This study examined the macro morphological,
proximate and phytochemical constituents of Heterotis rotundifolia (Sm.) Jac.-Fél. (=Dissotis rotundifolia [Sm.] Triana.) found in Melastomataceae, a prostrating to decumbent,
perennial dicot which roots in nodes, grows from stem fragments and from
seeds up to 50 ± 10 cm in height, notably found in damp places of the Niger
Delta Tropics. It is densely pubescent with a four angled reddish stem and
simple ovate-suborbicular to ovate-lanceolate petiolate leaves, 3 nerved,
having acute apex and rounded base, having ciliate margins and arranged in
opposite phyllotaxy, measuring up to 4 ± 2.5 cm long and 2 ± 1 cm wide. The
inflorescence is a cyme and flowers are solitary to paniculate with
inconspicuous deciduous bracts and terminal purple to pinkish pentamerous
corolla, large yellowish and purple stamens with connectives, the calyx tubes
covered with stalked stellate or plumose bristles which is 5 ± 2 mm in
length. The berry fruits are globular, measuring up to 1 cm in length
containing seeds having concentric ridges, pitted in the centre. The stem or
intermodal anatomy revealed, besides the usual peripheral ring arrangement of
vascular system, central phloem elements observed in pith region of plant.The phytochemical study revealed the
presence of: alkaloids, saponins and cardiac glycosides while steroidal
aglycones, was absent.Investigation
on proximate analysis revealed the following:0.47 ± 0.00 %
carbohydrate, 5.96 ± 0.46 % crude
fiber, 5.25 ± 0.00 % proteins, 2.60 ± 0.00 % lipids, 83.80 ± 0.28 % moisture
content and 1.93 ± 0.18 % ash respectively. These
information would assist for further delimitation of the species.
The weed Heterotis
rotundifolia (Sm.) Jacq.-Fel. is a member of the
family Melastomataceae which has about 188 genera and 5055 species Stevens
(2022). The genus Heterotis has about
23 species, distributed mainly in the tropics, formally treated as Dissotis rotundifolia (USDA-ARS., 2023).
The common names include: pink lady, Spanish shawl, Brunken (2008), rockrose Burkill
(1985), and Trailing Dissotis (Whistler,
2000); Awede in Yoruba and Nkpisi-nku in Igbo Wagner et al. (1990). It is also known for its ornamental and landscape
uses. It grows as weed on rocks or creeping and climbing among boulders and
along roadsides and waste dumpsites PROTA. (2013). In Nigeria, It is known as
Ebafo in Benin (Wagner et al., 1990;
Abere, et al., 2009). The foliar
morphology described as oval shaped and three-ribbed, which is ovate-lanceolate.
The floral morphology of D. rotundifolia
are solitary and the stalk of the flowers are covered with piloses like the
foliar ones and the petals are pink to pale purple coloration (Hutchinson and
Dalziel, 1954; Akobundu and Agyakwa, 1998).
In Africa it is
applied in remedying the following: rheumatism and diarrhea, cough, stomach
ache conjunctivitis, dysentery, bilharzia, prevents abortion, and tuberculosis
(PROTA, 2013; Abere, 2009; Kokwaso, 1976; Nguta, 2010; Abere, 2010). Abere
(2010) showed that clinical test using the leaves of H. rotundifolia have antimicrobial activities.
Proximate analysis
done by Charity et al. (2020) showed
the following; 1.89 % Ash, 1.30 % Fibre, 3.13 %Fat, 2.90 % Moisture, 9.9 %
proteins, 80.97 % carbohydrates. Cardiac
glycosides (5.65 ± 0.44 %) were the major active chemical component present in
the leaf of D. rotundifolia while
steroids were absent. Alkaloids, anthraquinone glycosides, flavonoids, phenols,
saponins, tannins and terpenes were present in this plant part (Ezeabara, 2022). The fat content was estimated to
be (72.53 ± 0.49 %) whereas ash (4.10 ± 1.05 %) was the least. Moreover, D. rotundifolia leaf extracts could be
regarded as good source of alkaloids, anthraquinone glycosides, cardiac
glucosides, flavonoids, phenols, saponins, tannins and terpenes used as
curatives in phytotherapy.
