Biocatalysis and Agricultural Biotechnology 30 (2020) 101862

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Biocatalysis and Agricultural Biotechnology

Phytochemical analysis and in vitro antioxidant and antimicrobial activities of

hydroalcoholic extracts of the leaves of Salacia kraussii
Hercílio E. Zimila a, * , Albertina L. Matsinhe a, Emma Malayikab , Átifa I. Sulemane a,
Vanina N.C. Saete b , Saquina C. Rugunate b , Paulo J. Cumbane b , Isabel Magaia b ,
François Munyemana a
a Departamento de Química, Faculdade de Ciências, Universidade Eduardo Mondlane (UEM), Av. Julius Nyerere No, 3453, P. O. Box 257, Maputo, Mozambique
b Instituto Superior de Ciência e Tecnologia de Moçambique (ISCTEM), Curso de Farmácia, Rua, 1394, Zona da FACIM, 322 - Maputo, Mozambique

ARTICLE INFO ABSTRACT

Keywords : The pre sent work was aimed at iden tifying the bioac tive com pounds and de termine the in vitro an tioxidant
Salacia kraussii and an timi cro bial ac tivities of the hy droethano lic ex tract of S. kraus sii leaves. Phy tochem ical analy sis was
Antioxidant activity
per formed us ing gas chro matog ra phy -mass spec trom e try (GC -MS) and clas sical col orimet ric meth ods.
Antimicrobial activity
Folin -Ciocalteu method, alu minum chlo ride pre cip itation, and ca sein pre cip itation were em ployed for
Phytochemistry
GC - MS
quan tifica tion of total phe nols, flavonoids, and tan nins, re spec tively. The an tioxidant ac tivity was es timated
by DPPH scav eng ing, phos pho molyb date (TAC), ABTS rad ical scav eng ing, and re duc ing power as says
(FRAP -1 and FRAP -2). An timi cro bial ac tivity was de termined by the disk dif fu sion method against Es -
cherichia coli, Staphy lo coccus aureus, Pro teus mirabilis, Kleb siella pneumo niae, and Candida al bi cans. Phy to-
chem ical tests re vealed the pres ence of flavonoids, quinones, alkaloids, steroids, tan nins, and saponins. GC -
MS analy sis iden tified 17 bi ologically im por tant com pounds be long ing to dif fer ent classes, in clud ing fatty
acids, car bo hy drates, and terpenoids. The ex tract showed rel atively low con tent of total phe nols, tan nins,
and flavonoids of 5.00 mgEAG g−1 , 0.08 mgEAT g−1 , and 0.67 ± 0.29 mgEQ g−1 , re spec tively. Its an tioxi-
dant po ten tial was con sid er ably stronger than that of the other Sala cia sp. with EC 50 values of
64.28 ± 0.01, 36.44 ± 0.67, 35.78 ± 0.09, 33.91 ± 0.12, and 2.22 ± 0.25 μg mL −1 as mea sured by
FRAP -1, TAC, DPPH, FRAP -2, and ABTS rad ical scav eng ing as says, re spec tively. Regard ing the an timi cro -
bial ac tivity, the ex tract in hib ited the growth of all the test organ isms with min imum in hibitory con cen tra-
tions of 125 μg mL −1 and 250 μg mL −1 for the bac teria and fun gus, re spec tively. This in sight ful study is a
signif icant step towards the ex ploita tion of S. kraus sii leaves as a cheap source of broad -spectrum an timi cro -
bial agents.

1. Introduction
Med icinal plants have been used for thou sands of years around the
world for the prevention and treat ment of different ailments as well as
a nat ural source of thera peutically ac tive metabo lites (Casuga et al.,
2016; Senkoro et al., 2012). Herbal remedies from medicinal plants are
more compat ible, effective, cheaper, and safer to the human body than
modern synthetic drugs (Prasad et al., 2012). More over, rural commu-
nities rely on herbal drugs for primary health care to overcome the low -
coverage of the conventional system (Conde et al., 2014). In Mozam -
bique, for instance, ap prox imately 90% of people in rural ar eas depend
on the diverse ar senal of the medicinal plants to meet primary health
needs (Conde et al., 2014; Senkoro et al., 2012).
Plant species belong ing to the genus Sala cia (Celas traceae ) are
among the widely used in tra ditional medicine across the globe
(Bandeira et al., 2001; Deepak et al., 2015). Dif ferent parts of Sala cia
sp. occurring in South east Asia, such as S. retic u lata and S. ob longa ,
have been used in the Ayurveda system of medicine to treat dia betes,
gon orrhea, rheumatism, asthma, leukemia, inflam ma tion, hemor-
rhoids, skin diseases, colic, itching, and ear diseases (Deepak et al.,
2015; Paarakh et al., 2008; Ramakrishna et al., 2015). Such tra ditional
uses are ascribed to the presence of a broad range of biolog ically ac tive

* Corresponding author.
