Release of bioactive substances from formulations containing "Arthrospira Platensis (Spirulina Platensis)"

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A c ta Poloniae Pharm aceu tica - D rug R esearch, V ol. 7 5 N o. 5 pp. 1 1 8 7 -1 1 9 9 , 2018 DO I: 10.32383/appdr/85111

IS S N 0001-6837 P o lish Ph arm aceu tical Society

RELEASE OF BIOACTIVE SUBSTANCES FROM FORMULATIONS CONTAINING ARTHROSPIRA PLATENSIS (SPIRULINA PLATENSIS)

B O Ż E N A M U S Z Y Ń S K A 1*, JA N L A Z U R 1, A G A T A K R A K O W S K A 2, B A R B A R A JĘ K O T 1, A G N IE S Z K A S Z E W C Z Y K 1, K A T A R Z Y N A S U Ł K O W S K A -Z IA JA 1, Ł U K A S Z Z IM M E R 3,

E W A P O L E S Z A K 3 and W Ł O D Z IM IE R Z O P O K A 2

d e p a r tm e n t o f P h arm aceu tical B otany, D e p a rtm e n t o f Inorganic and A n aly tical C hem istry, F aculty o f P harm acy, Jag iello n ian U niversity M ed ical C ollege, M ed y czn a 9, 30-688 K raków , P oland

3C hair and D ep artm en t o f A p p lied P harm acy, M ed ical U niversity o f L ublin, C hodźki 1, L ublin 20-093, P oland

A b stra c t: Arthrospira platensis (Spirulina platensis) is a w ell-know n m icroalga and has b een utilized as a m edicinal agent and foodstuff by hum ans since at least 16th century. The aim o f this study was to determ ine zinc co n ten t as w ell as determ ine phenolic and indole com pounds from com m ercial preparations containing Arthrospira p latensis (lyophilizate, tablets, and capsules) before and after extraction w ith m ethanol and incuba

tio n w ith artificial digestive juices. The secondary aim o f this study w as to evaluate the quality o f these p rep a

rations. The sam ples were incubated in artificial stom ach juice and in intestinal juice. The sam ples w ere m in eralized and their zinc(II) ions content was estim ated using flam e absorption atom ic spectroscopy (F-A A S). The m axim um zinc(II) ions content released into the digestive juices was found to be up to 1.6 m g/100 g o f the preparation. P henolic com pounds identified in the exam ined extracts are as follows: gallic acid; protocatechuic acid; 3,4-dihydroxyphenylacetic acid; p-hydroxybenzoic acid; syringic acid; cinnam ic acid; and quercetin.

Furtherm ore, indole com pounds identified were 5-hydroxy-L-tryptophan, 5-m ethyl-L-tryptophan, L-trypto- phan, tryptam ine, and 5-m ethyltryptam ine. C onsequently, it was also found th at the distributed Arthrospira p latensis in the form o f tablets does n o t disintegrate in the artificial digestive juices. A m ong the exam ined preparations, only hard capsules m et the requirem ents o f the European P harm acopeia 8 th ed.

K e y w o rd s: Arthrospira platensis, artificial digestive juices, indole com pounds, phenolic com pounds, zinc

Arthrospira p la te n sis (Spirulina p la ten sis) has b een u sed as a fo o d b y the A ztecs since the 16th ce n tury in M exico w hen it w as fish ed from Lake T exaco and then d ried and so ld in the form o f b is cuits as d escrib ed b y S panish soldiers. A . p la te n sis is currently cu ltiv ated and is u sed in m any countries as a dietary supplem ent b ecau se o f its substantial n u tri

tional value (1-3). A rthrospira m a xim a and Arthro- spira p la te n sis are the species c lassified as S p iru lin a , an d m o st co m m o n ly these species are u sed in d ietary supplem ents. The p ro tein co n ten t in A . p la te n sis is approxim ately 6 5 -7 9 % an d is a c o m  plete source o f p ro tein and am ino acids. A b o u t 7%

o f the w eig h t o f A . p la te n sis contains lipids, m ainly com prising y-linolenic acid, a -lin o le n ic acid, stearic acid, eicosapentaenoic acid, d o cosahexaenoic acid, and arachidonic acid. A . p la te n sis also contains sig  n ifican t am ounts o f y-linolenic acid (4, 5). A . p la ten - sis is a go o d source o f w ater-soluble v itam in s such

as C, PP (niacin) fro m group B (B 1, B 2, B 5, B 6, and B12); fat-soluble vitam ins, such as A , D, E, and K;

and eicosapentaenoic acid. It is also a go o d source o f m acro and m icroelem ents, such as P, Ca, K, N a, M g, Fe an d also contains inositol and d yes (phyco- cyanins, carotenoids, and ch lo ro p h y ll b) (6, 7, 8).

P hycocyanin, b elo n g in g to the w ater-soluble p ig m ents ph y co b ilin s (1% o f A . p la te n sis m ass), is m ainly resp o n sib le fo r its ability to neutralize free radicals. This b lu e dye gives A . p la te n sis its ch arac

teristic, d ark-turquoise color. It also contains carotenoids such as ß-carotene an d ß -cry p to x an th in as w ell as chlorophyll a an d b. A . p la te n sis is used in the p rev en tio n an d treatm en t o f vario u s diseases such as obesity, anem ia, h y pertension, hyperlipi- dem ia, diabetes, som e cancers, neurodegenerative diseases such as A lz h e im e r’s d isease, an d so on (9 -1 3 ). D ue to the antioxidant, anti-inflam m atory, antidepressant, and im m u n o stim u lato ry properties

* C orresponding author: e-m ail: m uchon@ poczta.fm

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o f A . p la te n sis d escrib ed earlier in the scientific lit

erature, it w as d ecid e d to determ ine its co n ten t of zinc, phenolic, and indole com pounds (1 4 -1 6 ).

Z inc is essential fo r the p ro p er d ev elo p m en t of hum an body. This elem en t is not only an activator b u t is also a co facto r o f about 300 enzym es. Z inc is resp o n sib le fo r the m etab o lism o f nucleic acids, p ro teins, lipids, and carbohydrates (17). It affects the ex p ressio n o f genes during the rep licatio n and tra n scription o f D N A and R N A (17). It is also resp o n si

b le fo r the synthesis o f re d b lo o d cells and affects the functions o f inter alia respiratory, reproductive, an d im m u n e system s (18, 19) and also dem onstrates an anti-inflam m atory, reg en eratin g and an tid ep res

sant activity.

P h e n o lic a n d in d o le co m p o u n d s p rim a rily e x h ib it an a n tio x id a n t, a n ti-in fla m m a to ry , an d im m u n o stim u lan t activity.

The aim o f this study w as to p erfo rm q u an tita

tive analysis o f zinc ions as w ell as p h en o lic and indole co m pounds in prep aratio n s o f com m ercial o rigin containing A . p la te n sis b efore an d after an ex tractio n using artificial digestive ju ices. Flam e a b sorption atom ic spectroscopy (F-A A S ) w as used to analyze zinc, w hereas p h en o lic an d indole c o m p o u n d s w ere d eterm in ed using rev erse phase-h ig h p erfo rm an ce liq u id ch ro m ato g rap h y (R P-H PL C ).

