Phytochemical Screening and Preliminary TLC Characterization of Alkaloids in Sabicea brevies Root
Sabicea brevipes (susu) plant root in Oghe Community medicine is of value in the management of male erectile dysfunction. The objective of the study was to evaluate the phytochemicals and the number of alkaloids present. Phytochemical screening of n-hexame, ethyl acetate and methanol crude extract of Sabicea brevipes (susu) root were evaluated. The extracts were subjected to qualitative chemical analysis for identification of various secondary metabolites or active principles. The extracts revealed the presence of steroids, alkaloids, gylcosides, saponins, tannins, triterpenoids, antracene, flavonoids and volatile oils. The Result of this study x-rayed valuable evidence in support of Sabicea brevipes plant as an enhancer of sexual function in men. Phyto-chemical investigation resulted in isolation and TLC identification of several alkaloids using five solvent system. Results showed that the yields of the extract were 0.43% and 0.29% for morphine and non-morphine alkaloids, respectively. There were seven spots observed in the chromatogram developed for morphine alkaloids while five spots were seen for the other (non-morphine) alkaloids in the various solvent system used, visualizing with dragendoffs location reagent. The result shows that the solvent systems A chloroform: ethanol (9:1), C methanol Ammonia (99:1) and E chloroform: Methanol: Ammonia (60:60:1) contain one alkaloid each for morphine and non morphine alkaloids with RF values 0.64 and 0.55 (solvent system A); 0.55 and 0.27 (solvent system C) and 0.55 and 05.2 (solvent system E)s, respectively. While solvent systems B chloroform: ethanol (85:15) had two spots for morphine alkaloids with RF value 0.34 and 0.84 and solvent system D, Methanol: ethyl acetate: ammonia (9.5:81:9.5) also had two spots for morphine alkaloids with RF of 0.27 and 0.95 and two spots for non-morphine alkaloid with RF 0.60 and 0.95, respectively. These results support the claim about the use of this herb in folk medicines.
Received: August 22, 2012;
Accepted: May 14, 2013;
Published: February 27, 2014
Herbal remedies from plants continue to provide a popular alternative for treating
known and emerging disease that have defiled many orthodox medical treatments.
Herbal medicines are also in great demand in the developing world for primary
health care because of their efficiency, safety and lesser side effects. According
to World Health Organization (WHO) traditional medicines are relied upon by
60-80% of the worlds population for their primary health care needs (WHO,
2002; Zhang, 2004). Most of these herbal remedies
have stood the test of time, particularly for the treatment of allergic, metabolic
and cardio-vascular diseases (Igoli et al., 2005).
Sabicea brevipes plant belongs to the rubivacea family that has more
than 6,500 species out of which 152 are members of the Sabicea genus
(Davis et al., 2009). It is an erect or climbing
shrub, which is usually 0.6-1.2 m in height. The shrub has a mass of red flowers
in the month of July. It is normally found in some part of Africa, Madgascar
There is a need for novel erectile dysfunction treatment drugs, as some of
the currently available drugs are frequently associated with lots of side effects.
A typical example is with Viagra known for abnormal vision, chest pain, diarrhea,
dyspepsia, flushing, headache, hypertension, indigestion, nausea, palpitation,
photophobia, priapism and temporary rash with possible severe side effects of
intraocular pressure, myocardinal infraction, severe hypotension, stroke, sudden
death and ventricular arrahytmias (Akash et al.,
2005). There is some evidence suggesting that Sabicea brevipes plant
root may help in remedying erectile dysfunction. Sabicea brevipes is
a herb used for centuries in Oghe community traditional folk medicine practice
as a remedy for erectile dysfunction. Indeed, more than 60 species are used
for more than 70 medicinal indications which include sexual weakness (Karou
et al., 2011). Maca remains one of the most popular natural remedies
in Peru for erectile dysfunction and as an overall male sexual stimulant and
libido aid (Oshima et al., 2003). Ashwagandtia,
tribulus berries, catuaba (Antunes et al., 2001);
Lepidum meyenii walp, daminana, Yohimbe (Pausinystalid yohimbe)
(Ernst and Pittler, 1998; Rowland
et al., 1997); Muria Puama (Lirisoma ovata), Ginkgo Biloba
(Folium ginkgoidis) all contain different kinds of alkaloids that act
as vasodilator and androgen precursor. Medical studies have shown that muria
puama and catuaba dilates blood vessels and are strong tonic and nervous system
fortifies due to the alkaloids present in them that enhance erectile function
(Hollman and Katan, 1999; Di Carlo
et al., 1999).
