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\DOI{10.5802/crchim.222}
\datereceived{2022-08-03}
\daterevised{2022-09-20}
\dateaccepted{2022-12-15}
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\begin{document}
\begin{noXML}
%\TopicEF{Materials and Clean Processes for Sustainable Energy and
%Environmental Applications}{Mat\'eriaux et proc\'ed\'es propres pour
%des applications \'energ\'etiques et environnementales}
\title{The use of D-Optimal Mixture Design in the development of date
stone and spirulina tablet formulation as a phycocyanin dietary
supplement}
\author{\firstname{Iness} \lastname{Jabri Karoui}\CDRorcid{0000-0002-0597-9460}}
\address{Laboratory of Materials Molecules and Applications,
Preparatory Institute for Scientific and Technical Studies,
Carthage University, B.P:51, La Marsa, 2075 Tunis, Tunisia}
\email[I. Jabri Karoui]{iness.karoui@yahoo.fr}
\author{\firstname{Majdi} \lastname{Hammami}\CDRorcid{0000-0002-4376-0638}\IsCorresp}
\address{Laboratory of Medicinal and
Aromatic Plants Biotechnology Centre of Borj-Cedria BP 901,
2050, Hammam-Lif, Tunisia}
\email[M. Hammami]{hammamimajdi@hotmail.com}
\author{\firstname{Manef} \lastname{Abderrabba}\CDRorcid{0000-0002-1129-0539}}
\addressSameAs{1}{Laboratory of Materials Molecules and Applications,
Preparatory Institute for Scientific and Technical Studies,
Carthage University, B.P:51, La Marsa, 2075 Tunis, Tunisia}
\email[M. Abderrabba]{abderrabbamanef@gmail.com}
\shortrunauthors
\keywords{\kwd{Date stone}
\kwd{Spirulina}
\kwd{Tablets}
\kwd{Antioxidant activity}
\kwd{Dietary supplement}
\kwd{Phycocyanin}}
\begin{abstract}
The aim of this work is in the formulation of a dietary supplement,
combining spirulina and date stone benefits. For this, a study was
done on these two ingredients by determining their approximate
composition and antioxidant potential. Following this, we
used a statistical method to determine the appropriate proportions of
ingredients to obtain a tablet with optimal physical properties. After
setting these parameters, we analysed this tablet's composition and
effectiveness by evaluating its physicochemical properties, antioxidant
potential, FTIR spectrum and studied
the controlled release of phycocyanin. Our results revealed that the
optimized formula showed high stability and richness in active
compounds.
\end{abstract}
\maketitle
\vspace*{35pt}
\twocolumngrid
\end{noXML}
\let\rmmu\upmu
\section{Introduction}\label{sec1}
Rediscovered in the 1950s by the scientific community, the
cyanobacterium \textit{Spirulina Platensis} or spirulina (blue-green
algae) revealed, through the analysis of its constituents, an exceptional
nutritional value due to its unequaled richness in proteins,
unsaturated lipids, antioxidant vitamins and minerals. It is commonly
used in nutrition and food industry {and} contains a wide range of
coloured
compounds and large amounts of {essential} elements for
developing and maintaining the human body~\cite{1}. Beyond its
interesting nutritional capacities, spirulina also contains many
proteins and molecules representing unrealised medical interest at
the moment, in particular phycocyanin~\cite{2}. Phycocyanin
is a component usually present in red algae and
cyanobacteria~\cite{3}.
\textit{Spirulina Platensis}
is the cyanobacterium richest in
this molecule and was therefore naturally chosen for extraction work
for scientific purposes. Phycocyanin is a pigment of very interesting
biomedical interest. It can be used as an anti-inflammatory~\cite{4}
or antibacterial adjuvant~\cite{5}. Studies also show an anti-cancer
effect~\cite{6}. It also has the characteristic of being a free radical
scavenger~\cite{7}. {On the other hand}, date stones were recently
valorised through the production of~activated carbon~\cite{8}, and
poultry nutrition~\cite{9} and in traditional medicine for their
antimicrobial and antiviral properties~\cite{10,11}. The
characterization of the date stones revealed a richness of various
valuable biochemical and mineral substances, namely dietary fibres
(22.5--94\%), proteins (2.3--6.4\%), ashes (0.9--1.8\%), sugars
(5--6\%), phenolic compounds (3102--4430~mg/100~g), and fat
(7--13\%)~\cite{12,13}. Also, date stone oil is known for its richness
in natural antioxidants such as polyphenols, sterols, tocopherols and
carotenoids~\cite{14}. In this work, we are interested in the
formulation of dietary supplement tablets of spirulina enriched with
date stone powder {as a diluent} by determining the ideal tablet
recipe based on\unskip\break
D-Optimal Mixture Design involving the physicochemical
characteristics of the spirulina and date stones powder and by
studying the effect of dissolution medium on the phycocyanin release in
simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid
(SIF, pH~6.8) and by analysing the Fourier Transform Infrared
(FTIR) spectrum of {{the date stone and spirulina tablets.}}
\section{Materials and methods}\label{sec2}
\subsection{Excipients preparation}\label{sec2.1}
The spirulina was procured from the Tunisian company ``Bio Algues''. The
date stones were extracted from the Tunisian ``Deglet Nour'' date
cultivar. The date stones were dried at 50~\textdegree C for 48~h,
crushed manually, and then all the biomass was ground using an electric
grinder to get a powder with a fine grain size
$({<}100~\rmmu\mathrm{m})$.
\subsection{Proximate composition of date stone and spirulina crude
powder}\label{sec2.2}
Moisture content and dry matter were evaluated according to Silva
\etal~\cite{15} by a Sartorius MA 45 desiccator (Sartorius AG,
Germany). Lipid content was determined as described by
Yuwana~\cite{16}. The dosage of total sugars was carried out according
to the phenol/sulphuric acid method described by
Pawar \mbox{\etal}~\cite{17}. The total protein content was evaluated
according to Hayes~\cite{18}. The fibre content was determined through
formic insoluble method~\cite{19}.