The analysis w as p erfo rm ed to evaluate the h ig h e s t am o u n ts o f b io e le m e n ts, p h e n o lic , and indole co m pounds and zinc (II) ions released am ong the prep aratio n s containing A . p la te n sis (lyophi- lizate, tablets, and capsules) after the extraction using artificial digestive ju ices. The final p ro d u ct w ill be a b etter source o f these substances fo r hum an consum ption. In addition, the n ex t aim o f the w ork w as to evaluate w h eth er the form u latio n containing A . p la te n sis is suitable fo r the effective release o f z in c an d p h e n o lic a n d in d o le c o m p o u n d s and w h eth er it m eets the p h arm aco p eia requirem ents.

E X P E R IM E N T A L

R eagen ts and standards

S tandard zinc (II) ions solution at a co n cen tra

tion o f 1000 pp m w as o b tain ed from O U M (Łódź, Poland); sub seq u en t dilutions o f 100, 10, and 1 ppm co n cen tratio n s w ere p rep ared from the above so lu tion. M g C l2 w as o btained from C hem pur (K raków , Poland); N aC l, K Cl, and N aH C O 3 w ere obtained from PPH G olpharm (K raków , Poland); p ep sin and b ile salts w ere o b tain ed from B T L (Ł ódź, Poland);

C aC l2 w as o btained from P harm a Z entrale G m bH (G erm any); pan creatic extract, H Cl, K Cl, co n cen trated H N O 3 Suprapur®, and K N O 3, Suprapur® w ere

o b ta in e d fro m M e rc k (D a rm sta d t, G erm a n y );

C6H 8O7, Z n S O 4, K H C O 3, N a2H P O 4, K 2H P O 4, and N aO H w ere p u rch ased from P olish C om pany of C hem istry (G liw ice, Poland). W a te r (qu ad ru p le-d is

tilled) w ith the co n d u ctiv ity o f less than 1 pS /cm w as o btained using an S2-97A 2 d istillatio n ap p ara

tus (C hem L and, S targard S zczecin, Poland). The follow ing standard p h en o lic co m pounds o f H PLC grade w ere p u rch ased from F luka C hem ie G m bh (S w itzerland): p -c o u m a ric acid; feru lic acid; p - h y droxybenzoic acid; van illic acid; and 3,4-dihy- d ro p h en y lacetate acid. C affeic acid, chlorogenic acid, cinnam ic acid, o-coum aric acid, protocatechuic acid, sinapic acid, gallic acid, and syringic acid an d quercetin and standards o f indole c o m p ounds, nam ely, L -tryptophan, 5 -hydroxy-L -tryptophan, 6 -m ethyl-L -tryptophan, serotonin, m elatonin, try p tam in e, a n d 5 -m e th y l-try p ta m in e w ere p u r c h ased from S igm a-A ldrich (St. L ouis, M O , U SA );

all w ere o f H PLC grade. M eth an o l, acetic acid, and p e tro le u m e th e r w ere p u rc h a s e d fro m M e rc k (D arm stadt, G erm any) w ere also o f H PL C grade.

R esearch m aterial

The studies w ere co n d u cte d on the dietary su p plem en ts containing Arthrospira p la te n sis (m icro al

gae fro m M icrocoleaceae fam ily), tw o preparations in the p o w d ered form and tw o in the tab let form , and one in capsules w ere evaluated. B oth, the m ethanolic extracts o f these prep aratio n s an d the extracts o btained after in cubation w ith artificial digestive ju ic e s w ere objects o f the experim ent. The selected prep aratio n s d iffered in p rep aratio n form and dosage an d w ere d e riv e d fro m d iffe re n t m an u factu rers (T able 1).

P reparation o f artificial d ig estiv e ju ices

A rtificial saliva (pH = 6.8.) w as prep ared according to the m eth o d o f A rv id so n (20). B riefly, 100 m L K H 2P O 4 at a concentration o f 25 m m ol/L , 100 m L N a 2H P O 4 at a concentration o f 24 m m ol/L , 100 m L K H C O 3 at a co n cen tratio n o f 150 m m ol/L , 100 m L M g C l2 at a concentration o f 1.5 m m ol/L , 6 m L citric acid at a concentration o f 25 m m ol/L , 100 m L C aC l2 at a co n cen tratio n o f 15 m m ol/L w ere added to the flask an d the v olum e w as m ade up to 1000 m L w ith four-tim es d istilled w ater.

A rtificial stom ach ju ic e (pH = 2.0) w as p re p a re d according to the m eth o d describ ed in P olish P harm aco p eia X. B riefly, 2.0 g N aC l an d 3.2 g p ep sin w ere d isso lv ed in fou r-tim es d istilled w ater.

T hen, 80 m L H C l at a co n cen tratio n o f 1 m ol/L w as added, an d the volu m e w as m ade up to 1 L using four-tim es d istilled w ater (21).

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Release o f bioactive substances from form ulations containing.. 1189

A rtificial intestinal ju ic e (pH = 8.0) w as p re p ared according to the m eth o d o f N eu m an n (22).

B riefly, 20 m g pan creatic extract, 120 m g bile salt, and 8.4 g N aH C O 3 w ere d isso lv ed in four-tim es d is tilled w ater and the v o lu m e w as m ade up to 1 L using four-tim es d istilled w ater.

S am p le preparation

R e le a se stu d ie s w ere p e rfo rm e d in the G astro el-2 0 1 4 apparatus, w hich w as co n stru cte d at the D ep artm en t o f Ino rg an ic and A n aly tical C h em is

try at the F acu lty o f P harm acy, M ed ical C ollege, Jag iello n ian U niversity. This apparatus allow s the study o f substances re le a se d into the artificial d ig e s

tive ju ices, im itates g astro in testin al m otions, and also pro v id es a co n stan t tem perature o f 37OC (23).

P re p a ra tio n s c o n ta in in g A . p la te n s is w ere w eig h ed (at 0.5 g) an d after p erform ing m in eraliza

tion three parts o f the sam ples w ere m ad e: one p art o f the sam ples w as u sed to determ ine zinc (II) ions content, the second fo r m ethanol ex tractio n (p h en o lic an d indole com pounds) w hile the other p art w as su b jected to in vitro digestion using G astroel-2014.

The second p a rt w as u sed fo r three tim es extraction b y 100 m L o f m ethanol using u ltraso u n d at a fre q u en cy o f 49 k H z during 30 m inutes (Sonic-2, P olsonic, Poland). F o r the last part, the w eighed sam ple w as tran sferred to 100 m L E rlen m ey er flasks and then w etted w ith 2 m L o f artificial saliva; su b sequently, 100 m L o f stom ach ju ice w as added. The flasks w ere clo sed w ith a stopper and p la c e d in the apparatus. The in cubation p ro cess w as co n tin u ed for 15, 30, an d 60 m in. The resulting solutions w ere fil

tered using a B ü ch n er funnel and a v acu u m set. The resid u e w as tran sferred to E rlen m ey er flasks to g eth er w ith the filter p a p e r an d 100 m L o f intestinal ju ice w as added (the extraction pro cess lasted 150 m in).

Then, the contents w ere filtered again. The sam ples w ere d iv id ed into tw o lots, one o f w hich w as d e sig n ated fo r zinc analysis an d the other fo r the analysis o f organic co m pounds (phenolic and indole c o m pounds).