It can be said that Sabicea brevipes when probably in central nervous
system increase blood flow to the penis. Men with penile circulatory problem
should typically get the best result with the root while psychological impotence
also fare well using the root product (Reid, 2004; Brown
1995). The overall objective of this study is an assessment of the extracts
of Sabice brevipes (susu) plant root samples for selected active ingredients
responsible for enhancement of erectile functioning in men and to isolate and
conduct preliminary TLC characterization of alkaloid extracted from the roots
of Nigeria Sabicea brevipes using selected solvent systems.
MATERIALS AND METHODS
Sample collection: The plant material (Sabicea brevipes root)
was collected from Oghe in Ezeagu Local Government Area of Enugu State Nigeria
on 10th July 2009 by 11.15 a.m. Presented and defended on September 2011. The
herbarilum specimen was identified by Prof. J.C. Okafor of Applied Biology and
Biotechnology, Enugu State University of Science and Technology, Enugu State,
Extraction and preparation of plant extract: The root was sun dried
for 14 days and pulverized with mechanical grinder. About 42.7 g the powdered
plant material was weighed and extracted successively and exhaustively with
n-hexane, ethyl acetate and methanol for 4 h, respectively using soxhlet extractor
technique. The extracts were concentrated on a reduced pressure to yield a brown-coloured
paste (n hexane extract 24.8 mg, ethyl acetate extract 19.4 mg and methanol
extract 27.3 mg, respectively). The crude root extracts were later screen qualitatively
for the phytochemical constituent utilizing standard methods of analysis (Vishnoi,
1979; Sofowora, 1993; Trease and Evans,
Phytochemical tests: To about 2.5 cm3 of each extract was
added to 10 cm3 of distilled water in a test tube and shaken vigorously
with 2 cm3 of olive oil. Fronting which persisted was taken as an
evidence for the presence of saponins (Sofowora, 1993).
Exactly 2.5 cm3 of each extract was added to 2.5 cm3 of
mixed fehling solution A and B in a test tube. The appearance of bluish green
precipitate indicates the presence of sugar bonded to saponin as a non-carbohydrate
moiety (Sofowora, 1993). Liebemann-Burchard Test for Steriod
and Triterpoenoids: To 2 cm3 of each extract was added to acetic
acid in a test tube and conc. sulphuric acid (H2SO4) was
carefully added. Colour development from violet to blue-green indicates the
presence of a steroidal ring (Sofowora, 1993). Diluted
sulphuric acid was added to each extract in a test tube and boiled for 15 min.
Then 10% sodium hydroxide and mixed Fehlings solution were added. The
formation of brick red precipitate indicates the presence of glycoside (Sofowora,
1993). A drop of ferric chloride was added to each extract in a test tube.
Then glacial acetic acid and conc. H2SO4¯ were added.
The development or appearance of blue layer indicates the presence of digitals
glycosides (Sofowora, 1993). Born-Traggers Test was used
to detect antracenes or anthraquinones. Two 2 cm3 of chloroform was
added to each extract in a test tube and allowed to separate. To the chloroform
layer was added 10% ammonia solution and vigorously shaken and kept to separate.
The formation of brick-red precipitate indicates the presence of antracenes
or anthraquinones (Sofowora, 1993). A mixture of 4 cm3
of each extract and 4 cm3 of distilled water was stirred very well
in a test tube and 3 drops of ferric chloride added. The occurrence of a blue-black,
green or blue-green precipitate indicates the presence of tannins (Trease
and Evans, 2002). About 4 cm3 of 10% ammonia solution was added
to 4 cm3 of each extract in a test tube and shaken very well. The
formation of an emulsion indicates the presence of hydrollisable tannins (Sofowora,
1993). A matchstick was dropped into a 3 cm3 of each extract
in a test tube and 2 drops of conc. HCl added. Then the resulting mixture was
left undisturbed for 5 min. The development of a dark purple colouration indicates
the presence of pseudotannins (Sofowora, 1993). Shinodas
test for Flavonoids a small quantity of magnesium chips was dropped into 3 cm3
of each extract in a test tube and 5 drops of conc. HCl added. A pink, orange,
or red to purple colouration indicates the presence of flavonoids (Trease
and Evans, 2002). Resins was detected by adding 2 cm3 of acetic
anhydride to 2 cm3 of each extract in a test tube. Then 2 drops of
conc. H2SO4 was added to the mixture. The appearance of
violet colouration indicates the presence of resins (Sofowora,
1993). The presence of alkaloids was confirmed thus: 2 cm3 of
each extract was taken individually into 4 test tubes. To the test-tubes, 2
drops of Dragendorffs reagent, Mayers reagent, Wagnerss reagent
and 10% (w/v) tannic acid were added, respectively. Occurrence of orange-red
precipitate, appearance of blue-coloured precipitate, observation of a deep
brown precipitate and development of cream colouration is a positive tests,
respectively (Sofowora, 1993; Trease
and Evans, 1978, 1989). Six drops of Ferric chloride
was added to a mixture of 2 cm3 of each extract and 2 cm3
of 90% (v/v) ethanol in a test tube. The observation of green colouration indicates
the presence of volatile oil (Sofowora, 1993).