\subsection{Biochemical characterization of date stone and spirulina
crude powders}\label{sec2.3}
The phenolic fraction extraction was carried out according to the
method of Msaada \etal~\cite{20}; 2~g of dry powder was extracted
with 10~ml of 80\% methanol, stirred for 60~min and then kept for
24~h at 4~\textdegree C. The extracts were filtered with a Whatman
filter paper (Grade 4), and then stored at
${-}20~\text{\textdegree}\mathrm{C}$. Total phenolic content (TPC) was
determined by the Folin--Ciocalteu assay according to Msaada
\etal~\cite{20} and expressed as mg Gallic acid and quercetin
equivalents per g dry weight
$(\mathrm{mgEQ}{\cdot}\mathrm{g}^{-1}\mathrm{DW})$. The antioxidants
activity was conducted according to the
2,2-diphenyl-1-picrylhydrazyl (DPPH)
radical scavenging assay
according to Msaada \etal~\cite{20} and expressed as
$\mathrm{IC}_{50}~(\rmmu\mathrm{g}{\cdot} \mathrm{ml}^{-1})$, the
concentration required to scavenge 50\% of DPPH free radicals;
butylhydroxytoluene (BHT) was used as the positive control. The total
chlorophyll content was determined according to Li \etal~\cite{21}.
The total carotenoid content was extracted using the method of Vila
\etal~\cite{22}. All experiments were carried out in triplicates,
and the results were reported as mean $\pm$ standard deviation.
\subsection{Phycocyanin extraction and quantification}\label{sec2.4}
UV-Visible spectrophotometry was used to measure phycocyanin utilising
an external calibration method. The phycocyanin extraction and dosing
were performed using the colorimetric method described by Park
\etal~\cite{23}. In brief, 200~mg of powdered spirulina were
pulverised with sea sand and extracted by sonication for 30~min, each
with 60~ml of phosphate buffer saline (pH 7.0). After centrifuging, the
supernatant was filtered through a $0.45~\rmmu\mathrm{m}$ membrane
filter. Two hundred microliters of each extract were transferred to a
96 well plate, where the absorbance at 620~nm was measured using a
microplate reader. Working calibration solutions in
$100\ndash1000~\rmmu\mathrm{g}{\cdot} \mathrm{ml}^{-1}$ were made by
diluting the phycocyanin stock solution with PBS buffer. To avoid
pigment deterioration, all processes were carried out in low-light
conditions.
\vspace*{-2pt}
\subsection{Preparation of date stone and spirulina tablets using
D-Optimal Mixture Design}\label{sec2.5}
According
to the literature~\cite{24}, the formulation of a tablet
requires the use of various excipients. Excipients, also called
``vehicles'' or ``adjuvants'', are substances which are inactive by
themselves, but which facilitate the administration and preservation of
the medicinal principle. In our case, date stone powder was used as a
natural diluent and binder agent~\cite{25}. In a similar study, Adiba
\etal~\cite{26} found that tablets formulated with 100\% spirulina had
the most fragile texture with a friability of 1.058\% and a relatively
long disintegration time $({>}120~\mathrm{min})$, hence the need for
the addition of other ingredients to ensure the success of the
formulation and therefore to ensure the delivery of the active
ingredient targeted in our tablet. The galenic formulation of the
tablets has been widely discussed in the literature. Recently, the
optimization of this formulation has become easier, given the
availability of computer tools such as experiment plans and mixing
plans~\cite{27}; in this context, we will optimize the formulation of
the spirulina tablets using date stone powder as a carrier by the
D-Optimal Mixture Design method with the three independent
factors (Table~\ref{tab1}): the
amount of date stone powder (X1), the
amount of spirulina powder (X2),
and the
amount of water (X3). The experimental matrix was
prepared by mixing 15 ingredients in different quantities (in g). The
different mixtures obtained were subjected to hardness, friability and
disintegration time
assays
(Table~\ref{tab2}).\looseness=-1
\vspace*{-2pt}
\begin{table}
\caption{\label{tab1}Parameters studied in the preparation of date
stone and spirulina tablets using D-Optimal Mixture Design}
\tabcolsep=4pt
\begin{tabular}{cccc}
\thead
Code & Parameters & Low level & High level \\
\endthead
X1 & Date stone powder & (0) 8~g & (1) 19~g\\
X2 & Spirulina powder & (0) 80~g & (1) 90~g \\
X3 & Water content & (0) 1~g & (1) 2~g
\botline
\end{tabular}
\tabnote{Independent factors levels are expressed in coded
values (in brackets) and experimental values {{in g}}.}
\vspace*{-2pt}
\end{table}
\begin{table*}
\caption{\label{tab2} The experimental matrix of the preparation of
date stone and spirulina tablets using D-Optimal Mixture Design}
\begin{tabular}{ccccccc}
\thead
\xmorerows{2}{Formula} & \multicolumn{3}{c}{Independent factors} &
\multicolumn{3}{c}{Responses}\\\cline{2-4}\cline{5-7}
& X1 & X2 & X3 & Y1 & Y2 & Y3 \\
& \parbox[t]{4pc}{\centering Date stone powder (g)} &
\parbox[t]{4pc}{\centering Spirulina powder (g)} &
\parbox[t]{4pc}{\centering Water content (g)} &
\parbox[t]{4pc}{\centering Hardness (Kp)} &
\parbox[t]{4pc}{\centering Friability (\%)} &
\parbox[t]{4.7pc}{\centering Disintegration time (s)} \vspace*{2pt}\\
\endthead
{F1} & {1 (19)} & {0 (80)} & {0 (1)} & {12.24} & {0.16} & \0{627} \\
{F2} & {0 (9)} & {1 (90)} & {0 (1)} & {18.61} & {0.28} & {1026} \\
{F3} & {0 (9)} & {0 (80)} & {1 (2)} & {14.02} & {0.17} & \0{594} \\
{F4} & {0.5 (14)} & {0.5 (85)} & {0 (1)} & {18.61} & {0.28} & {1026} \\
{F5} & {0.5 (14)} & {0 (80)} & {0.5 (1.5)} & {15.03} & {0.17} & \0{620} \\
{F6} & {0 (9)} & {0.5 (85)} & {0.5 (1.5)} & {18.80} & {0.31} & {1117} \\
{F7} & {0.75 (16.5)} & {0.25 (82.5)} & {0 (1)} & {18.60} & {0.28} & {1022} \\
{F8} & {0.25 (11.5)} & {0.75 (87.5)} & {0 (1)} & {19.24} & {0.32} & {1395} \\
{F9} & {0.75 (16.5)} & {0 (80)} & {0.25 (1.25)} & {16.17} & {0.19} & \0{667} \\
{F10} & {0.5 (14)} & {0.25 (82.5)} & {0.25 (1.25)} & {18.44} & {0.27} & \0{965} \\
{F11} & {0.25 (11.5)} & {0.5 (85)} & {0.25 (1.25)} & {19.07} & {0.36} & {1308} \\
{F12} & {0 (9)} & {0.75 (87.5)} & {0.25 (1.25)} & {19.16} & {0.39} & {1392} \\
{F13} & {0.25 (11.5)} & {0.25 (82.5)} & {0.5 (1.5)} & {18.02} & {0.24} & \0{858} \\
{F14} & {0.25 (11.5)} & {0 (80)} & {0.75 (1.75)} & {14.65} & {0.17} & \0{609} \\
{F15} & {0 (9)} & {0.25 (82.5)} & {0.75 (1.75)} & {16.96} & {0.24} & \0{867}
\botline
\end{tabular}
\xxtabnote{Independent factors are expressed in coded values and
experimental values in g (in brackets), All the experiments were
carried out in triplicates.}
\vspace*{-4pt}
\end{table*}
\subsection{Tablets preparation}\label{sec2.6}
According to Obaidat and Obaidat~\cite{28}, a portion of date stone
and spirulina powder combination (F1 to F15 in Table~\ref{tab2})
was\vadjust{\pagebreak}
mixed with water as a diluent for 20~min. The mixture was passed
through a 0.25~mm sieve, stirred, and blended in a plastic bag. The
mixture was subsequently compressed using a single punch tablet press
(ERWEKA GmbH, Germany) and 9~mm diameter circular punches with flat faces
under $2\times10^3~\mathrm{kg}$ compression. The machine parameters
were changed to create tablets with the same hardness and mass; 15
formulations with a target mass of 400~mg were generated.
