|Year : 2017 | Volume
| Issue : 1 | Page : 36-44
Phytochemical analysis, toxicity profile, and hemomodulatory properties of Annona muricata (Soursop)
Kingsley C Agu PhD 1, Ngozi P Okolie2, Ikechi Eze3, John C Anionye1, Abiodun Falodun4
1 Department of Medical Biochemistry, University of Benin, Benin, Nigeria
2 Department of Biochemistry, University of Benin, Benin, Nigeria
3 Department of Anatomy, University of Benin, Benin, Nigeria
4 Department of Pharmaceutical Chemistry, University of Benin, Benin, Nigeria
|Date of Submission||31-Aug-2016|
|Date of Acceptance||17-Nov-2016|
|Date of Web Publication||18-May-2017|
Kingsley C Agu
Department of Medical Biochemistry, College of Medical Sciences, University of Benin, 300001, Benin City
Source of Support: None, Conflict of Interest: None
Background A wide array of ethnomedicinal values have been attributed to the different parts of Annona muricata, and indigenous communities in Nigeria, Africa, and South America extensively use this plant to augment conventional drugs.
Aim The beneficial effects of A. muricata on the hematological profile have also been widely reported and this research sought to validate these claims.
Design Adult albino Wistar rats were used in this study. Methanolic extracts of the various parts of the plant were used, with which we determined the 50% lethal dose (LD50) and acute toxicity status of the plant before hematological studies for a duration of 28 days (subchronic studies).
Materials and methods During the subchronic studies, 100, 200, 400, 600, and 800 mg/kg of the fruit, leaf, stem-bark, and root-bark methanolic extracts were administered to groups 2–6, respectively, whereas group received 2 ml of distilled water and served as control. At the end of the administration period, the rats were killed and blood samples collected for onward hematological studies. Phytochemicals were quantified using standard procedures.
Results The obtained results showed that both the leaf and fruit extracts had LD50 of 1918.33 mg/kg, whereas the stem-bark and root-bark both had LD50 above 5000 mg/kg. Subchronic observations were also made, including increased heart rates and diarrhea. The fruit and stem-bark extracts recorded a dose-dependent increase in CD4+ cells, especially from the 200 mg/kg dose. Also, the fruit, leaf, and root-bark extracts showed a dose-dependent increase in white blood cells and lymphocytes. The extracts of the various parts of the plant apart from the stem-bark recorded marked increase in platelet levels. The various extracts of the plant parts recorded striking increase in red blood cells, hemoglobin concentration, and packed cell volume. These observations could be linked to the remarkable quantity of alkaloids, flavonoids, and phenols present in the leaf and fruit fractions.
Conclusion Thus, the obtained data suggest and validate the reported hemomodulatory and wound-healing properties of A. muricata, especially the fruit and leaf.
Keywords: Annona muricata, hematological parameters, hemomodulation, phytochemicals, wound healing
|How to cite this article:|
Agu KC, Okolie NP, Eze I, Anionye JC, Falodun A. Phytochemical analysis, toxicity profile, and hemomodulatory properties of Annona muricata (Soursop). Egypt J Haematol 2017;42:36-44
|How to cite this URL:|
Agu KC, Okolie NP, Eze I, Anionye JC, Falodun A. Phytochemical analysis, toxicity profile, and hemomodulatory properties of Annona muricata (Soursop). Egypt J Haematol [serial online] 2017 [cited 2017 Dec 12];42:36-44. Available from: http://www.ehj.eg.net/text.asp?2017/42/1/36/206431
| Introduction|| |
According to Moghadamtousi et al. , the fruit of Annona muricata is extensively used to prepare syrups, candies, beverages, ice creams, and shakes locally. A wide array of ethnomedicinal values have been attributed to the different parts of A. muricata, and indigenous communities in Africa and South America extensively use this plant as medicinal replacement for conventional drugs. Numerous scientific investigations have substantiated these activities, including anticancer, anticonvulsant, antiarthritic, antiparasitic, antimalarial, hepatoprotective, and antidiabetic activities. Phytochemical studies reveal that alkaloids, flavonoids, carbohydrates, cardiac glycosides, saponins, tannins, phytosterols, terpenoids, proteins, essential oils, and annonaceous acetogenins are the major constituents of A. muricata. Over 120 annonaceous acetogenins have been isolated from the leaves, stem-bark, seeds, root-bark, and fruits of A. muricata. Syahida et al.  and Usunomena  have reported the beneficial effects of A. muricata on the hematological status of rats, especially on the synthetic ability of some of the important plasma proteins, including albumin and platelets. However, in this study we investigated the influence of the methanolic leaf extract of A. muricata on the hematological status of treated Wistar rats.
| Materials and methods|| |
The rats used were adult albino male Wistar rats weighing between 100 and 150 g. The rats were supplied by Mr Silvanus Innih of the Anatomy Department, University of Benin, Benin City, housed in the Department of Biochemistry Animal House, and acclimatized for 1 week before the administration procedure begun. They were fed standard rat chow and water ad libitum (livestock feed). The research ethics committee guideline principles on the handling of animals of the College of Medicine, University of Benin (CMR/REC/2014/57), was adopted and strictly adhered to.
