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ORIGINAL ARTICLE
Year : 2017  |  Volume : 36  |  Issue : 4  |  Page : 191-195

Traditional consumption of the fruit pulp of Chrysophyllum albidum (Sapotaceae) in pregnancy may be serving as an intermittent preventive therapy against malaria infection


1 Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria
2 Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Nigeria

Date of Web Publication28-Nov-2017

Correspondence Address:
Chibueze Peter Ihekwereme
Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0257-7941.219363

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  Abstract 


Background: The bark of Chrysophyllum albidum is reported to possess antimalarial property. The fruit pulp of C. albidum consumed by pregnant women of south eastern Nigeria may also possess antimalarial activity. The present preliminary study investigated the antimalarial potential of the pulp juice and seed of C. albidum. Methods: Schizonticidal activity was evaluated using the Peter's 4-day suppressive test. The prophylactic and curative antimalarial activities of the extracts were evaluated in Albino mice inoculated with Plasmodium berghei. Results: The oral acute toxic dose of the pulp extract is beyond 5000 mg/kg. The seed and pulp possess both suppressive and curative properties. The seed extract suppressed early infection by 72.97% and 97.30%, at 500 and 1000 mg/kg, respectively. The pulp juice recorded 72.97% and 81.08%, at 500 and 1000 mg/kg, respectively. At 500 mg/kg dose, the level of parasite control on Day 7 was the same (96.10%) for both seed and pulp. Conclusion: This study demonstrates the presence of antimalarial constituents in the chemically uncharacterized samples (fruit pulp and seed) of C. albidum. Its ethnomedicinal use may be valuable in pregnancy where it may possibly serve as an intermittent preventive therapy against malaria.

Keywords: Chrysophyllum albidum, intermittent preventive therapy, malaria, parasitemia, pregnancy


How to cite this article:
Ihekwereme CP, Okoye FK, Agu SC, Oli AN. Traditional consumption of the fruit pulp of Chrysophyllum albidum (Sapotaceae) in pregnancy may be serving as an intermittent preventive therapy against malaria infection. Ancient Sci Life 2017;36:191-5

How to cite this URL:
Ihekwereme CP, Okoye FK, Agu SC, Oli AN. Traditional consumption of the fruit pulp of Chrysophyllum albidum (Sapotaceae) in pregnancy may be serving as an intermittent preventive therapy against malaria infection. Ancient Sci Life [serial online] 2017 [cited 2019 Jul 21];36:191-5. Available from: http://www.ancientscienceoflife.org/text.asp?2017/36/4/191/219363




  Introduction Top


There is a need to tame malaria for any meaningful development in countries where it flourishes. Malaria affects fertility, population growth, savings and investment, worker productivity, absenteeism, premature mortality and medical cost.[1] Malaria primarily affects human health especially in pregnancy where it is detrimental to both mother and unborn child. Commonly, malaria causes anemia in the mother, and low birth weight in the fetus. The risk inherent from untreated malaria in pregnancy outweigh the adverse effects arising from treatment.[2],[3],[4],[5]P. falciparum causes more maternal and fetal morbidity and mortality than non-falciparum infections.[6]

Intermittent preventive therapy (IPT) is the administration of antimalarial doses to pregnant women in malaria endemic countries who have not shown obvious signs of malaria infection. Such women are given full curative regimen whenever malaria is evident. Generally, treatment of severe malaria includes parasite clearance and supportive measures such as monitoring of blood sugar and potassium levels, as well as management of high fever and the seizures it may provoke.[7] Antimalarial resistance and treatment costs are among the current issues that necessitate an urgent need for malaria control especially in endemic regions.[8]

Chrysophyllum albidum is a forest tree native across much of tropical Africa. Several other components of the tree including the roots and leaves are used for medicinal purposes.[9] The pulp of the fruit is commonly consumed by all in South East Nigeria, especially pregnant women. The leaves are frequently used to treat malaria, blood pressure, anemia, skin eruptions, diarrhea and stomach ache.[9] The roots are used in the treatment of sterility, sexual asthenia, and asthma; while its seeds are mostly used to treat intestinal worms and hemorrhoids. The bark is used against cough, icterus, yellow fever and also malaria, while the fruit is used for the treatment of dental decay. Eleagnine, an alkaloid isolated from C. albidum seed cotyledon has been reported to have antinociceptive, anti-inflammatory and antioxidant activities.[10] Studies have shown that the leaf extract of C. albidum contains antiplatelet, hypoglycemic,[11] and antibacterial compounds.[9] The methanol bark extract contains antiplasmodial substances.[12] In spite of its vast use, there is a dearth of scientific information on its antimalarial potential.

