Abstract: Background and Objective: Amaranthus hybridus and Amaranthus spinosus are widely distributed in Nigeria and are being used as medicinal plants. The comparative morphological, anatomical and phytochemical studies were carried out on 2 species of the genus Amaranthus L. (A. hybridus and A. spinosus) to determine their differences to easy their identification and potential sources of raw materials for pharmaceuticals. Materials and Methods: Standard HPLC method was used for phytochemical screening. For anatomical study, fresh specimens were dehydrated, wax embedded, sectioned, mounted and micro-photographed using Optika B-1000 FL LED fitted with digital camera and morphological study was done by visual observation. Results: The presence of spines on A. spinosus distinguishes it from A. hybridus. Also, the stem of A. spinosus is reddish-brown while that of A. hybridus is light green. The number of vascular bundles in the midrib, petiole, stem and root were different and could be used to differentiate them. Data obtained from the quantitative phytochemical analysis showed that their concentrations varied among the 2 species. Amaranthus hybridus had 72.56 and 61.79% vitamin A in the root and leaves, respectively while compared to A. spinosus. Leaves of A. hybridus had higher total flavonoids concentration of 56.46% while A. spinosus had the highest phenolics of 19.27 g/100 g and 18.63 g/100 g in the leaves and roots, respectively. Also, the roots of A. spinosus had the highest concentration of alkaloids, glycosides and phenolics. These explain why they are used in ethno-botany in Nigeria. Conclusion: Based on the phytochemical constituents, both plants could be valuable sources of dietary vitamins and potential sources of phytochemicals if properly exploited.
INTRODUCTION
From the origin, plants have provided a variety of resources that contribute to the fundamental needs of food, clothing and shelter to man. Among plants of economic importance, medicinal and aromatic plants have played vital roles in alleviating human suffering. Plants are utilized as therapeutic agents since time immemorial in both organized and unorganized (folk, tribal, native)1,2. The genus Amaranthus L. belongs to the family Amaranthaceae in the order Caryophyllales and is native to tropical America and Africa. Members of this family are mainly herbs, but also include vines, shrubs and trees and comprised approximately 800 species represented by more than 60 genera and is broadly divided into 2 sub-families Amaranthoideae and Gomphrenoideae3. The cultivated grain Amaranths include A. caudatus, A. cruentus and A. hypochondriacus and their parental wild species are thought to be A. hybridus L., A. quitensis and A. powellii S. Other Amaranthus species like A. dubious L., A. hybridus and A. ticolor L. are consumed as leafy vegetables. Meanwhile, A. retro exus L. (redroot pigweed), A. albus (tumbleweed), A. palmeri S. Wats. (Palmer amaranth) and A. spinosus (spiny amaranthus) represent weed species4.
According to Akubugwo et al.5 leaves extract of A. spinosus produces about 73% inhibition of prostaglandin biosynthesis in vitro. It has been discovered that the leaves and roots boiled together are also used traditionally as a diuretic, anti-diabetic, antipyretic, anti-snake venom, antileprotic and antigonorrheal6. The plant has a long history of usage in traditional medicine against various ailments around the World7. Traditionally boiled leaves and roots of A. spinosus are given to children as a laxative. Leaves and stems are collected, cooked, steamed or fried and consumed. It is used as forage for livestock. The ash is a tenderizer in cooking vegetables and pigeon peas. The root is effective to treat gonorrhea, applied externally to treat eczema, burns wounds, boils, earache, sores, ophthalmia and convulsions6. Amaranthus spinosus is a potential agent for accumulation and translocation of heavy metals8. In Nigeria, A. hybridus leaves combined with condiments are used to prepare soup9. Leaves are also eaten as green vegetables. Amaranthus hybridus grown on dumpsites possessed a higher concentration of heavy metals10.
Amaranthus hyridus also known as ‘green’ in Nigeria, is mostly cultivated and used as leafy vegetables to prepare various meals such as; yam and soup in southern Nigeria9. Other species of Amaranthus such as; A. spinosus is also eaten in Nigeria and it is also used for other ethnobotanical purposes by the local communities. Despite the economic importance of these species, only a few or little information is available about the potentials of these species and the taxonomy of some Amaranthus still problematic, although some methods have been employed to classify them11-16. Also, proper identification of these species is important. This work aimed at conducting morpho-anatomical studies of two Amaranthus species (A. hybridus and A. spinosus) widely distributed in Nigeria for easy identification and to analyze and compare the phytochemical and vitamin contents of both species as potential raw materials for pharmaceutical industries.