Preparation of A. muricata crude extract stock solution
A large quantity of fresh parts of the plant were collected from trees from household gardens in Benin City and around the University of Benin, Edo state, Nigeria. The plant was identified by Dr Bamidele of the Department of Plant Biology and Biotechnology, University of Benin, and authenticated by Professor Idu of the same department. A voucher specimen number, UBHa 0205, was deposited at the Herbarium of Department of Plant Biology and Biotechnology, University of Benin. The properly washed plant samples were pulverized after drying at room temperature (about 25°C) for 4 weeks. The pulverized plant samples were macerated in methanol using jar bottles for 48 h after which they were subjected to filtration using cheese cloth. The obtained extracts were then concentrated in vacuo using a rotary evaporator to obtain viscous gels that were air-dried to gel-like solids. The gel-like crude methanolic extracts obtained from the various parts of the plant were reconstituted to obtain a stock solution using distilled–deionized water as solvent. The reconstituted crude extract was stored in small-capped plastic containers in a refrigerator at −4°C until use.
Administration of extracts
The extracts were administered with the aid of a gavage acting as an orogastric tube. Utmost care was taken not to inflict oral or esophageal injuries on the rats.
Determination of 50% lethal dose and acute toxicity
50% lethal dose (LD50) and acute toxicity were determined using modified Lorke’s  method with 180 male and 35 female rats of Wistar strain after 1 week of acclimatization to standard animal cage conditions followed by an overnight fast. In phase 1, male rats were randomly placed into three groups of five rats each and administered methanolic extracts of A. muricata (fruit, leaf, stem-bark, and root-bark) orally at 100, 400, and 1000 mg/kg body weight (b.w.), respectively. Female rats in separate groups as above also received oral administration of all the extracts at the same doses as that of phase 1 male rats. All rats were observed for 24 h for signs of toxicity (morbidity and mortality). In the second phase, another set of five male rats in three groups of five per group were administered orally methanolic extracts of the plant parts at doses of 1200, 1600, 2900, and 5000 mg/kg b.w. in a similar manner as above. Again the rats were observed for 24 h for any sign of toxicity. The geometric mean lethal dose (LD50) was determined after the third phase for fruit and leaf extracts. The fruit and leaf extracts were administered to four sets of five rats each at 1600, 2300, 2500, and 2900 mg/kg b.w., respectively.
Subchronic toxicity assessment
During the period of usage, extracts were administered to the rats on the basis of the calculated doses per weight of rat (i.e. equivalent volume). The dose calculations were performed weekly per weight of rats, which was recorded weekly on days 0, 7, 14, 21, and 28. Group 1 served as the control group and was administered 2 ml of distilled water.
Observations in subchronic assessment (clinical signs and mortality)
The rats were observed for signs of weakness, increased or decreased appetite, weight loss, and other physiological changes including mortality. Clinical signs to be assessed before dosing, immediately, and 4 h after dosing included the level of sedation, restlessness, change in the nature of stool, urine and eye color, excretion of worms, diarrhea, hematuria, uncoordinated muscle movements, etc. The animals were observed for toxic symptoms such as weakness or aggressiveness, food refusal, loss of weight, diarrhea, discharge from the eyes and ears, noisy breathing, and mortality ,.
Experimental protocol for subchronic toxicity studies
Various methanolic extracts of the plant parts were administered at increasing doses starting from 100 mg/kg (group 2), 200 mg/kg (group 3), 400 mg/kg (group 4), 600 mg/kg (group 5), to 800 mg/kg (group 6). Group 1 rats were given 2 ml of distilled water and served as controls.
Blood sample collection for subchronic assessment
At the end of the experimental period of 28 days, the rats were killed. Thereafter, whole blood (5 ml) was collected into EDTA tubes for hematological analysis. The EDTA whole blood was immediately taken to the University of Benin Teaching Hospital Hematological Department for determining the full hematological profile using the automated blood cell count analyzer.