\vspace*{-2pt}
\subsection{Tablets physical evaluation}\label{sec2.7}
Tablet hardness measurement was performed using the ERWEKA TBH 30 MD
equipment (ERWEKA GmbH, Germany). Hardness was expressed in kiloponds
(kp)~\cite{29}. The tablets' friability was tested using ERWEKA TA
40 (ERWEKA GmbH, Germany) at 20~rpm for four minutes and stated as a
percentage~\cite{30}. Disintegration time was performed according to
Shiyani \etal~\cite{31}. Disintegration time was carried out in 800~ml
of the various media at $37\pm0.5~\text{\textdegree}\mathrm{C}$. The
swelling assay was realised by weight measurement. The experiments were
performed in a USP 23 dissolution apparatus II according to
Pharmacopoeia~\cite{32}. The tablets were placed in a dissolution
flask and immersed in the phosphate buffer pH 5.8 or gastric fluid at
$37\pm0.5~\text{\textdegree}\mathrm{C}$ for 5, 15, 25, 60, 75 and
90~min. The erosion assay followed immediately the swelling test. This
test consists of determining the dry weight of a wet tablet after
incubation {{at 80~\textdegree C for
24~h}}~\cite{33}. All tests were run in triplicate using
ten tablets for each one.\looseness=-1
\vspace*{-2pt}
\subsection{Kinetics release of the phycocyanin}\label{sec2.8}
The release investigations were carried out using a USP dissolving
apparatus II~\cite{32} equipped with paddles and operated at a speed
of 50~rpm to investigate the effects of release medium on drug release.
Nine hundred millilitres of either standard simulated gastric fluid (SGF, pH
1.2) or standard simulated Intestinal Fluid (SIF, pH~6.8)
was placed in the glass vessel as the dissolution medium,
{{the apparatus}} was then assembled and maintained
at $37\pm0.5~\text{\textdegree}\mathrm{C}$. The amount of
phycocyanin
released
was calculated as a percent (recovered content)
from 3 to 33~min~\cite{34}.
\vspace*{-2pt}
\subsection{Statistical analysis}\label{sec2.9}
One-way and multivariate analysis of variance (ANOVA) followed
by
Duncan's multiple range test and Tukey's test were performed by SPSS 15
(SPSS Inc. Chicago, IL, USA). D-optimal Mixture Design was performed by
NemrodW (LPRAI, version 2000) software.
\begin{table*}
\caption{\label{tab3}Chemical composition of date stone and spirulina
powder}
\begin{tabular}{ccc}
\thead
& Date stone & Spirulina \\
\endthead
Moisture content (\%) & $10.78^{\mathrm{a}}\pm0.23$ & $5.85^{\mathrm{b}}\pm0.11$ \\
Carbohydrate (\%) & $60.46^{\mathrm{a}}\pm5.6$ & $8.74^{\mathrm{b}}\pm0.24$ \\
Proteins (\%) & $4.63^{\mathrm{b}}\pm0.03$ & $63.73^{\mathrm{a}}\pm0.02$ \\
Lipids (\%) & $3.78^{\mathrm{b}}\pm0.07$ & $5.92^{\mathrm{a}}\pm0.18$ \\
Fibbers (\%) & $32.95^{\mathrm{a}}\pm1.44$ & $3.55^{\mathrm{b}}\pm0.23$ \\
Carotenoids (mg/100~gDW) & $0.025^{\mathrm{b}}\pm0.7$ & $451.38^{\mathrm{a}}\pm12.5$ \\
Chlorophylls-a (mg/gDW) & - & $4.59\pm0.15$\\
Chlorophylls-b (mg/gDW) & - & $0.47\pm0.01$ \\
Chlorophylls-c (mg/gDW) & - & $1.13\pm0.02$ \\
Phycocyanin (mg/gDW) & - & $104.36\pm5.1$ \\
Phenolic content $(\mathrm{mgEAG}{\cdot}\mathrm{g}^{-1}\mathrm{DW})$ &
$4.74^{\mathrm{a}}\pm0.17$ & $36.4^{\mathrm{b}}\pm2.08$ \\
DPPH $\mathrm{IC}_{50}\;(\rmmu\mathrm{g}{\cdot}\mathrm{ml}^{-1})$ &
$86.5^{\mathrm{a}}\pm2.04$ & $27.71^{\mathrm{b}}\pm1.4$
\botline
\end{tabular}
\xxxtabnote{The values shown in this table were the mean of triplicates
and given as mean ${\pm}$ SD $(n=3)$. A one-way ANOVA followed by
Duncan's multiple range test was used.
