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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 44  |  Issue : 1  |  Page : 65-71

In-vitro anti-sickling and membrane stability potentials of Mishenland polyherbal extract on sickle red blood cells


1 Department of Medical Laboratory Science, College of Health Sciences, Ladoke Akintola University of Technology, Osogbo, Nigeria
2 Department of Anatomy, Olabisi Onabanjo University, Ago-Iwoye, Nigeria
3 Department of Oral Pathology, DUH, King Saudi University Medical City, Riyadh, Saudi Arabia
4 Department of Morbid Anatomy and Histopathology, Ladoke Akintola University of Technology Teaching Hospital, Osogbo, Nigeria
5 Department of Hematology and Blood Transfusion, Ladoke Akintola University of Technology Teaching Hospital, Osogbo, Nigeria

Date of Submission15-Sep-2018
Date of Acceptance19-Nov-2018
Date of Web Publication27-Sep-2019

Correspondence Address:
Musiliu A Oyenike
Department of Medical Laboratory Science, College of Health Sciences, Ladoke Akintola University of Technology, P.M.B. 4400, Osogbo
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_33_18

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  Abstract 


Background Sickle cell disease is a genetic disorder that causes stiff, rod-like sickle-shaped hemoglobin in red blood cells (RBCs) and consequently poses serious health complications.
Aim We investigated an in-vitro anti-sickling potential of a novel Mishenland polyherbal formula (MPF) for possible ameliorative effects.
Materials and methods Sickling of RBCs induced with 2% sodium metabisulfite was followed by treatment with MPF mixtures in different saline concentrations (7, 9, 14, and 28 mg/ml). The red cell morphology was examined microscopically. Percentage sickling was assessed at 30 min intervals at 37°C for 2 h. The effect of the MPF on membrane stability of RBCs was analyzed using osmotic fragility testing.
Results Qualitative phytochemical screening demonstrated the presence of some secondary metabolites namely alkaloids, glycosides, phenols, saponin, tannin, and terpenoids. Sickling of RBCs induced with metabisulfite was inhibited by MPF. This anti-sickling effect was directly proportional to the concentration of the MPF, dose dependently. There was a significant difference (P<0.05) between MPF-treated and untreated sickled red cell counts. Osmotic fragility curves obtained from MPF-treated RBCs showed leftward shifts against the untreated control, indicative of increased RBC membrane stabilization and hemolytic resistance, while the mean corpuscular fragility also showed a significant difference (P<0.05).
Conclusion MPF demonstrated significant anti-sickling and erythrocyte membrane stability properties. These effects under hypoxia signified a promising effect of the bioactive components as probable drug candidates against sickling of red cells.

Keywords: erythrocyte membrane stability, in-vitro anti-sickling, phytochemical screening, polyherbal formula, sickle cell disease


How to cite this article:
Oyenike MA, Akpan HB, Otulana OJ, Adefule AK, Adedokun KA, Oluogun WA, Muhibi MA, Ojokuku HO. In-vitro anti-sickling and membrane stability potentials of Mishenland polyherbal extract on sickle red blood cells. Egypt J Haematol 2019;44:65-71

How to cite this URL:
Oyenike MA, Akpan HB, Otulana OJ, Adefule AK, Adedokun KA, Oluogun WA, Muhibi MA, Ojokuku HO. In-vitro anti-sickling and membrane stability potentials of Mishenland polyherbal extract on sickle red blood cells. Egypt J Haematol [serial online] 2019 [cited 2019 Oct 23];44:65-71. Available from: http://www.ehj.eg.net/text.asp?2019/44/1/65/268002


  Introduction Top


Sickle cell disease (SCD) is a genetic disorder that causes stiff, rod-like sickle-shaped hemoglobin in red blood cells (RBCs) and consequently poses serious health complications [1],[2]. SCD as a genetic disorder is occasioned by abnormal β-globin gene. This gene defect causes substitution of β-6 glutamic acid for valine thereby leading to severe reduction in hemoglobin solubility in sickle cells when oxygenated [3],[4]. Likewise, at low oxygen tension, hemoglobin S (HbS) goes through polymerization, resulting in deformation of RBCs, which assume a typical sickle shape [5].