A nalysis o f Z n con ten t before and after in cu b a  tion w ith artificial d ig estiv e ju ic e s usin g th e F- A A S m ethod

The co n cen tratio n o f zinc (II) w as d eterm ined using the F-A A S m ethod. M in eralizatio n o f the prep aratio n s containing A . p la te n sis w as p erfo rm ed in the M ag n u m II m icrow ave m in eralizer E R T E C (Poland). M in eralizatio n w as p erfo rm ed fo r 1 h in three m ag n etro n cycles: 15 m in at 60% pow er, 15 m in at 80% pow er, an d 30 m in at 100% pow er.

M in eralizatio n o f solutions after in cu b atio n w ith artificial digestive ju ic e s using G astroel-2014 w as p erfo rm ed in the U V R -8 M in eral P o lan d m in eraliz

er. T his p rocess w as p erfo rm ed b y U V irrad iatio n of the m in eralized test solution in a quartz reactio n v e s sel in 5 cycles o f 6 - 8 h.

T herm o S cientific A A S pectro m eter iC E 3000 SE R IES (U SA ) w as u sed in all the m easurem ents of zinc.

R ev erse-p h a se h ig h p erform an ce liq u id ch ro

m atography (R P -H P L C ) o f p h en olic com p ou n d s The extracts o btained from m eth an o l an d after incubation w ith artificial digestive ju ic e s w ere an a

ly zed fo r p h en o lic co m p o u n d s using the m eth o d o f R P-H PL C , w hich w as p erfo rm ed b ased on the p ro cedure d ev elo p e d b y S u lk o w sk a-Z iaja (24). The analysis w as p erfo rm ed at 25OC, w ith a m obile phase consisting o f A - m eth an o l and B - m ethanol:

0.5% acetic acid 1 : 4 (v/v). The follow ing gradient w as applied: 100% B fo r 0 - 2 0 m in; 1 0 0 -8 0 % B for 2 0 -3 5 m in; 8 0 -6 0 % B fo r 3 5 -5 5 m in; 6 0 -0 % B for 5 5 -7 0 m in; 0% B fo r 7 0 -7 5 m in; 0 -1 0 0 % B for 7 5 -8 0 m in; 100% B fo r 8 0 -9 0 m in at a flow rate 1 m L /m in, X = 254 nm (phenolic acids), X = 370 nm (flavonoids). Q uantification w as p erfo rm ed b y the m easu rem en t o f p e a k area w ith referen ce to the stan d a rd c u rv e d e riv e d fro m fiv e c o n c e n tra tio n s (0 .0 3 1 2 5 -0 .5 m g/m L ). The quantitative analysis of phenolic co m p o u n d s w as p erfo rm ed using a c alib ra

tion curve w ith the assum ption o f the lin ear size o f the area u n d er the p e a k and the co n cen tratio n o f the

Table 1. D ietary supplem ents containing A rthrospira platensis w hich were used for determ ination o f zinc con

ten t after an extraction to artificial digestive juices.

Name of the preparation Form Expiry date

Spirulina B Powder 11.2016

Spirulina P Powder 07.2012

Spirulina M Capsules 10.2016

Spirulina N Tablets 08.2016

Spirulina O Tablets 03.2017

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referen ce standard. The results w ere e x p ressed in m g/100 g o f dry w eig h t (d.w.).

R P -H P L C an alysis o f in d o le com p ou n d s

The extracts o btained from m ethanol and after in cubation w ith digestive ju ices w ere evap o rated to d ryness u n d er p ressu re o f 200 m B a at 40OC (BUchi evaporator, G erm any). The co n cen trated analyte w as d is so lv e d in m e th a n o l tra n sfe rre d th ro u g h W h atm an N o. 3 filter paper. The extracts w ere q u an titatively disso lv ed in 1.5 m L o f so lv en t system (m ethanol : w ater : am m onium acetate at 15 : 14 : 1 v /v/v) and su b jected to separation b y R P -H P L C using the H itachi H PL C (M erck, Japan) equipped w ith a p u m p type L -7100, the Purospher® R P-18 (4 X 200 m m , 5 pm ) colum n k e p t at 25OC and UV d etec to r o p erated at X = 280 nm . The isocratic sep a

ratio n w as as follow s: m ethanol/w ater/am m onium acetate (15 : 14 : 1 v/v/v) at a flow rate o f 1 m L/m in.

The qu an titativ e analysis o f indole co m pounds w as p e rfo rm e d u sin g a c a lib ra tio n c u rv e w ith the a ssum ption o f the linear size o f the area u n d er the p e a k and the concentration o f the referen ce stan dard. Q uantification w as p erfo rm ed by the m easu re

m en t o f p e a k area w ith referen ce to the standard curve d eriv ed from five concentrations (0 .0 6 2 5 -1 m g/m L ). The resu lts w ere ex p ressed in m g/100 g o f d.w .

Properties o f tablets

The tablets w ere ev alu ated as p e r standard p ro cedure according to E uropean P h arm acopeia 8th ed fo r u n ifo rm ity o f w eight, hardness, friability, and disin teg ratio n tim e (25). T ablets w ere also tested for v ariation in thickness to determ ine any variab ility a ssociated w ith the tab let p ress or the m eth o d o f p reparation.

The average w eig h t w as o btained according to p h arm aco p eia lim its b y w eighing ran d o m ly selected 20 ta b le ts on an a n a ly tic a l b a la n c e (O H A U S A d v en tu rer Pro). The h ardness o f the tablets w as

d eterm in ed fo r at le a st 10 tablets using the E rw eka TBH 20 h ardness tester (E rw eka G m bH ), and adopt

ing a m inim um h ardness o f 40 N as the acceptance criterion. F o r each form ula, friab ility w as evaluated from the p ercen tag e w eig h t lo ss o f 20 tablets tu m b le d in E rw e k a T A R 120 fria b ila to r (E rw ek a G m bH , H ausenstam m , G erm any) at 25 rpm for 4 m in. The tablets w ere d ed u sted and the loss in w e ig h t cau sed by fracture or abrasion w as reco rd ed as the percen tag e w e ig h t loss. F riab ility < 1% w as c o n sid e re d accep tab le. R esp ectiv e d isin teg ratio n tim es o f the p rep ared tablets w ere m easu red in 900 m L o f p u rified w ater w ith disks at 37OC using an E rw eka Z T 222 tester (E rw eka G m bH , H ausen

stam m , G erm any). Six tablets w ere ran d o m ly select

ed from each form ulation and w ere p u t into b a sk e t

rack. The d isintegration tim e w as reco rd ed till all the fragm ents o f the d isin teg rated tab let p assed through the screen o f the basket. F o r n o n -m o d ified tablets, the d isintegration tim e sh ould not be lo n g er than 15 m in. The thickness o f the tablets w as d eterm in ed for 20 tablets using digim atic V ern ier calip er (0 -1 5 0 m m ).

S ta tistica l analysis

Statistical analysis o f the data w as p erfo rm ed using one-w ay A N O V A w ith T ukey -K ram er p o st hoc analysis o f m ultiple com parisons. A value p <

0.05 w as accep ted as the level o f statistical sig n ifi

cance.

R E SU L T S A N D D IS C U S S IO N

T his is the first study w here the co n ten t o f zinc, p h en o lic an d in d o le co m p o u n d s in co m m ercial prep aratio n s o f A . p la te n sis from d ifferen t m an u fac

turers w ere analyzed. F urtherm ore, the lev el o f these co m pounds release w as in v estig ated using G astroel- 2014 apparatus w hich im itates co n d itio n s in the hum an digestive tract. S elected co m m ercially avail

able prep aratio n s as dietary supplem ents d iffered in

Table 2. Z inc content in selected preparations containing A rthrospira platensis.