{Values with different letter
were significantly different at $P<0.05$}.}
\vspace*{-5pt}
\end{table*}
\section{Results and discussion}\label{sec3}
\subsection{Characterization of date stone powder}\label{sec3.1}
Table~\ref{tab2}
summarizes
the characterization of date stone powder; the
moisture content is a crucial parameter for powder manipulation. It
affects the rheological properties during the formulation of the
tablet~\cite{35}. The moisture content of the date stone powder was
$10.78\pm0.24\%$. This value is compatible with that found by Azodi
\etal~\cite{36} with a percentage of 10.5\%, and it is also close to the
value found by Hamada \etal~\cite{37} (9.375\%). The fat content of
date stone powder was $3.78\pm0.07\%$ (Table~\ref{tab3}). This value
is close to the results given by Juhaimi \etal~\cite{38}, which vary
between 4.68\% and 7.96\%. In a study carried out on Tunisian varieties
(Mabsili, Um-Salah and Shahal), they found values between 5 and
6\%~\cite{39}. The total carbohydrate content of the date stones powder
was $60.46\pm5.6\%$ (Table~\ref{tab3}). This value is very close to
that of Rahman \etal~\cite{40} (62.31\%). In addition, Besbes
\etal~\cite{14} determined the total sugar content of two varieties of
Tunisian date stones. They found 81\% for Allig and 83.1\% for the
Deglet Nour variety. Therefore, the percentage of total sugars depends
on the variety of dates studied. The amount of nitrogenous matter in
the date stones powder was $4.63\pm0.03\%$ (Table~\ref{tab3}).
According to literature, Hussein and Alhadrami~\cite{41} determined
the protein percentage of date stones of 23 varieties. They found
values that vary between 2.3\% and 6.9\%. Lecheb \etal~\cite{39}
confirmed that the variety of
dates
might influence the variation
in protein content. Other authors studied the chemical composition of
date stones of 7 varieties. They found a protein content
value between
3.71\% and 5.47\%~\cite{38}. Compared to other by-products, date stones
have the highest nitrogen\unskip\break
content
(4.30\% in date stalks)~\cite{42}.
The cellulose and lignin content in the date stone
powder was $32.95\pm1.44\%$ (Table~\ref{tab3}). Several
authors
reported the richness of date stones in dietary fibres. AL-Kahtani
\etal~\cite{43} reported that the total dietary fibre in date stone was
58\%, of which 53\% was insoluble dietary fibre (hemicellulose,
cellulose, and lignin). These results agreed with those reported in our
work. The difference may be due to the varieties studied, the process
for obtaining the powder, or the dietary fibre assay method. Alamri
\etal~\cite{44} indicated that date stones have high percentages of
dietary fibre which vary between 22.50 and 80.20\%. Dietary fibre
can
help
prevention against hypertension, coronary heart disease, high
cholesterol, cancers and intestinal disorders~\cite{45,46,47,48,49}.
The carotenoid content in the
date stones
was
$25\pm0.7~\rmmu\mathrm{g}/100~\mathrm{g}$ of powder (Table~\ref{tab3});
this value is lower than that obtained by Boudries~\cite{50}, which
varies between 0.051 and 0.145~mg/100~g of fresh matter. This
difference may be due to the use of different varieties of date stone
powder\unskip\break
(4 varieties).
\subsection{Characterization of spirulina powder}\label{sec3.2}
The characterization of spirulina powder is given in Table~\ref{tab3}.
The moisture content\vadjust{\vspace*{9.8pt}\pagebreak}
of powders is one of the main factors
that limit
the manufacture\unskip\break
of tablets. It facilitates their quality of production
by referring to the design of the compression process~\cite{51}. The
moisture content of spirulina powder was about $5.85\pm0.11\%$.
This value was found to be in the range of 4 to 7\% by Sguera~\cite{52}
and is also equivalent to that reported by Branger \etal~\cite{53}:
5.4\%. The percentage of lipids content was equal to $5.92\pm0.18\%$
(Table~\ref{tab3}). This value is higher than that given by the
``French Oil Institute'' and found by Agustini \etal~\cite{54}; these
authors studied the composition of spirulina powder in lipids and they
gave a value of 2.86\%~\cite{55}. The estimated value of sugar content
was $8.74\pm0.24\%$ (Table~\ref{tab3}), which was slightly lower than
that found by Gershwin and Belay~\cite{56} (15--25\%), and it is also
close to that provided by N'Djamena~\cite{57}. The results given by
these authors are between 13.8\% and 14.2\%. This variation
in
total sugar rate depends on
the
spirulina's production site. In
addition, other researchers have found a rate very close to our
results. Agustini \etal~\cite{54} have shown that the percentage of
carbohydrates contained in spirulina is between 11.74 and 12.73\%. The
remarkable protein content of spirulina (60 to 70\%) makes it an
exceptional food. Numerous analyses have indeed revealed particularly
interesting nutritional properties: balanced protein composition,
presence of rare essential lipids, numerous minerals and
vitamins~\cite{58}. The protein content in biomass was
determined\unskip\break
using
the Kjeldahl method~\cite{59} using a correction factor of 5.22
specific for microalgae. As seen in Table~\ref{tab3},
protein
content in the spirulina powder was $63.73\pm0.02\%$ of powder. This
observed value was in accordance with previous found content (60 to
70\%)~\cite{60}. Moreover, Sharoba~\cite{61} gave a value
of 62.84\%
very close to
our results. The quantification of spirulina
pigments is
given in Table~\ref{tab3}:
4.59~mg chlorophyll-a, 0.468~mg chlorophyll-b
and 1.13~mg chlorophyll-c in 100~g of spirulina powder.
Sguera~\cite{52} showed that the chlorophyll-a content in spirulina
powder varies between 6.1 and 7.4~g/kg. On the other hand, Park
\etal~\cite{23}
accomplished
the quantification of different
chlorophyll-a from commercial spirulina and it gave total chlorophyll
content between 2.6 and 10.8~mg/g. Concerning the carotenoid content,
a value of $451.38\pm12.5~\mathrm{mg}/100~\mathrm{g}$ dry matter was
found with our powder,
a
value
in
agreement
with the results
found by Park \etal~\cite{23}. The
differences may
be due to the growth factors and to the efficiency of the method used
for the extraction and separation of the different pigments~\cite{62}.