Sickling of red cells increases the risk of blood flow obstruction thereby resulting in various serious complications, such as sickle cell crisis (sudden and severe pain), severe hemolytic anemia and multiple organ damage involving the heart, brain, lungs, kidneys, skin, penis, skeleton, eyes, and the spleen [6]. SCD affects millions of people worldwide. About 75% of all patients live in sub-Saharan Africa [7]. Annually, Nigeria records the highest incidence of this disorder worldwide, with ∼91 011 birth defects [8] and about 100 000 infant deaths [9].

Meanwhile, many drugs that are currently used in the management of SCD today are associated with cytotoxic effects. Most times, these drugs are expensive and often less effective. As a result, further investigation urgently becomes necessary to search for better, nontoxic, efficacious, and affordably accessible therapies from the available phytochemical agents [10]. Previously, many studies have shown positive outcomes from the use of various phytochemical agents for managing SCDs [2],[10],[11]. Precisely, tannin from ethanolic extract of root of Uvaria afzelii [12], flavonoids, and cardiac glycosides from extracts of leaves, stem, root, and fruit of Uvaria chamae [13] have been proved to possess anti-sickling activity under hypoxic conditions. However, there are still more to be discovered and availability of quality and genuine varieties may not yet be exhaustively tapped out [14].

In this present study, a combined medicinal plants’ product named the Mishenland polyherbal formula was put into consideration following consultation with local herbalists. Mishenland contains six different medicinal plant species namely: U. afzelii (Gbogbonse or Anikan wogba-arun in Yoruba), Securidaca longipedunculata (Ipeta in Yoruba), Sorghum bicolor (Poroporo Okababa in Yoruba), Momordica charantia (Ejinrin in Yoruba), Phyllanthus amarus (Eyin Olobe in Yoruba), and Dialium guineense (Awin in Yoruba). We investigated anti-sickling and erythrocyte membrane stability effects of the recipe from this novel polyherbal formulation and scientifically examined its validation ex vivo.


  Materials and methods Top


Plant materials

The following plant species with different parts were selected for anti-sickling preparation: U. afzelii roots, S. longepedunculat root barks, S. bicolor leaves, M. charantia leaves and seeds, P. amarus leaves and D. guineense leaves and barks. The herbs were obtained fresh from different parts of Ogun and Osun States in Nigeria. They were taxonomically identified and authenticated at the Herbarium, Department of Botany, Olabisi Onabanjo University, Ago-Iwoye, Nigeria.

Preparation of Mishenland polyherbal formula

Mishenland polyherbal formula (MPF) or recipe was prepared combining various parts of respective plant materials previously dried in shed for 3 weeks. Each dried herb was crushed, coarsely powdered, and then finely blended using mortar and pestle and an electric blender to obtain a desired consistency following a previous method [15].

Similar to the usual mode of preparation stipulated by the local herbalists, best described as decoction in clean water, 100 g each of the individual herb powder was then weighed for a similar preparation. The herb substances were mixed geometrically using a blender and dissolved in 1.5 l of 50% ethanol–water solution in a conical flask. The mixtures were left for 72 h under intermittent stirring with a clean spatula, following two modified methods [12],[16]. The extract was filtered using Whatman filter paper no. 42 (125 mm) and concentrated to dryness. The percentage yield was 7.3%. A working solution of 28% (w/v) of the plant extract was prepared with distilled water and then stored at 4°C until the time of analyses.

Ethical approval

A written consent was obtained from the participants prior to their recruitment. Also, the study protocol was approved by the Ethics Committee of Olabisi Onabanjo University, Ago-Iwoye and was duly followed as approved. Ethical clearance on the use of sickle cell (HbSS) blood was strictly observed according to the international rules.