Preparation Zinc content in preparations containing spirulina (mg/100 g ± SD)

Spirulina B 1.87 ± 0.05

Spirulina P 0.38 ± 0.02

Spirulina M 0.63 ± 0.03

Spirulina N 1.17 ± 0.06

Spirulina O 1.15 ± 0.07

The data presented was m ean ± SD (standard deviation); n = 6

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Table 3. The content o f zinc released to artificial digestive juices from selected Arthrospira p latensis - containing preparations.

Release o f bioactive substances from form ulations containing... 1191

Digestive

juice Stomach juice Intestinal juice (150 min)

Time

[min] 15 30 60 after 15 in

stomach juice

after 30 in stomach juice

after 60 in stomach juice Amount of Zn released (mg 100 g-1 of preparation)

Spirulina B (powder)

0.60 ± 0.04a 1.56 ± 0.50a 0.33 ± 0.02a 0.14 ± 0.05a 0.14 ± 0.05a 0.39 ± 0.03a Spirulina P (powder)

0.91 ± 0.07abc 1.14 ± 0.04abcd 0.67 ± 0.04abAd 0.16 ± 0.01bd 0.20 ± 0.00a,c,d 1.05 ± 0.01abAd Spirulina M (capsules)

0.50 ± 0.17b 0.62 ± 0.04a,b 0.35 ± 0.06a,b 0.24 ± 0.0a,b 0.16 ± 0.02b 0.12 ± 0.00ab Spirulina N (tablets)

0.26 ± 0.03ab-c 0.27 ± 0.02a,c 0.403 ± 0.08b,c 0.13 ± 0.00bc 0.15 ± 0.02c 0.15 ± 0.00a-b-c Spirulina O (tablets)

0.21 ± 0.02abd 0.40 ± 0.08cAd 0.22 ± 0.02a,b,c,d 0.27 ± 0.04a-c-d 0.05 ± 0.01abAd 0.09 ± 0.02abAd

D ata are presented as the m ean ± standard deviation (SD); n = 6 repetitions T u k ey -K ram er te st w as used to reveal the differences betw een paired groups o f zinc in row s, the sam e letters (a, b, c, d, e) are m arked for the co n ten t w hose differences are statistically significant (for p values < 0.05) (G raphPad InStat)

S p ir u lin a sp e c ie s, p re p a ra tio n fo rm (p o w d e re d lyophilizates, capsules, an d tablets), dosage, and w ere d eriv ed fro m d ifferen t m an u factu rers.

F-A A S w as u sed to evaluate the co n ten t o f zinc (II) ions in A . p la te n sis-c o n ta in in g prep aratio n s that w ere in cu b ated w ith artificial digestive ju ices. T he elab o rated co n d itio n s o f ly o p h ilized m aterial m in e r

alization an d the analytical m eth o d allo w ed to d eter

m ine the zinc(II) ions concentration in preparations and extracts o f in cu b ated preparations.

Z inc content in the exam in ed preparations was fo u n d to be in the range o f 0.38-1.87 m g/100 g of preparation (T able 2). The h ig h est co n ten t o f zinc (II) ions w as d eterm ined in the p o w d er form o f preparation (1.87 m g/100 g o f preparation). The am ount o f zinc (II) ions released fo r artificial d ig es

tive juice varies w ithin a narrow range o f values and it depends on the degree o f pow dering the raw m ate

rial in preparations. The content o f zinc (II) ions in the dry m atter o f A . p la te n sis according to previous studies has b een rep o rted as 2 m g/100 g (26). B ut, in this study, w as fou n d a considerably lo w er co n ten t o f zinc (II) ions, 0.38 m g/100 g o f preparation, in the po w d ered A . p la te n sis (cell lyophilizate). F o r tablets, very sim ilar am ounts w ere determ ined, that is, in the range o f 1.1 5 -1 .1 7 m g/100 g o f preparation.

A l-D habi (2013) d eterm in ed zinc (II) ions in A.

p la te n sis-c o n ta in in g prep aratio n s and o btained the resu lts at a sim ilar level (0 .0 5 -0 .6 m g/100 g of preparation) (27).

In order to estim ate the actual quantities o f this elem en t available to the h um an body, in cu b atio n o f p rep aratio n s co ntaining A . p la te n sis w ith artificial digestive ju ic e s (in G astroel-2014 apparatus) w as p erfo rm ed u n d er con d itio n s im itating those in the h um an bo d y (tem perature 37OC an d m ovem ents m im icking p eristalsis o f the h um an digestive tract).

O n the b asis o f F-A A S analysis w as fo u n d th a t zinc (II) ions are b etter released from ly o p h ilized A . p la te n sis th an th a t from tablets or capsules. The am ount o f zinc (II) ions released into the artificial digestive ju ic e s ran g ed from 0.05 to 1.56 m g/100 g o f the p rep aratio n (Figs. 1a, b). The hig h est am ount o f zinc (II) d eterm in ed in artificial stom ach ju ic e w as in the ran g e o f 0.2 to 1.6 m g/100 g o f the p re p a ration, after 30 m in o f in cubation (T able 3). In ad d i

tion, these am ounts in artificial intestinal ju ice w ere significantly low er, reg ard less o f the in cubation tim e in the stom ach ju ic e , w hich ran g ed from 0.1 to 0.4 m g /1 0 0 g o f th e p re p a ra tio n . T he h ig h est am ounts w ere d eterm in ed fo r S pirulina B, th a t is, p rep aratio n in the form o f a cell ly o p h ilizate (p o w der), and 1.6 m g/100 g o f p rep aratio n w as released into the stom ach ju ice after 30 m in o f in cubation (Figs. 1a, b). T his p rep aratio n p ro v ed to be the m ost optim al due to the am o u n t o f zinc (II) ions fo u n d in b o th types o f artificial digestive ju ic e s (stom ach and intestinal) and in any tim e variant. Z inc (II) ions w ere m ore efficien tly released from capsules than th at from tablets, despite the d em o n strated low er

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co n ten t o f this elem en t in the p ro d u ct before in c u b a tion w ith the artificial d igestive ju ices. The degree of z in c (II) ions released into the artificial stom ach ju ic e w as fo u n d to be lo w est in stom ach ju ic e (in case o f 15 m in in cu b atio n ). T he d aily hum an d em an d fo r this elem en t is about 1 2 m g , th a t is, the am o u n t released from the p rep aratio n can constitute zinc su p p lem en t to the h um an d iet (28).

P h en o lic and indole co m pounds w ere d eter

m in ed using R P -H P L C an d the resu lts o f the ca lc u latio n s w ere co n v erted to the am o u n t o f com pounds released from 100 g o f the preparation.