E -mail address: herciliozimila@gmail.com (H.E. Zimila).

https://doi.org/10.1016/j.bcab.2020.101862
Received 3 August 2020; Received in revised form 6 October 2020; Accepted 8 November 2020
Available online 12 November 2020
1878-8181/© 2020 Published by Elsevier Ltd.
H.E. Zimila et al.
principles (comprising terpenoids, flavonoids, phenolic compounds,
steroids, triterpenoids, al ka loids, and friedo- oleanones) with an tidia -
betic, hepato protective, an tiox idant, cy to toxic, an timalar ial, and an -
timicrobial ac tiv ities (Deepak et al., 2015; Oda et al., 2015; Paarakh et
al., 2008). Sala cinol, ko ta lanol, neosala cinol, and neoko ta lanol are the
typ ical ac tive compounds isolated from Asian Sala cia sp. (Oda et al.,
2015).
In South ern Africa, par ticularly in Mozam bique and South Africa,
S. kraus sii is among the most abun dant members of Sala cia . It is a small
shrub grow ing on the dunes and bushy steppes, whose roots are used to
treat bilharzia, dysentery, and malar ial infections in Mozam bique
(Bandeira et al., 2001; Figueiredo et al., 1998). More over, Tra ditional
Med icine Practitioners in Matu tuine district, Mozam bique, have used
for mula tions contain ing extracts of roots and stem bark of S. kraus sii
to cure tuberculosis and diar rhea (unpublished personal communica -
tion). Besides thera peutic uses, the sweet fruits and seed kernels of S.
kraus sii have contributed to food and nutrition security in rural com-
munities, ow ing to the high content of car bohydrates, amino acids, and
minerals (Magaia, 2015).
An understand ing of the associa tion between the medicinal uses and
the ac tive components present of S. kraus sii , would be of para mount
importance for the development of more effective thera peutic al terna -
tives for a wide range of diseases plagu ing human beings. How ever, de-
spite its proven pharma colog ical importance in folk lore medicine, the
scientific back ground behind the cura tive properties of S. kraus sii is still
lack ing in the main litera ture. As far as we know, the only avail able
study on bioac tiv ity S. kraus sii was car ried out by Figueiredo et al.
(1998), who linked the an timalar ial ac tiv ity of the roots to the presence
of quinone methides and celastroloids. Therefore, the present work was
aimed to identify the biolog ically ac tive metabo lites and investigate
the in vitro an tiox idant and an timicrobial properties of hydroethano lic
extract of S. kraus sii leaves.
2. Materials and methods
2.1. Chemicals
Ascorbic acid (99.5%), tan nic acid (33%), 1,1- diphenyl- 2-
picrylhydrazyl (85%) (DPPH), 2,2′- azino - bis(3- ethylbenzothiazoline-
6- sulfonic acid) (ABTS) (99%), (±)6- hydroxy - 2,5,7,8-
tetramethylchromane - 2- carboxylic acid (Trolox) (97%) and gal lic acid
(98%) were obtained from Sigma - Aldrich, South Africa. Quercetin
(95%), 2,4,6- tris(pyridyl)- s- triazine (TPTZ) (≥98%) and Folin-
Ciocalteu reagent were purchased from Acros Organ ics (South Africa),
Alfa Aesar (Germany) and LNS labs (South Africa), respectively. The
remaining chemicals were of an a lyt ical grade, unless oth erwise men-
tioned.
2.2. Sample collection, preparation, and extraction
Sala cia kraus sii leaves were collected from Matu tuine district, Ma -
puto Province – Mozam bique, on July 29th, 2018, and au thenticated
at the Herbarium of the Eduardo Mond lane University, Mozam bique.
The samples were washed thoroughly with tap wa ter to remove dust
and air- dried in shade at room tempera ture (25 °C). The dried ma terial
was subsequently pow dered through a Ham mer Mill and stored at
4 °C.
The extrac tion was performed ac cording to Sahayam et al. (2014),
with minor modifica tions. Briefly, ap prox imately 50 g of the pow der
was successively mac erated for 72 h with two 150 mL aliquots of 80%
hydroethano lic solution. The resulting extracts were combined and fil-
tered through a What man 113 filter pa per and rotary evap orated to
dryness at 45–50 °C. The dried extract was stored at 4 °C.
2
Biocatalysis and Agricultural Biotechnology 30 (2020) 101862
2.3. Phytochemical screening and GC - MS
The hydroethano lic extract was subjected to phyto chemical screen-
ing fol low ing the methodology described by Silva et al. (2018), with
some modifica tions.