The justification is
directed to enhance more information on existing literature of the plant.
Therefore, the objective of the study focusses on providing extensive and more
current taxonomic characteristics on
Heterotis rotundifolia.
MATERIALS
AND METHODS
Plant
Collection
The
plant material used in this study was collected fresh from the Centre for
Ecological Studies, University of Port Harcourt, Rivers (405214411North,
605512011East). It was identified at the
University of Port Harcourt Herbarium domicile at the Department of Plant
Science and Biotechnology.
Macro
morphological Studies
For
the macro morphological study, visual observation of plant parts was done and
verified using the aid of Akobundu and Agyakwa (1998). The meter ruler was used
for measurement involving plant height from the root-collar to the terminal
bud, the leaf length from the leaf tip to the petiole base and the leaf width
across the leaf lamina, from one margin to another at the widest region.
Anatomical Study
The
plant grew in the wild. The harvested stems and other plant parts were
dehydrated in alcohol solutions of 70% and absolute alcohol and sectioned using
free hand method. Microphotographs were taken from good preparations using Sony
camera of 7.2 Mega pixels having 2.411 LCD monitor and High sensitivity ISO
1250.
Phytochemical
Study
The
leaves ofthe Heterotis rotundifolia were sun dried for 72 hours and later
weighed. Fifty grams (50 g) of the dried leaves were macerated in 96 % ethanol
with a pestle and a mortar. The extract was filtered and then evaporated to
dryness using a rotary evaporator set at 45 0C. Residue yields were
noted and a portion used for the phytochemical investigation.
Test
for alkaloids
This
involved using 0.5 g of the plant extract, stirred with 5 ml of 1 % aqueous
hydrochloric acid on a water bath; 1 ml of the filtrate was treated with few
drops of Mayer’s reagent and a second 1 ml portion was treated in same way with
Dragendorff’s reagent. The third 1 ml was treated with Wagner’s reagent.
Turbidity or precipitation with these reagents was taken as preliminary
evidence for the presence of alkaloids (Harborne, 1973; Trease and Evans, 1989).
A modified thin-layer chromatography (TLC) method as described by Farnsworth
(1962) was used. One gram (1 g) of the extract was treated with 40 % calcium
hydroxide solution until the extract was distinctly alkaline to litmus paper,
and then treated twice with 10 ml of chloroform. The extracts were combined and
concentrated to 5 ml. The chloroform extract was spotted on thin-layer plates.
Four different solvent systems were used to develop each plant extract. The
presence of alkaloids in the developed chromatograms was detected by spraying
the chromatograms with freshly prepared Dragendorff’s spray reagent. A positive
reaction on the chromatograms (indicated by an orange or darker colored spot
against a pale yellow background) was used as confirmatory evidence for the
presence of alkaloid.
Test
for flavonoids
Shinoda reduction test: 5 g of the pulverized sample
was boiled in 5ml of distilled water for 5 minutes on water bath and filtered
while hot. Magnesium (Mg) was added to the filtrate and few drops of conc.H2SO4
were carefully introduced into the mixture. The formation of orange, red,
crimson or magenta was taken as evidence of preliminary presence of flavonoid.
Lead acetate test: 5 g of pulverized sample was
boiled in 5ml of distilled water for 5 minutes in water bath and filtered while
hot. 2 ml of 10 % lead acetate was added to the filtrate and observed. Yellow
precipitate indicated presence of flavonoids.
Test
for tannins
Ferric
chloride test (FeCl3)
5 g of the prepared sample was boiled in 5 mls of
distilled water for 5minutes on water bath. This was filtered while
hot. 1 ml of 5 % FeCl3 was added to the filtrate and observed.
Blue-black, green or blue-green precipitate was taken as tannins present in the
sample (Trease
and Evans, 1989).
