By Wahua, C; Ekeke, C; Emenaka, EW (2024). Greener Journal of Biological Sciences, 14(1): 7-13.
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Table of Contents
Vol. 14(1), pp. 7-13, 2024
ISSN: 2276-7762
Copyright ©2024, Creative Commons Attribution 4.0 International.
https://gjournals.org/GJBS
1, 2, 3 Department of Plant Science and Biotechnology, University of Port Harcourt, Choba, P.M.B. 5323, Rivers State, Nigeria.
ABSTRACT
Article No.: 012824018
Type: Research
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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.
Published: 26/02/2024
Chika Wahua
E-mail: chika.wahua@uniport.edu.ng, ekeke.uche@uniport.edu.ng
Tel.: + (234)8064043448
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.
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 of the 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 5 minutes 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 (Shoppe 1964).
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:
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
Perennial
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.
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
+ve
Tannins
Key: – Absent
+ Present
Proximate Constituent of Heterotis rotundifolia
Table 3: Quantitative Proximate Constituent of Heterotis rotundifolia, leaf extract
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 Heterotis rotundifolia 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.
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.
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.
We acknowledge the effort of Pere, T. who did the initial collection of sample and some Laboratory work.
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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.
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