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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.
Keywords: Starchyterpheta cayennensis, gas chromatography, pathogens, phytochemicals, minimum inhibition
concentration.
INTRODUCTION
Stachytarpheta cayannensis (Rich) Vahl belongs to the cayennensis in the treatment of sleeping disorders. This is
genus Stachytarpheta, of the family Verbenaceae. This so because oleamide accumulates in the cerebrospinal
genus has other species recognized in Nigeria which fluid during sleep deprivation and thus induces sleep in
include; Stachytarpheta angustifolia(Mill.) Vahl, and S. animals. It has been studied as a potential medicinal
indica (Linn.) Vahl and S. jamaicennesis. They are treatment for mood and sleep disorders. Olayiwole et al,
generally known as herbs, shrubs or vines and sometimes
trees with leaves usually opposite or whorled, simple or
palmately compound. Stachytarpheta cayennesis,
commonly known as blue rat tail is a predominating tropical *Corresponding Author: Iwu Irenus Chinonye,
plant exhibiting a wide range of growth habit. Ezeabara Department of Chemistry, Federal University of
and Eze, (2015), Lillyama and Shah, (1987). Previous Technology Owerri, Nigeria. Tel.: +2348032444212. E-
work on the root of this plant by Iwu et al, (2018a) has led mail: iwu.chinonye@yahoo.com. Co-Author Email:
2
to the identification of 9-Octadecenamide (Oleamide), an ucheonuh2018@gmail.com, Tel.: +2348037676079;
3ukaoma.adanma@yahoo.com, Tel.: +2348038743752;
amide. The presence of this compound has been shown to
4fideconwumere2@gmail.com, Tel.: +2347038824111
be responsible for the use of the roots of Starchytarpheta
The leaves of Starchytarpheta cayennensis were obtained 20cm3 of the water extract was treated with Fehling
in Eziobodo community in Owerri West L.G.A of Imo state, solutions of A and B in equal amount and boiled. A
Nigeria. They were then room dried for a period of one brownish red precipitate indicates the presence of
month before been milled into powder using a milling glycoside. Iwu et al, (2018b)
machine. The milled sample was then stored in airtight
container till required for analysis, Iwu and Onu, (2018). Preparation of Samples for GC-MS Analysis
Frothing test for Saponins Two hundred grams of the leaf sample was soaked in
ethanol for 48 hours and then extracted. The extract was
This test is based on the ability of the saponins to produce re-extracted using chloroform to obtain chloroform soluble
froth in aqueous solution. 5g of the leaf sample was extract. This was centrifuged at 10,000 rpm for 20 minutes
weighed into a test tube and 50cm 3 of water was added and the clear supernatant oil was subjected to GCMS
and extracted after4hours. The water extract was shaken analysis.
vigorously in a conical flask. The production of a persistent
froth when allowed to stand for 5 minutes indicates the GC-MS Experimental Procedures
presence of saponins in the sample.Iwu et al,(2016b)
GC-MS analysis was carried out with SHIMAZU Japan
Test for Flavonoids Gas Chromatography 5890-11 with a fused GC column
OV 101 coated with polymethyl silicon (0.25 mm x 50 m)
5g of the sample was soaked with 50cm3 of water and then and the as follows: Temperature programming from 80 –
filtered. To the filtrate were added drops of ammonia and 200oC held at 80oC for 1 minute, the rate is 5oC/min and at
3cm3 of concentrated H2SO4 was added. A yellow 200oC for 20 minutes. FID Temperature of 300oC, injection
precipitate which disappears on storage indicates the temperature of250oC, carrier gas is Nitrogen at a flow rate
presence of flavonoids. Okwu and Okwu, (2004) of 1 cm3/min and split ratio of 1:75. GC-MS Gas
(chromatography, Mass spectrum) analysis was
Test for Alkaloids conducted using GC-MS QP 2010 Plus Shimadzu Japan
with injector Temperature at 230oC and carrier gas
5g of the sample was extracted using 20% acetic acid in pressure of 100kpa. The column length was 30 m with a
ethanol .5cm3 of the extract was treated with 3drops of diameter of 0.25 mm and the flow rate of 50m/min. The
Wagner’s reagent (iodine crystals and KI). A yellowish eluents were automatically passed into the Mass
brown precipitate indicates the presence of alkaloids. Iwu Spectrometer with a detector voltage set at 1.5kv and
et al, (2018b). sampling rate of 0.2 seconds. The Mass Spectrometer was
also equipped with a computer fed Mass Spectra data
bank, HERMCE Z 233 M-Z centrifuge Germany was used. RESULTS AND DISCUSSION
Reagents and solvents such as Ethanol, Chloroform,
Diethyl ether, hexane all of analytics grade was obtained PHYTOCHEMICAL ANALYSIS
from Merck Germany (Iwu et al 2016a, b.)
