2) 19.79 - 24.78 22.37 ± 2.02a 19.84 - 24.56 22.08 ± 1.99a 19.75 - 24.75 22.28 ± 2.11a

SP (×10 mmHg) 9 - 12 11.00 ± 1.02a 10 - 12 11.06 ± 0.76a 9 - 12 10.38 ± 0.87b

DP (×10 mmHg) 6-9 7.18 ± 0.82a 4-7 5.81 ± 1.15b 6-9 6.87 ± 0.94a

Values are means ± SD (n = 32). Means in the same row followed by different letters differ significantly at 5% probability. SD = standard deviation,
BMI = body mass index, SP = systolic pressure, DP = diastolic pressure.

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G. Maffo Tazoho et al.

BMI were 22.37 ± 2.02, 22.08 ± 1.99 and 22.28 ± 2.11 kg/m2 for D0, D7 and D14 respectively (p ˃ 0.05). The
systolic pressure decreased significantly (p < 0.05) between D0 and D14 while there was a no significant de-
crease in diastolic pressure (p ˃ 0.05) between D0 and D14. The mean values of systolic pressure were 11.00 ±
1.02, 11.06 ± 0.76 and 10.38 ± 0.87 (×10 mmHg) respectively for D0, D7 and D14 while the mean values of di-
astolic pressure were 7.18 ± 0.82, 5.81 ± 1.15 and 6.87 ± 0.94 (×10 mmHg) respectively for D0, D7 and D14. In
spite of the variation observed on blood pressure of study subjects, the values were within the normal range that
is <14 (×10 mmHg) for Systolic pressure and <9 (×10 mmHg) for diastolic pressure.

3.1.4. Effect of Hibiscus sabdariffa L. beverage Consumption on Hematological Parameters in

Human Subjects
The results of the effect of consumption of H. sabdariffa L. beverage on hematological parameters are show in
Table 5. The H. sabdariffa L. dried calyces drink has a remarkable effect on red blood cells (RBC), Haemoglo-
bin (Hb), pack cell volume (PCV) and white blood cells (WBC) as the mean values of these parameters changed
significantly (p < 0.05) during the study period. The mean values of RBC were 3.59 ± 0.69, 4.22 ± 1.20 and 4.62
± 1.02 (×106/μL) for D0, D7 and D14 respectively. As for Hb, the mean values were 11.45 ± 2.45, 13.51 ± 3.86
and 14.80 ± 3.49 (g/dL) for D0, D7 and D14 respectively while the mean values for PCV were 22.95 ± 6.32,
28.29 ± 10.26 and 31.94 ± 8.47 (%) for D0, D7 and D14 respectively. There was not significant change (p ˃
0.05) on mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglo-
bin concentration (MCHC), granulocytes, lymphocytes, platelet and mean platelet volume (MPV) values during
the study period. However, the mean values of white blood cells (WBC) and MID cells decreased significantly
(p < 0.05). For WBC, mean values of 5.34 ± 1.17, 4.91 ± 1.44 and 4.37 ± 0.79 (×103/μL) were obtained respec-
tively for D0, D7 and D14 while for MID cells the mean values were 9.58 ± 2.08, 9.12 ± 1.85 and 8.37 ± 2.06
(%) for D0, D7 and D14 respectively.

3.1.5. Effect of Hibiscus sabdariffa L. Beverage Consumption on Biochemical Parameters in Human

Subjects
Biochemical parameters were assessed through the determination of the effect of H. sabdariffa L. beverage
consumption on lipid profile, blood glucose, serum iron, and serum enzymes on the study subjects (Table 6). A
complete lipid profile including total cholesterol (TC), triglycerides (TG), high density lipid cholesterol (HDL-C)

Table 5. Effect of Hibiscus sabdariffa L. tea drinking on hematological parameters in human.