Preparation of fractions for quantification of phytochemicals
The crude methanolic extracts of various parts of the plant were subjected to solvent–solvent partition chromatography (SSPC) to obtain fractions using petroleum–ether, trichloromethane, ethyl acetate, methanol, and methanol–water (90 : 10), in the order of increasing polarity. The fractions were subsequently subjected to in-vacuo drying using the rotary evaporator.
Quantification of phytochemical
The presence of alkaloid was confirmed by the method of Trease and Evans , total flavonoids by the method of Harborne , and total phenol by the method of Kaur and Kapoor . However, alkaloidal content was quantified using the bromocresol green method described by Zaree et al. , total phenolic content by the method described by Kaur and Kapoor , and total flavonoid content by the method described by Chang et al. .
Values are presented as mean±SEM for five determinations. Data were analyzed with the statistical package for the social sciences (SPSS, version 21.0; IBM Corp., 2012, version 21.0, Armonk, NY, USA), using the paired-sample Student’s t-test at a confidence interval of 95% (P=0.05).
| Results|| |
The modified Lorke’s  method was used to ascertain the lethality of the extracts. There was no recorded lethality above 5000 mg/kg for the stem-bark and root-bark methanolic extracts. However, leaf and fruit methanolic extracts gave similar outcomes as described in [Table 1].
|Table 1 50% lethal dose investigation of Annona muricata fruit and leaf methanolic extracts|
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CD4+ cells and platelets significantly increased, whereas WBCs rose steadily; i.e. as the concentration of the extract administered increased, the levels of CD4+, platelets, and WBCs increased concomitantly ([Table 2]).
|Table 2 CD4+ cells, platelets, white blood cells, and differentials of rats administered methanolic fruit extract of Annona muricata|
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Red blood cells (RBCs) increased in group 4 compared with that in the control group (group 1), but decreased progressively through group 6. This is an indication that as the extract concentration increased (400 mg/kg), the RBCs increased but decreased with higher concentrations. Hemoglobin concentration (HC), mean cell volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) followed a similar trend ([Table 3]).
|Table 3 Red blood cell differential of rats administered fruit extract of Annona muricata|
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CD4+ did not show any significant increase. With the administration of higher concentrations − that is 600 and 800 mg/kg − CD4+ level dropped significantly. WBCs recorded the highest concentration at the highest administered concentration. Platelet level increased significantly compared with the control level ([Table 4]).
|Table 4 CD4+ cells, platelets, white blood cells, and differentials of rats administered methanolic leaf extract of Annona muricata|
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RBC and HC did not vary significantly. However, the highest extract concentration (800 mg/kg) caused a significant increase in packed cell volume (PCV), MCV, and MCHC levels ([Table 5]).
|Table 5 Red blood cell differential of rats administered leaf extract of Annona muricata|
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CD4+ level was increased significantly at 200 and 800 mg/kg, but WBCs recorded contrary levels at same concentrations. Lymphocytes did not show any significant change as concentration was increased. However, plasma platelet level increased at the extract dose of 400 mg/kg but decreased subsequently ([Table 6]).
|Table 6 CD4+ cells, platelets, white blood cells, and differentials of rats administered methanolic stem-bark extract of Annona muricata|
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RBC and HC did not show any significant change. The other RBC differentials did not show significant variations as well ([Table 7]).
|Table 7 Red blood cell differential of rats administered stem-bark extract of Annona muricata|
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CD4+ increased with the concentration of the administered extract, but declined at 600 mg/kg. WBCs recorded the highest level at the highest extract dose. Plasma lymphocyte showed significant increase at 400 mg/kg, which progressed as the concentration increased. Platelet level increased with concentration ([Table 8]).
|Table 8 CD4+ cells, platelets, white blood cells, and differentials of rats administered methanolic root-bark extract of Annona muricata|
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The RBC level, HC, PCV MCV, MCH, and MCHC did not show significant variations ([Table 9]).
|Table 9 Red blood cell differential of rats administered root-bark extract of Annona muricata|
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[Figure 1] shows quantities of alkaloids, phenols and flavonoids present in the various fractions of the parts of Annona muricata.
|Figure 1 (a) Histogram for alkaloid content of various solvent partition chromatography (SSPC) fractions of Annona muricata fruit, leaf, stem-bark, and root-bark. (b) Histogram for total phenol content of various SSPC fractions of Annona muricata fruit, leaf, stem-bark, and root-bark. (c) Histogram for total flavonoid content of various SSPC fractions of Annona muricata.|
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Acute toxicity studies
The following observations were made (especially for the leaf and fruit extracts above 2900 mg/kg).