The chromato graphic analy sis was car ried out in an Agilent GC
(Agilent GC 106 system 7820 A) equipped with an MSD (Agilent Mass
Spectrometry detector 107 5977 B), ac cording to Painuli et al. (2015)
with some modifica tions. The extracts were dissolved in ab solute
ethanol, filtered through membrane filters (nylon 0.45 μm), and dried
over MgSO 4. One microliter of the sample was injected into an HP- 5MS
IU column (30 m × 250 μm x 0.25 μm) in split mode at a 1:10 ra tio.
The system was equipped with an au toinjector and the injector temper-
a ture was 250 °C, while the interface tempera ture was 260 °C. The
oven tempera ture initially held at 80 °C for 3 min, and then increased
to 280 °C at 10 °C min−1. Helium (>99.99%) at a vol umetric flow rate
of 1 mL min−1 was used as the car rier gas. The eluates were ionized
through electron impact (70 eV) and identified by compar ing the eluate
mass spectra with those of the reference compounds in the NIST14. LIB
library.
2.4. Estimation of total phenols, flavonoids, and tannins
The content of to tal phenols, flavonoids, and tan nins was deter-
mined in triplicate ac cording to de Amorim et al. (2012), with minor
modifica tions.
Folin- Ciocalteu method was used to quan tify the to tal phenols. Ba -
sically, 500 μL of 10% Folin- Ciocalteu reagent were mixed with an
equal vol ume of 1 mg mL−1 extract in methanol in a 10 mL vol umetric
flask. The mixture was al lowed to settle for 5 min and 1 mL of 7.5%
(w/ v) Na 2CO 3 was added. The vol ume was made up with distilled wa -
ter and the homog enized solution was incubated in the dark at room
tempera ture for 2 h. The ab sorbance of the solution was read at
760 nm in a UV/Vis spectropho tometer (Aqua mate UV–Vis). The blank
consisted of methanol and all other reagents, except the extract. The
to tal phenols content was obtained from the cal ibra tion curve of gal lic
acid at 2–10 μg mL−1. The result was expressed as milligrams of gal lic
acid equiva lent per gram of dry extract (mgGAE g −1).
To estimate the to tal flavonoids, an assay mixture contain ing
500 μL of 1 mg mL−1 extract in methanol, 500 μL of 60% glacial acetic
acid, 2 mL of 20% pyridine in ethanol, and 1 mL of 5% aluminum chlo-
ride (methanol solution) was added into a 10 mL vol umetric flask. The
vol ume was made up with 80% methanol, homog enized and incubated
at room tempera ture for 30 min. Next, the ab sorbance was recorded at
420 nm. Methanol and all other reagents, except the extract, were used
as blank. The flavonoid content was obtained using the cal ibra tion
curve of quercetin at 2–10 μg mL−1. The result was expressed as mil-
ligrams of quercetin equiva lents per gram of dry extract (mgQE g −1).
For tan nins quan tifi ca tion, a mixture of around 500 mg of ca sein,
500 μL of 2 mg mL−1 extract in methanol, and 5 mL of distilled wa ter
was prepared in a 25 mL Erlenmeyer flask. Then, it was stirred at
600 rpm through a mag netic stirrer for 2 h (time required for the com-
plexing of the tan nins to the to tal protein). Next, the extract was fil-
tered through a What man 113 filter pa per to a 10 mL vol umetric flask,
and vol ume was ad justed with distilled wa ter. Sub sequently, a 500 μL
aliquot of this solution was placed into a 10 mL vol umetric flask and
proceeded ac cording to the above - described Folin- Ciocalteau method
for determina tion of to tal phenols. The difference of ab sorbances be-
fore and af ter ca sein precipita tion was used for cal cula tions. The to tal
tan nin content was estimated using the cal ibra tion curve of tan nic acid
at 2–10 μg mL−1 and expressed as milligrams of tan nic acid equiva lents
per gram of dry extract (mgTAE g −1).
H.E. Zimila et al.