MATERIALS AND METHODS
Study area: The study was carried out at Department of Plant Science and Biotechnology, University of Port Harcourt, Nigeria from April, 2018-August, 2019.
Sample collection: Materials used for this research work were sourced from in and around the University of Port Harcourt. Fresh plant parts from A. spinosus were collected from the botanical garden of the Faculty of Agriculture, University of Port Harcourt while A. hybridus was collected from Igbogo road in Choba community, Obio-Akpor Local Government, Rivers State. The plant specimens were properly identified, confirmed and deposited in the Department of Plant Science and Biotechnology Herbarium, University of Port Harcourt with herbarium number A. spinosus (UPH/V/1354) and A. hybridus (UPH/V/1403). The mature vegetative parts including the stem root and leaves of both species were examined for morphological characteristics, anatomical studies and phytochemicals (flavonoids, alkaloids, glycoside and phenolic) properties.
Morphology: Morphological studies were carried out by visual observation of the vegetative parts, stem and roots of A. spinosus and A. hybridus growing in the wild. The morphological observations were described.
Epidermal investigation: Fresh samples of the mature leaves were obtained. Adaxial and abaxial epidermal peels were peeled and were fixed by passing through alcohol series (30, 50, 70 and 100%) solution for 2 h each after which they were washed with distilled water. The strips were first stained with safranin O and counter stained with Alcian blue, it was mounted on a slide and a drop of glycerine was put on it then it was covered with a coverslip and viewed under the microscope using ×40. Photomicrographs were taken with the aid of a camera.
Anatomical investigation: The fresh parts (stem, petiole, midrib and root) were fixed in Formalin Acetic Acid (FAA) for 24 h after which they passed through alcohol series (30,50, 70 and 100%) solution and stored in 100% ethanol until use. These specimens were embedded in paraffin wax and sectioned. Thin sections of the stem, petiole, midrib and root were obtained by free-hand sectioning using a new razor blade. These selected thin sections were de-waxed, stained with safranin O and counter stained with Alcian blue, rinsed with distilled water and mounted on a clean slide with a drop of glycerine then covered with a coverslip and viewed under microscope. Photographs of selected good sections were taken using camera.
Phytochemical investigation
Alkaloids determination: Five grams of the sample was weighed into a flask. Hundred milliliters of 12% alcohol was added, shake and filtered. Thereafter, washed with 20 mL of industrial alcohol. The extracted residue was washed into flasks with 50 mL of ammonia-water (i.e., use ultrapure water), heated in boiling water for 20 min and cooled. Diastase (0.1 g+water) was added and the temperature maintained at 50-55°C for 2 h. At the expiration of 2 h, the sample was allowed to cool and made up to 250 mL with ultrapure water, swirled and filtered. Filtrate (200 mL) was mixed with 20 mL hydrochloric acid (sp.g. 1.125), heated in boiling water for 3 h, cooled, neutralize with sodium hydroxide solution, made up to 250 mL, shook, centrifuged and decanted. The supernatant was used for alkaloid determination using water 616/626 HPLC with the nitrogen gas flow rate of 40 mL min–1, detector temperature of 170°C, injection port temperature of 190°C and column temperature17 of 125°C.
Phenolics determination: Sample (2 g) was weighed into a set of test tubes. Three milliliters of 70% acetone and water were added to the test tube, placed in an ultrasonic water bath at 10°C for 5 min. The sample was stirred occasionally with a glass rod and filtered through a 50-60 μ Gooch crucible into a 50 mL Erlenmeyer flask. Steps 2 and 3 were repeated 3 times and the test tubes with the final rinsed with a 3 mL portion of 70% acetone in water and emptied into the test tubes. About 2 mL of 0.1 M acetate and 15 mL of 0.1 M triethylamine (TEA) reagent were added into the filtrate. Thereafter, the contents of the test tube were transferred into a volumetric flask, closed with a rubber stopper, swirled, shook for 20 min and allowed to settle for 4 h. The supernatants were collected for analysis using HPLC (Water 616/626) with the argon gas flow rate of 60 mL min–1, detector temperature of 120°C, injection port temperature of 155°C and column temperature18 of 117°C.