\subsection{Phycocyanin content}\label{sec3.3}
Phycocyanin is the only natural blue food colorant authorized in Europe
and it is also used in some cosmetic products~\cite{63}. This
phytopigment fluorescent protein absorbs and captures photons then
transforms this light energy into electro-biochemical
energy~\cite{64}. The bilin group that constitutes it is very close to
human bile pigments, which could explain its detoxifying and
hepatoprotective activity~\cite{65}. Phycocyanin has the ability to
develop beneficial properties for the health of consumers, demonstrated
during numerous experiments carried out \textit{in vitro} and
\textit{in vivo} in different animal models. The liquid extract of
fresh spirulina allows to have a total bio-availability of the
molecules in their native forms. Several studies show that
spirulina or its extracts can prevent or inhibit cancers in humans or
animals~\cite{6}. Quantitative analysis of phycocyanin in spirulina
powder shows that it is a major pigment with a rate of
$104.36\pm5.1~\mathrm{mg}{\cdot}\mathrm{g}^{-1}\mathrm{DW}$
(Table~\ref{tab3}). These results are in agreement with the literature
which presumes that phycocyanin is the only abundant pigment present in
blue-green algae. Our results were comparable to that given by Park
\etal~\cite{23} who found that the commercial spirulina powder can
contain 153.3 mg phycocyanin per gDW. However, the quantity of these
pigments varies according to the culture conditions, where the light
intensity to which the cells are exposed, constitutes an important
parameter~\cite{66}. Likewise, a study conducted by Hoi \etal~\cite{67}
demonstrates that the pigment concentration is
limited by the nitrogen level in the culture medium.
\begin{figure*}
\includegraphics{fig01}
\vspace*{5pt}
\caption{\label{fig1}Probability versus residues values for hardness,
friability and disintegration time.}
\vspace*{4pt}
\end{figure*}
\subsection{Phenolic composition and antioxidant\newline
potential}\label{sec3.4}
The dosage of total phenolic content gives us an overall estimate of
the content of different classes of phenolic compounds contained in the
powders' methanolic extract. As shown in Table~\ref{tab3}, the average
phenolic content in date stones powder was
$4.74\pm0.17~\mathrm{mgEAG}{\cdot}\mathrm{g}^{-1}\mathrm{DW}$. Date seeds
of 14 Iranian date varieties have previously been examined for total phenol
content and it was reported that the content ranged from 459~to
$3284~\mathrm{mgEAG}{\cdot}100~\mathrm{g}^{-1}\mathrm{DW}$~\cite{68}.
Similarly, Metoui \etal~\cite{69} studied the total phenol contents of
11 varieties of Tunisian date seeds and their results vary between
5.224 and $9.532~\mathrm{gEAG}{\cdot}
100~\mathrm{g}^{-1}\mathrm{DW}$. For spirulina powder, total phenolic
content was $36.4\pm2.08~\mathrm{mgEAG}{\cdot}\mathrm{g}^{-1}\mathrm{DW}$.
Similar contents have been reported concerning the total phenolic content
in \textit{Spirulina platensis} by {\c{S}}ahin~\cite{70} and they found
it to be about $34.22~\mathrm{mgEAG}{\cdot}\mathrm{g}^{-1}\mathrm{DW}$.
DPPH assay is given in Table~\ref{tab3}. The results
show that there are
significant differences between examined biomass. Regarding the date stone
extract, the results reveal that the methanolic seeds extract confirms an
$\mathrm{IC}_{50}$ of $86.5\pm2.04~\rmmu\mathrm{g}{\cdot}\mathrm{ml}^{-1}$;
on the other hand, the spirulina extract had an $\mathrm{IC}_{50}$ of
$27.71\pm1.4~\rmmu\mathrm{g}{\cdot} \mathrm{ml}^{-1}$. We note that
only spirulina had an activity close to ascorbic acid which has an
$\mathrm{IC}_{50}$ of $23.22~\rmmu\mathrm{g}{\cdot}\mathrm{ml}^{-1}$.
According to Metoui \etal~\cite{69}, it was found that the DPPH
radical inhibition could attain 55.47\% for the Korkobi date variety
when using palm date seeds extract at a concentration of
$23.6~\rmmu\mathrm{g}{\cdot}\mathrm{ml}^{-1}$. In an Algerian study
(Djaoudene \protect\etal \protect\cite{71}) about date seeds
(\textit{Phoenix dactylifera} L.), the Ouaouchet and Ourous
cultivars~extracts displayed the most potent antioxidant capacity
against DPPH free radicals $(\mathrm{IC}_{50}=
37.30~\rmmu\mathrm{g}{\cdot} \mathrm{ml}^{-1})$. Our results were in
accordance with those found by Agustini \etal~\cite{54}, who found
that the $\mathrm{IC}_{50}$ of spirulina
extract was equal to
$33.07~\rmmu\mathrm{g}{\cdot}\mathrm{ml}^{-1}$.
\vspace*{-4pt}
\subsection{Experimental design methodology}\label{sec3.5}
In order to determine the effect of the percentage of date stone,
spirulina powder and water and their interactions on the quality of our
tablets, we used the D-Optimal Mixture Design in Optimizing 3-factor
design. Table~\ref{tab2} illustrates the 15 tests results carried out
according to the factorial model describing the combination between the
different levels of the\unskip\break
factors.
The response model proposed by the D-Optimal Mixture Design is written
as follows:
{\begin{eqnarray*}
\mathrm{Y} &=&
\mathrm{b}1*\mathrm{X}1+\mathrm{b}2*\mathrm{X}2+
\mathrm{b}3*\mathrm{X}3+\mathrm{b}12*(\mathrm{X}1*\mathrm{X}2)\\
&&+\,\mathrm{b}13*(\mathrm{X}1*\mathrm{X}3)+
\mathrm{b}23*(\mathrm{X}2*\mathrm{X}3)
\end{eqnarray*}}\unskip
with:
Y: the studied response
b1, b2, b3:
coefficient linked to the following factors: percentage of date stone
powder, spirulina powder and water content.
b12, b13 and b23:
coefficients linked to the crossing of the three factors.
\subsubsection{D-Optimal Mixture Design validation}\label{sec3.5.1}
The validation of this model was based on the verification of three
statistical tests; the
model's
linear Pearson coefficient of determination
$(R^{2})$, the analysis of variance and the analysis of
residuals. The coefficient of determination was 0.91, 0.90 and 0.89 for
hardness, friability and disintegration time, respectively.
Statistically,
$R^{2}$
is a measure of the quality of the prediction of a linear
regression. The closer the
coefficient
of determination
is
to 0, the
more the cloud of points is dispersed around the regression line. On
the contrary, the more $R^{2}$ tends towards 1, the more the cloud
of points tightens around the regression line~\cite{72}. In our study,
all the coefficients were close to 1, which confirms the good
distribution of hardness, friability and disintegration time
responses according to the proposed model. The adjusted $R$-squared
$(R^2\cdot\mathrm{adj})$ for hardness, friability, and disintegration
time were 0.86, 0.84, and 0.82, respectively. Their values are very
close to the values of
$R^2$. This
proves that all significant terms have been included in the empirical
models.
The analysis of variance is illustrated in Table~\ref{tab4}. The ratio
of the sum of squares and mean square for hardness, friability and
disintegration time (17.83, 15.89 and 13.9, respectively) were superior
to 4.77 extracted from the Fisher--Snedecor Law Table at $\rmalpha =
5\%$ for (5,9) as degrees of freedom~\cite{73}.