The concept behind this study is that the discovery of antimalarial constituents in the bark of C. albidum could be a pointer that the fruit pulp, commonly consumed by pregnant women may also have antiplasmodial property. This preliminary study, therefore, was designed to investigate the presence of antimalarial principles in the pulp juice and seed of the plant.


  Methods Top


Plant material source

Fresh whole fruits of C. albidum collected in February 2015 from its natural habitat in Agulu area of Anambra State, Nigeria, were authenticated by Mr. Alfred Ozioko, a plant taxonomist. A voucher specimen was deposited in the Herbarium of the Department of Pharmacognosy and Traditional Medicine, Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University, Awka, Agulu.

Preparation of extracts and fractions

The seeds were air-dried at room temperature under shade for 14 days, after which their shells were broken and discarded. The shell contents were air-dried at room temperature under shade for another 7 days and pulverized using a blender, after which 400 g was macerated in 2 L of ethanol (EtOH) for 24 h, sieved using a porcelain cloth, and filtered through filter paper (Whatman ® no. 1). Ethanol was removed using a rotary evaporator at 40°C under reduced pressure. The extract was further concentrated in a hot water bath at a temperature of 50°C and stored at 4°C until use. The yield value was determined.

The pericarp of the fruit was discarded; exposing the fleshy pulp of the fruit which was collected wet and immediately ground using a blender. A fixed volume (750 ml) of water was used to grind each 250 g of the fleshy pulp. The resulting material was sieved using a porcelain cloth. The filtrate was stored as stock at 4°C until use. Before administration to animals, an aliquot of the filtrate was withdrawn and allowed to warm up to room temperature. Phytochemical screening was the only physicochemical characterization done on the pulp juice and seed samples.

Animals

Swiss albino mice (16 – 26 g, 4 – 6 weeks old) of either sex were used for the study. The experimental animals were housed under standard environmental conditions of temperature at 22 – 29°C under a 12 h dark–light cycle and allowed free access to drinking water and standard pellet diet. The experimental protocols used in this study were approved by the Ethics Committee of the Faculty of Pharmaceutical Sciences, Nnamdi Azikiwe University Awka Nigeria.

Acute toxicity and phytochemical screening

The acute toxicity test was carried out on the expressed juice using Lorke's methods.[13] The ethanol seed extract and the expressed juice from the pulp were tested for the presence of alkaloids, flavonoids, glycosides, tannins, saponins, and steroids using Draggendorff's reagent, ammonium test, Fehling's reagent, lead subacetate reagent, frothing test, and Salkowski's test standard procedures, respectively.[14]

Parasite inoculation

All animals were quarantined for 7 days prior to infection. Blood drawn from a mice infected with P. berghei was used to infect the animals used for the experiment. Standard inoculums of 1 × 107 P. berghei infected erythrocytes in 0.2 ml were prepared by diluting the infected blood with 0.9% normal saline. Each mouse was inoculated by intraperitoneal injection with a blood suspension (0.2 ml) containing 1 × 107 parasitized erythrocytes.[15] The parasite was maintained by serial passage of blood from infected to non-infected mice on a weekly basis.

Evaluation of schizonticidal activity in early infection

The standard four-day suppressive method was used in screening of the plant extracts and fractions.[16] Experimental animals were in groups (n = 5) and treatment was by administration of single daily oral administration of test samples (500 mg/kg, 1000 mg/kg) which began 3 h post infection and lasted from Day 0 to Day 3. There were 4 test groups which were administered 500 mg/kg and 1000 mg/kg doses of both the seed and pulp extracts of C. albidum. Three control groups were used namely, normal (uninfected and untreated group), negative (infected and treated with 0.09 ml distilled water), and positive (infected and treated with 10 mg/kg Artesunate [Mekophar Chemical Pharmaceutical, Vietnam]). Thin smears were prepared from blood samples collected from the tail tips of each mouse on Day 4. The doses in this preliminary study were chosen in consideration of doses used in the index study.[12]

Evaluation of schizonticidal activity in established infection (curative or Rane test)

At 72 h post infection, when the level of parasitemia was observed to be >4%, the animals were divided into groups (n = 5). There were 4 test groups which orally received either 500 mg/kg or 1000 mg/kg doses of the seed or pulp extracts of Chrysophyllum albidum. Three control groups were used namely, normal (uninfected and untreated group), negative (infected and treated with 0.09 ml distilled water), and positive (infected and treated with 10 mg/kg Artesunate [Mekophar Pharmaceuticals, Nigeria]). Blood samples were collected from the tips of the tails of the animals on Day 4 and Day 7 post treatment.