Glycosides determination: Half (0.5 g) of the sample was weighed into a set of digestive tubes. About 5 mL of 0.1 M HCl was added and warm gently for 15 min at 105°C and transferred into a 50 mL volumetric flask. Steps 1 and 2 above were repeated twice, rinsed with 2-3 additional aliquots, allowed for complete filtration and the filtrate made up to 100 mL mark with the extractant solution and mixed thoroughly. Extract (5 mL) solution from the 100 mL flask was purified by running it through a 2 cm layer (the resin is packed on a macro pipette tip) cation exchange resin (CEC). The glycoside compounds were eluded with 10 mL of absolute ethanol, the ethanol washed from the column with ultrapure water (10 mL), supernatant transferred to a sample vial and ran on HPLC (Water 616/626) with the nitrogen gas flow rate of 38 mL min–1, detector temperature of 167°C, injection port temperature of 183°C and column temperature18 of 130°C.
Flavonoids determination: Sample (1.5 g) was weighed into a set of extraction tube. Boiled (20 mL) ultra-pure water dispensed into each extraction tubes, allowed to stand for 1½ h, a vertex for 5 min and transferred to a set of centrifuge tubes, shook for 15 min and centrifuged for 5 min at 3000 rpm. Thereafter, the supernatant was transferred to a set of vials and determined on water 616/626 HPLC with the nitrogen gas flow rate of 60 mL min–1, detector temperature of 147°C, injection port temperature of 166°C and column temperature19 of 115°C.
Determination of vitamins: The extraction and determination of vitamin A, B2, B6, B12 and E were performed according to the method described by Ezeonu and Ejikeme20 and Gaafar et al.21 while vitamin C was determined using the titrimetric method22.
Vitamin A (extraction and determination): Plant sample (0.5 g) was weighed into a conical flask, 20 mL of 0.2 N HCl dispensed and allowed to stand for 1.5 h. The solution was cooled and the pH adjusted to pH 6, using NaOH. Also, 1 N HCl added to lower the pH to 4.5. The solution was made up to 50 mL, shook and centrifuged for 10 min at 3000 rpm. The supernatant was separated, 1 mL of acetic acid (CH3COOH) added and mixed properly. Also, 0.5 mL of 3% H2O2 added and mixed well. Finally, 20 mg of sodium hydrogen sulphate was added and then shook properly. The extract was run on HPLC (Waters 616/626). Water 616/626 accessories used had Merck Lichrospher WOCH-18/2 (5 μm) at 40°C column (stationary phase) and mobile phase (Solvent ‘A’ was 30 mM sodium acetate, pH 6.5 containing 5% dimethylformamide and solvent ‘B’ was acetonitrile) with fluorescence detector, range of working standard (0, 2, 4, 6 and 8 ppm) and determination was carried out at a wavelength of 328 nm.
Vitamin B1, B2, B3 , B6, B9, B12 (extraction and determination): Plant sample (2.5 g) was weighed into a set of digestion tubes and an extraction solution (Ultra-pure water: HCl: 0.1N H2SO4 in the ratio 5:2:3) dispensed. The tube was warmed at the temperature of 40°C for 2 h, allowed to cool to room temperature and transferred to a set of plastic centrifuged tubes. The latter was shaken for 10 min and centrifuged at 3000 rpm. The supernatant was set in auto-analyser tubes and ran on HPLC. Water 616/626 accessories used had Merck Lichrospher WOCH-18/2 (5 μm) at 40°C column (Stationary phase) and mobile phase (Solvent ‘A’ was 30 mM sodium acetate, pH 6.5 containing 5% dimethylformamide and solvent ‘B’ was acetonitrile) with fluorescence detector, range of working standard (0, 0.2, 0.4, 0.6 and 0.8 ppm) and determination was carried out at wavelength range of 240- 465 nm.