C om m ercial prep aratio n s o f A . p la te n sis are a g o o d source of p h en o lic an d indole com pounds. In this study, w e id en tified seven p h en o lic com pounds (gallic acid; p ro to catech u ic acid; 3,4-dihydroxy- p h en y lac etic acid; p -hydroxybenzoic acid; syringic acid; cinnam ic acid; and quercetin) in alm ost all co m m ercial prep aratio n s (T able 4). Furtherm ore, 3 ,4 -d ih y d ro x y -p h en y lacetic acid an d g allic acid w ere fo u n d to be hig h est am ong the ex am in ed p h e nolic com pounds. It w as b e st ex tracted fro m the p o w d er form o f p rep aratio n released in the artificial stom ach ju ic e , w hich w as up to 38.1 m g/100 g of p reparation. In addition, gallic acid w as fo u n d to be released hig h est in the intestinal ju ice from capsules (S pirulina M ) containing ly o p h ilized A . p la te n sis, up to a m ax im u m o f 7.3 m g/100 g o f preparation. O ther p h en o lic com pounds w ere d eterm in ed in b o th sto m ach an d intestinal ju ic e s at a sim ilar level o f 0.01 to

2.2 m g/100 g o f preparation. It is estim ated th a t 0.1 to 1.0 g daily dose o f p h en o lic com pounds is req u ired in the hum an diet. This ran g e is b ro a d and depends larg ely on the diet, including the am o u n t of co n su m ed fruits, vegetables, coffee, or tea (29, 30).

A ll the analyzed prep aratio n s w ere fo u n d to be a source o f p h en o lic com pounds. A fter con v ersio n of the reco m m en d ed daily dosage b y m anufacturers for the product, the total am o u n t o f p h en o lic c o m po u n d s w as determ ined, and on this basis, w as d em o n strated th at the prep aratio n s m ay be a source o f p h en o lic com pounds up to 60.7 m g/day for S pirulina P (lyophilizate).

The indole co m pounds d eterm in ed in the p re p aratio n s include 5 -h y droxy-L -tryptophan, trypta- m ine, 5 -m e th y ltry p ta m in e , 6 -m e th y l-try p to p h a n , and L -tryptophan. T hey w ere d eterm in ed in a sig

n ifican tly lo w er n u m b er o f ex p erim en tal v ariants than p henolic com pounds. C om p ared to m ethanolic extracts, indole co m p o u n d s w ere d eterm in ed at h ig h er concentrations in artificial digestive ju ices (T able 5). In addition, 5-hy d ro x y -L -try p to p h an w as indole co m p o u n d fo u n d in the hig h est am ounts in the d igestive ju ices, up to 156.2 m g/100 g o f p rep a

ra tio n . D u e to th e ir n u m e ro u s h e a lth b e n e fits (a n tio x id a n t, a n ti-in fla m m a to ry , a n tid e p re s siv e etc.), indole co m pounds should be supplem ented.

The daily doses of indole co m pounds supplied from A . p la te n sis - containing prep aratio n s (converted to the doses reco m m en d ed b y m anufacturers) w ere the

Figure 1. a) Total zinc content (m g/100 g o f preparation) after extraction to stom ach juice over the tim e o f 15, 30, and 60 m in. b) Total zinc content (m g/100 g o f preparation) after extraction to intestinal juice over the tim e o f 15, 30, and 60 m in incubation in stom ach juice

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Table 4. The content o f phenolic com pounds released to artificial digestive juices from selected Arthrospira p /atensis-containing prepara

tions.

Artificial juice Artificial stomach juice (mg/100 g of preparation)

Artificial intestinal juice (mg/100 g of preparation)

^''''UTime [min] 15 30 60 150 150 150

Preparation''''.. (after 1 min incubation in artificial saliva) (after incubation in artificial stomach juice) Extract to artificial

digestive juice

Methanolic extract (control) Gallic acid

Spirulina B 0.12 ± 0.05a 0.14 ± 0.00a 0.13 ± 0.00a 0.66 ± 0.00a 0.12 ± 0.00a 0.33 ± 0.00a 0.79 ± 0.02a Spirulina P 0.18 ± 0.00b 0.38 ± 0.00a,b 0.07 ± 0.2a-b 2.13 ± 0.33b 0.89 ± 0.09b 1.38 ± 0.00a 0.67 ± 0.04b Spirulina M 0.33 ± 0 .0 3 * 0.69 ± 0 .0 4 * 0.10 ± 0 .0 1 * 7.22 ± 1.11abc 4.56 ± 1.78abc 1.41 ± 0.30ac 2.44 ± 0 .1 7 * Spirulina N 0.01 ± 0 .0 0 * 0.02 ± 0 .0 3 * 0.01 ± 0.00 0.62 ± 0.58 0.59 ± 0.61 0.42 ± 0.40 1.06 ± 0.06 Spirulina O 0.10 ± 0.00 0.15 ± 0.00 - 7.31 ± 0 .8 1 * 2.32 ± 0.38a-c 2.09 ± 0.74a,d 0.81 ± 0.02c,d

Protocatechuic acid

Spirulina B 1.46 ± 0.14a 0.28 ± 0.05a 1.97 ± 0.05a 0.93 ± 0.10a 0.03 ± 0.00a 0.16 ± 0.06a 0.01 ± 0.001a Spirulina P 1.44 ± 0.14b 0.01 ± 0.00a,b 0.71 ± 0.05a,b 0.01 ± 0.00a 0.30 ± 0.02b 0.50 ± 0.05a,b 0.01 ± 0.00b Spirulina M 0.40 ± 0.03a,b,c 0.39 ± 0.05b,c 2.22 ± 0.60b,c 0.04 ± 0.005 0.01 ± 0.001 0.01 ± 0.01 0.01 ± 0.01 Spirulina N 0.20 ± 0.002a,b,d 0.25 ± 0.06b,d 0.64 ± 0.04a-c 0.05 ± 0.001a 0.05 ± 0.003b,c 0.01 ± 0.00a,b 0.05 ± 0.003c Spirulina O 1.25 ± 0.27c-d 0.79 ± 0 .1 3 * d 0.93 ± 0.0a-c 0.02 ± 0.002a 0.06 ± 0 .0 0 1 * 0.01 ± 0.00a,b 0.01 ± 0.002d

3,4-Dihydroxyphenylacetic acid

Spirulina B 26.47 ± 8.94a 20.27 ± 6.01a 20.90 ± 2.66a 2.05 ± 0.08a 2.29 ± 0.25 4.49 ± 0.20a 1.13 ± 0.11a Spirulina P 38.14 ± 2.25db 1.83 ± 0.00a 7.05 ± 0.13a-b 2.27 ± 0.19b 2.80 ± 0.30b 9.48 ± 2.11ab 0.71 ± 0.07b Spirulina M 5.27 ± 0.89a-b 6.55 ± 1.27a 25.15 ± 2.12b-c 3.86 ± 0 .2 1 * 2.39 ± 0.2 1.95 ± 0.04db 1.61 ± 0.13c Spirulina N 2.05 ± 0.00a,b 3.15 ± 0.77a 7.96 ± 0.24a-c 2.16 ± 0.00c-d 2.00 ± 0.16b 2.14 ± 0.28db 5.77 ± 0.38a,c,d Spirulina O 6.62 ± 1.30a-b 8.60 ± 1.72a 7.64 ± 1.24ac 3.26 ± 0 .1 5 * 2.23 ± 0.15 1.99 ± 0.34db 0.71 ± 0.01b,c,d

p-Hydroxybenzoic acid

Spirulina B 0.16 ± 0.01a 0.12 ± 0.06a 0.11 ± 0.05a 0.09 ± 0.01a 0.09 ± 0.01a 0.11 ± 0.04a 0.03 ± 0.01a Spirulina P 0.45 ± 0.07a-b 0.22 ± 0.03b 0.29 ± 002a-b 0.09 ± 0.01b 0.12 ± 0.03 0.22 ± 0.05a,b 0.05 ± 0.01b Spirulina M 0.11 ± 0.01bc 0.13 ± 0.05c 0.14 ± 0.02b 0.22 ± 0 .0 3 * 0.11 ± 0.03c 0.08 ± 0.00db 0.09 ± 0.01a-bc Spirulina N 0.09 ± 0.01b-d 0.08 ± 0.00b,d 0.18 ± 0.06 0.34 ± 0 .0 2 * d 0.20 ± 0.19 0.07 ± 0.01b 0.37 ± 0.01a-b-c-d Spirulina O 0.30 ± 0.04a,b,c,d 0.44 ± 0 .0 1 * d 0.25 ± 0.06a 0.08 ± 0.01c-d 0.10 ± 0.05d 0.09 ± 0.01b 0.05 ± 0.01c-d

