The GC/MS analysis, phytochemical and antimicrobial properties of the leaf extract of Stachytarpheta cayennesis was carried out in the laboratory as a part of our probe into the usefulness of the plant in medicinal applications. GC-MS analysis was obtained by the use of SHIMADZU Japan Gas Chromatography 5890-11 with a fused GC column OV 101 coated with polymethyl silicon (0.25 mm x 50 m). Results obtained revealed 13 absorption peaks; Peak 1 occurred at m/z 128 which corresponds to the molecular formula C10H8 and is identified as Azulene.Similarly Peak 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, occurred at m/z 220, 200,242, 240, 268, 270, 256 ,296 ,296, 282, 281, 252 respectively corresponding to molecular formulas; C15H24O, C12H24O2, C15H30O2, C17H36, C19H40, C17H34O2, C16H32O2, C19H36O2, C20H40O C18H34O2, and C18H35NO. They were identified as Butylated Hydroxytoluene, Dodecanoic acid, Methyl tetradecanoate, Heptadecane, Nonadecane, Hexadecanoic acid methyl ester, n-Hexadecanoic acid, 9-octadecanoic acid methyl ester, Phytol, Octadec-9-enoic acid, 9-octadecenamide, and 11-tetradecyl-1-ol acetate respectively. At concentrations of 100mg/cm3 the extract showed inhibition of Staphylococcus aureus, 8mm, Klesiella spp 9mm, Proteus mirabilis 5mm and Pseudomonas aureginosa 5mm. The minimum inhibition concentrations are 25mm/cm3 for Staphylococcus aureus, 12.5mg/cm3 for Klebsiella spp, 25mg/cm3 for Proteus mirabilis and 12.5mg/cm3 for Pseudomonas aureginosa. These result are very close to those obtained when standard antibiotics levofloxacin and streptomycin.
2. Characterization of the Volatile Components of the Leaf of Starchytarphetacayennesis (Rich) Vahl
Iwu et al. 042
(2013). Stachytarpheta cayennensis has shown anti-
plasmodial activities against Plasmodium bergheiberghei.
Studies by other researchers revealed the anti-
inflammatory and ulcerogenic properties of S.
cayennensis, Ezeanyi et al, (2016), Penido et al, (2006).
They were able to provide evidence for the anti-
inflammatory and gastro-protective properties of the pant
which includes its anti ulcerogenic properties Okoye et al,
(2014). Other researchers have reported the anti-microbial
and antispasmodic activities of the leaf of the plant. Their
work seems to provide evidence for the use of the plant in
wound healing and gastrointestinal ulceration treatment
Olayiwole and Ibikunle, (2013). Other researchers have
reported the antipsychotic effect of the leaves of S.
cayennensis, their result gives credence to the use of the
extract in treatment of mental illness. Similarly, Ramanuj
et al, (2014) has also reported the anti tyrosinase activities
of S. cayennensis and showed that the plant can be used
as a skin whitening and anti-ageing agent. Most plants
found in Africa have one or more anti malaria properties
.
MATERIALS AND METHODS
Sample Collection
The leaves of Starchytarpheta cayennensis were obtained
in Eziobodo community in Owerri West L.G.A of Imo state,
Nigeria. They were then room dried for a period of one
month before been milled into powder using a milling
machine. The milled sample was then stored in airtight
container till required for analysis, Iwu and Onu, (2018).
Frothing test for Saponins
This test is based on the ability of the saponins to produce
froth in aqueous solution. 5g of the leaf sample was
weighed into a test tube and 50cm3 of water was added
and extracted after4hours. The water extract was shaken
vigorously in a conical flask. The production of a persistent
froth when allowed to stand for 5 minutes indicates the
presence of saponins in the sample.Iwu et al,(2016b)
Test for Flavonoids
5g of the sample was soaked with 50cm3 of water and then
filtered. To the filtrate were added drops of ammonia and
3cm3 of concentrated H2SO4 was added. A yellow
precipitate which disappears on storage indicates the
presence of flavonoids. Okwu and Okwu, (2004)
Test for Alkaloids
5g of the sample was extracted using 20% acetic acid in
ethanol .5cm3 of the extract was treated with 3drops of
Wagner’s reagent (iodine crystals and KI). A yellowish
brown precipitate indicates the presence of alkaloids. Iwu
et al, (2018b).