Test
for anthraquinones
Borntrager’s
test:Five grams (5 g) of each plant
extract was shaken with 10 ml benzene, filtered and 5 ml of 10 % ammonia
solution added to the filtrate. The mixture was shaken and the presence of a
pink, red, or violet color in the ammonia (lower) phase indicated that free
hydroxyanthraquinones were present.
Test
for combined anthraquinones
Five
grams (5 g) of each plant extract was boiled with 10 ml aqueous sulphuric acid
and filtered hot. The filtrate was shaken with 5 ml of benzene, the benzene
layer separated and half its own volume of 10 % ammonia solution added. A pink,
red or violet coloration in the ammonia phase (lower layer) indicated that
anthraquinone derivatives were present in the extract (Trease and Evans, 1989).
Test
for phlobatannins
The
deposition of a red precipitate when an aqueous extract of the plant part was
boiled with 1 % aqueous hydrochloric acid was accepted as evidence that
phlobatannins were present in the sample (Trease and Evans, 1989).
Test
for cardiac glycosides
Lieberman’s test
0.5
g of the extract was dissolved in 2 ml of acetic anhydride and cooled in ice. One
milliliter (1 ml) of Sulphuric acid was added in drops until a color change
from violet to blue to green indicating that steroidal aglycones were present
in the extract (Shoppe1964).
Test
for Saponins
Frothing tests:
Preliminary
following the method described by Wall (1952) was observed. The ability of
saponins to produce frothing in aqueous solution and to haemolyse red blood
cells was observed as screening test for saponins. 0.5 g of the plant extract
was shaken with water in a test tube. Frothing which continued on warming was
taken as preliminary evidence for that saponins were present in the sample. The
disc was then washed in ether, dried and placed on a 7 % blood nutrient agar.
Complete haemolysis of red blood cells around the disc after about 6 hours was
taken as further evidence that saponins presence in sample.
Proximate
Properties
Proteins
(Kjeldahl method)
Stage 1: 0.1 g of sample was weighed into a
conical flask of 250 ml capacity, 3 g of digestion catalyst was placed into the
flask and 20 ml conc.H2SO4 added and heated to digest.
Color change observed from black to sky-blue, cooled to room temperature and
then diluted to 100 ml with distilled water.
Stage 2: 20 ml diluted digest was measured
into a distillation flask and held in place on hot plate. The distillation
flask was attached to a Liebig condenser connected to a receiver containing 10 ml
of 2 % boric acid indicator. 40 ml NaOH was injected into the digest, and
heated to boiling and the distilled ammonia gas via the condenser into the
receiver beaker. The color of the boric acid change from purple to green as
ammonia distillate was introduced into the boric acid.
Stage 3: The distillate was titrated with
standard 0.1 N HCl solution back to purple from greenish. The volume of HCl
added to effect this change was recorded as titre value.
Thus,
Where 1.4 = Nitrogen equivalent to the
normality of the HCl used in the titration 0.1 N
100 = the total volume of digest dilution
100 = percentage factor
0.1 g of the sample
1000 = conversion from gram to milligram
20 = integral volume of digits analyzed or
distilled
0.1 g = the weight of sample in gram digested
Carbohydrate
(Cleg Anthrone Method)
0.1 g of the sample was weighed into 25 ml volumetric
flask, 1 ml distilled water and 1.3 ml of 62 % perchloric acid was added and
stirred for a period of 20 minutes to homogenize completely. The flask was made
up to 25 ml mark with distilled water. The solution was filtered with a glass
filter paper and allowed to sediment and then decanted. 1 ml of the filtrate
was collected and transferred into a 10 ml test tube which was diluted to
volume with distilled water. 1 ml of the working solution was pipette into a
test tube and made up to volume with distilled water. 1 ml of working solution
was pipette into a test tube and 5 ml anthrone reagent added. 1 ml distilled
water was added and 5 ml anthrone reagent mixed. Similarly, the whole mixture was
read at 630 nm wavelength using the 1 ml distilled water and 5 ml anthrone
reagent prepared as blank. 0.1 ml glucose was also prepared and was treated as
the sample with anthrone reagent.