Isolation and purification of alkaloids: The roots of the harvested
plant sample were cut off, dried and ground using a mechanical grinder. About
180 g of ground plant material was acidified with alcoholic solution of tartaric
acid (15 g tartaric acid dissolved in 900 cm3 of 95% ethanol) and
warmed to 50°C. This was left in 2000 cm3 conical flask to stand
overnight and thereafter filtered. The filtrate was partially evaporated and
filtered again to remove fat and protein before the evaporation was completed.
The residue was dissolved in boiling 100 cm3 of 95% (v/v) ethanol
and the solution cooled overnight in a refrigerator. The alcoholic extract was
filtered and the filtrate evaporated to dryness under reduced pressure. The
residue was dissolved in 50 cm3 of 2% sulphuric acid (v/v) and the
fat extracted with light petroleum ether (40-60°C). The light petroleum
ether extract was washed with 200 cm3 of 2% sulphric acid (v/v) and
the washing added to the aqueous solution and later neutralized extract was
evaporated to dryness and the resulting residue dissolved in 100 cm3
sodium carbonate. The neutralized extract was evaporated to dryness and the
resulting residue dissolved in 100 cm3 of absolute ethanol and filtered
and the filtrate evaporated to dryness under reduced pressure. The residue was
dissolved in 100 cm3 of 1 Mol dm-3 acetic acid and 50
cm3 of 0.25 mol dm-3 aqueous solution of lead acetate
and warned. The coagulated precipitate was filtered off. The excess lead was
removed by cautiously adding in drops dilute sulphuric acid and the lead sulphate
filtered off. At this stage non-basic poisons were removed by extracting twice
with 30 cm3 petroleum ether. The aqueous liquid was made alkaline
with 2 mol dm-3 sodium hydroxide and the alkaloids except morphine
were extracted three times by shaking with 25 cm3 of chloroform.
The chloroform extract was later evaporated to dryness in a water bath and the
alkaloid weighed and the weight recorded.
For the morphine alkaloid analysis the procedure was thus: the aqueous liquid
was acidified with 2 mol dm-3 of H2SO4 and
then made alkaline with ammonia and equal volume of methanol (110 cm3)
added. The morphine alkaloid was extracted three times with 25 cm3
of chloroform and the extracts evaporated in a water bath and the morphine alkaloid
weighed and the weight recorded (Allport, 1951; Dickel
et al., 1958; Grinkerich and Safronich, 1983).
TLC analysis for alkaloids: All thin-layer chromatographic analysis
was performed using TLC plates pre-coated aluminium oxide F-254 (Type E) with
layer thickness of 0.25 mm made in Germany by E. Merck, Darmstadt.
TLC plate (F-254, type E) was used to separate the active compounds present
in the extracts (Hashmi et al., 1968). The TLC
sheets after development are visualized by spraying with Dragendoffs reagent.
RESULTS AND DISCUSSION
The result of the phytochemical screening tests conducted on the crude extracts
of Sabicea brevipes roots shows the presence of saponins, tannins, glycosides,
alkaloids, steroids and triterpenoids as well as volatile oils as shown in Table
The phytochemical test of the crude n-Hexane, ethyl acetate and methanol extracts
of Sabicea brevipes roots revealed the presence of steroids, glycosides,
alkaloids, saponins, tannins and volatile oil (Table 1). The
active principles identified in the n-hexane extract (HE) were steroids, alkaloids
(in Wagners and Dragendorffs
reagents). Surprisingly, the Mayers and Tannic Acids tests for alkaloids were
negative. This observation for alkaloids also occurred in the Ethyl Acetate
Extract (EA) and Methanol Extract (ME).