Syringic acid

Spirulina B 0.37 ± 0.03a 0.21 ± 0.02a 0.28 ± 0.07a 0.10 ± 0.01a 0.08 ± 0.01a 0.07 ± 0.005a 0.02 ± 0.001a Spirulina P 0.40 ± 0.02b 0.38 ± 0.02a,b 0.29 ± 0.05b 0.07 ± 0.02b 0.17 ± 0.01a-b 0.21 ± 0.01ab 0.03 ± 0.00ab Spirulina M 0.04 ± 0.0a-b 0.21 ± 0 .0 6 * 0.45 ± 0 .0 6 * 0.29 ± 0 .0 0 1 * 0.14 ± 0.01a-c 0.05 ± 0.005b,c 0.05 ± 0.01a-bc Spirulina N 0.02 ± 0.00a-b 0.02 ± 0 .0 0 * 0.05 ± 0 .0 1 * 0.19 ± 0 .0 2 * d 0.09 ± 0 .0 1 * 0.05 ± 0.01bd 0.07 ± 0.01a-b-c-d Spirulina O 0.04 ± 0.00a-b 0.05 ± 0.0a,b,c 0.05 ± 0.01abc 0.31 ± 0 .0 1 * 0.15 ± 0.03a-d 0.13 ± 0.04db-c-d 0.01 ± 0.00b,c,d

Cinnamic acid

Spirulina B 0.11 ± 0.01a 0.01 ± 0.00a 0.07 ± 0.01a 0.03 ± 0.00a 0.08 ± 0.005a 0.10 ± 0.01a 0.01 ± 0.00a Spirulina P 0.04 ± 0.005a,b 0.07 ± 0.00 0.05 ± 0.00b 0.11 ± 0.003a-b 0.06 ± 0.01a-b 0.06 ± 0.001a-b 0.01 ± 0.00b Spirulina M 0.07 ± 0 .0 0 3 * 0.04 ± 0.001c 0.11 ± 0 .0 2 * 0.11 ± 0.01ac 0.07 ± 0.005c 0.07 ± 0.00a,c 0.04 ± 0.002a-b-c Spirulina N 0.09 ± 0 .0 0 2 * 0.12 ± 0.005dc 0.07 ± 0.00c,d 0.01 ± 0.00a,b,c,d 0.01 ± 0.00a,b,c,d 0.05 ± 0.001a-c-d 0.08 ± 0.005a,b,c,d Spirulina O 0.04 ± 0.005a,c,d 0.12 ± 0.06a-c 0.03 ± 0.003a,c,d 0.04 ± 0.005* 0.05 ± 0.005a,c,d 0.07 ± 0.00dd 0.02 ± 0 .0 0 * d

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Table 4. C ontinued.

^ ' ' \ T i m e [min] 15 30 60 150 150 150

Preparation'''''^^ (after 1 min incubation in artificial saliva) (after incubation in artificial stomach juice) Extract to artificial

digestive juice

Methanolic extract (control) Quercetin

Spirulina B 0.06 ± 0.01a 0.03 ± 0.00a 0.04 ± 0.00a 0.01 ± 0.00a 0.02 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a Spirulina P 0.04 ± 0.01a-b 0.21 ± 0.02bb 0.11 ± 0.04bb 0.01 ± 0.00b 0.03 ± 0.005a,b 0.18 ± 0.001a-b 0.03 ± 0.00a-b Spirulina M 0.02 ± 0.00a-b 0.03 ± 0.002b 0.11 ± 0.02a-c 0.02 ± 0.002b-c 0.01 ± 0.00bb 0.01 ± 0.00b 0.03 ± 0.02a-c Spirulina N 0.01 ± 0.00a-b 0.01 ± 0.00b 0.01 ± 0.00b-c 0.01 ± 0.00c 0.01 ± 0.00bb 0.01 ± 0.00b 0.11 ± 0.00*dd Spirulina O 0.01 ± 0.00a-b 0.01 ± 0.00b 0.02 ± 0.00b-c 0.01 ± 0.000c 0.01 ± 0.00bb 0.01 ± 0.00b 0.02 ± 0.00*cd D ata are presented as the m ean ± standard deviation (SD); n = 6 repetitions T u k ey -K ram er test was used to reveal the differences betw een paired groups o f phenolic com pounds in row s, the same letters (a, b, c, d) are m arked for the content w hose differences are statistically sig

n ificant (for p values < 0.05) (G raphPad InStat)

h ig h est fo r capsules (up to 6.8 m g/day) an d the lo w est fo r ly o p h ilizate (up to 2.6 m g/day).

In sum m ary, the release o f zinc and organic co m pounds from prep aratio n s co ntaining A . p la ten - sis d ep en d ed on the tim e o f incubation an d the degree o f degradation in artificial digestive juices.

A rtificial digestive ju ic e s did not b re a k dow n A.

p la te n sis-containing tablets even after a m axim um in cubation p erio d o f 60 m in in artificial stom ach ju ic e an d 150 m in in artificial intestinal juice.

D u rin g extractions w as o bserved the form ation o f a g elatin o u s lay er on the surface to p rev en t liq u id p e n etration into the in terio r an d further disintegration o f the tab let m ass. A fter the tab let w as c u t into tw o p arts, the co m p letely dry core w as clearly visible inside.

T hereby, w as d ecid ed to study the p hysical p ro p erties o f tablets containing A . p la te n sis acco rd  ing to E uropean P harm acopeia 8th ed (25). The p h y s

ical properties o f p rep ared tablets are show n in T able 6.

The co n tro lled tablets w ere eleg a n t in ap p ear

ance. The thickness o f the tablets ra n g e d from 4.74 to 4.99 m m . F or form u latio n S pirulina O p ercent d ev iatio n s o f thickness ex ceed ed 5% (acceptable ran g e o f thickness is ± 5% ). A verage w eight, h ard n ess, an d fria b ility w ere w ith in p h a rm a c o p e ia specification. The v ariation in w eig h t ran g ed from 255.3 to 401.5 m g (acceptable ran g e o f w e ig h t v a ri

ation is ± 5% ). The hardness o f tablets ran g ed from 62.4 to 64.3 N (acceptable range o f hardness is > 40 N ) an d friab ility ran g ed from 0.15 to 0.22% (accep t

able ran g e o f friability is < 1%). The disintegration tim es o f in v estig ated tablets ex ceed 15 m in. T ablet

fo rm ulations h ad excessively lo n g disintegration tim es w hich am ounted from 72.3 to 81.2 m in. The average disintegration tim e o f in v estig ated tablets w as fo u n d to be up to 76.7 m in. A ccording to E uropean P harm aco p eia 8th ed., the disintegration tim es fo r these prep aratio n s should be less than 15 m in. H ence, on the b asis o f the co n d u cte d studies, w as fou n d th a t A . p la te n sis in the form o f tablets can n o t be a go o d source o f zinc and organic c o m po u n d s in the hum an diet. O f the three preparations containing A . p la te n sis tested in accordance w ith the pharm aco p eia reg u latio n s, only capsule form c o m p lied w ith the p h arm aco p eia standards. The d ev ia

tions o f w eig h t in capsule ran g ed from 0 .2 to 3.6%

and the d isintegration tim e w as fo u n d to be 11.5 m in.