Absorbance of the
standard glucose was read and the value of carbohydrate as glucose was
calculated as shown below:
Moisture (Air Oven Method)
1 g of the sample was weighed in to a
porcelain evaporating dish. This was placed in an oven set at 105 0C
for 6 hours. The evaporating dish was cooled in the desiccator to room
temperature and reweighed. Thus, the calculation of % moisture was as shown
below:
Lipid
(Soxhlet Extraction Method)
2 g of sample was inserted into a filter
paper and was introduced into a soxhlet extractor. The extractor was placed
into a pre-weighed dried distillation flask. Then the solvent (acetone) was added
to the distillation flask through the condenser end attached to the soxhlet
extractor. The set-up was held in place with a stand clamp and cooled water jet
was allowed to flow into the condenser and the heated solvent was refluxed as a
result. The lipid in the solvent chamber was extracted in the process of
continuous refluxing. When the liquid was observably extracted completely from
the sample, the condenser and the extractor were disconnected and the solvent
was evaporated to concentrate the lipid. The flask was then dried in the air
oven to constant and reweighed to obtain the weight of the lipid as thus
calculated below:
Ash
(Furnace Method)
1 g of dried sample was weighed in to a
porcelain crucible which was previously preheated and weighed. The crucible was
inserted into a muffle furnace set at 630 0C for 3 hours and allowed
to cool to room temperature and reweighed. Thus % ash was calculated as shown
below:
Crude
Fibre
Crude fibre observed as the insoluble,
combustible organic residue which remained after a sample was treated with
light petroleum ether, diluted acid and alkali (Association of Official
Analytical Chemists, 1990).
About 2 g of sample
was extracted with petroleum ether (W1). Sample was boiled under
reflux for 30 minutes with 200 ml of dilute HCl and filtered. The residue was
thoroughly washed with water until acid-free. The residue was transferred into
a baker and boiled for about 30 minutes with 200 ml of dilute NaOH solution, filtered
and transferred into ignition crucible. The residue was washed 3 times with 20
ml ethanol and 2 times with 10 ml ether. The residue was dried in an oven and cooled
and weighed (W2). The dried residue was transferred into a furnace
and ignited, cooled and weighed (W3). Thus % crude fibre was
calculated as shown below:
RESULTS
Morphological
Study
Heterotis
rotundifolia (Sm.)
Jac.-Fél. (=Dissotis rotundifolia
[Sm.] Triana.) belongs to Melastomataceae. It is a prostrating to decumbent,
perennial dicotyledonous subshrub which roots in the nodes, grows from stem
fragments and from seeds, up to 50 ± 10
cm in height, notably found in damp places. It is densely pubescent with a four
angled reddish stem and simple ovate-suborborbicular to ovate-lanceolate
petiolate leaves which are covered with more or less dense setose-piloses on
both surfaces (extending distinctly to the petiole) 3 nerved, having acute apex
and rounded base with ciliate margins, arranged in opposite phyllotaxy,
measuring up to 4 ± 2.5 cm long and 2 ± 1 cm wide. The inflorescence is a cyme containing
2 to 4 actinomorphic flowers which are solitary and terminal having purple to
pinkish pentamerous corolla, large yellowish and purple stamens, with more or
less campanulate calyx tubes covered with plumose bristles, 5 ± 2 mm in length
with accessory linear lobes between sepals, covered with distinctly reddish and
4 to 5 celled ovary. The berry fruits are globular, measuring up to 1cm in
length (Plates 1 A to 1 D) and (Table 1).
Table
1: Summary of macro morphological characteristics of Heterotis rotundifolia
Characters
Heterotis
rotundifolia
Habit
Sub shrub
Duration
Perennial
Root
Roots from the nodes
Stem Description
Trailing to decumbent pilose stem up to 50 ±
10 cm in height
Leaf type
Simple and petiolate covered with more or
less dense setose-piloses on both surfaces, 3 nerved, having acute apex and
rounded base, arranged in opposite phyllotaxy.