Vitamin E (extraction and determination): Plant sample (0.5 g) each was weighed into a set of digestion tubes, 20 mL of diluted Hydrochloric Acid (HCl) added and shook vigorously for 2 h. The extract was further treated with phosphatase to liberate free vitamin E into the solution. The extract was purified by passing through base exchange silicate alkaline column to remove interfering compounds. Thereafter, the extract was stored in a set of vials for analysis using HPLC. Water 616/626 accessories used had Merck Lichrospher WOCH-18/2 (5 μm) at 40°C column (stationary phase) and mobile phase (Solvent ‘A’ was 30 mM sodium acetate, pH 6.5 containing 5% dimethylformamide and solvent ‘B’ was acetonitrile) with fluorescence detector, range of working standard (0, 0.2, 0.4, 0.6 and 0.8 ppm) and determination was carried out at a wavelength of 356 nm.
RESULTS
Macro-morphology: The results showed that both species have similar leaf shape, leaf arrangement, leaf type, leaf venation, root colour and hairy leaves. However, they differ in the presence of spines in A. spinosus and absent in A. hybridus and in stem colour which is reddish-brown in A. spinosus and light green in A. hybridus (Table 1).
Micro-morphological (anatomy) characteristics
Epidermal studies: The results of the epidermal investigation in Table 2 showed that both species have similar epidermal wall and cell shape in both adaxial and abaxial surfaces and amphistomatic, however, they differ in their stomata type. The adaxial surface of A. hybridus has tetracyclic, anisocytic and contiguous stomata while A. spinosus have stomata of anisocytic and isotricytic type. The abaxial surface is the same in both A. hybridus and A. spinosus with regards to stomata types as shown in Fig 1.
Midrib and petiole anatomy: The midrib cuticle in A. hybridus is V-shaped while the cuticle outline of A. spinosus is relatively straight or flat (Table 3, Fig. 2(a-d). The result of the petiole as seen in Table 3 and Fig. 2 showed that both species have similar vascular bundle types and shape of parenchyma. The adaxial cuticle region of A. hybridus has pronounced collenchymatous cells (Fig. 2d) while that of A. spinosus is not visible (Fig. 2c). The petiolar protuberances in A. spinosus have thick layers of collenchymatous cells (Fig. 2c) while this feature is minimized or absent in A. hybridus.
| Table 1: | Results for macro-morphological characteristics of A. spinosus and A. hybridus |
| Table 2: | Summary of the epidermal attributes of A. hybridus and A. spinosus |
| Fig. 1(a-d): | Leaf epidermal characteristics of Amarathus, (a) Abaxial surface of A. hybridus, (b) Adaxial surface of A. hybridus, (c) Abaxial surface of A. spinosus and (d) Leaf adaxial surface of A. spinosus |
| Table 3: | Summary of the anatomy of petiole and midrib of A. hybridus and A. spinosus |
The leaf trace in both species comprised of bundle sheaths. Also, the leaf lamina of both species has bundle sheath (kranz) anatomy. Druse (calcium oxalate) crystals were conspicuous in A. spinosus than A. hybridus.
Stem and root anatomy: The stem and root of both species studied have similar anatomical features (Table 4) but the stem of A. spinosus has narrow cambial ring while the cambial ring is pronounced in A. hybridus (Fig. 3). Also, the number of vascular bundle in the stem of A. spinosus was 52 while that of A. hybridus is less than 52. Furthermore, the root of A. spinosus has 16 vascular bundles while that of A. hybridus has 18 vascular bundles.
Vitamins: The results of vitamins in different parts of the plants studied are shown in Table 5. The vitamins (A, B1, B2, B3, B6, B12, C and E) occurred in the leaves and roots of both A. hybridus and A. spinosus; however, the concentration varied. The concentration of vitamin A varied from 27.46% in A. spinosus root to 72.56% in A. hybridus root and from 32.70% in A. spinosus leaves to 61.80% in A. hybridus leaves.
| Fig. 2(a-d): | Midrib and petiole anatomy, (a) Midrib of A. hybridus, (b) Midrib A. spinosus, (c) Petiole of A. spinosus and (d) Petiole of A. hybridus |
| Vb: Vascular bundle, dr: Druses, tr: Trichome, Lt: Leaf traces | |
| Table 4: | Summary of root and stem anatomy |
| Table 5: | Quantitative vitamin composition in A. hybridus and A. spinosus |
These concentrations of vitamin A are relatively high. The percentage vitamins B1, B2, B3, B6, B12, C and E in the species are fairly similar.