Test for Tannins
5g of the leaf sample was weighed into a beaker and
50cm3 of water was added and allowed to soak properly
for 4 hours and extracted.10cm3of the leaf extract was
treated with 3 drops of ferric chloride. A blue-black
precipitate indicates the presence of tannins Iwu et al
(2018c)
Test for Steroids
5cm3 of the water extract was treated with concentrated
H2SO4 in acetic anhydride. The formation of a blue-green
color indicates the presence of steroids. Iwu and Onu,(
2018)
Test for Phenols
20cm3 of the water extract was treated with 5cm3 of
concentrated sulphuric acid and drops of sodium nitrate
(NaNO3). 2cm3 of sodium hydroxide was added to the
mixture. A blue precipitate indicated the presence of
phenols. Iwu et al, (2018a)
Test for Glycosides
20cm3 of the water extract was treated with Fehling
solutions of A and B in equal amount and boiled. A
brownish red precipitate indicates the presence of
glycoside. Iwu et al, (2018b)
Preparation of Samples for GC-MS Analysis
Two hundred grams of the leaf sample was soaked in
ethanol for 48 hours and then extracted. The extract was
re-extracted using chloroform to obtain chloroform soluble
extract. This was centrifuged at 10,000 rpm for 20 minutes
and the clear supernatant oil was subjected to GCMS
analysis.
GC-MS Experimental Procedures
GC-MS analysis was carried out with SHIMAZU Japan
Gas Chromatography 5890-11 with a fused GC column
OV 101 coated with polymethyl silicon (0.25 mm x 50 m)
and the as follows: Temperature programming from 80 –
200oC held at 80oC for 1 minute, the rate is 5oC/min and at
200oC for 20 minutes. FID Temperature of 300oC, injection
temperature of250oC, carrier gas is Nitrogen at a flow rate
of 1 cm3/min and split ratio of 1:75. GC-MS Gas
(chromatography, Mass spectrum) analysis was
conducted using GC-MS QP 2010 Plus Shimadzu Japan
with injector Temperature at 230oC and carrier gas
pressure of 100kpa. The column length was 30 m with a
diameter of 0.25 mm and the flow rate of 50m/min. The
eluents were automatically passed into the Mass
Spectrometer with a detector voltage set at 1.5kv and
sampling rate of 0.2 seconds. The Mass Spectrometer was
also equipped with a computer fed Mass Spectra data
3. Characterization of the Volatile Components of the Leaf of Starchytarphetacayennesis (Rich) Vahl
Int. J. Herbs, Spices Med. Plants 043
bank, HERMCE Z 233 M-Z centrifuge Germany was used.
Reagents and solvents such as Ethanol, Chloroform,
Diethyl ether, hexane all of analytics grade was obtained
from Merck Germany (Iwu et al 2016a, b.)
Antimicrobial Analysis
The microorganisms; Staphylococcus aureus,
Streptococcus spp, Klebsiella spp, Proteus spp and
Pseudomonas spp were used for the analysis. They are
clinical isolates of human pathogens obtained from the
Federal Medical Centre Umuahia and were brought to the
laboratory and resuscitated in buffered peptone broth
(Secharian chemie) and thereafter into nutrient agar
medium and incubated at 37oC for 24 hrs Iwu et al (2016b).
Antibacterial Assay
The test solution of each extract was prepared by
dissolving 0.1 g of the plant extract separately. 1.0cm3 of
dimethyl sulphoxide (DMSO) to get a concentration of
100mg/cm3. The antibacterial activity was performed by
filter paper disc diffusion technique. Filter paper disc
(Whatman No 1.6 mm diameter) were placed in glass
petridish and sterilized in hot air oven. Iwu et al 2018b, the
media (10g nutrient Agar in 200cm3 distilled water,
autoclaved at115oC for 30 minutes) was cooled to 50oC.