The results of the phytochemical screening of the leaf of of
Antimicrobial Analysis Stachytarpheta cayennensis are given in table 1 below.
The table revealed the presence of flavonoids, saponnins,
The microorganisms; Staphylococcus aureus, tannins, phenols, steroids, cardiac glycosides and
Streptococcus spp, Klebsiella spp, Proteus spp and alkaloids
Pseudomonas spp were used for the analysis. They are
clinical isolates of human pathogens obtained from the Table 1: Phytochemicals screening test result of sample
Federal Medical Centre Umuahia and were brought to the test of the leaf of S.cayennesis
laboratory and resuscitated in buffered peptone broth Phytochemicals Inference
(Secharian chemie) and thereafter into nutrient agar Flavoloids ++
medium and incubated at 37oC for 24 hrs Iwu et al (2016b). Saponins ++
Tannins ++
Antibacterial Assay Phenols ++
Steroids ++
The test solution of each extract was prepared by Glycosides ++
dissolving 0.1 g of the plant extract separately. 1.0cm3 of Alkaloids ++
dimethyl sulphoxide (DMSO) to get a concentration of Key; ++ present in reasonable quantity
100mg/cm3. The antibacterial activity was performed by .
filter paper disc diffusion technique. Filter paper disc Alkaloids are vast and vary a lot in their activity when
(Whatman No 1.6 mm diameter) were placed in glass ingested by man and livestock. Some alkaloids are useful
petridish and sterilized in hot air oven. Iwu et al 2018b, the and important in medicine and constitute most of the
media (10g nutrient Agar in 200cm 3 distilled water, valuable drugs currently used by humans. They are
autoclaved at115oC for 30 minutes) was cooled to 50 oC. reported to have marked physiological effect on animals
The sterile nutrient Agar media were poured into the sterile Edeoga and Eriata, (2001).
petridish and allowed to solidify. The bacteria were
swabbed with a sterile wire loop. Each disc was Phenolics are broadly distributed in plants and are the
impregnated with 0.2cm 3 of plant extract. Standard most abundant secondary metabolites of plants. Plant
antibiotic Ciprofloxacin was used as a control on a disc phenolic have drawn increasing attention due to their
with DMSO 100mg/cm 3. The discs were used after drying potent anti-oxidant properties and their marked effects in
them in an incubator at 40oC to remove any trace of the prevention of various oxidative stress associated
solvent. Discs were introduced into the surface of the diseases such as cancer. In the last few years, the
medium. The plates were microbated at 37oC for 24 hours identification and development of phenolic compounds or
to obtain zones of inhibition. The experiments were extracts from different plants has become a major area of
repeated three times for each extract and twice for health and medical-related research.
reference antibiotics to minimize error and the average of
these values were recorded. Flavonoids have been shown to be highly effective
scavengers of most oxidizing molecules. Tukappa and
Minimum Inhibitory Concentration (MIC) Londonkar, (2013). In addition, tannin was found in the
plant at a concentration range. Plant leaves with high
The minimum inhibitory concentration of the extract was tannin content have been used successfully as hops
determined by incorporating constant volume of 0.2cm3 of alternative in beer Hutchinson and Dalziel, (1963).
each diluents of the extract into the perforated disc on a
seeded nutrient agar plate as described in the anti- Flavonoids are polyphenolic compounds based on a C15
microbial susceptibility test section. 0.1g of each extract (C6C3C6) framework. They contain a chroman ring (C-ring)
was dissolved in 1cm 3 of DMSO to obtain 100mg/cm 3. This with a second aromatic ring (B-ring) at the C2, C3 and C4
concentration of DMSO was then doubled to obtain position. The heterocyclic six-membered C-ring is
50mg/cm3 then doubled again to obtain 12.5mg/cm 3 and sometimes replaced by a 5-membered ring. The oxidation
again to obtain6.25mg/cm 3. Each concentration was then state of the C-rings is used to classify flavonoids into
used in the method earlier described to obtain zone of different categories of which typical examples are Flavan-
inhibition. The least concentration that showed inhibitory 3-ols, flavanones, flavones, isoflavones and flavanols.