{{Consequently, the significant variables (X1, X2, X3), applied
to intricate the three response
models, were highly significant on their responses}}
(hardness, friability and disintegration time).
For residues analysis, Figure~\ref{fig1} shows
the distribution of the residues versus
probability for the three responses. The points were virtually randomly
dispersed, showing that the errors found by our model are not
systematic.
\begin{table*}
\caption{\label{tab4}Proposed model validation parameters}
\begin{tabular}{ccccccc}
\thead
& & Sum of squares & Degrees of freedom & Mean square &
Ratio & Signification \\
\endthead
\multicolumn{7}{c}{Hardness}\\
Regression && 62.89 & \05 & 12.58 & 17.83 & $p<0.001^{***}$\\
R{\'{e}}sidus && 6.35 & \09 & 0.71 & & \\
Total && 69.23 & 14 & & & \\
$R^2$ & 0.91 &&&&& \\
$R^2\cdot\mathrm{adj}$ & 0.86 &&&&&
\vspace*{5pt}\\
\multicolumn{7}{c}{Friability} \\
Regression && 0.07 & \05 & 0.01 & 15.89 & $p<0.001^{***}$\\
R{\'{e}}sidus && 0.01 & \09 & 0 & & \\
Total && 0.07 & 14 & & & \\
$R^2$ & 0.90 &&&&& \\
$R^2\cdot\mathrm{adj}$ & 0.84 &&&&&
\vspace*{5pt}\\
\multicolumn{7}{c}{Disintegration time} \\
Regression && 989,104 & \05 & 197,821 & 13.9\0 & $p<0.001^{***}$\\
R{\'{e}}sidus && 128,060 & \09 & 14228.9 & & \\
Total && 1,117,160 & 14 & & & \\
$R^2$ & 0.89 &&&&& \\
$R^2\cdot\mathrm{adj}$ & 0.82 &&&&&
\botline
\end{tabular}
\vspace*{4pt}
\end{table*}
\subsubsection{Significance of the factors for the three responses
hardness, friability and disintegration time}\label{sec3.5.2}
The results of the present study (Table~\ref{tab5}) show
that the
three factors (X1, X2 and X3) exert a highly significant
influence on hardness, friability and disintegration time of the
tablets. For the interaction effect, the results obtained
(Table~\ref{tab5}) show that the interaction b12 (date
stones---Spirulina) was the most significant with a positive
coefficient of 13.96 for the\unskip\break
hardness. This
translates physically into the fact that the hardness of the
tablets increases with the date stone and spirulina
powder content. For friability, the
interaction b23 was the most significant one, with a positive
coefficient 0.42. The same trend was found for disintegration time with
1295.23 as a coefficient. This is physically explained by the increase
in friability and disintegration time following the increase in water
and spirulina powder. By replacing the
significant coefficients in the
equations of the model, we can describe for each factor its own
equation:
{\begin{eqnarray*}
&&\mathrm{Y}_{(\mathrm{Hardness})}=
13.38*\mathrm{X}1+18.24*\mathrm{X}2
+13.72*\mathrm{X}3\\
&&\quad +\,13.96*(\mathrm{X}1*\mathrm{X}2)
+8.14*(\mathrm{X}1*\mathrm{X}3)\\
&&\quad +\,10.93*(\mathrm{X}2*\mathrm{X}3) \\
&& \mathrm{Y}_{(\mathrm{Friability})} =
0.18*\mathrm{X}1+0.30*\mathrm{X}2
+\,0.15*\mathrm{X}3\\
&&\quad +\,0.28*
(\mathrm{X}1*\mathrm{X}2)
+0.42*(\mathrm{X}2*\mathrm{X}3) \\
&&\mathrm{Y}_{(\text{Disintegration time})}=
632.13*\mathrm{X}1+1157.69*\mathrm{X}2\\
&&\quad +\,540.44*\mathrm{X}3
+1174.20*(\mathrm{X}1*\mathrm{X}2)\\
&&\quad +\,1295.23*
(\mathrm{X}2*\mathrm{X}3)
\end{eqnarray*}}\unskip
With these models, it is possible to calculate all the responses
in the study area by assigning values to levels X1, X2 and X3 to
immediately obtain the content of each response.
\begin{table*}
\caption{\label{tab5}Coefficients values and their statistical
significance for different responses}
\begin{tabular}{ccccccc}
\thead
\xmorerows{1}{Coefficient} & \multicolumn{2}{c}{Hardness} &
\multicolumn{2}{c}{Friability} & \multicolumn{2}{c}{Disintegration time} \\
\cline{2-3}\cline{4-5}\cline{6-7}
& Value & Signif (\%) & Value & Signif (\%) & Value & Signif (\%)\\
\endthead
\multicolumn{7}{c}{Linear effects}\\
b1 & 13.38 & $^{***}$ & 0.18 & $^{***}$ & \0632.13 & $^{***}$ \\
b2 & 18.24 & $^{***}$ & 0.30 & $^{***}$ & 1157.69 & $^{***}$ \\
b3 & 13.72 & $^{***}$ & 0.15 & $^{***}$ & \0540.44 & $^{***}$
\vspace*{5pt}\\
\multicolumn{7}{c}{Quadratic effects}\\
b12 & 13.96 & $^{**}$ & 0.28 & $^{*}$ & 1174.20 & $^{*}$\\
b13 & \08.14 & $^{*}$ & 0.06 & 62.40\% & \0141.77 & 75.30\% \\
b23 & 10.93 & $^{**}$ & 0.42 & $^{**}$ & 1295.23 & $^{*}$
\botline
\end{tabular}
\tabnote{Signif, \%: $p$-value $<$ 0.05 ($^*$), $p<0.01\;(^{**})$,
$p<0.001\;(^{***})$ mean significant, very significant and strongly
significant, respectively.}
\end{table*}
\begin{table*}
\caption{\label{tab6}Desirability results and optimal formulation
parameters}
\begin{tabular}{cccccc}
\thead
Responses & Values & D \% & Factors & Coded values & Real values (g)\\
\endthead
Hardness & \015.75 & 100.00 & Date stone powder & 0.53 & 13.83 \\
Friability & \0\00.18 & \091.27 & Spirulina powder & 0.12 & 81.2\0 \\
Disintegration time & 642.25 & 100.00 & Water content & 0.4\0 & \01.4\0 \\
\multicolumn{6}{l}{Total Desirability: 97\%}
\botline
\end{tabular}
\vspace*{-6pt}
\end{table*}
\subsubsection{Analysis and optimization of the response
surface}\label{sec3.5.3}
According to Mohamad Zen \etal~\cite{30}, in statistical methods, the
response surface design is used to discover the effects of process
variables on the specific responses of a system. In our study, the
surfaces
for
the three responses hardness, friability and
disintegration time, are shown in Figure~\ref{fig2}. The contour
plots of the mixture show that
hardness decreased significantly
with the addition of water and date stones powder.