Phytochemical screening of Mishenland formula

Qualitative analyses of phytochemical constituents of the MPF extract were carried out using modified standard procedures [17],[18] as described in [Table 1].
Table 1 Qualitative analysis of phytochemical constituents for Mishenland polyherbal extract

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Preparation of biological material

Fresh blood was collected from antecubital vein of the stable sickle cell patients (participants) without crises at the time of recruitment. The ages ranged between 18 and 25 years, and both sexes were included. Blood samples were preserved in EDTA bottles for the investigations. We further confirmed their hemoglobin phenotype using the electrophoresis method following their medical history.

Isolation of red blood cells

Serum and buffy coats were separated by centrifugation at 5000 rpm for 10 min. The RBCs were washed three times in isotonic PBS (IPBS) (pH 7.4; 140 mmol/l NaCl, 5 mmol/l KCl and 5 mmol/l glucose in 10 mmol/l PBS concentration using 1 : 5 v/v) as previously reported [16]. The RBCs were then resuspended in IPBS solution (1 : 1 v/v; 50% suspension). The red cell suspensions used in this study were freshly prepared on a daily basis.

Polymerization inhibition (anti-sickling) test

Principle

When sickle red blood cells (SS-RBCs) are deoxygenated by the addition of a reducing agent, the RBCs turn sickle and maintain this abnormal shape until re-exposed to sufficient oxygen. The ability of a drug to prevent or inhibit sickling in this deoxygenated state is then determined by counting the number of sickle cells over a period of time.

Procedure

The suspended RBCs (100 μl) on incubation in IPBS (at 37°C for≥30 min in a water bath) were mixed with graded dilutions/concentrations of the MPF (7, 9, 14, and 28 mg/ml) in test tubes. The samples were taken from different mixtures at varying time intervals (30, 60, 90, and 120 min) and were incubated at 37°C, while shaking intermittently.

In-vitro induction of red cell sickling

A measure of 100 μl of 2% sodium metabisulfite was added to the mixtures of preincubated RBCs in MPF solutions, mixed thoroughly and sealed with liquid paraffin to keep out air (maintaining hypoxia). The samples were then taken from different mixtures, after which the samples were further reincubated at 37°C. The samples were taken at 30 min intervals until four readings were recorded in a row. Both the SS-RBCs and total cells (including normal and abnormal) were enumerated from five different fields randomly selected across the slide after counting at least 100 cells altogether. The procedure was repeated five times each for every sample mixture while the average counts were reported. Normal saline (NaCl 0.9%) without the extract was used for positive control.

Morphological evaluation of red blood cells

The RBCs were observed, counted, and analyzed under an Olympian light microscope. The procedure for smear preparation and counting of sickled and unsickled cells followed a modified method [12]. The counting was done by a Principal Medical Laboratory Scientist majoring in hematology. Normal cells were considered for the RBCs that reasonably resembled the shape of a classical biconcave disk and including those with central pallor, while abnormal cells were considered as those cells (drepanocytes) with elongated, crescent, star-like, rod-like, wrinkle, or bolt-shaped characteristics.

The percentage inhibition of sickling was calculated using Huntsman’s formula [19]:



Osmotic fragility test

The fragility of RBCs (ability of RBCs to resist hemolysis) was determined by mixing fresh RBCs (1 : 1 v/v; 50% suspension in PBS) with graded concentrations of hypotonic saline solutions (pH 7.4) at a blood-hypotonic solution ratio of 1 : 200. Two tubes were used each for dilutions labeled as treated and untreated of every blood sample, respectively. Concentrations ranging from 0.2 to 0.8% NaCl were made up in 5 ml pipettes. A separate mixture of standard isotonic solution (0.9% NaCl w/v) was used as control.

Untreated preparation

A 10 µl sample of washed SS-RBCs was added to 1990 µl (after removing 10 µl away from 2000 µl) of each graded hypotonic saline solution and immediately mixed by inverting several times. The tubes were allowed to stand for 150 min at room temperature.