2.5. Antioxidant activity
2.5.1. DPPH radical scavenging assay
The DPPH rad ical scav enging ability was determined as described
by Alhakmani et al. (2013), with some modifica tions. Briefly, 200 μL
aliquots of extract solutions or stan dard an tiox idants (ascor bic acid
and quercetin) at 25–150 μg mL−1 were added to 1 mL aliquots of
DPPH (0.3 mM methanol solution) in Eppendorf tubes. The mixture
was immediately incubated in dark at room tempera ture for 30 min. A
solution contain ing only 200 μL of methanol and 1 mL of DPPH solu-
tion was used as a control. The ab sorbance was recorded at 518 nm
and the inhibition of the DPPH rad ical was cal culated as fol lows: DPPH
rad ical inhibition (%) = [(Acontrol – Asample/reference
) x 100]/Acontrol
2.5.2. Total antioxidant capacity (TAC) assay
The to tal an tiox idant ca pac ity was determined by the method of
Prieto et al. (1999), with some modifica tions. To 200 μL of the sample
at different concentra tions (25–150 μg mL−1) was added 3 mL of the
phosphomolyb denum reagent (prepared by mixing 0.495 g sodium
phosphate, 0.4 g am monium molyb date, and 3.136 mL H2SO 4 and the
vol ume ad justed to 100 mL with distilled wa ter). The tubes were incu-
bated in a wa ter bath at 95 °C for 90 min. Ascorbic acid and quercetin
were the reference an tiox idants. The ab sorbance of cooled samples was
read at 695 nm and the to tal an tiox idant ca pac ity was cal culated as
fol lows: TAC (%) = [(Acontrol–Asample)x 100]/Acontrol
2.5.3. Ferric reducing antioxidant power (FRAP) assay
Method 1 (FRAP - 1). The reducing an tiox idant power was ac -
cording to Saraswathi et al. (2018) with some modifica tions. Briefly,
200 μL of extract (25–150 μg mL−1) was mixed with 1.5 mL of 0.2 M
phosphate buffer (pH 6.6) and 1.5 mL of 1% potas sium ferrocyanide.
The tubes were then incubated at 50 °C for 20 min, cooled, acid ified
with 1.5 mL of 10% trichloroacetic acid, and centrifuged (Nahita
2698 centrifuge) at 3000 rpm for 10 min. Next, 1.5 mL of the super-
natant was mixed with 1.5 mL of distilled wa ter and 500 μL of 0.1%
ferric chloride. Ascorbic acid and quercetin at the same concentra tion
levels as the extract were used as the reference an tiox idants. The ab -
sorbance was measured at 700 nm against control, which consisted of
all reagents with out the extract/ stan dard. The reducing power was
cal culated as fol lows: FRAP (%) = [(Asample–Acontrol)x 100]/Asample.
Method 2 (FRAP - 2). The reducing power was determined ac cord-
ing to Benzie and Strain (1996). FRAP reagent was prepared just be-
fore use by mixing 25 mL of 300 mM ac etate buffer (3.1 g of sodium
ac etate trihydrate and 16 mL of glacial acetic acid per liter of buffer
solution), 2.5 mL of TPTZ (10 mM solution in 40 mM HCl) and 2.5 mL
of 20 mM hexa hydrate ferric chloride. Sam ple extract or quercetin at
different concentra tions was added to 2 mL of FRAP reagent, homog e-
nized and incubated at 37 °C in a wa ter bath for 30 min. The ab -
sorbances were recorded at 593 nm and quercetin at 50 μg mL−1 was
used as a reference (control). The reducing power was cal culated as
fol lows: FRAP (%) = 100–[(Acontrol–Asample)x 100]/Acontrol.
2.5.4. ABTS radical - scavenging assay
The ABTS rad ical - scavenging ac tiv ity was measured as described
by Adebiyi et al. (2017). Firstly, the ABTS rad ical - cation was prepared
by react ing 5 mL of ABTS (7 mM aque ous solution) with 5 mL of
2.45 mM potas sium persulfate solution. The mixture was kept in the
dark at room tempera ture (25 °C) for 16 h. Afterward, the solution was
diluted with wa ter to obtain an ab sorbance of 0.700 ± 0.020 at
734 nm and equilibrated at room tempera ture (25 °C). Then, 30 μL of
Trolox at 100–2000 μM (0.25–5 μg mL−1) or sample extract at
5–25 μg mL−1 was treated with 3 mL of the ABTS rad ical - cation. The
ab sorbance was recorded at 734 nm af ter 6 min of incuba tion at room
tempera ture. The an tiox idant ca pac ity was cal culated as fol lows:
3
Biocatalysis and Agricultural Biotechnology 30 (2020) 101862
ABTS rad ical scav enging ac tiv ity (%) = [(Acontrol –
Asample)x 100]/Acontrol
2.6. Antimicrobial activity
The an timicrobial ac tiv ity was assessed against a fun gus (Can dida
al bicans (ATCC 10231)), Gram - negative (Escherichia coli (ATCC
25922), Pro teus mirabilis (ATCC 25933), Klebsiella pneu mo niae (ATCC
700603)), and Gram - positive (Staphy lo coc cus au reus (ATCC 249619))
bac terial strains. These stan dard strains were obtained from the Lab o-
ra tory of Mi crobiology of Ma puto Central Hospital (HCM), Mozam -
bique.