This
observation was confirmed for
friability and disintegration time.
The graphical search for the optimal zeta potential showed that the
percentage of spirulina powder clearly correlated with the increase
in
all responses.
Indeed, these observations were consolidated by the
analysis of the coefficients of Table~\ref{tab5}, the interaction
b12 and b23 was\unskip\break
distinguished by a positive coefficient for all the
responses. The effect of moisture on tablet formulation has been widely
studied, and it has been demonstrated that increased
moisture {{causes}}
deterioration of tablets by increasing the percent friability~\cite{74}.
\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig02}
\vspace*{-4pt}
\caption{\label{fig2}3D and 2D graphical study of response surface
optimization for hardness, friability and disintegration time.}
\vspace*{-4pt}
\end{figure*}
\subsubsection{Desirability study}\label{sec3.5.4}
The NemrodW software for D-Optimal Mixture Design processing made it
possible to simultaneously optimize the three responses, as it is an
entirely numerical procedure which allows the mathematical search for a
combination of formulation parameters for which the desired responses
are optimal.
The software used uses these functions for each
response, a curve profile of the desirability function is chosen
(Figure~\ref{fig3}). The desirability (D) is zero for an unsuitable
answer and is maximum when the given answer is very satisfactory. The
desirability prediction was programmed to be at 100\% for
increasing
the hardness to the superior limit found in the
experimental matrix (19~kp), friability was minimized to 0.16\%, and
disintegration time to 600~s. These \mbox{limits} represent the best values
found in our previous section. Table~\ref{tab6} gives the constituent
values of the tablet formulation with a D of 97\% which gives us the
optimal tablet formula with the ingredient proposals proposed by the
function. The optimal formula was 13.83~g date stone powder, 81.2~g
spirulina powder and 1.4~g water.
\vspace*{-3pt}
\begin{figure*}
\includegraphics{fig03}
\vspace*{-4pt}
\caption{\label{fig3}Desirability study for hardness, friability and
disintegration time.}
\vspace*{-5pt}
\end{figure*}
\subsection{Physicochemical characterization of tablets}\label{sec3.6}
\vspace*{-1pt}
The results of the physicochemical properties of the tablets
formulated according to the results of previous section are summarized
in
Table~\ref{tab7}. The physical parameters
of the tablets showed
that the
optimal formulation gave a
uniform and stable tablet. This was
confirmed by
friability measurement which was comparable to that
predicted by the statistical analysis $(0.18\pm0.03\%)$. Likewise, for
the disintegration time $(625\pm12~\mathrm{s})$. The composition
analysis revealed a richness in carbohydrates, proteins, lipids and
fibres ($36.01\pm5.36$, $32.68\pm5.02$, $4.88\pm0.22$ and
$16.25\pm1.82\%$, respectively). The antioxidant activity was
characterized with a DPPH $\mathrm{IC}_{50}$ of
$47.31\pm3.4~\rmmu\mathrm{g}{\cdot}\mathrm{ml}^{-1}$ with a polyphenol
content of \mbox{$18.75\pm3.01~\mathrm{mgEAG}/\mathrm{tablet}$.} Phycocyanin
was also dosed and an amount of
$417.44\pm19.32~\rmmu\mathrm{g}/\mathrm{tablet}$ has
been
recovered.
\begin{table*}
\caption{\label{tab7}Physicochemical characteristics of the tablets\vspace*{-2pt}}
\begin{tabular}{cc}
\thead
Characteristics & Values\\
\endthead
Weight (mg) & \;\0$402\pm5.20$ \\
Hardness (Kp) & $13.69\pm2.01$ \\
Thickness (mm) & \0$4.04\pm0.08$\\
Diameter (mm) & \;\0\0\0$9\pm0.36$ \\
Friability (\%) & \0$0.18\pm0.03$ \\
Disintegration time (sec) & $625\pm12$ \\
Humidity (\%) & \0$1.77\pm0.12$ \\
Carbohydrate & $36.01\pm5.36$ \\
Proteins & $32.68\pm5.02$ \\
Lipids & \0$4.88\pm0.22$ \\
Fibres & $16.25\pm1.82$ \\
Total polyphenol content (mgEAG/tablet) &
$18.75\pm3.01$ \\
Antioxidant activity:
DPPH $\mathrm{IC}_{50}\;(\rmmu\mathrm{g}{\cdot}\mathrm{ml}^{-1})$ &
$47.31\pm3.4$\0 \\
Phycocyanin ($\rmmu\mathrm{g}$/tablet) & $417.44\pm19.32$
\botline
\end{tabular}
\xxxtabnote{The values shown in this table were the mean of triplicates
and given as mean $\pm$ SD $(n=3)$.\vspace*{-4pt}}
\end{table*}
\subsection{Swelling and erosion test}\label{sec3.7}
Once consumed, the tablets enter the digestive tract or the stomach and
will undergo a spontaneous process of adsorption by macromolecules
accompanied by a significant increase in volume. In order to study the
rheological behaviour of the constituents of two solid matrices (powder
of date stones and spirulina) at the level of the digestive system,
\mbox{simulated} \mbox{gastric} fluid (SGF, pH 1.2) and simulated intestinal fluid
(SIF, pH~6.8) were used. As shown in Figures~\ref{fig4}A and~B,
the rate of swelling and erosion
depended on
the ingestion time and type of medium. The tablet placed in distilled
water was the least swollen, followed by
SIF and then
SGF.