Treated preparation

To determine the effect of the formulation, a saline solution containing the MPF extract was prepared. Hundred microliters (100 µl) of MPF (28 and 14 mg/ml) was added to 1900 µl of each hypotonic saline solution (by 1 : 20 v/v) separately. Then, 10 µl of RBCs was added (after removing 10 µl away from 2000 µl of the mixture) following a modified method of Mpiana et al. [20].

Standard preparation

A 10 µl sample of washed SS-RBCs was added to 1990 µl of isotonic saline solutions; 0.9% NaCl (by 1 : 200 v/v) was mixed accordingly following the same method for both treated and untreated preparations. The number of nonhemolyzed RBCs in each saline concentration was counted manually using a hemocytometer (Neubauer’s cell counter) under an eyepiece lead microscope (Olympus BX43, Olympus (China) Inc., Ltd. Beijing, China) using 40× objective power. Hemolysis was calculated using the following equation:



Statistical analysis

The results were reported as mean±SEM of five determinations. SPSS program-version 10.0 (SPSS Inc., Chicago, IL. USA) was used for data analysis. The statistical significance of difference was calculated using Kruskal–Wallis and Mann–Whitney U-tests. P value of up to 0.05 was considered as significant.


  Results Top


Blood samples from SCD stable patients were obtained to assess the potency of MPF in preventing sickling of RBCs. The results are presented in tables, micrographs, and in a chart below.

Qualitative phytochemical screening demonstrated the presence of some secondary metabolites, namely, alkaloids, glycosides, phenols, saponin, tannin and terpenoids. Among those tested negative and probably absent were anthraquinones, flavonoids, phlobotanins, quinones, and steroids.

There were significant differences (P<0.05) in the numbers of SS-RBCs count comparing the effect of various dilutions of MPF extract against the control (normal saline preparation). RBC counts were recorded using the microscopy method at 30 min (P=0.001), 60 min (P<0.001), 90 min (P=0.002), and 120 min (P=0.001), respectively ([Table 2]).
Table 2 Time course of the sickling of sickle red blood cells in extract-treated preparations by red cell count

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The mean corpuscular fragility (MCF) was determined from the concentration of saline causing 50% hemolysis of the RBCs [21]. MCF index was derived through interpolation from osmotic fragility curves of hemolysis (%) versus NaCl concentrations (w/v%), for both treated and untreated groups. The results were presented by graphical representation in [Figure 1]. There were significant differences (P=0.005 and 0.008) comparing MCF index from the plots of MPF-treated (28 and 14 mg/ml) in varying hypotonic saline solutions compared with untreated hypotonic solutions, correspondingly ([Table 3]).
Figure 1 (A) Photomicrograph shows morphology of sickle red blood cells (SS-RBCs) in a positive control slide. Mag.X400. The micrograph illustrates numerous abnormal shape of red cells (white arrows) after treatment with a reducing agent (2% Na2S2O4) in normal saline at the end of 120 mins. The blue arrows point toward classical biconcave disks of normal red cells. (B) Photomicrograph shows effect of MPF-extract on SS-RBC morphology. Mag.X400. It shows how pretreatment of SS-RBCs with MPF/normal saline dilution inhibited sickling of RBCs on exposure to low oxygen tension milieu (after induction with 2% Na2S2O4).The micrograph reveals more of classical biconcave disks of normal red cells and cells with central parlor (blue arrows) as against the abnormal sickle cells (white arrows).

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Table 3 Determination of mean corpuscular fragility indices as an effect of Mishenland polyherbal drug-extract on sickle red blood cells

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  Discussion Top


The use of folklore medicine in the treatment of ailments has been acknowledged since time immemorial. Recently, plant-derived pharmaceutical substances became of great interest [22]. Various approaches have been tailored toward the discovery of drug candidates that can inhibit the %Hemolysis polymerization of HbS or increase the oxygen affinity of red cells and thus prevent or reduce the occurrence of SCD crises [23].