The susceptibility test was assessed by the Kirby- Bauer disc diffu -
sion method ac cording to Ejiofor et al. (2018). Firstly, the stan dard ized
culture of strain was prepared by inoculat ing cultures in a test tube
contain ing nutrient broth and incubat ing at 37 °C for 24 h for growth
and obtain ing a turbidity equiva lent to 0.5 on the Mc Far land scale. A
swab was soaked from each inoculum and passed evenly to cover the
entire surface of Mueller Hinton agar, prepared ac cording to the man u-
fac turer's specifica tions in a Petri dish. Each Petri dish was divided into
four parts, and a filter pa per with 6 mm in diam eter was put in the cen-
ter, under aseptic conditions, and impregnated with 10 μL of the ex-
tract (500 μg mL−1 in 10% dimethyl sulfox ide). The disk impregnated
with 10% dimethyl sulfox ide was used as the nega tive control, whereas
ciprofloxacin 5 μg and flucona zole 25 μg were used as positive controls
for the bac terial and fun gal strains, respectively. The disks were incu-
bated at 35 °C for 72 h. The sensitiv ity of the strains was determined by
measuring the inhibition zone created by the sample/control. All tests
were done in triplicate and strains in which the mean diam eter of the
inhibition zone was equal to or greater than 8 mm were considered sen-
sitive.
The minimum inhibitory concentra tion (MIC) was assessed as
above - described, but and the fol low ing concentra tions of the extract
were tested: 250; 125; 62.5; 31.25, and 15.625 μg mL−1.
2.7. Statistical analysis
Experimental data were expressed as the mean of three repli-
cates ± stan dard devia tion (SD). Data analy sis was car ried out using
Minitab® 17.1.0 sta tistical pack age. One- way analy sis of vari ance
(ANOVA) with Fisher's LSD tests or paired t- test, at a 95% confidence
level, were used to exam ine the sta tistical significance of the differences
among the effective concentra tions of the tested drugs that give a half -
maximal response (EC50).
3. Results
3.1. Phytochemical composition and GC - MS
The qual ita tive preliminary phyto chemical screening of S. kraus sii
leaves extract revealed the presence of al ka loids, flavonoids (flavones),
cat echins, saponins, steroids, triterpenoids, condensed tan nins, and
quinones. Neither hydrolyzable tan nins nor flavonols were detected in
the extract. On the other hand, the GC - MS analy sis led to the identifi-
ca tion of 17 phyto chemical constituents whose chromatogram is
shown in Fig. 1.
Squa lene and n - hexadecanoic acid were the ma jor chemical compo-
nents identified by GC - MS in the S. kraus sii leaves extract. The detailed
list of metabo lites the respective retention times and molecular for mu-
lae are presented in Table 1.
3.2. Content of phenolic compounds and in vitro antioxidant activity
The to tal contents of phenols, flavonoids and tan nins in the extract
were determined using the cal ibra tion curves of gal lic acid
H.E. Zimila et al. Biocatalysis and Agricultural Biotechnology 30 (2020) 101862

Fig. 1. Typ i cal GC-MS chro matogram of the hy droethano lic ex tract of S. kraus sii leaves.

Table 1 Table 2
Com pounds iden tified in the hy droethano lic ex tract of S. kraus sii by GC - EC 50 values ob tained from DPPH rad ical scav eng ing ac tivity, total an tioxi-
MS. dant ca pac ity (TAC), re duc ing an tioxidant power (FRAP -1 and FRAP -2),
and ABTS rad ical scav eng ing as say of hy droethano lic ex tracts of S. kraus sii
Peak Name RT Molecular
# (min) formula leaves.
Method EC 50 (μg mL −1)
1 2- Ethylhexyl octanoate 5.375 C 16H 32O 2
2 d - Glycero - d - ido - heptose 12.293 C 7H 14O 7 S. kraussii Quercetin Ascorbic acid Trolox
3 (Z)- Tetradec - 10 - en- 1- yl acetate 10.914 C 16H 30O 2
4 Methyl 8- [(2′- hexyl - 1,1′ - bi (cyclopropyl) - 2- yl] - 15.215 C 21H 38O 2 DPPH 35.78 ± 0.09 a 34.25 ± 0.89 b 32.24 ± 0.85 c –
octanoate scavenging
5 2,3,4,6 - Tetrahydroxy - 5- methoxy - N, N- 15.299 C 9H 19NO 6 TAC 36.44 ± 0.67 a 29.73 ± 0.71 b 28.27 ± 0.35 c –
dimethylhexanamide FRAP - 1 64.28 ± 0.01 a 41.98 ± 0.11 b 37.93 ± 0.24 c –
6 3- [2 - Diethylaminoethyl] - 2,4 - pentanedione 16.145 C 11H 21NO 2 FRAP - 2 33.91 ± 0.12 a 28.38 ± 0.03 b – –
7 10 - Methyl - (11 E)- 11 - tridecen - 1- ol propionate 16.200 C 17H 32O 2 ABTS radical 2.22 ± 0.25 a – – 0.99 ± 0.04 b
8 Ethyl hexadecanoate 17.300 C 18H 36O 2 scavenging
9 n - Hexadecanoic acid 16.940 C 16H 32O 2
10 Melezitose 17.735 C 18H 32O 16
11 Phytol 18.632 C 20H 40O 3.3. Antimicrobial activity
12 1,2 –15,16 - Diepoxyhexadecane 18.885 C 16H 30O 2
13 (9Z,12Z) - 9,12 - Octadecadienoic acid 18.885 C 18H 32O 2 The an timicrobial ac tiv ity of hydroethano lic extract of S. kraus sii
14 Octadecanoic acid 19.300 C 18H 36O 2 leaves was determined against the fun gus C. al bicans and var ious
15 4,8,12,16 - Tetramethylheptadecan - 4- olide 21.021 C 21H 40O 2 Gram - negative and Gram - positive path ogenic bac terial strains. The re-
16 Squalene 24.604 C 30H 50
sults in Table 3 demonstrate that the hydroethano lic extract of S.