The active ingredients found in the ME were saponins, saponin glycosides, tannins,
pseudo tannins, steroids and volatile oils.
|| Phytochemical constituents of root of Sabicea brevipes
|+: Present, -: Absent, HE: N-Hexane extract, EA: Ethyl acetate
extract, ME: Methanol extract
The root extracts in the three solvents-HE, EA and ME-show no presence (negative
results) for the following active principles: hydrolysable tannins, pseudo tannins,
digitalis glycosides, anthracenes, Mayers
test alkaloids, tannins acid test alkaloid, flavonoids and triterpenoids.
This positive observations help in providing chemotaxonomic evidence for the
classification of the species since Sabicea brevipes belongs to the rubiaceea
family, which has been reported to contain these compounds (Stary
and Storchova, 1991). The identification of these family of compounds further
supports claims of the use of the root as a traditional medicine as these compounds
have valuable amoebicidal, anti-fungal and anti-inflammatory properties (Opakunle
and Jayicoba, 1988). They have stimulating and tonifying effects on the
muscles when consumed and this probably accounts for their use in enhancing
male potency. Furthermore, the metabolites: alkaloids, steroids, glycosides,
saponins and tannins found in the root extracts are known to have curative activity
against several pathogens and therefore the plant root is use traditionally
for the treatment of various illnesses (Hassan et al.,
2004; Usman and Osuji, 2007).
Probably the constituents of Sabicea brevipes from which its male potency
enhancing properties emanated are based upon the actions of certain steroidal
alkaloid and glycosides. A study has implicated saponin component of plants
in enhancing aphrodisiac properties due to its androgen increasing property
(Gauthaman et al., 2002). Saponins present in
the methanol extract of this plant might have assisted in stimulating an increase
in the body natural endogenous testosterone levels by raising the level of Leutinizing
Hormones (LH). The LH release normally by the pituitary gland helps to maintain
testosterone levels, as LH increases, so does the testesterone (Gauthaman
et al., 2002). The increase in testosterone seemed to have translated
into the male sexual competence. Furthermore, this study suggests that the aphrodisiac
action may be mediated through a change in the blood testosterone level (Yakubu
et al., 2005).
Analysis of Sabicea brevipes root using aluminium oxide TLC, eluted
with chloroform/ethanol (A) solvent system followed by staining with Dragendorff
reagent, showed one orange brown sport with RF values 0.64 and 0.55
each for morphine alkaloids and non-morphine alkaloids, respectively.
|| Yield, solvent system composition and RF values
of the various alkaloids
|NF: Not found
Organ brown spot (RF value of 0.84 and 0.34) were observed only
for morphine alkaloids using the chloroform/ethanol (B) solvent system. The
solvent system methanol/ammonia (C) solvent system showed one spot
for morphine alkaloids (RF value 0.55) and also one spot for morphine
alkaloids (RF value 0.55) and also one spot each for non-morphine
alkaloids (RF value 0.27). Solvent system D (methanol/ethyl acetate/ammonia)
show two spots for morphine alkaloids (RF values 0.27 and 0.95) and
also two spots for the non-morphine alkaloids (RF value 0.60 and
0.95). Solvent system E (choroform/metahno/ammonia) gave one spot each for the
morphine and non-morphine alkaloids with RF value of 0.55 and 0.52,
respectively. Their RF values were quite close compared with other
solvent systems (Table 2).
Results show that morphine alkaloids contain highest number of spots and percent
yield which are 7 sports and 0.433%, respectively; while the non morphine (other)
alkaloids had 5 sports and 0.25% yield (Table 2). These findings
supported the view that alkaloids are commonly present in Sabicea brevipes
(Stary and Storchova, 1991). Therefore further studies
are needed to isolate these active compounds and elucidate their chemical structures
and highlight their bioefficacies.
The results of this study shows that the three extracts of the root of Sabicea
brevipes indicates the presence of alkaloids in all the extracts and the
presence of saponins, saponin glycosides, tannins and volatile oils only in
methanol extracts. While steroids/triterpenoids are only present in n hexane
The results also revealed that morphine alkaloids is the main alkaloids (0.43%)
in Sabicea brevipes than non-morphine alkaloids.
The plant parts studied here has also been seen as a potential source of useful
drugs. Further studies are going on in order to isolate, identify characteristics
and elucidate the structure of the bioactive compounds. Male potency enhancing
activities of the plant root for the treatment of erectile dysfunction as claimed
by traditional healers are also being comprehensively investigated.
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