The present study showed an increased number of SS-RBCs in positive controls left untreated with Mishenland polyherbal extract on exposure to low oxygen tension ([Figure 1]a). In another way, treatment of the same SS-RBCs with an extract of MPF inhibited the purported sickling by large difference. The treatment with this novel phytomedical product greatly demonstrated a reduced number of sickle cells ([Figure 1]b). This anti-sickling effect was also shown to have a direct relationship with concentrations of the extract, thus suggesting the potency of MPF to be dose dependent.

Our report is consistent with previous studies [2],[12]. Although, we put different plant species into trial, however, the presence of similar pharmaceutically active secondary metabolites such as alkaloids and phenolics in previous studies [10],[12],[24] indicate possible similar mechanisms of reaction.

Besides, in her report, Gbadamosi et al. [25] highlighted various traditional recipes used for the management of SCD. Majority of these recipes incorporated at least one similar plant species formulated in the MPF recipe of the present study. Pills made of fresh shoot of P. amarus denoting mono-plant recipe and a powder of another recipe involving leaves of S. bicolor were said to be good anti-sickling phytomedicines.

Accordingly, the outcomes of this present study are also in concordance with previous reports where U. afzelii roots [12], S. longipedunculata root barks [26], S. bicolor leaves, M. charantia leaves [11], P. amarus leaves [27], and D. guineense [28] have been screened for the presence of phytochemicals such as alkaloids, flavonoids, tannins, saponins, glycosides, and tannins. In addition, application of phytochemicals such as glycosides, in some plant species for the inhibition or prevention of red cell sickling has also been reported [29]. The presence of these active constituents in MPF extract of the present study may be an indication for anti-sickling property.Furthermore, the effect of MPF on erythrocyte membrane stabilization and their ability to resist hemolysis was further evaluated by comparing the hemolysis rates between the MPF-treated and untreated SS-RBCs using the osmotic fragility test at varying NaCl concentrations. MPF showed appreciable erythrocyte membrane protection with reduced MCF index. That is, osmotic fragility curves showed a leftward shift for the two most effective concentrations (14 and 28 mg/ml) against the untreated control and thus indicative of increased RBC membrane stabilization ([Figure 2]).
Figure 2 Osmotic fragility curve of Mishenland polyherbal formula (MDF) treated against untreated saline dilutions of sickle cells.

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When the osmotic fragility of a cell reduces, osmotic resistance increases and vice versa [10]. The ability of all these plant extracts to increase resistance to hemolysis as presented in this study is consistent with several earlier reports that showed the efficacy of medicinal plants on the reduction of osmotic fragility of RBCs [24],[30].

This stabilization effect could be explained by the fact that MPF rendered the SS-RBCs capable of withstanding higher concentrations of NaCl by increasing the volume of the RBCs, reverting the sickling to produce biconcave cells, and thereby, maintaining membrane integrity. This may possibly indicate that MPF effected mechanistically at the cell membrane level and not through direct interaction with HbS molecules against the earlier assumption of inhibition of Hb polymerization.


  Conclusion Top


MPF extract contains some pharmaceutically important phytochemicals and secondary metabolites. These constituents were able to prevent a good percentage number of sickled cells, during induced hypoxia. The observed anti-sickling effect of MPF, through its active metabolites, controlled the hypoxia-induced sickling in vitro, and hence may protect sickle cell patients from the effects of low oxygen tension, especially from crisis. Mechanistically, the MPF extract showed great potential by possibly inhibiting HbS polymerization or increase the hemoglobin oxygen affinity indicated by a shift in the oxygen dissociation curve leftwards. This effect was dose dependent under hypoxia, a promising result of its bioactive components as probable drug candidates.

Acknowledgements

The authors appreciate the support received from students of the postgraduate school, Department of Anatomy, Olabisi Onabanjo University, for committing their time in participation during patients’ recruitment and laboratory analyses.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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