17 2,2 - Dimethyl - 3- (3,7,12,16,20 - pentamethyl - 25.347 C 30H 50O
3E,7E,11E,15E,19E - 3,7,11,15,19 - kraus sii leaves exhibited the highest an timicrobial ac tiv ity against the
heneicosapentaenyl) oxirane bac terial strain P. mirabilis (inhibition zone 16.5 ± 1.0 mm), fol lowed
by K. pneu mo niae (15.8 ± 1.5), S. au reus (13.9 ± 1.2), and E. coli
(y = 0.038x + 0.180; R2 = 0.9985), quercetin (y = 0.058x – 0.013; (12.1 ± 1.0). The fun gus C. al bicans was the least susceptible strain
R2 = 0.9997) and tan nic acid (y = 0.041x + 0.281; R2 = 0.9988), with an inhibition zone diam eter of 10.5 ± 1.0 mm. Ciprofloxacin and
and the respective contents were 5.000 ± 0.269 mgGAE g −1, flucona zole were ac tive against all strains with inhibition zones of
0.670 ± 0.291 mgQE g −1 and 0.034 ± 0.014 mgTAE g −1. 15–29 mm and 19 mm, respectively. The minimum inhibitory concen-
The an tiox idant ac tiv ity was assessed through five complementary tra tions against the bac terial strains fun gus were 125 μg mL−1 and
assays (DPPH rad ical scav enging, to tal an tiox idant ca pac ity (TAC), 250 μg mL−1, respectively.Where: NG – microbial growth was not ob-
and ferric reducing an tiox idant power (FRAP- 1 and FRAP- 2) and ABTS served, G – microbial growth was observed.
rad ical scav enging assay) and was compared to that of the stan dards
ascor bic acid, quercetin, and/or Trolox. Both leaves extract and stan - 4. Discussion
dards showed a dose- dependent increase in an tiox idant ac tiv ity, and
the regression equa tions were used to compute the EC50 val ues (Table The diverse pharma colog ical properties of Sala cia sp. are closely re-
2). lated to their biolog ically ac tive metabo lites and their detection may
Values are expressed as mean ± SD of three replicates. In each lead to drug discov ery and improvement (Pakkirisamy et al., 2017).
method, the sample was an a lyzed in par al lel with one or two stan dards Preliminary phyto chemical analy sis of the hydroethano lic extracts of
(quercetin, ascor bic acid, and/or Trolox). The compar ison was done ei- S. kraus sii revealed the presence of al ka loids, flavones, cat echins,
ther by ANOVA (three means) or t- test (two means) and means that do saponins, steroids, triterpenoids, condensed tan nins, and quinones.
not share a letter in a row are significantly different (P < 0.05). These compounds are reported to present an timicrobial, an tiox idant,
Sig nificant differences among the EC50 of the drugs (P < 0.05) were an tidia betic, anti - inflammatory, an titumoral, and anx iolytic ac tiv ities
detected by ANOVA. Hy droethano lic extract of S. kraus sii leaves pre- (Al- Owaisi et al., 2014). These findings are consistent with the existing
sented higher EC50 val ues than the reference stan dards in all the meth- studies on the bioac tiv ity of other Sala cia sp. (Basu et al., 2013;
ods. Likewise, the results illustrated that the EC50 for ascor bic acid Deepak et al., 2015; Paarakh et al., 2008).
quercetin. The GC - MS chromatogram indicated that S. kraus sii leaves contain
numerous bioac tive phyto constituents, but only 17 were identified.
Surprisingly, none of these compounds was reported in Sala cia sp.

4
H.E. Zimila et al.
Table 3
An timi cro bial ac tivity and min imum in hibitory con cen tration of hy droethano lic ex tracts of S. kraus sii leaves against test mi croor gan isms.