zones was taken as the MIC. Ekundayo and Ezeogu, Flavonoids are the major nutraceutical ingredients that are
(2006). 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
protecting the body against reactive oxygen species The presence of Phenolic compounds in the leaf of
(ROS). Antibacterial activity has been displayed by a Starchytarpheta cayennesis indicates that this plant might
number of flavonoids, Quercetin has been reported to be an anti-microbial agent. This is because phenols and
completely inhibit the growth of Staphylococcus aureus. phenolic compounds have been extensively used in
Havsteen, (1983). Flavonoids also possesses anti- disinfections and remains the standard with which other
inflammatory and analgesic effect as well as anti- bactericides are compared. Phenolic compounds act as
ulcerogenic activity. Shahid et al 1998. electron donors and are readily oxidized to form phenolate
ions. This gives rise to protonated phenol which is used as
The infusions of S. cayennesisis taken as a remedy for a cleaning agent. Extracts from leaves of Starchytarpheta
gonorrhea and jaundice. Draghon, (2004), Mohammed et cayennesis therefore have potent antiseptic or bactericidal
al, (2013). This is probably due to antibacterial action of properties. This finding supported the use of extracts of the
saponins. Saponins are foam forming in nature and have leaves in treating wounds that not only heal fast but also
been implicated as a bioactive antibacterial agent of plant, prevent the formation of infection. Okwu and Okwu,
Thomas-Barbera et al, (1990). Saponins are also potential, (2004). Phenols have antioxidant properties. Thomas-
sometimes for utilization in foods that need sustained foam Barberan et al (1990) The presence of phenol further
volume such as ice-creams. Saponins are a class of indicates that Stachytarpheta cayennesis could act as anti-
chemical compounds, more specifically, they are inflammatory, anti-clotting, immune enhancers and
amphipathic glycosides grouped, in terms of hormone modulators.
phenomenology by the soap-like foaming they produce
when shaken in aqueous solution and in terms of structure Glycosides are molecules in which a sugar is bound to
by their composition of one or more hydrophilic glycosides another functional group via a glycosidic bond. Glycosides
combined with a lipophilic triterpene derivative. In plants play numerous important roles in living organisms. Many
saponins may serve as anti-feedants to protect the plant plant store chemicals in form of inactive glycosides. Many
against microbes and fungi. Some plant saponins may such plant glycosides are used as medications. Some
enhance nutrient absorption and aid in animal digestion. glycosides have shown some evidence of pharmacological
Saponins have been used as a pharmacological and/or effects in patients with hypertension or with type-2
immunological agent that modifies the effect of other diabetes but concluded that further study was required to
agents in vaccines. Saponins from plants have been determine proper dosage.
shown to significantly augment the cytotoxicity of
immunotoxins and other target toxins directed against ANTIMICROBIAL ACTIVITY OF THE LEAF EXTRACT
human cancer cells. OF STARCHYTARPHETA CAYENNESIS
Tannins are astringent, bitter plant polyphenol compounds The anti-microbial activity of the leaf extract of
that bind to and precipitate proteins and various other Starchytarpheta cayennesis are summarized as shown in
organic compounds including amino acids and alkaloids. table 2.
The tannin compounds are widely distributed in many
species of plants where they play a role in protection from The leaf extract showed marked inhibition of some of the
predation and perhaps also as pesticides and in plant selected pathogens, at concentrations of 100mg/cm 3,the
growth regulation Urqiuaga and Leighton 2000. The extract showed inhibition of Staphylococcus aureus, with
astringency from tannin is what causes the dry puckery inhibition diameter of 8mm, Klebsiella spp, 9mm, Proteus
feeling in the mouth following the consumption of unripe mirabilis, 5mm and Pseudomonas aureginosa 5mm. The
fruits or red wine. Tannins are important ingredients used minimum inhibition concentrations are 25mm/cm3 for
in process of making tannin leather. Medicinally, tannins staphylococcus aureus,12.5mg/cm3 for Klebsiella spp,
are used as anti-diarrhea, hemostatic and anti-hemorrhoid 25mg/cm3 for Proteus mirabilis and 12.5mg/cm3 for
compounds.
Pseudomonas aureginosa (table 2). These results are very Antibiotics sensitivity test is usually carried out to
close to those obtained when standard antibiotics determine the antibiotic that will be the most effective
Levoflxacin and Streptomycin were used. Staphylococcus against specific bacteria and fungi infecting in an
aureus is a gram positive coccus that causes skin infection individual. Standard drugs were used to inhibit the
such as; pimples, impetigo, boils, cellulitis, folliculitis, pathogens in table 3 and some were found to be very
carbuncles, scalded skin syndrome, abscesses, effective in inhibiting all strains of the bacteria amongst
pneumonia, toxic shock syndrome, bacteremia and sepsis. which were Ciproflox, Streptomycin, Rifampicin and
Pseudomonas aureginosa is a gram negative gamma- Levofloxacin. They were used as control in the anti-
proteobacteria which belong to the family microbial test carried out
Pseudomonaceae. It causes bacteremia, pneumonia,
foliculitis, swimmer ear which is an ear infection GC/MS ANALYSIS
accompanied with swelling, ear pus. Itching, discharge
and difficulty in hearing, eye inflammation with associated The GC/MS analysis gave a spectrum with 13 absorption
pains, pus, swelling redness and impaired vision. peaks (fig1)
Klebsiella, a non-motile gram negative, oxidase rod
shaped bacteria which causes infectious wounds, The interpretation of the chromatogram obtained was done
pneumonia, Okonkwo et al (2012), blood stream infection with the aid of data obtained from a computer fed Mass
and urinary tract infection. Proteus mirabilis is a gram Spectra data bank, HERMCE Z 233 M-Z. From these data,
negative facultatively anaerobic rod shaped bacterium the molecular weights, structures, molecular formulas and
implicated in urinary tract infection (UTIs), bacteremia, name of the compounds were obtained, (table 4).