Parasitemia monitoring

Parasitemia was monitored using a previously described method.[15] Blood samples were collected from the tip of the tails of the animals. Thin blood films were dried, and fixed (for 15 min) using methanol, and subsequently stained with 10% Giemsa for 25 min. The stained film was washed off using phosphate buffer, pH 7.2 and allowed to dry. The film was immersed in oil and viewed at 100× magnification. The parasitemia level was determined by counting the number of parasitized erythrocytes out of 100 erythrocytes in random fields of the microscope.[11]

Data analysis

The results were presented as the mean ± SEM (standard error of mean) for each group of experiments. The test groups were compared with the normal control group using one-way analysis of variance (ANOVA). All data were analyzed at a 95% confidence interval. Average percentage parasitemia was calculated using the formula:



Average percentage of chemosuppression was calculated using the formula:




  Results Top


Preparation of extracts and fractions

The yield value of the seed was 0.92%.

Acute toxicity and phytochemical screening

The acute toxic dose of the pulp extract is beyond 5000 mg/kg per oral since all the animals survived even at 5000 mg/kg. Both the ethanol seed extract and the expressed juice from the pulp showed the presence of alkaloids, flavonoids, cardiac glycosides, and tannins. In addition, the ethanol seed extract also revealed the presence of saponins and steroids.

Evaluation of schizonticidal activity in early infection

The seed extract of C. albidum suppressed early infection by 72.97% and 97.30% at 500 and 1000 mg/kg, respectively [Table 1]. These were significant (P< 0.01) when compared with the negative control. Similarly, the pulp juice suppressed early infection by 72.97% and 81.08% at 500 and 1000 mg/kg, respectively. Artesunate (positive control) recorded 86.48% suppression. There were no statistical differences between the percentage suppressions of Artesunate and the seed or pulp extracts.
Table 1: Schizonticidal activity of expressed juice of the pulp and ethanol extract of the seed of Chrysophyllum albidum in early infection in Plasmodium berghei-infected mice in vivo

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Evaluation of schizonticidal activity in established infection (curative or Rane test)

The result of the curative study on Day 4 [Table 2] shows that there is not much difference in the percentage parasitemia at 500 and 1000 mg/kg doses, both for the seed (92.31% and 94.87%) and the pulp (88.46% and 92.31%), respectively. At 1000 mg/kg, the seed (97.30%) was found to be more active than the pulp (81.08%). The seed (92.31% and 94.87%) showed more activity than the pulp (88.46% and 92.31%) at both doses (500 and 1000 mg/kg) respectively. The Plasmodial load of the positive control on Day 7 was similar to the values obtained for the seed at both doses. There were no statistical differences between the percentage suppressions of Artesunate and the seed or pulp extracts.
Table 2: Curative effect of expressed juice of the pulp and ethanol extract of the seed of Chrysophyllum albidum in Plasmodium berghei-infected mice on day 4

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On Day 7 [Table 3], it was observed that at 500 mg/kg dose, the level of parasite control was the same (96.10%) for both seed and pulp. At 1000 mg/kg, there was a marginal difference between the seed (98.70%) and the pulp (97.40%). The Plasmodial load of the positive control on Day 7 was similar to the values obtained from the seed and pulp at both doses. On further analysis, there were no statistical differences between the percentage suppressions of Artesunate and the seed or pulp extracts.
Table 3: Curative effect of expressed juice of the pulp and ethanol extract of the seed of Chrysophyllum albidum in Plasmodium berghei-infected mice on day 7

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Day 7 was seen to have the highest % inhibition. On comparison, Artesunate produced an average percentage reduction of 93.24% on day 4 and 98.64% on day 7, similar to that of 1000 mg/kg of C. albidum seed extract.

At 500 mg/kg, plasmodial control was higher in the curative experiment than in the suppressive experiment. Between Day 4 and Day 7, control values were higher on Day 7. Similar observation was also made at 1000 mg/kg. There were no statistical differences between the percentage suppressions of Artesunate and the seed or pulp extracts.