Day 0 Day 7 Day 14

RBC (×10 /μL)

6
3.59 ± 0.69 b
4.22 ± 1.20 a
4.62 ± 1.02a

Hb (g/dL) 11.45 ± 2.45b 13.51 ± 3.86a 14.80 ± 3.49a

PCV (%) 22.95 ± 6.32b 28.29 ± 10.26a 31.94 ± 8.47a

MCV (fl) 88.39 ± 2.99a 88.61 ± 3.66a 88.64 ± 3.17a

MCH (pg) 32.34 ± 1.75a 32.07 ± 1.58a 32.41 ± 1.69a

MCHC (g/dL) 36.67 ± 0.99a 36.20 ± 1.17a 36.64 ± 1.27a

WBC (×103/μL) 5.34 ± 1.17a 4.91 ± 1.44ab 4.37 ± 0.79b

Lymphocytes (%) 48.56 ± 10.18a 47.67 ± 13.19a 47.14 ± 9.98a

MID cells(%) 9.58 ± 2.08a 9.12 ± 1.85ab 8.37 ± 2.06b

Granulocytes (%) 41.86 ± 10.46a 43.15 ± 14.21a 45.52 ± 15.00a

Platelet (×103/μL) 178.5 ± 47.54a 155.3 ± 57.94a 147.2 ± 54.31a

MPV (fl) 10.19 ± 0.53ab 9.99 ± 0.53b 10.33 ± 0.43a

Values are means ± SD (n = 32). Means in the same row followed by different letters differ significantly at 5% probability. SD = standard deviation,
RBC = red blood cells, Hb = hemoglobin, PCV = pack cell volume (hematocrit), MCV = mean corpuscular volume, MCH = mean corpuscular he-
moglobin, MCHC = mean corpuscular hemoglobin concentration, WBC = white blood cells, MPV = mean platelet volume.

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Table 6. Effect of Hibiscus sabdariffa L. tea consumption on some biochemical parameters in human.

Day 0 Day 7 Day 14

a a
TC (mg/dL) 134.1 ± 40.32 130.5 ± 37.25 101.7 ± 19.57b

HDL-C (mg/dL) 32.05 ± 8.94b 36.00 ± 7.89b 42.14 ± 9.99a

TG (mg/dL) 46.80 ± 33.40c 75.72 ± 39.37b 128.5 ± 57.30a

LDL-C (mg/dL) 84.70 ± 34.94a 84.66 ± 43.08a 37.97 ± 16.10b

TC/HDL-C 4.63 ± 2.49a 3.82 ± 1.55a 2.56 ± 1.68b

LDL-C/HDL-C 2.99 ± 1.99a 2.56 ± 1.68a 0.93 ± 0.44b

Serum iron (μg/dL) 101.1 ± 33.50a 100.8 ± 43.17a 108.6 ± 35.77a

Blood glucose (g/L) 0.99 ± 0.09a 0.99 ± 0.08a 0.99 ± 0.09a

ASAT (UI /L) 7.01 ± 2.24a 6.87 ± 2.27a 6.94 ± 2.52a

ALAT (UI/L) 3.67 ± 1.64a 4.33 ± 1.99a 4.55 ± 1.79a

ASAT/ALAT 2.36 ± 1.46a 1.94 ± 0.91ab 1.68 ± 0.68b

Creatinine (mg/dL) 0.52 ± 0.14b 0.69 ± 0.12a 0.71 ± 0.14a

Urea (mg/dL) 19.06 ± 7.00b 23.29 ± 6.55a 20.80 ± 6.45ab

Values are means ± SD (n = 32). Means in the same row followed by different letters differ significantly at 5% probability. SD = standard deviation,
TC = total cholesterol, HDL-C = high density lipoprotein cholesterol, TG = triglycerides, LDL-C = low density lipoprotein cholesterol, ASAT = as-
partate aminotransferase, ALAT = alanine aminotransferase.

was investigated and low density lipid cholesterol (LDL-C) as well as the TC/HDL-C and LDL-C/HDL-C ratios
was calculated afterward. The TC, LDL-C, TC/HDL-C and LDL-C/HDL-C decreased significantly (p < 0.05)
during the study period. The average values of TC were 134.1 ± 40.32, 130.5 ± 37.25 and 101.7 ± 19.57 mg/dL
for D0, D7 and D14 respectively. On D0, D7 and D14 the mean values were respectively 84.70 ± 34.94, 84.66 ±
43.08 and 37.97 ± 16.10 mg/dL for LDL-C. The average serum TG level significantly increases from 46.80 ±
33.40 mg/dL on D0 to 128.5 ± 57.30 mg/dL on D14 (p < 0.05). Besides, the HDL-C level also increases signifi-
cantly (p < 0.05) from 32.05 ± 8.94 mg/dL on D0 to 42.14 ± 9.99 mg/dL on D14. The results of serum iron,
glycaemia, aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and urea have shown no sig-
nificant change (p ˃ 0.05) during the test period. The ratio ASAT/ALAT and creatinine mean values were re-
spectively decreasing and increasing significantly (p < 0.05) during the study. For the ratio ASAT/ALAT, we
obtained mean values of 2.36 ± 1.46, 1.94 ± 0.91 and 1.68 ± 0.68 for D0, D7 and D14 respectively. The mean
values of serum creatinine were 0.52 ± 0.14, 0.69 ± 0.12 and 0.71 ± 0.14 mg/dL respectively for D0, D7 and
D14.