- The rats were curled inwardly.
- They became less active with disorganized movements.
- They developed increased heart rate (rapid breathing).
- There was convulsion before death.
- Anal observation of the dead rats showed passing out of watery stool, probably the occurrence of diarrhea, with a characteristic odor for the rats administered leaf extract.
| Discussion|| |
Qualitative and quantitative phytochemical analyses
Some phytochemicals were quantified − namely, alkaloids, total phenols, and flavonoids − to identify their comparative constituents in the four parts of the plant, and also to have a numerical picture of the spread of these phytoconstituents with varying solvent polarities, from more polar solvent (methanol), through ethyl acetate and chloroform, to the nonpolar solvent, petroleum–ether (decreasing order of solvent polarity). Alkaloidal content was generally highest in the leaf SSPC fractions compared with the other parts of the plant, followed by the root-bark SSPC fractions. The leaf is the major site of synthesis and storage of phytonutrients and metabolites, whereas the root may show seemingly increased levels of some of these phytonutrients compared with the stem-bark and fruit due to downward capillary pull by gravity and the presence of more phytonutrient synthesizing cells. Also, preference and discriminatory storage and synthetic sites for these phytoconstituents may be major deciding factors of the distribution of these phytochemicals. Alkaloids may be preferentially synthesized and stored in the leaf compared with the other parts of the plant.
However, the petroleum–ether SSPC fraction of the leaf had the highest alkaloid content (36.5×10−2 mg ascorbic acid equivalence (ASE)/g), followed by the leaf methanolic fraction (25.9×10−2 mg ASE/g). This means that the major types of alkaloids present in the leaf of A. muricata are of polar extremes, that is very polar and very nonpolar alkaloids ,. Total phenolic content was generally highest for the root-bark fractions, followed by the stem-bark, fruit, and leaf fractions in decreasing order. The more polar solvents (methanol and methanol–water) had higher total phenolic contents, as expected . The results obtained for total flavonoid content showed that the fruit fractions had the highest comparative levels, followed by the leaf, stem-bark and root-bark in decreasing order. The fruit chloroform fraction recorded the highest (248.7 mg quercetin equivalence (QE)/ml), followed by the leaf petroleum–ether fraction (197.9 mg QE/ml) and the fruit methanol–water fraction (139.9 mg QE/ml).
Fifty% lethal dose determination and acute toxicity studies
This experimental phase was aimed at evaluating and establishing the LD50, acute and subchronic toxicity profiles of A. muricata Linn methanolic crude extracts (AMC). The various parts of the plant were studied − that is, fruit pulp, leaf, stem-bark, and root-bark. The LD50 of the various plant parts was determined using the modified Lorke’s  method. Phase 1 (100, 400, and 1000 mg/kg) and phase 2 (1200, 1600, 2900, and 5000 mg/kg) were carried out (five rats per dose administration) with no recorded lethality above 5000 mg/kg for stem-bark and root-bark. The fruit and leaf AMC recorded deaths from 2900 mg/kg, so that there was an extension to phase 3 of the studies (1600, 2300, 2500, and 2900 mg/kg) .
Lethality was recorded from 2300 mg/kg above, and the geometric means were determined to be 1918.33 mg/kg. However, on careful examination of the rats (immediately, 4, 8, 12, 24, and 48 h later) after administration of the fruit and leaf (including the stem-bark and root-bark) AMC extracts, the rats were observed to have curled inward, to be less active, and to have developed increased heart rates at higher doses. Where deaths were observed, the rats had passed out watery stool (possibly a sign of diarrhea) and urine had a characteristic foul odor, especially in the group administered the leaf extract. The anus of the rats that died were observed to be slightly distended than normal. On the basis of the information obtained from the acute studies, the subchronic studies were carried out .
Hematological parameters: white blood cells
To study the influence of these extracts on immunity and hematological profiles, CD4+, WBCs, and RBC statuses were assayed. The data obtained for CD4+ showed that the groups administered the fruit and stem-bark AMC extracts demonstrated fluctuating patterns with a final significant rise with the highest dose, whereas the groups administered leaf and root-bark AMC extract increased significantly in group 4 (400 mg/kg) with subsequent steady dose-dependent decrease.
WBCs and lymphocytes increased in the groups administered fruit, leaf, and root-bark AMC extracts (higher doses compared with that in the control group), whereas the group administered stem-bark AMC extract showed a significant decrease in comparison with the higher doses of the control. Platelet levels showed significant increases in all groups administered the AMC extracts, but in the group administered the stem-bark AMC extract the platelet level decreased steadily, starting from group 5 (600 mg/kg) and then to group 6 (800 mg/kg) compared with the control, after an initial increase.