C h em om etric analysis

C hem om etric tools w ere u sed to obtain deeper inform ation o f the ob tain ed d ataset (results o f in d o le and p h en o lic co m pounds p erfo rm ed fo r p rep ara

tions co ntaining A . p la te n sis). The analysis w as b ased on T able 4. and 5. The objects o f analysis w ere prep aratio n s co ntaining A . p la te n sis in various form s (pow der, tablets, and capsules), d escrib ed by p aram eters - analyzed indole an d ph en o lic c o m pounds. C hem om etric analysis fo r such a dataset, c h aracterized by a w ide range o f variability, allow ed the extraction o f additional inform ation on the co r

relatio n s th at o ccur betw een the analyzed objects prep aratio n s containing A . p la te n s is . Tw o m ethods fo r chem om etric analysis w ere u sed in the study:

c lu s te r an aly sis (C A ) an d p rin c ip a l c o m p o n en t analysis (PC A ). C A m eth o d in d icated the sim ilarity

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Table 5. The content o f indole com pounds released to artificial digestive juices from selected Arthrospira platensis - containing preparations.

* \ T i m e [min] 15 30 60 150 150 150

Preparation (after 1 min incubation in artificial saliva) (after incubation in artificial stomach juice) Control 5-Hydroxy-L-tryptophan

Extract Control

Spirulina B 124.14 ± 10.3d 18.10 ± 0.81* 88.73 ± 12.0- 45.74 ± 1.79‘ 35.56 ± 4.21‘ 50.02 ± 8.78- 11.47 ± 0.71‘

Spirulina P 117.35 ± 2.41d-b - 25.02 ± 1.21ab 33.81 ± 5.67b 29.07 ± 4.31b 21.25 ± 1.41ab 9.54 ± 0.15b Spirulina M 27.17 ± 2.98db 80.28 ± 1 .6 0 * - 156.20 ± 17.2b 96.69 ± 11.06-1c 28.70 ± 1.71a - Spirulina N 22.07 ± 1.93ab 3.47 ± 0 .0 4 * 46.46 ± 3 .9 6 * 1 33.02 ± 3.97c 60.51 ± 5 .8 4 * 23.39 ± 1.18‘ 29.49 ± 1.61dbd Spirulina O 27.19 ± 1.02ab 42.23 ± 3 .6 2 * 27.71 ± 2.43"a 42.03 ± 2.24c 34.56 ± 3.43°-d 33.29 ± 2.92a,b -

Tryptamine

Spirulina B - - - 1.54 ± 0.00- 1.57 ± 0.01- 5.37 ± 0.80a -

Spirulina P 1.66 ± 0.01 - - 1.57 ± 0.00b 1.83 ± 0.03b 1.71 ± 0.20‘-b -

Spirulina M - 2.37 ± 0.00 - 3.66 ± 0 .3 8 * 2.94 ± 0.38d-bb 4.23 ± 0.17dAd -

Spirulina N - 1.68 ± 0.15 1.36 ± 0.01 - - - -

Spirulina O - - - 3.18 ± 0 .0 8 * d 2.37 ± 0.37‘-d 2.49 ± 0 .0 9 * -

5-Methyltryptamine

Spirulina B - - 3.27 ± 0.00 3.43 ± 0.01 3.21 ± 0.01 3.21 ± 0.01 0.69 ± 0.01

Spirulina P - - - - - - -

Spirulina M - - - - - - -

Spirulina N - - - - - - -

Spirulina O - - - - - - -

6-Methyl-L-tryptophan

Spirulina B 1.57 ± 0.08" 1.03 ± 0.00- 1.40 ± 0.10- 1.12 ± 0.01 0.92 ± 0.08- - -

Spirulina P 3.71 ± 0.18ab 3.92 ± 0.21‘-b 1.41 ± 0.22b 1.92 ± 0.33d-b 2.94 ± 0.11bb 5.00 ± 0.26‘-b 0.76 ± 0.04b

Spirulina M 3.06 ± 0 .2 1 * 10.23 ± 1 .7 4 * 4.91 ± 0.08‘-b - - 1.04 ± 0.01d-bb -

Spirulina N 2.44 ± 0 .1 1 * d - - 0.26 ± 0.01bb 0.74 ± 0.09b,c,d - 1.38 ± 0.01b

Spirulina O 1.00 ± 0 .0 8 * d 1.88 ± 0.83c 1.13 ± 0.20°-d - 0.74 ± 0.04a,b,d 0.58 ± 0.04bbbd - L-Tryptophan

Spirulina B 2.39 ± 0.78- 27.7 ± 0.32 1.69 ± 0.42 1.50 ± 0.10 0.50 ± 0.01 - -

Spirulina P 2.61 ± 0.25b 12.03 ± 0.46‘-b 0.46 ± 0.00d-b - - - -

Spirulina M 9.60 ± 0 .7 8 * 18.45 ± 1 .7 5 * 1.26 ± 0 .0 5 * 2.92 ± 0.00 1.08 ± 0.10 - -

Spirulina N 0.76 ± 0 .0 6 * - 0.83 ± 0 .0 4 * - - - -

Spirulina O - 1.09 ± 0.40bb 0.75 ± 0 .0 3 * - - - -

D ata are presented as the m ean ± standard deviation (SD); n = 6 repetitions T u k ey -K ram er te st w as used to reveal the differences betw een paired groups o f phenolic com pounds in row s, the same letters (a, b, c, d) are m arked for the co n ten t w hose differences are statistically sig

n ificant (for p values < 0.05) (G raphPad InStat)

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b etw een the ex am in ed objects (preparations c o n tain ing A . p la ten sis). In case o f this analysis, the sim i

larity o f objects is d em o n strated b y their close re la tive p o sitio n in m u ltid im en sio n al space. T he g rap h ical im age o f the C A analysis is the d endrogram (Fig. 2), in w hich the objects characterized b y c o n sid e ra b le s im ila rity fo rm th e c lu s te rs . In this instance, the x and у -axes do n o t co rresp o n d to the C artesian num erical axes (31, 32). The analyzed o bjects (A. p la te n sis preparations) w ere m ark ed on the x -axis, w hereas the distance b etw een the ex a m  in ed objects c alc u lated on the b asis o f W a rd ’s a gglom eration m eth o d on the у -axis. T hree m ajor clu sters w ere o b serv ed b a se d on the sim ilarity analysis (C A - Fig. 2). The first c lu ster form ed p rep aratio n s in the fo rm o f capsules, w hile the rem ain in g clusters w ere fo rm ed from prep aratio n s in

the form o f tablets or pow ders. T heir m em bership in particu lar clusters indicates their sim ilarity w ithin particu lar clusters. This sim ilarity m ay be related to the com p o sitio n o f the exam ined prep aratio n s (co n ten t o f indole and ph en o lic com pounds) as w ell as the d egree o f release o f the exam in ed m etabolites into the digestive ju ices.