3.2. Discussion
3.2.1. Proximate and Mineral Composition of Hibiscus sabdariffa L. Dried Calyces
The chemical analysis of dried calyces harvested in the Adamaoua and West regions of Cameroon have shown
that dry matter, crude protein, crude lipid, crude fiber and ash values respectively ranged from 93.09 - 94.72,
4.57 - 5.98, 10.10 - 11.33, 20.39 - 22.30 and 9.15% - 10.38%. These results were higher than those of Morton
[16] who obtained the values 90.8, 1.145, 2.61, 12.0 and 6.90% respectively for dry matter, crude protein, crude
lipid, crude fiber and ash. Adanlawo and Ajibade [17] and Amed [18] from their studies on the calyces from
Nigeria have obtained the values of 12.24 and 94.9% for Ash and dry matter respectively. These values were
higher than those we obtained. Bahaeldeen et al. [19] have found that the calyx from Sudan contain 89, 7.88,
0.16, 13.20 and 10.60% of dry matter, crude protein, crude lipid, crude fiber and ash respectively. These values
were lower than those obtained in this study. The crude protein, crude fiber and ash levels of calyces harvested
in the West region were significantly higher (p < 0.05) than those of Adamaoua region of Cameroon while the
dry matter and total lipid of calyces from Adamaoua region were significantly higher (p < 0.05) than those of

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West region of Cameroon. These results might be due to the variable agro climatic conditions of the subject re-
gions. The crude protein of H. sabdariffa L. calyx from the Western high plateaus zone of Cameroon was sig-
nificantly higher (p < 0.05) than that of high Guinean savanna zone. This result is in line with the findings of
Modi [20] who reported that cool environmental conditions are associated with high total protein in leafy vege-
tables while hot temperature had a significant decrease in leaf protein content. Therefore, the higher tempera-
tures and the long periods of drought in high Guinean savanna zone decrease the calyces crude protein levels
harvested in this agro ecological zone of Cameroon. Hibiscus calyces harvested in the Western high plateaus
zone recorded lower mean dry matter content of 93.09% than that of high Guinean savanna zone of 94.72%. The
high dry matter value recorded in calyx from high Guinean savanna zone may be due to the high temperature
and long drought period which may be associated to high rate of evapotranspiration. In fact, during this period,
the atmospheric humidity becomes very low in high Guinean savanna zone. The difference in relative humidity
around the stomata and adjacent air regulates transpiration rates and pulls water up through the xylem tissues
[21].
The results of mineral analysis have shown that the values ranged between 512.0 - 740.6, 77.8 - 177.7, 52.84 -
52.85, 1.10 - 2.10, 41.2 - 119.5, 3.25 - 8.20 and 0.56 - 17.5 mg/100g respectively for calcium (Ca), magnesium
(Mg), potassium (K), sodium (Na), phosphorus (P), iron (Fe) and zinc (Zn). These results were lower than the
values 1583, 316, 2060, 5.5, 37.8 and 6.5 mg/100g respectively for Ca, Mg, K, Na, Fe and Zn obtained by Mady
et al. [22] with the calyx cultivated in Nigeria. However, except the Na content (96.66 mg/100g), these results
were higher than the values 49.35, 12.65, 38.65, 3.22, 12.22 and 36.30 mg/100g obtained by Adanlawo and
Ajibade [17] respectively for K, Ca, Mg, Fe, Zn and P. The effect of agro ecological area is observable with Hi-
biscus sabdariffa L. calyx mineral composition. Except the K and Ca content, the Mg, Na, P, Fe and Zn levels of
Hibiscus sabdariffa L. calyx harvested in the West region of Cameroon (Western high plateaus zone) were
higher than those of Adamaoua region of Cameroon (high Guinean savanna zone). This may be attributed to the
variable uptake of minerals by plant material and variable agro climatic conditions of study areas. The long dry
season in the high Guinean savanna makes soils dry for longer periods of time inhibiting the absorption of soil
nutrients for plant development [23].