This shows that the capacity of our tablet to adsorb water depends on
the pH and it was higher with an acidic pH. This observation can be
explained by the protonation of the compounds which constitute our
tablet rich in carbohydrates and fibres. Protonation \mbox{promotes} the
formation of hydrogen bonds which stabilizes the swelling. Our~\mbox{results}
were in agreement with that found by Akhgari \etal~\cite{75} that the
swelling and erosion of a pectin-based tablet depends on the pH of the
medium, and that this biopolymer decreases the rate of swelling which can
influence tablet stability.
\begin{figure*}
\includegraphics{fig04}
\caption{\label{fig4}Swelling (A) and erosion (B) test of the spirulina
tablets in distilled water (D water), simulated gastric fluid (SGF) and
simulated intestinal fluid (SIF), (C) Phycocyanin release rate as a
function of immersion time in different solutions of spirulina
tablets.}
\end{figure*}
\vspace*{-2pt}
\subsection{\textit{In vitro} phycocyanin release study}\label{sec3.8}
The ability of tablets to dissolve and release their active ingredients
is naturally affected by how well they can break apart and disintegrate
in the body. If a tablet cannot erode or disintegrate effectively, it
may not release its active ingredients as intended, which could affect
its effectiveness~\cite{76}. The \textit{in vitro} phycocyanin release
profiles from the tablets prepared with date stone and spirulina
powders were studied in SGF (pH 1.2) and SIF (pH 7.4) using USP
dissolution apparatus according to Pharmacopoeia~\cite{32}. As seen in
Figure~\ref{fig4}C, the release rate of phycocyanin from formulated
tablets increases significantly in distilled water
(100\% in 30~min) compared to the other {{media}}. Thus,
tablets showed a slower release rate in the acidic medium (56.20\% in
30~min). This variation may due to the structure of the carrier used
in tablet formulation~\cite{77}. In SIF, the dissolution of the tablet
favoured a slow diffusion of the active \mbox{principle} towards the external
environment. Sustained release forms are designed to reduce the
frequency of \mbox{administration} of drugs with short elimination half-lives
and short duration of action~\cite{78}. These forms also limit
fluctuations in the plasma concentration of the drug, ensuring a more
regular therapeutic effect while minimizing side effects~\cite{79}.
The sustained release of antioxidants and anti-inflammatories has been
very effective and in high demand by drug developers~\cite{77}.
\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5}Fourier transform infrared (FTIR) spectroscopic
spectrum of the {date stone and spirulina tablets}.}
\end{figure*}
\vspace*{-2pt}
\subsection{Fourier transform infrared (FTIR) spectroscopic analysis of
tablets}\label{sec3.9}
As seen in Figure~\ref{fig5}, the
following functional categories are
represented by the FTIR analyses of the date stone and the spirulina
tablets.
The O--H stretching vibration, representing the carbohydrates,
and the presence of amino acids were all represented by the frequency
range from 3700 to $3200~\mathrm{cm}^{-1}$ in the FTIR spectrum. The
existence of secondary amines (protein, lipid) in the N--H stretching
vibration was represented by frequency ranges from 3300 to
$3000~\mathrm{cm}^{-1}$,
while the presence of aliphatic C--H
stretching vibration is represented by frequency ranges from 2980 to
$2830~\mathrm{cm}^{-1}$.
The frequency ranges between 1750 and
$1710~\mathrm{cm}^{-1}$ are a representation of the vibration of
C${=}$O (ester and amino acid). The N--H bending vibration in the
$\rmbeta$-carbonyl unsaturated keto-amide is made up of the
$1640\ndash1590~\mathrm{cm}^{-1}$. The frequency ranges from
$1445\ndash1400~\mathrm{cm}^{-1}$
resulting from $-\mathrm{CH}_2$
bending vibration $(\mathrm{CH}_2\ndash\mathrm{CO}\ndash)$ indicated
the presence of carbonyl compounds. The particular frequency ranging
$1360\ndash1250~\mathrm{cm}^{-1}$ characterized the C--O
stretching.
O--H bending vibration confirmed the presence of alcohol.
The frequency ranges from $1310\ndash1240~\mathrm{cm}^{-1}$ confirmed
presence of C--O asymmetric C--O--C stretching (presence of esters),
the {{bond}} with frequency value
representing the $1070\ndash1000~\mathrm{cm}^{-1}$
range corresponds to the symmetric stretching vibration of
{{$\ndash\mathrm{SO}_3$}}, indicating the presence of
sulphonate bonds. The S--O stretching vibration of sulphonic components
can be detected by
frequencies ranging from 700 to $600~\mathrm{cm}^{-1}$. The
observations of the protein, lipid, sulphonate and amino acid content
were to categorize spirulina powder. The results obtained after
the functional study confirmed by FTIR showed the dominance of the
specific functions of spirulina. Its richness in proteins was confirmed
by the carbonyl beta-unsaturated keto-amide (C${=}$N) groups, and the
sulfated polysaccharides were confirmed by R--$\mathrm{SO}_3$
functions. Our study agrees with the work
of Dotto \etal~\cite{80} where they found that the main intensity bands
of spirulina were located at 3269, 2918, 1660, 1627, 1548, 1409, 1028,
and $850~\mathrm{cm}^{-1}$. At $3269~\mathrm{cm}^{-1}$, the O--H bond
stretching combined with the $\mathrm{NH}_2$ group may be seen. The
peak of $2918~\mathrm{cm}^{-1}$ is related to the stretching of
$\mathrm{CH}_2$. At $1660~\mathrm{cm}^{-1}$, $\mathrm{NH}_2$ groups
may be seen scissor bending. C${=}$C stretching may be seen at 1627 and
$1548~\mathrm{cm}^{-1}$. The bands at 1409 and $1028~\mathrm{cm}^{-1}$
are perhaps --S--O and --P--O, respectively. Similarly, Venkatesan
\etal~\cite{81}
recorded
spirulina
FTIR spectra
in the region of
$3428\ndash3320~\mathrm{cm}^{-1}$
to
$620\ndash490~\mathrm{cm}^{-1}$
in the different frequency ranges.
\section{Conclusion}\label{sec4}
In conclusion, our work has demonstrated that spirulina powder and date
stones are a potential source of essential nutrients and have active
ingredients such as phenolic compounds and phycocyanin. The
valorisation of these compounds was made by the formulation of a food
supplement in the form of a tablet having the purpose of delivering
phycocyanin. The final formula was optimized by a statistical model
(D-Optimal Mixture Design) involving the physicochemical parameters of
the tablets. The final formula was effective in terms of controlled
release in simulated gastric and
intestinal fluids.\unskip\break
According to the
results of the FTIR study,
our tablet comprises protein, lipid,
carbohydrate, aliphatic (C--H), carbonyl (esters and acid), carbonyl
beta unsaturated ketone amide (C${=}$N), ester, symmetric C--H
stretching vibration, and sulfated chemicals
$(\mathrm{R}\ndash\mathrm{SO}_{3})$. Thus, more distinctive
characteristics may be seen in the FTIR spectrum. These phytochemicals
may have some significant biological effects.
\vspace*{2pt}
\section*{Conflicts of interest}
\vspace*{2pt}
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.
\vspace*{2pt}
\back{}
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