S hort d endrogram arm s in case o f preparations in the form o f tablets show the h ig h est correlation sim ilarity w ithin these p rep aratio n s (31, 33). In addition, the differences b etw een the preparations (capsules and tablets and p o w d ered form ) belonging to other clusters m ay resu lt from u n eq u al conditions o f A . p la te n sis culture and depend on the type o f A.

p la te n sis used in the preparations; th erefore, there are d iscrepancies fo r m etabolites released into the digestive juices.

Figure 2. D endrogram presenting sim ilarity betw een the objects (preparations containing Arthrospira p latensis) depending on the form of preparation (C ity-Block, W ard ’s algorithm ). The analysis w as perform ed in Statgraphics C enturion X V II program

F igure 3. Scatterplot - sim ilarity o f the analyzed preparation w ith Arthrospira p latensis w ith respect to the site o f release phenolic and indole com pounds (s. j - stom ach juice, i. j - intestinal juice). The analysis was perform ed in Statgraphics C enturion X V II program

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Figure 4 . B iplot diagram presenting the correlation betw een the analyzed organic com pound p resent in the preparations containing Arthrospira p latensis and the site o f their release in h u m an digestive tract (s. j - stom ach juice, i. j - intestinal juice). The analysis was p er

form ed in Statgraphics C enturion X V II program

Table 6. E valuation o f physical properties o f prepared tablets (m ean ± standard deviation).

Formulation Weight variation (mg)

Thickness (mm)

Friability (%)

Breaking force (N)

Disintegration time (min)

Spirulina M 443.0 ± 4.62 7.5 ± 1.10 - - - 11.5 ± 1.05

Spirulina N 401.45 ± 4.21 4.74 ± 0.03 0.219 62.4 ± 7.06 81.17 ± 5.56

Spirulina O 255.3 ± 5.09 4.99 ± 0.12 0.15 64.3 ± 6.38 72.33 ± 4.59

Table 7. Factor loads for three first m ain principal com ponents (PC1, PC 2, and PC3).

Compounds PC1 PC2 PC3

3,4-Dihydroxyphenylacetic acid 0.450 0.217 -0.170

5-Hydroxy-L-tryptophan 0.061 0.581 -0.090

6-methyl-D,L-Tryptophan 0.314 -0.070 0.415

Cinnamic acid -0.038 0.226 -0.173

Gallic acid -0.242 0.495 0.249

L-Tryptophan 0.186 -0.137 0.528

p-Hydroxybenzoic acid 0.279 0.047 -0.277

Protocatechuic acid 0.422 0.084 -0.355

Quercetin 0.326 -0.061 0.333

Syringic acid 0.381 0.352 0.240

Tryptamine -0.303 0.397 0.216

The second m eth o d com p lem en tary to the CA u sed in this study is P C A . P C A allow s the red u ctio n o f the m easu rem en t data space to the m in im u m n e c essary to characterize the interactions b etw een them . A large n u m b er o f p aram eters (m easu red co n ten t of indole and p h en o lic com pounds) describing an a

ly z e d objects (preparations w ith A . p la te n sis) m ade the d irect v isu alizatio n o f m u ltid im en sio n al data space com plicated. In order to facilitate it, the n u m b er o f data w as red u ced b y correlating them . O utput p aram eters w ere re p la c e d b y new variab les - p ri

m ary com ponents. P C A analysis d em o n strated that

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61.92% o f the variations in the exam ined data set can b e described by the three prim ary com ponents (PC1, PC 2, and PC3). C onsequently, the rem aining com p o nents w ere not considered for further analysis. The three prim ary com ponents (PC1, PC 2, and PC 3) are a linear com bination o f prim ary variables m ultiplied by corresponding charges. The values o f the charges that correspond to the correlation coefficient w ith the p ri

m ary variables are sum m arized in Table 7. F or the PC1 com ponent, its m agnitude is considerably affect

e d b y the m easured value ofthe phenolic com pound, th at is, 3,4-dihydroxyphenylacetic acid. The other m ain com ponents, P C 2 and PC 3, can be interpreted in the sam e w ay. C onsequently, the reduction o f the input data area to the three prim ary com ponents allow ed for analysis in tw o-dim ensional space (Fig.

3) (34, 35). Tw o distinct clusters w ere distinguished analyzing the exam ined objects (preparations w ith A.

platensis) in relation to the site in the digestive sys

tem , w here phenolic and indole com pounds w ere released (Fig. 3). These com pounds are to a various degree released into an artificial stom ach and intes

tinal juices, as evidenced by their belonging to d is

tinct groups. Such division indicates that the ex am i

n ed organic com ponents are released into the d iges

tive ju ices to varying degrees, w hich in turn, depends on the release site in the condition im itated hum an digestive tract. In addition, considering the B iplot dia

gram (Fig. 4) b ased on the tw o prim ary com ponents (PC1 and PC3), the preference o f com ponents to the site w here they w ere released into the artificial d iges

tive tract w as analyzed. On this basis, w as found that the release o f indole and phenolic com pounds from preparations containing A . p la ten sis into artificial digestive ju ices depended on the type o f artificial digestive juice. The organic com pounds from the preparations w ere to the highest degree released into stom ach juices, w hich confirm s the direction o f the arm s in the B iplot diagram (Fig. 4). O nly sparse p h e

nolic com pounds (gallic acid and cinnam ic acid) and indole ones (tryptam ine) w ere released from the preparations m ostly to intestinal juice.

C O N C L U S IO N S

The ex p erim en t p re se n te d th a t the active su b stances in the co m m ercial p rep aratio n s are n o t c o m p letely re le a se d into the artificial d ig estiv e ju ic e s an d hen ce m ay n o t be p o te n tia lly av ailab le to hum an. The q u an tities o f re le a se d zinc (II) ions fro m p rep aratio n s co n tain in g A . p la te n sis are in s u f

fic ie n t fo r h u m an b o d y according to the daily re q u ire m e n t fo r this elem ent. In the case o f p h e n o lic com p o u n d s, th eir am ounts w ere fo u n d to be

h ig h er in th e ex tracts o f artificial d ig estiv e ju ic e s than th a t o f m eth an o lic ex tracts o f the tested p re p a ratio n s. In th is w ay, it has b een ad eq u ately show n th a t the co n te n t o f active substance in m ed icin al p rep aratio n s is n o t the sam e as its am o u n t released into d ig estiv e ju ic e s w hich su g g est the n eed for such analyzes. T he e x p erim en t fu rth er sh o w ed th at only h ard cap su les m e t the E uro p ean P h arm aco p eia 8th e d stan d ard s w h ereas tab lets d id not. D u e to this fact, an im p o rtan t ele m e n t o f research on d ietary su p p lem en ts is the ev alu a tio n o f th eir fo rm u la tio n s’

q u ality an d the d efin itio n o f a form th a t allow s the release o f b io lo g ically active co m p o u n d s in the m o st effectiv e m anner.

A ck n ow led gm en ts

T h is p ro je c t w as p a rtia lly su p p o rte d b y Jagiellonian U niversity M ed ical C ollege project:

K /Z D S /005619.

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