3.2.2. Phytochemical Composition of Aqueous and Methanol Extracts of H. Sabdariffa L. Dried

Calyces
The Phytochemical screening performed on aqueous and methanol extracts has shown the presence of different
classes of chemical compounds such as anthocyanins, alkaloids, flavonoids, tannins and phenols. Most phyto-
chemical compounds are known to have positive effect on health and general wellbeing. Anthocyanins may pro-
vide protection from DNA cleavage, altering development of hormone-dependent disease symptoms, boosting
production of cytokines, anti-inflammatory activity, antimicrobial infections, oxidative stress protection and in-
hibition of both body weight and adipose tissue increases [24]. Beside, tannins in plants have been shown to
confer antidiarrheal and anti-hemorrhagic properties [25], while saponins have been reported to have antifungal
properties [26]. Polyphenols can chelate pro-oxidant metal ions, such as iron and copper, thus preventing free
radical formation from these pro-oxidants [27], though flavonoids are known to be synthesized by plants in re-
sponse to microbial infection and it should not be surprising that they have been found in vitro to be effective
antimicrobial substances against a wide array of microorganisms [28]. Moreover, flavonoids are known to have
antioxidant effects and have been shown to inhibit the initiation, promotion, and progression of tumors [29].
Therefore, most of medicinal properties (antioxidant, antibacterial, anti-inflammatory, cardio protective, antihy-
pertensive, glucose blood lowering effects, etc.) affected to H. sabdariffa L. calyx might be possible favor to
phytochemical compounds present in this plant.

3.2.3. Anthropometric Data of the Study Population

One of the specific objectives of this study was to evaluate the effect of drinking H. sabdariffa L. calyxes’ be-
verageon blood pressure and body mass index (BMI) of the participant. At the beginning (day 0) of the study,
the mean values of weight, BMI, SP (systolic pressure) and DP (diastolic pressure) were respectively 69.23 ±
6.87 kg, 22.37 ± 2.02 kg∙m2, 11.00 ± 1.02 (×10 mmHg) and 7.18 ± 0.82 (×10 mmHg). At the end of the study
(day 14), the observations have shown a significant decrease of SP (p < 0.05) and a decrease of DP, BMI and
weight (p ˃ 0.05) with the respective mean values of 10.38 ± 0.87 (×10 mmHg), 6.87 ± 0.94 (×10 mmHg), 22.28
± 2.11 kg∙m2 and 68.75 ± 7.22 kg. These results were similar to those obtained by Mckay et al. [6], who used a

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randomized, double-blind, placebo-controlled clinical trial to test the effect of H. sabdariffa L. tea (tisane) in
prehypertensive and mildly hypertensive 65 adults aged between 30 - 70 years. Each subject consumed 1.25 g H.
sabdariffa L./240mL boiled water three times day during six weeks. They observed that the change in SP among
participants who consumed the hibiscus tea was greater than in participants who consumed the placebo beverage.
The change in DP after treatment was not significant between the placebo and hibiscus groups. Besides, Mozaf-
fari-Khosravi et al. [30] tested 27 persons with mean age of 55.37 ± 8.6 years who consumed 2 g hibiscus
tea/240mL boiling water with 5 g sugar twice day during 30 days. They observed a statistically significant de-
crease of SP and a non-significant decrease of DP, weight and BMI during the test period. Their values were
ranged between 134.4 - 112.7 (mmHg), 81.6 - 80.5 (mmHg), 28.3 - 28.0 (kg∙m2) and 70.4 - 69.9 kg respectively
for SP, DP, BMI and weight from basal to day 30. Furthermore, in the study of Haji and Haji [31], two groups of
patients suffering from hypertension consumed sour tea (ST) and black tea (BT) for 15 days. The patient’s mean
age was 52 years and the treatment group consumed 1 glass daily of H. sabdariffa L. tea prepared with 2 spoon-
ful’s of blended tea per glass brewed in boiling water for 20 - 30 min. This study showed that 12 days after the
intervention, SP and DP in the ST consumers decreased respectively by 11.2% and 10.7% and after three days of
withdrawal, the SP and DP increased by 7.6 and 5.6% respectively. Hibiscus tea possesses an antihypertensive
activity and its consumption could not significantly affect body weight changes.