Leaf pigmentation
Green
Leaf venation type
Reticulate
Leaf margins
Ciliate
Phyllotaxy
Oppositely arranged
Leaf outline or shape
Ovate-suborborbicular to ovate-lanceolate
Leaf margin
Entire to slightly serrated
Length of leaf (cm)4 ± 2.5 cm long
Range
1.5 to 6.5 cm in height
Breadth of leaf (cm)
2 ± 1 cm wide
Range1 to 3 cm
Stipules
Paired
Flower description
Pinkish and of radial symmetry, 5 cm across
Inflorescence
Terminal
Fruit description
Simple and globular, covered with
spine-like piloses
Anatomical Study
The stem or intermodal anatomy revealed,
besides the usual peripheral ring arrangement of vascular system, central
phloem elements observed in pith region of plant.Plates 2 A to 2 C.
Plate 2: Stem anatomy; 1 A
arrows revealed central vascularized regions; 1 B ‘circled region’ showed the
enlarged vascularized region in 1 C (with arrows showcasing the specific
areas). Pith is made of Parenchyma.
The
stem section has at its opposite ends, 4 main horn-like protuberances and
multicellular trichomes.
Phytochemical
properties.
The phytochemical
properties and proximate analysis are as shown below, see table 2 and 3.
Table 2:Phytochemical screening result of methanolic leaf extract of Heterotis
Phyochemicals
Heterotis
rotundifolia leaf extract
Saponins
+ve
Tannins
+ve
Phenols
+ve
Terpenoids
+ve
Steroids
-ve
Alkaloids
+ve
Cardiac
Glycosides
+ve
Anthraquinone Glycosides
+ve
Key: - Absent
+Present
Proximate Constituent of Heterotisrotundifolia
Table 3: Quantitative Proximate Constituent
of Heterotis rotundifolia, leaf
extract
Parameters
Results (%)
Moisture
83.80 ± 0.28
Ash
1.93 ± 0.18
Protein
5.25 ± 0.00
Crude Fibre
5.96 ± 0.46
Carbohydrate
0.47 ± 0.00
Lipids
2.60 ± 0.00
DISCUSSION
Macro morphological
features of Heterotis
rotundifolia (Sm.)
Jac.-Fél. (= Dissotis rotundifolia
[Sm.] Triana.) described here conformed to those of Hutchinson
and Dalziel. (1954) and Akonbudu and Agyakwa (1978).The anatomical studies revealed
that the stem has an arranged and open vascular bundle connected by an
interfasicular cambium ring which show that the plant undergoes secondary
growth, and unique central phloem tissues are observed coupled with absence of
intercellular spaces in the stem sections. Piloses were also seen in the
Leaves, stem and petioles. Phytochemical investigation of the methanolic
extract of the leaf of Heterotisrotundifolia revealed the
presence of secondary metabolites such as phenols, saponins, cardiac
glycosides, alkaloids, terpenoids, anthraquinones and flavonoids as seen in
Table 3.1 supports the work of Ezeabara et
al. (2022) with absence of steroids. Moisture content estimated to be very
high in contrast to those of Ezeabara et
al. (2022) and the ash content estimated to be rather of lower value.
CONCLUSION
Heterotis
rotundifolia is
a common weed in Nigeria. It is eaten as vegetable in some ethnic groups, but
also used as antidote fibroid remedy. However, more research investigations on
the plant are needed due to the potential economic uses. Other areas which may
necessitate research findings include: the quantitative aspect of
phytochemistry, DNA barcodes and proximate analysis.
Recommendation
I recommend that further research should be
done on Heterotis rotundifolia using molecular approaches for
appropriate classification of the species. More phytochemical screening should
be carried out to discover the potential pharmacological and biological uses of
the species.
Acknowledgement
We acknowledge the
effort of Pere,
T. who did the initial collection of sample and some Laboratory work.
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Cite this Article: Wahua, C; Ekeke, C; Emenaka,
EW (2024). Macro morphological,
Anatomical, Proximate and Phytochemical Constituents of Heterotis rotundifolia (Sm.) Jac.-Fél., of Melastomataceae.
Greener Journal of Biological Sciences, 14(1): 7-13.