The sterile nutrient Agar media were poured into the sterile
petridish and allowed to solidify. The bacteria were
swabbed with a sterile wire loop. Each disc was
impregnated with 0.2cm3 of plant extract. Standard
antibiotic Ciprofloxacin was used as a control on a disc
with DMSO 100mg/cm3. The discs were used after drying
them in an incubator at 40oC to remove any trace of
solvent. Discs were introduced into the surface of the
medium. The plates were microbated at 37oC for 24 hours
to obtain zones of inhibition. The experiments were
repeated three times for each extract and twice for
reference antibiotics to minimize error and the average of
these values were recorded.
Minimum Inhibitory Concentration (MIC)
The minimum inhibitory concentration of the extract was
determined by incorporating constant volume of 0.2cm3 of
each diluents of the extract into the perforated disc on a
seeded nutrient agar plate as described in the anti-
microbial susceptibility test section. 0.1g of each extract
was dissolved in 1cm3 of DMSO to obtain 100mg/cm3. This
concentration of DMSO was then doubled to obtain
50mg/cm3 then doubled again to obtain 12.5mg/cm3 and
again to obtain6.25mg/cm3. Each concentration was then
used in the method earlier described to obtain zone of
inhibition. The least concentration that showed inhibitory
zones was taken as the MIC. Ekundayo and Ezeogu,
(2006).
RESULTS AND DISCUSSION
PHYTOCHEMICAL ANALYSIS
The results of the phytochemical screening of the leaf of of
Stachytarpheta cayennensis are given in table 1 below.
The table revealed the presence of flavonoids, saponnins,
tannins, phenols, steroids, cardiac glycosides and
alkaloids
Table 1: Phytochemicals screening test result of sample
test of the leaf of S.cayennesis
Phytochemicals Inference
Flavoloids ++
Saponins ++
Tannins ++
Phenols ++
Steroids ++
Glycosides ++
Alkaloids ++
Key; ++ present in reasonable quantity
.
Alkaloids are vast and vary a lot in their activity when
ingested by man and livestock. Some alkaloids are useful
and important in medicine and constitute most of the
valuable drugs currently used by humans. They are
reported to have marked physiological effect on animals
Edeoga and Eriata, (2001).
Phenolics are broadly distributed in plants and are the
most abundant secondary metabolites of plants. Plant
phenolic have drawn increasing attention due to their
potent anti-oxidant properties and their marked effects in
the prevention of various oxidative stress associated
diseases such as cancer. In the last few years, the
identification and development of phenolic compounds or
extracts from different plants has become a major area of
health and medical-related research.
Flavonoids have been shown to be highly effective
scavengers of most oxidizing molecules. Tukappa and
Londonkar, (2013). In addition, tannin was found in the
plant at a concentration range. Plant leaves with high
tannin content have been used successfully as hops
alternative in beer Hutchinson and Dalziel, (1963).
Flavonoids are polyphenolic compounds based on a C15
(C6C3C6) framework. They contain a chroman ring (C-ring)
with a second aromatic ring (B-ring) at the C2, C3 and C4
position. The heterocyclic six-membered C-ring is
sometimes replaced by a 5-membered ring. The oxidation
state of the C-rings is used to classify flavonoids into
different categories of which typical examples are Flavan-
3-ols, flavanones, flavones, isoflavones and flavanols.
Flavonoids are the major nutraceutical ingredients that are
in plants. The best described property of almost every
group of flavonoids is their capacity to act as anti-oxidants.
The flavones seem to be the most powerful flavonoid for
4. Characterization of the Volatile Components of the Leaf of Starchytarphetacayennesis (Rich) Vahl
Iwu et al. 044
Table 2: Result of the antimicrobial activities of the leaf extract of S. cayennesis
Samples Conc. Of test
substance mg[cm3)
Diameter of Zone of Inhibition
Leaf
extract
Staphylococcus
aureus
Streptococcus
spp.
Klebisella spp. Proteus
mirabilis
Pseudomonas
aureginosa
100 8.0 -- 9.0 5.0 5.0
50 4.0 -- 5.0 3.0 2.5
25 2.0 -- 2.0 1.0 1.8
12.5 -- -- 1.2 -- 1.0
LEV 0.02 20 12 20 10 8
S 0.03 8 4 20 8 6
Key Lev – Levofloxacin S –Stretomycin
protecting the body against reactive oxygen species
(ROS). Antibacterial activity has been displayed by a
number of flavonoids, Quercetin has been reported to
completely inhibit the growth of Staphylococcus aureus.