3.2.4. Effect of H. sabdariffa L. Beverage Drinking on Hematological Parameters in Human Subjects

The assessment of hematological parameters could be used to reveal the benefit or deleterious effects of foreign
compounds like hibiscus tea on the blood constituents of animal or human subjects. The significant (p < 0.05)
elevation observed in the levels of RBC, Hb and PCV clearly indicates that the beverage contains phytochemical
compounds that stimulate the formation of erythropoietin in the stem cells (erythropoiesis) as well as the de-
creasing in the destruction of matured RBC during the study period. Erythropoietin is a glycoprotein which sti-
mulates stem cells in bone marrow to produce red blood cells [32]. The Hibiscus calyx extract have been shown
to contain anthocyanins as it is known to induce the renal secretion of erythropoietin [33] [34]. These results are
similar to those of Ejere et al. [35] who observed significant (p < 0.05) increase in RBC, Hb and PCV values in
rats following their study on the effect of aqueous extract of H. sabdariffa L. calyces on the hematological pro-
file of normal male albino rats. The increase of Hb and PCV is indicative of the fact that oxygen uptake and
transfer was very adequate in the patients [36]. The significant increase (p < 0.05) of Hb value also corroborate
our previous results [7]. There was no significant (p ˃ 0.05) variation on MCH, MCV and MCHC values during
the study period. This may be explained by the fact that MCHC, MCV and MCH relate to individual red blood
cells while PCV, Hb and RBC count relate to the total population of red blood cells in the blood [37]. These re-
sults were at variance with those of Ejere et al. [35] who state that the effects of aqueous extract of Hibiscus
sabdariffa calyces on hematological characteristics of rattus norvergicus shown significant increase (p < 0.05)
on MCHC, MCV and MCH. Besides, the effect of consumption of Hibiscus tea have no significant change on
platelet, MPV, granulocytes and lymphocytes but a significant decrease (p < 0.05) on WBC and MID cells val-
ues. The WBC count and indices have been known to play important roles in improving the immune system and
also in protection of body cells against pathogens. However, In spite of the significant decrease of WBC and
MID cells during the study period, the values of these parameters were in the normal range that is 4 - 10
(×103/μL) and 1.0% - 15.0% respectively for WBC and MID cells. The results indicate that the participant re-
mained healthy because decrease in number of WBC below the normal range is an indication of allergic condi-
tions and certain parasitism or presence of foreign body in circulating system [38]. But none of this was ob-
served during this study. These results not corroborated the findings of Ejere et al. [35] who observed a signifi-
cant increase (p < 0.05) of WBC value of rats following treatment with aqueous H. sabdariffa calyx L. extracts.
Barger [39] reported that any increase in MCV, MCH and decrease in MCHC above or below the normal range
indicate macrocytic and hypochromic anemia, probably due to the increase activity of bone marrow and defi-
ciency of some hematopoietic factors which are useful index of the capacity of bone marrow to produce red
blood cells. In this study, even though the MCV and MCH values increased and MCHC value decreased, these
values were in the normal range that is 80 - 99 fl, 26 - 32 pg and 32 - 36 g/dL respectively for MCV, MCH and
MCHC. In short, H. sabdariffa L. calyx extracts from Cameroon might be beneficial to the hematopoietic system.

3.2.5. Effect of H. sabdariffa L. Drinking Beverage on Biochemical Parameters in Human Subjects

Plasma lipid disorders characterized by hypercholesterolemia are the most prevalent indicator for susceptibility