Phytochemical results: The data obtained for the phytochemical investigation showed that they are similar in the composition, however, they differ in their concentrations. The total alkaloids, flavonoids, phenolics and glycosides are presented in Table 6. The total alkaloids contents varied form 29.69 g/100 g in the root of A. hybridus to 50.89 g/100 g in the root of A. spinosus.
| Fig. 3(a-f): | Stem and root anatomy, (a) Stem of A. hybridus, (b-c) Stem of A. spinosus, (d) Root of A. hybridus and (e-f) Root of A. spinosus |
| Table 6: | Summary of phytochemicals in A. hybridus and A. spinosus |
It is worthy to note that there is relatively no variation in the concentration of the alkaloids in the leaves of the species. Flavonoids varied from 16.70 g/100 g in A. spinosus root to 23.08 g/100 g in A. hybridus. Also, the concentration of phenolics ranged from 12.29 g/100 g in A. hybridus to 19.27 g/100g in A. spinosus (Table 6).
Anti-nutrients: The anti-nutrient contents of the plant species studied is presented in Table 6. The oxalate maximum (2.935 ppm) and minimum (2.009 ppm) contents were recorded in the root and leaves of A. hybridus, respectively. The concentrations of the tannin, saponin and Trypsin-inhibitor followed the same sequence. Generally, the concentrations of the anti-nutrients in the leaves are lower than the concentrations in the roots.
DISCUSSION
Comparative studies carried out on 2 species of the genus Amaranthus (A. hybridus and A. spinosus) for their morphological, phytochemical and anatomical characteristics. The macro-morphology evaluation of these species showed that they are monoecious erect herbs which grow up to 1-2 m. These species showed major similarities in the leaf arrangement, root colour, leaf type and leaf shape. They both have glabrous on leaves. One major distinctive difference in the macro-morphology of these species is the presence of spines. Amaranthus spinosus possess spines on the stem at the base of the petiole while A. hybridus does not. Thus the results obtained correspond with the morphological studies on different Amaranthus species23,24.
Most of the members of Amaranthaceae are amphistomatic25-31 and many stomata types have been reported among the members of this family such as; Anomocytic and diacytic in A. braslilina32, anisocytic, paracytic and anomocytic in A. spinosus and A. hybridus26. The epidermal characteristic of A. hybridus and A. spinosus studied showed that both species are amphistomatic with similar epidermal cell shape and wall pattern. However, adaxial surface of A. hybridus had anisocytic, tetracytic and contiguous stomata while A. spinosus had anisocytic and isotricytic. The abaxial surfaces of both species are similar having anisocytic and tetracytic stomata with irregular shape and undulating walls. This study agreed with the stomata types26-31, observed on the leaves of the members of this family but differed in his report on the anticlinal cell wall pattern26.
Amaranthus species are used in traditional medicine due to their phytochemical constituent properties33. Pharmacological analysis has shown that most Amaranthus species have anti-cancer, anti-bacterial, anti-fungal, antioxidant and, anti-inflammatory properties33, analgesic and antipyretic activity34, antioxidant activity35, immunological effects36, antidepressant activity37, antifertility activity38, hepatoprotective activity39, antiulcer activity40-43, haematological activity44 and diuretic activity45,46.
Amaranthus spinosusis an extremely interesting crop because its vegetable and seeds are known to be highly nutritious and, therefore, consumed by human as well as animals, as nearly all essential nutrients for humans are available in plants47-49 and high in vitamins50. Other species in this genus are edible and some are cultivated for their leaves51. In Nepal, Amaranth seeds are eaten as gruel called "sattoo" or milled into a flour to make chappatis52 and Amaranthus leaves contain 3 times more vitamin C, calcium and niacin than spinach. In Ethiopia, the root of A. caudatus and A. sylvestris are used as a laxative and the seed for expelling tapeworms and for treating eye diseases, amoebic dysentery and breast complaints53. Research showed that A. caudatus plants grown on dump sites contain a higher concentration of heavy metals54. Amaranthus tricolor has a high cadmium-accumulating ability55. Amaranthus viridis can concentrate heavy metals especially Pb and Cd in its tissues56. This suggested that Amaranthus species can serve as phyto-accumulators of heavy metals and can be used for the purpose of phytoremediation56.