Havsteen, (1983). Flavonoids also possesses anti-
inflammatory and analgesic effect as well as anti-
ulcerogenic activity. Shahid et al 1998.
The infusions of S. cayennesisis taken as a remedy for
gonorrhea and jaundice. Draghon, (2004), Mohammed et
al, (2013). This is probably due to antibacterial action of
saponins. Saponins are foam forming in nature and have
been implicated as a bioactive antibacterial agent of plant,
Thomas-Barbera et al, (1990). Saponins are also potential,
sometimes for utilization in foods that need sustained foam
volume such as ice-creams. Saponins are a class of
chemical compounds, more specifically, they are
amphipathic glycosides grouped, in terms of
phenomenology by the soap-like foaming they produce
when shaken in aqueous solution and in terms of structure
by their composition of one or more hydrophilic glycosides
combined with a lipophilic triterpene derivative. In plants
saponins may serve as anti-feedants to protect the plant
against microbes and fungi. Some plant saponins may
enhance nutrient absorption and aid in animal digestion.
Saponins have been used as a pharmacological and/or
immunological agent that modifies the effect of other
agents in vaccines. Saponins from plants have been
shown to significantly augment the cytotoxicity of
immunotoxins and other target toxins directed against
human cancer cells.
Tannins are astringent, bitter plant polyphenol compounds
that bind to and precipitate proteins and various other
organic compounds including amino acids and alkaloids.
The tannin compounds are widely distributed in many
species of plants where they play a role in protection from
predation and perhaps also as pesticides and in plant
growth regulation Urqiuaga and Leighton 2000. The
astringency from tannin is what causes the dry puckery
feeling in the mouth following the consumption of unripe
fruits or red wine. Tannins are important ingredients used
in process of making tannin leather. Medicinally, tannins
are used as anti-diarrhea, hemostatic and anti-hemorrhoid
compounds.
The presence of Phenolic compounds in the leaf of
Starchytarpheta cayennesis indicates that this plant might
be an anti-microbial agent. This is because phenols and
phenolic compounds have been extensively used in
disinfections and remains the standard with which other
bactericides are compared. Phenolic compounds act as
electron donors and are readily oxidized to form phenolate
ions. This gives rise to protonated phenol which is used as
a cleaning agent. Extracts from leaves of Starchytarpheta
cayennesis therefore have potent antiseptic or bactericidal
properties. This finding supported the use of extracts of the
leaves in treating wounds that not only heal fast but also
prevent the formation of infection. Okwu and Okwu,
(2004). Phenols have antioxidant properties. Thomas-
Barberan et al (1990) The presence of phenol further
indicates that Stachytarpheta cayennesis could act as anti-
inflammatory, anti-clotting, immune enhancers and
hormone modulators.
Glycosides are molecules in which a sugar is bound to
another functional group via a glycosidic bond. Glycosides
play numerous important roles in living organisms. Many
plant store chemicals in form of inactive glycosides. Many
such plant glycosides are used as medications. Some
glycosides have shown some evidence of pharmacological
effects in patients with hypertension or with type-2
diabetes but concluded that further study was required to
determine proper dosage.
ANTIMICROBIAL ACTIVITY OF THE LEAF EXTRACT
OF STARCHYTARPHETA CAYENNESIS
The anti-microbial activity of the leaf extract of
Starchytarpheta cayennesis are summarized as shown in
table 2.
The leaf extract showed marked inhibition of some of the
selected pathogens, at concentrations of 100mg/cm3,the
extract showed inhibition of Staphylococcus aureus, with
inhibition diameter of 8mm, Klebsiella spp, 9mm, Proteus
mirabilis, 5mm and Pseudomonas aureginosa 5mm. The
minimum inhibition concentrations are 25mm/cm3 for
staphylococcus aureus,12.5mg/cm3 for Klebsiella spp,
25mg/cm3 for Proteus mirabilis and 12.5mg/cm3 for
5. Characterization of the Volatile Components of the Leaf of Starchytarphetacayennesis (Rich) Vahl
Int. J. Herbs, Spices Med. Plants 045
Pseudomonas aureginosa (table 2). These results are very
close to those obtained when standard antibiotics
Levoflxacin and Streptomycin were used. Staphylococcus
aureus is a gram positive coccus that causes skin infection
such as; pimples, impetigo, boils, cellulitis, folliculitis,
carbuncles, scalded skin syndrome, abscesses,
pneumonia, toxic shock syndrome, bacteremia and sepsis.