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to cardiovascular diseases. It is characterized by alterations occurring in serum lipid and lipoprotein profile due
to increase concentrations of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), very low den-
sity lipoprotein cholesterol (VLDL-C) and triglycerides (TG) with a concomitant decrease in the concentrations
of high density lipoprotein cholesterol (HDL-C) in the blood circulation [40]. During this study, there were a
significant decrease (p < 0.05) of TC and LDL-C values and a significant increase (p < 0.05) of HDL-C and TG
values. The result of TC is similar to that of Tzu-Li et al. [41] who investigated the cholesterol-lowering poten-
tial of Hibiscus extract in human subject, a clinical study using oral preparation of Hibiscus capsules and using
42 volunteers aged between 18 - 75 years during 4 weeks. They observed that a dosage of two capsules of Hi-
biscus extract for one month significantly lower the serum cholesterol level. Pooja and Priscilla [42] also ob-
served in their study that the groups of rats treated with extract of calyces and leaves of H. sabdariffa L. showed
a significant decrease in the serum TC, LDL-C, VLDL-C and TG values along with an increase in serum
HDL-C levels. Furthermore, in this study we observed a significant decrease (p < 0.05) of TC/HDL-C and
LDL-C/HDL-C ratios which are also the best indicators of cardiovascular diseases risk. The normal values are
<5 and <3.5 for TC/HDL-C and LDL-C/HDL-C ratios respectively. The result of artherogenic index, LDL-C/
HDL-C was similar to that of Pooja and Priscilla [42]. Therefore the H. sabdariffa L. drink may be used in the
prevention of cardiovascular diseases.
Iron is the micronutrient necessary for respiratory gas transport via hemoglobin. In this study we observed an
increase (p ˃ 0.05) of serum iron value. As this value increased, it follows the same trend like hemoglobin con-
centration; therefore the profit is the organism capacity to transport oxygen and to increase vital capacity of H.
sabdariffa L. consumers. Besides, the blood glucose level of the studied subjects was permanent during the
study. Therefore, there is no fear that the drink could have hyperglycemic property because of the sugar used in
its preparation. Adrian et al. [43] in order to investigate the efficacy of H. Sabdariffa L. beverage as a means of
reducing post-prandial blood glucose levels in human subjects have shown in their study that H. sabdariffa L.
tea appears to have an effect in terms of slowing the rate of rise in blood glucose following consumption of high
glycemic index food. The hypoglycemia effect of Hibiscus tea might be linked to the fact that H. sabdariffa L.
tea has been shown to have a pancreatic α-amylase inhibitory action, which appears to be attributable to its hi-
biscus acid content [44].
ASAT and ALAT are enzymes generally used in the diagnostic of liver function integrity while creatinine and
urea are used for kidney. The increase in these enzymes is a sign of deterioration of these organs. During this
study, we observed a decrease (p ˃ 0.05) in ASAT, an increase (p ˃ 0.05) of ALAT, urea and a significant in-
crease (p < 0.05) in creatinine. In spite of the variations observed, all the values of these parameters were in the
normal range for men that is <35, <45 (UI/L), 0.7 - 1.20 and 15 - 45 mg/dL respectively for ASAT, ALAT,
creatinine and urea; therefore the consumption of this tea could have some beneficial effect for liver and kidney
functions. Ologundudu et al. [45] in their study have shown that the Hibiscus anthocyanins extract significantly
lowered the serum levels of the hepatic enzymes ASAT and ALAT and also reduced oxidative liver damage.
Moreover, Onyenekwe et al. [46] have showed that water extract of Hibiscus calyx decrease serum creatinine
levels in spontaneously hypertensive and normotensive rats. The ASAT/ALAT ratio is a diagnostic marker of
liver disease. The value <1 suggested nonalcoholic steatohepatitis while a ratio ≥2 strongly suggest an alcoholic
liver disease [47]. In this study there was a significant decrease (p < 0.05) in ASAT/ALAT ratio from 2.36 ±
1.46 to 1.68 ± 0.68. The decrease in this ratio is indicative of hepatoprotective role of H. sabdariffa L. drink tea
in studied subjects.

4. Conclusion
The present study indicated that H. sabdariffa L. calyces from Cameroon are important source of crude fiber and
minerals. Phytochemical screening test has revealed the presence of bioactive compounds such as alkaloid, fla-
vonoids, tannins, phenols and anthocyanins. The in vivo studies have shown that consumption of H. sabdariffa
L. aqueous extract might have an antihypertensive activity and boost the hematopoietic system therefore is use-
ful in the prevention of anemia and also might have a cardio protective effect. There were no risks on immune
system function and also on liver and kidney functions as well as serum enzymes were concerned.

Acknowledgements
The authors thank the volunteer participants for their consent.

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Conflict of Interest
The authors declare no conflict of interest.

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