Test Inhibition zone diameter (mm) Susceptibility of strains to different concentrations of S. kraussii leaves extract (μg
organism mL −1)
S. kraussii Ciprofloxacin Fluconazol 250
(500 μg mL −1) (5 μg) (25 μg)
P. mirabilis 16.5 ± 1.0 21.3 ± 1.2 – NG
K. 15.8 ± 1.5 23.1 ± 0.7 – NG
pneumoniae
E. coli 12.1 ± 1.0 29.0 ± 1.3 – NG
S. aureus 13.9 ± 1.2 15.1 ± 1.0 – NG
C. albicans 8.0 ± 1.0 – 19.0 ± 1.0 NG
(Figueiredo et al., 1998; Oda et al., 2015), but in many other plant
species. This can be ascribed to several reasons, including differences in
the an a lyt ical technique (GC - MS was used instead of HPLC), part of
the plant (leaves instead of roots), and the spectral data base limita tion.
Furthermore, the genetic differences and the ecogeo graphic prove-
nances are perhaps the underlying reasons behind the non- detection of
the metabo lites reported by Oda et al. (2015).
Squa lene, the ma jor compound found in the present study, is a
triterpene and precursor of cholesterol biosynthesis (Huang et al.,
2009). It is a highly effective oxy gen- scavenging agent (an tiox idant),
hence exhibits a chemoprotective ac tiv ity for the human body against
ox idative dam age (Huang et al., 2009). The diterpenoid phytol, in turn,
is the synthetic precursor of vit a mins K and E. Through mecha nisms in-
volv ing perox isome prolifera tor - activated receptors and tumor necrosis
fac tor - alpha, phytol exerts a wide range of biolog ical ac tiv ities, includ-
ing anx iolytic, metab olism- modulation, cy to toxic, an tiox idant, au -
tophagy - and apop to sis- induction, an tinociceptive, anti -
inflammatory, immune- modulating, and an timicrobial effects (Islam et
al., 2018). The car bohydrates melezitose (a nat ural trisaccha ride and
non- reducing sugar) and d- glyc ero - d- ido- heptose were reported to ex-
hibit an tibac terial (Tsao et al., 2003) and anti - inflammatory properties
(Hussein et al., 2016), respectively.
The fatty acids (hexa decanoic acid, octade canoic acid and (Z,Z)-
9,12- octadecadienoic acid) and the esters (ethyl hexa decanoate and
10- methyl- (11 E)- 11- tridecen- 1- ol propionate) were reported to ex-
hibit numerous biolog ical ac tiv ities, including an timicrobial, anti -
inflammatory, nemati cide, an tiox idant, and hepato protective ac tiv i-
ties (Hussein et al., 2016; Omoruyi et al., 2014). 2,2- Dimethyl - 3-
(3,7,12,16,20- pentamethyl - (3E,7E,11E,15E, 19E)- 2,7,11,15,19-
heneicosapentaenyl)oxirane and methyl 8- [(2′ - hexyl - 1,1′- bi (cy clo-
propyl)- 2- yl] octanoate were reported to have anti - cancer properties
(Banakar and Jayaraj, 2018; Hameed et al., 2016). The remaining
compounds are rarely reported in the litera ture.
Phenolic compounds have been implicated as ma jor an tiox idants in
nat ural products as they possess an ideal structural pat tern for free rad -
ical sta bilization (Alhakmani et al., 2013). Priya et al. (2019) reported
that crude methanol extracts of the leaves of seven species belong ing to
the genus Sala cia (namely S. bed domei, S. chi nen sis, S. fru ticose, S.
gam bleana, S. macros perma, S. mal abar ica, and S. ob longa ) are rich in
phenolic compounds, including to tal phenols (49.40–85.38 mg g −1),
tan nins (51.41–83.97 mg g −1), and flavonoids (50.74–84.96 mg g −1).
How ever, our study revealed that the content of phenolic compounds in
hydroethano lic extract of S. kraus sii leaves is more than a 9- fold lower
than the previously found in other Sala cia sp. The genetic distance
among the species, the differences in the geograph ical origin of the
plants, and the dissimilarity between the extrac tion solvent properties
are perhaps the most relevant underlying reasons behind the observed
discrepan cies (Yusuf et al., 2018).
Antiox idants play a huge role in prevent ing ox idative stress that
trig gers several diseases, including dia betes, rheumatoid arthritis, car -
diovas cular diseases, ath erosclerosis, neurodegenera tive diseases
(Parkinson, Alzheimer, and Huntington), can cer, and ag ing (Jemli et
5
Biocatalysis and Agricultural Biotechnology 30 (2020) 101862
125 62.5 31.25 15.625
NG G G G
NG G G G
NG G G G
NG G G G
G G G G
al., 2016). In this study, the hydroethano lic extract of S. kraus sii leaves
exhibited weaker an tiox idant ac tiv ity than all the stan dards eventu-
ally due to the low content of phenolic compounds. The an tiox idant ac -
tiv ity observed may be ascribed to the presence of the non- phenolic
constituents (such as unsat urated fatty acids, phytol, and squa lene) de-
tected by GC - MS and preliminary phyto chemical tests.