Pseudomonas aureginosa is a gram negative gamma-
proteobacteria which belong to the family
Pseudomonaceae. It causes bacteremia, pneumonia,
foliculitis, swimmer ear which is an ear infection
accompanied with swelling, ear pus. Itching, discharge
and difficulty in hearing, eye inflammation with associated
pains, pus, swelling redness and impaired vision.
Klebsiella, a non-motile gram negative, oxidase rod
shaped bacteria which causes infectious wounds,
pneumonia, Okonkwo et al (2012), blood stream infection
and urinary tract infection. Proteus mirabilis is a gram
negative facultatively anaerobic rod shaped bacterium
implicated in urinary tract infection (UTIs), bacteremia,
type 2 diabetes, cystitis, pyelonephritis, urosepsis and
urinary stone (urolithiasis). Stachyterpheta cayennensis
has been used as a remedy for syphilis, gonorrhea, catarrh
condition, skin wounds and sores in children Hutchinson
(1963). The extracts exhibited some level of inhibitory
effects against some of the studied pathogens which have
been implicated in one bacterial infection to the other in
human and plant. Klebsiella specie causes pneumonia. In
plant, Pseudomonas spp causes bacterial blight in guinea
corn. The sensitivity test for most antibiotics against
certain pathogens are shown in table 3. These
microorganisms are inhibited by the leaf extract of S.
cayennesis implying that the extract may be used to treat
diseases associated with these organisms.
Table 3: Sensitivity test carried out on the pathogens by
certain antibiotics used to evaluate the inhibitory properties
of S.cayennesis
Antibiotics Staphylo-
coccus
aureus
Strepto-
coccus
spp.
Klebsiella
spp.
Proteus
spp.
Pseudo-
monas
spp.
CPX +++ ++ ++ ++ +
NB +++ -- + +++ --
CN ++ -- +++ ++ --
AMX -- -- ++ ++ --
S +++ + +++ ++ ++
RD + + +++ + ++
E ++ -- -- +++ +
CH -- -- +++ ++ +++
APX -- -- +++ -- --
LEV +++ +++ +++ +++ +++
Key: CPX=Ciproflox; NB=Norfloxacin; CN= Gentamycin;
AMX=Amoxil; S=Streptomycin; RD=Rifampicin;
E=-Erythromycin; CH=Chloramphenicol; APX=Ampiclox;
LEV= Levofloxacin
+ fairly sensitive, ++ very sensitive. +++ highly sensitive –
none sensitive
Antibiotics sensitivity test is usually carried out to
determine the antibiotic that will be the most effective
against specific bacteria and fungi infecting in an
individual. Standard drugs were used to inhibit the
pathogens in table 3 and some were found to be very
effective in inhibiting all strains of the bacteria amongst
which were Ciproflox, Streptomycin, Rifampicin and
Levofloxacin. They were used as control in the anti-
microbial test carried out
GC/MS ANALYSIS
The GC/MS analysis gave a spectrum with 13 absorption
peaks (fig1)
The interpretation of the chromatogram obtained was done
with the aid of data obtained from a computer fed Mass
Spectra data bank, HERMCE Z 233 M-Z. From these data,
the molecular weights, structures, molecular formulas and
name of the compounds were obtained, (table 4).
Peak 1 occurred at m/z 128 which corresponds to the
molecular formula C10H8 and is identified as Azulene.