Phenolic compounds have anti-cancer, anti-bacterial and anti-fungal properties. Also, it has antioxidant, anti-inflammatory, antiallergic, hepatoprotective, antithrombotic, antiviral, anticarcinogenic and vasodilatory actions57,58. Flavonoids (phenolic compounds) have been reported to have antioxidant, protect cell from degradation, stress, act as signaling molecules, phytoalexins, detoxifying agents, reduce toxic effects and stimulants, triggers the production of natural enzymes that fight disease, hence reduce the risk of certain cancers, heart disease and age-related degenerative diseases and play chemopreventive role in cancer59,60. Catechin a naturally occurring phenolic compound has anti-oxidant activity and has the potential to reduce cardiovascular disease, stroke, obesity and cancer.
Although high intake of flavonols is associated with reduced risk of cancer and stroke, promote bone health, prevent osteoporosis and have anti-inflammatory properties60,61. Also, flavonols promote a healthy brain as they possess neuroprotective properties. Hence, the consumption of food rich in flavonols is associated with long-term health benefits58.
Alkaloids are used in drug production and has antifungal and bactericidal properties62 can inactivate enzymes, block ion channels, interfere with neurotransmission and cause loss of electrical coordination (ataxia) in affected organisms63 and have anticancer, antibacterial, antiviral and antifungal properties64.
Cardiac glycosides induce strong specific effects on the myocardium and enhance the strength of cardiac contractions65,66. Tannins have anti-malaria, antimicrobial, antifungal, allelopathic activities, dyes and spices67 and astringents68. Saponins are immunostimulant and possess phytoanticipins or phytoprotectants properties69.
Phytochemical studies revealed that the plant A. spinosus has several active constituents like alkaloids, flavonoids, glycosides, phenolic acids, terpenoids, tannins and saponins. It also shows that kaempferol glycosides, amaranthoside, a lignan glycoside, etc. are also contained in the stem bark of A. spinosus70. Barku et al.11 also stated that the roots contain α-spinasterol octacosanoate and saponin and have antibacterial activity71.
From present study, A. spinosus and A. hybridus have similar phytochemical constituents though the concentrations varied. According to Walton et al.72 and Wang73, A. hybridus and A. spinosus contain similar phytochemicals as reported in this current work i.e., vitamins (vitamin A, B1, B2, B3, B6, B12, C and E) and secondary metabolites (alkaloids, flavonoids, glycosides and phenolics). These species showed major difference amongst them. Amaranthus hybridus had a higher concentration of vitamin A compared to A. Spinosus in both leaves and root. Also, the leaves A. hybridus had a higher concentration of flavonoids than A. spinosus while phenolics are higher in A. spinosus than A. hybridus. Also in the roots, A. spinosus had a higher concentration of alkaloids, glycosides and phenolics than A. hybridus while flavonoids are higher in A. hybridus than A. spinosus. Presence of these phytochemicals demonstrates while they are used as food47-49 and different pharmacological purposes52,73. Therefore, these species can be used for antimicrobial, antioxidant, antidiuretic, etc. activities due to the presence of these phytochemical properties. Amaranthus hybridus leaves contain appreciable values of β-carotene, thiamine, riboflavin, niacin, pyridoxine and ascorbic acid but low value of β-tocopherol and as such are good sources of these vitamins74. This could be the reason why A. hybridus is used more domestically compared to A. spinosus. Therefore, both plants could be a valuable source of dietary vitamins in human nutrition.
CONCLUSION
Based on this study, it is evident that A. hybridus and A. spinosus have similar morphological, anatomical and phytochemical characters. The number of vascular bundles in the midrib, petiole, stem and root are different and could be used to differentiate them. The data obtained from the quantitative phytochemical analysis showed that their concentrations varied among the 2 species. They are potentials sources of phytochemicals if properly exploited.
SIGNIFICANCE STATEMENT
This study discovered the anatomical difference among the two species for easy identification and high vitamin contents and other phytochemicals in the plant species that can be beneficial. These made A. hybridus and A. spinosus potential sources of raw materials for pharmaceutical industries.