Notwith stand ing the weaker an tiox idant ca pa bility compared to
the stan dards, the an tiox idant potential of hydroethano lic extracts of
S. kraus sii leaves in the present work is pretty stronger than that of the
species recently studied by Priya et al. (2019). These au thors assessed
the an tiox idant ac tiv ity of the leaves of S. bed domei , S. chi nen sis, S.
fru ticosa , S. gam bleana , S. macros perma , S. mal abar ica , and S. ob longa ,
and found EC50 val ues in the range of 100–108 μg mL−1and
138–183 μg mL−1 for DPPH and ABTS scav enging methods, respec-
tively.
Sala cia kraus sii leaves extract showed considerable an timicrobial
ac tiv ity against all the test organ isms (E. coli, S. au reus, P. mirabilis,
K. pneu monae, and C. al bicans ) with MIC of 125 μg mL−1 and
250 μg mL−1. This is perhaps the most significant finding in this
study and could be explained by the presence of many an timicrobial
agents, including phytol, fatty acids, and esters, and other class of
metabo lites found in phyto chemical analy sis. The observa tions agree
with the results of Kannaiyan et al. (2012) and Deepa and Bai
(2004), who reported a significant an timicrobial potential of several
extracts of S. chi nen sis against S. au reus, C. al bicans, P. mirabilis , E.
coli , S. au reus, and K. pneu monae . This striking an timicrobial ac tiv ity
against fungi and bac teria, suggests that the hydroethano lic extract
of S. kraus sii leaves is a broad - spectrum herbal medicine that can be
explored in the treat ment of var ious fun gal and bac terial infections.
5. Conclusion
The hydroethano lic extract of S. kraus sii leaves contains several
bioac tive secondary metabo lites that may be responsible for the an -
tiox idant and an timicrobial properties. It has a low content of phenolic
compounds, hence its an tiox idant ac tiv ity is weaker than the stan -
dards. Nev ertheless, the an tiox idant potential was stronger than the
other Sala cia species. The results in the present work suggest that S.
kraus sii leaves are an unexploited nat ural source of broad - spectrum
drugs for safe treat ment of a wide range of bac terial and fun gal dis-
eases.
CRediT authorship contribution statement
Her cílio E. Zim ila: Con ceptual iza tion, Su pervision, Project ad -
ministra tion, Writing - original draft, Funding ac quisition. Al bertina
L. Matsinhe: Data ac quisition, Formal analy sis, Writing - original
draft. Emma Malayika: Data ac quisition, Formal analy sis. Átifa I.
Sule mane: Con ceptual iza tion, Su pervision, Project ad ministra tion,
Writing - review & editing, Funding ac quisition. Van ina N.C. Saete:
Con ceptual iza tion, Su pervision, Writing - review & editing, Data ac -
quisition, Formal analy sis. Saquina C. Ru gu nate: Con ceptual iza -
H.E. Zimila et al.
tion, Su pervision, Funding ac quisition. Paulo J. Cum bane: Con cep-
tual iza tion, Su pervision, Writing - review & editing, Funding ac quisi-
tion. Is abel Ma gaia: Con ceptual iza tion, Data ac quisition, Funding
ac quisition. François Mun yemana: Con ceptual iza tion, Writing - re-
view & editing, Funding ac quisition.
Declaration of competing interest
The au thors declare no conflict of interest.
Acknowledgments
The au thors sincerely ac knowl edge the finan cial support from
Fundo Na cional de In vestigação (FNI ) through the Project on Assess-
ment of Bioac tiv ity and Tox icity of Med icinal Plants used in Matu tuine
district, Ma puto province, Mozam bique. We would like to express our
deepest grat itude to the Lab ora tory of Mi crobiology of Ma puto Cen-
tral Hospital for providing us with the strains used to conduct an timi-
crobial tests. We are thank the Min istry of Science, Technology, Higher
and Technical Voca tional Educa tion for award ing the Scientific Initia -
tion Schol ar ship to Albertina Matsinhe. We are also thank ful to Mr.
Fran cisco Ma panga and Mr. Amon Tikoko for au thenticat ing the plant
species and lan guage edition, respectively.
Funding
This work was supported by Fundo Na cional de In vestigação
(UEM - FC ASDI – PLANTAS MED I CI NAIS – Grant Number 21 A),
Mozam bique.
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