These compounds are isomers of naphthalene which is a
cyclic aromatic hydrocarbon. Peak 2 appeared at m/z 220
with molecular formula C15H24O and is identified as
Butylated Hydroxytoluene, an aromatic hydroxyl. Peak 3
appeared at m/z 200 with formula C12H24O2 which is called
Dodecanoic acid, a fatty acid. Peak 4 appeared at m/z 242
with molecular formula C15H30O2 and its name is Methyl
tetradecanoate, an ester. Peak 5 occurred at m/z 240 with
molecular formula C17H36 named Heptadecane. Peak 6
occurred at m/z 268 with formula C19H40 called
Nonadecane. Peak 7 appeared at m/z 270 with molecular
formula C17H34O2 and is named Hexadecanoic acid methyl
ester. Peak 8 occurred at m/z 256 with a molecular formula
C16H32O2 and is a carboxylic acid called n-Hexadecanoic
acid, a fatty acid. Peak 9 appeared at m/z 296 with formula
C19H36O2 with name 9-octadecanoic acid methyl ester.
Peak 10 appeared at m/z 296 with formula C20H40O and
the name of the compound is Phytol. Phytols are
isoprenoids, they are acyclic diterpene alcohols used to
manufacture vitamin E and K1. They are constituents of
chlorophyll and are important building blocks of
chlorophyll. Peak 11 occurred at m/z 282 with
corresponding molecular formula C18H34O2 and name is
Octadec-9-enoic acid a fatty acid, peak 12 which occurred
at m/z 281 with molecular formula C18H35NO an amide
called 9-octadecenamide. This compound is an oleamide,
an amide derived from oleic acid biosynthesis from N-
Oleoylglycine. This compound has earlier been reported in
the root of of this plant Iwu et al (2018a) Peak 13 occurred
at m/z 252 with molecular formula C16H28O2 and is
identified as 11-tetradecyl-1-ol acetate an ester. One or
both oxygen atoms in the compound can be replaced by
sulphur giving a thio acid or dithio acid respectively. Thio
acids react readily with alcohols to form thio ester. Thio
esters play important role in the break down and synthesis
of lipids and steroids in living tissues.
6. Characterization of the Volatile Components of the Leaf of Starchytarphetacayennesis (Rich) Vahl
Iwu et al. 046
Fig 1. GC/MS spectrum of stachytarpheta cayenneses leaf extract
7. Characterization of the Volatile Components of the Leaf of Starchytarphetacayennesis (Rich) Vahl
Int. J. Herbs, Spices Med. Plants 047
Table 4: Molecular formula of 12 chromatographic peaks and their structures for the GC-MS analysis of the leaf extract
of S. cayennesis
Chromatographic
peak
Molecular
formula
Molecular
weight
Molecular structure Name of compound
1. C10H8 128 Azulene
2. C15H24O 220 Butylated Hydroxy Toluene
3. C12H24O2 200 Duodecanoic acid
4. C15H30O2 242 Methyl tetradecanoate
5. C17H36 240 Heptadecane
6. C19H40 268 Nonadecane
7. C17H34O2 270 Hexadecanoic acid methyl
ester
8. C16H32O2 256 n-Hexadecanoic acid
9. C19H36O2
296
9-octadecanoic acid methyl
ester
10. C20H40O 296 Phytol
11. C18H34O2 282 Octadec-9-enoic acid
12 C18H35NO 281 a-octadecenamide
13 C16H2802 252 11-Tetradecyn-1-ol acetate
CONCLUSION
Results so far obtained have shown the leaf extract of the
plant to be full of phytonutrients which have potent
medicinal applications. The leaf extract of S .cayennesis
has proven health benefits for both the treatment and
prevention of diseases and one can therefore understand
why traditional medicine practitioners use the leaf extract
for the treatment of host of diseases caused by both gram
positive and gram negative bacteria. The leaf extract is
used for the treatment of most body infections ranging from
kidney infections to wound and burns infections and also
in the treatment of pneumonia. The GC/MS result revealed
vital volatile chemical substances in the plants, Iwu et al,
(2018d). One important compound identified is 9-
octadecenamide (oleamide) a sleep inducing compound.
We could understand why traditional medical healers use
the plant extract to treat insomnia and mental disorder.
This study has thrown more light into the use of different
parts of S. cayennensis for the treatment of insomnia and
mental disorder Iwu et al ,(2018c)
8. Characterization of the Volatile Components of the Leaf of Starchytarphetacayennesis (Rich) Vahl
Iwu et al. 048
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