type 2 diabetes, cystitis, pyelonephritis, urosepsis and
urinary stone (urolithiasis). Stachyterpheta cayennensis Peak 1 occurred at m/z 128 which corresponds to the
has been used as a remedy for syphilis, gonorrhea, catarrh molecular formula C10H8 and is identified as Azulene.
condition, skin wounds and sores in children Hutchinson These compounds are isomers of naphthalene which is a
(1963). The extracts exhibited some level of inhibitory cyclic aromatic hydrocarbon. Peak 2 appeared at m/z 220
effects against some of the studied pathogens which have with molecular formula C15H24O and is identified as
been implicated in one bacterial infection to the other in Butylated Hydroxytoluene, an aromatic hydroxyl. Peak 3
human and plant. Klebsiella specie causes pneumonia. In appeared at m/z 200 with formula C12H24O2 which is called
plant, Pseudomonas spp causes bacterial blight in guinea Dodecanoic acid, a fatty acid. Peak 4 appeared at m/z 242
corn. The sensitivity test for most antibiotics against with molecular formula C15H30O2 and its name is Methyl
certain pathogens are shown in table 3. These tetradecanoate, an ester. Peak 5 occurred at m/z 240 with
microorganisms are inhibited by the leaf extract of S. molecular formula C17H36 named Heptadecane. Peak 6
cayennesis implying that the extract may be used to treat occurred at m/z 268 with formula C19H40 called
diseases associated with these organisms. Nonadecane. Peak 7 appeared at m/z 270 with molecular
formula C17H34O2 and is named Hexadecanoic acid methyl
Table 3: Sensitivity test carried out on the pathogens by ester. Peak 8 occurred at m/z 256 with a molecular formula
certain antibiotics used to evaluate the inhibitory properties C16H32O2 and is a carboxylic acid called n-Hexadecanoic
of S.cayennesis acid, a fatty acid. Peak 9 appeared at m/z 296 with formula
Antibiotics Staphylo- Strepto- Klebsiella Proteus Pseudo- C19H36O2 with name 9-octadecanoic acid methyl ester.
coccus coccus spp. spp. monas Peak 10 appeared at m/z 296 with formula C20H40O and
aureus spp. spp. the name of the compound is Phytol. Phytols are
CPX +++ ++ ++ ++ + isoprenoids, they are acyclic diterpene alcohols used to
NB +++ -- + +++ -- manufacture vitamin E and K1. They are constituents of
CN ++ -- +++ ++ -- chlorophyll and are important building blocks of
AMX -- -- ++ ++ -- chlorophyll. Peak 11 occurred at m/z 282 with
S +++ + +++ ++ ++ corresponding molecular formula C18H34O2 and name is
RD + + +++ + ++ Octadec-9-enoic acid a fatty acid, peak 12 which occurred
E ++ -- -- +++ + at m/z 281 with molecular formula C18H35NO an amide
CH -- -- +++ ++ +++ called 9-octadecenamide. This compound is an oleamide,
an amide derived from oleic acid biosynthesis from N-
APX -- -- +++ -- --
Oleoylglycine. This compound has earlier been reported in
LEV +++ +++ +++ +++ +++
the root of of this plant Iwu et al (2018a) Peak 13 occurred
Key: CPX=Ciproflox; NB=Norfloxacin; CN= Gentamycin;
at m/z 252 with molecular formula C 16H28O2 and is
AMX=Amoxil; S=Streptomycin; RD=Rifampicin;
identified as 11-tetradecyl-1-ol acetate an ester. One or
E=-Erythromycin; CH=Chloramphenicol; APX=Ampiclox;
both oxygen atoms in the compound can be replaced by
LEV= Levofloxacin
sulphur giving a thio acid or dithio acid respectively. Thio
+ fairly sensitive, ++ very sensitive. +++ highly sensitive –
acids react readily with alcohols to form thio ester. Thio
none sensitive
esters play important role in the break down and synthesis
of lipids and steroids in living tissues.
Table 4: Molecular formula of 12 chromatographic peaks and their structures for the GC-MS analysis of the leaf extract
of S. cayennesis
Chromatographic Molecular Molecular Molecular structure Name of compound
peak formula weight
1. C10H8 128 Azulene