The wound-healing potential of A. muricata has been identified, and some of the possible mechanisms behind this potential have been reported. Moghadamtousi et al.  reported the relationship between the antioxidant capacities and wound healing. According to Muthu and Duraira , platelets can adhere to the walls of the blood vessels, release bioactive compounds, and aggregate to each other. These properties increase to a well-established level in conditions of arterial thrombosis and atherogenesis ,.
However, they were able to establish the inhibitory effects of A. muricata on this platelet aggregation and hemolysis, thus stimulating an interest on a possible interaction between the components of A. muricata and platelets. On the basis of what Muthu and Duraira  suggested, we were tempted to hypothesize that these links and interactions between A. muricata and platelets could include stimulating increases in bone marrow platelet production, increased mobilization, and sensitivity to the injury site, whether in the endothelium, ground tissue, or epithelial sites of the body, as well as direct modulatory interactions with biomolecules synthesized, store, or released by the platelets. It is possible that these active phytomolecules of A. muricata possess a modulatory influence on thromboxane A and ADP, the two important biomolecules that are secreted by the platelets to enhance stickiness of these platelets to each other during coagulation and blood clotting . It is also possible that there could be a direct molecular interaction of these potent phytomolecules with membrane-bound glycoproteins of the platelets. These membrane-bound glycoproteins repulse adherence of these platelets to the normal endothelium and yet enhance adherence to injured areas of the blood vessel wall (endothelial cells as well as exposed vascular and tissue collagen). However, the platelets house an important growth factor that causes vascular endothelial cells, vascular smooth muscles, and fibroblasts to multiply and grow, thus causing cellular growth that eventually helps to repair damaged or injured vascular walls and tissues ,,.
This growth factor could be another platelet-based biomolecule that the A. muricata phytomolecules interact with to initiate wound healing. Pathak et al.  reported a possible link between the antimicrobial activities of A. muricata and wound healing. They reported the presence of numerous bioactive compounds that possess the ability to ward off microbes on the wound surface, thus initiating healing. This claim is supported by the increase in WBCs and lymphocytes observed with the administered AMC extracts in this experiment ,.
However, platelets play a major role in blood-clot formation during tissue damage, preventing blood loss and microbial infection, as well as reinforcing the functions of immune components, and further serves as dead-tissue covering for the regeneration of new cells and tissues to cover up the wound and initiate healing. Thus, if A. muricata can boost the formation of platelets as well as the immune components in a dose-dependent manner as seen in this research, it can as well play a key role in the wound-healing process.
Red blood cells
On observation of the influence of these extracts of AMC on the RBCs, HC, and PCV, the group administered fruit AMC extract demonstrated a dose-dependent increase in RBCs and HC, with an initial decrease in PCV (from 41.5% in controls to 37.3% in group 3, i.e. 200 mg/kg; P>0.05), which was brought almost to the control level in the highest dose group − that is 800 mg/kg (P>0.05). The group administered the leaf and stem-bark AMC extracts showed increases in RBCs and HC, as well as significant increases in PCV in a dose-dependent manner, compared with the control ,. This drift in the PCV status for the leaf and stem-bark groups compared with the pattern observed in the group administered fruit AMC could be associated with hemoconcentration due to dehydration (fluid loss) of the rats as a result of excessive urination and diarrhea, especially in the group administered the leaf extract. The observation corroborates the results of the acute toxicity studies (where diarrhea and anal distention were observed in the rats that died), and also that of the subchronic phase where excessive urination with characteristic foul odor was noted. The group administered the root-bark extract showed an increase in RBCs, whereas HC and PCV increased but decreased significantly from group 5 (600 mg/kg) to group 6 (800 mg/kg) in a dose-dependent manner.
| Conclusion|| |
From the obtained data, the fruit and leaf methanolic extracts of A. muricata demonstrated remarkable ability to boost immunity through CD4+ cells as well as WBCs and lymphocytes. The acclaimed wound-healing properties of A. muricata were established and linked to the influence on blood platelets and also possible immunity against microorganisms that prevent wound healing. RBCs and HC were also elevated, suggesting that the plant has hemomodulatory properties.
However, considering the high contents of alkaloids, flavonoids, and phenols in the methanolic fractions of the fruit and leaf, this blood platelet-boosting properties of A. muricata can be said to be due to the high amounts of these phytochemicals. Apart from boosting the production of platelets, these phytochemicals could also be attributed with potent abilities to act at the wound surface as antioxidants and antimicrobial agents.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]