  Discussion Top


When commonly consumed food substances in malaria endemic zones possess anti-plasmodial property, it could enhance control of malaria in such milieu. This may be advantageous since such food could work in synergy with already acquired immunity to mitigate the infection. Furthermore, it would occupy a unique position in the public health structure of such community as disease-causing organisms may not easily develop resistance to whole herbal extracts.

The result of our study shows that both the seed and pulp possess both suppressive and curative anti-plasmodial property. Previously, methanol bark extract of C. albidum was reported to suppress early plasmodial infection (per oral) by 74.20% and 62.90% at 1000 and 1500 mg/kg/day respectively.[12] Reported percentage reductions for the curative tests on Day 7 were 82.86%, 97.14%, and 77.14% at 750, 1000 and 1500 mg/kg respectively.[12] On comparison with equal doses (at 1000 mg/kg/day), it becomes obvious that both the ethanol seed extract (97.30%) and the pulp juice (81.08%) have higher chemosuppressive potency than the methanol bark extract (74.20%). On the curative effect, the result from the bark (97.14%) is comparable with that of seed extract (94.87%) and the pulp juice (92.31%). Even though the statistical relevance of the comparison between our results and the previously published data remains to be verified, our results seem to suggest the seed has more antimalarial principles than the pulp. Interestingly, when the positive control was statistically compared with either the pulp or seed at 500 mg/kg, it was observed that there were no significant differences suggesting that the plant material was as effective as the control.

The sampled plant parts can be used for treatment and prevention of malaria both in pregnant and non-pregnant individuals. It also shows the plant has a potential of serving as IPT against malaria in pregnancy. Pregnancy predisposes to malaria infection, and IPT was initiated to control the menace of the infection to both the mother and the unborn child. Traditional consumption of the pulp by pregnant women does not suggest any harm to the mothers or their babies. Also, the results of the oral acute toxicity test show the pulp to be considered safe. This opinion is strengthened by the experience of the consuming public since they have not associated it with any untoward effects. Conversely, LD50 of the plant methanol bark extract in mice was calculated to be 1850 mg/kg intraperitoneal, suggesting the bark might be toxic at clinically effective doses.[13] Perhaps, this may also offer some explanation why the methanol bark extract demonstrated a higher antimalarial activity at a lower oral dose of 1000 mg/kg/day, than at 1500 mg/kg/day per oral. Irrespective of the above, it is still necessary to scientifically establish safety using appropriate pharmacological models.

It is important to isolate and characterize the compound(s) responsible for this activity. Phytochemicals present in both the ethanol seed extract and the expressed juice from the pulp include alkaloids, flavonoids, cardiac glycosides, and tannins. In addition, the ethanol seed extract also revealed the presence of saponins and steroids. Previous studies found alkaloids, anthraquinones, saponins, cardenolides and tannins in the leaf, stem and bark of the plant.[12] Alkaloids, flavonoids, triterpenoids, and saponins have been associated with antimalarial property. Saponins are detergents and might alter cell membrane properties of protozoans through their surface activity.[18] Alkaloids are a major class of compounds possessing antimalarial activity; quinine is one of the most important and oldest antimalarial drugs. Eleagnine, tetrahydro- 2 - methylharman and skatole have been isolated from the seeds of C. albidum.[11],[19] Eleagnine, an alkaloid has antimicrobial, antinociceptive, anti-inflammatory and antioxidant activities.[10]

The compounds responsible for the activity may possibly have short half-lives. A comparison of the level of suppression on Day 4 and Day 7 shows no statistical difference suggesting there was no continued activity after Day 4. Drugs with long half-lives such as Mefloquine, Pyrimethamine, Piperaquine, and Lumefantrine continue to act many more days even after discontinuation of treatment.

There are two key conclusions of this study. Consumption of the fruit pulp should be encouraged especially in pregnancy. Secondly, it provides an avenue to explore possibilities of obtaining an antimalarial drug molecule.


  Conclusion Top


This study demonstrates the presence of antimalarial constituents in chemically uncharacterized samples (fruit pulp and seed) of C. albidum. The fruit pulp, seeds, and bark of the plant have antimalarial property. Consequently, consumption of the fruit pulp by pregnant women may offer some protection against malaria, and may possibly serve as IPT against the infection. Furthermore, these findings suggest these plant parts have the potential for isolation of lead compounds from their extracts. Hence, further studies should be carried out on the plant to harness its antimalarial property.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3]



 

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