Probable mechanism of action and Gas Chromatography - Mass Spectroscopy Profile of the antimalarial fractions of the hexane seed extract of Garcinia kola (GUTTIFERAE)

Tejumade S. Ujomua,b*, Oyindamola O. Abioduna,b, Gabriel O. Adegokec,Grace O. Gbotoshoa,b,d

aMalaria Research Laboratories, Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, 200223, Nigeria

bDepartment of Pharmacology and Therapeutics, College of Medicine, University of Ibadan, 200223, Nigeria

cDepartment of Food Science and Technology, University of Ibadan, 200223, Nigeria

dDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, University of Ibadan, 200223, Nigeria

Received: 29-Aug-2020 , Accepted: 09-Dec-2020

Keywords: Malaria, Garcinia kola seeds, Beta heamatin, Gas chromatography-mass Spectrometry

DOI: http://dx.doi.org/10.20510/ukjpb/8/i6/1607693474

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Abstract

To access the antimalarial activity, mechanism of action and constituents of the hexane extract of Garcinia kola (GK)seeds and its column-fractions. The in vivo antimalarial activity of the hexane extract of GK and its column-fractions against Chloroquine-resistant Plasmodium berghei (ANKA) strain, the mechanism of action and the chemical constituents of the column fractions was determined using the Peters’ 4-day suppressive test, the beta hematin assay and Gas chromatography- mass spectrometry respectively. In this study, Fractions 4,5,15 and 95 obtained from the hexane extract of Garcinia kola seeds using column chromatography produced chemosuppression values ≥ 59.6% at 200 mg/kg. These fractions inhibited the conversion of heme to heamozoin and contained arenes following Gas chromatography-mass spectrometry. The observed antimalarial activity justifies the use of Garcinia kola seeds in the treatment of febrile illnesses.

1 Introduction

There has been a substantial reduction in the incidence and mortality rates attributable to malaria between 2000 and 2015 1,2. Unfortunately, the emergence and spread of artemisinin resistant Plasmodium falciparum in Southeast Asia threatens the gains and the potential prospects for the elimination of malaria3.  There is an urgent need for the discovery and development of new chemotherapeutic agents that could serve as alternative drugs or leads for the synthesis of new antimalarial agents readily available for use.  Medicinal plants might be a possible source for such safe, effective and affordable alternatives.

Garcinia kola Heckel (Guttifereae) seeds also known as bitter kola, is a nut-bearing tropical forest tree found in the regions of West and Central Africa. The seed and leaves of Garcinia kola enjoy a folk reputation in Africa as a poison antidote4. Garcinia kola is reported to possess hypoglycemic, antiparasitic, antimicrobial, antiviral, anti-inflammatory, antihepatotoxic and aphrodisiac properties 4,5. The seeds and leaf of the plant are used in folk medicine for the treatment of gram positive and gram-negative bacteria, Ebola virus infections, dysentery and diarrhea4.  Recently, the antimalarial activity of the hexane extract of Garcinia kola seeds was reported6.

Based on a previous study that showed that the hexane extract of Garcinia kola seeds possessed notable in vivo antimalarial activity6, efforts in this study were devoted to the evaluation of the in vivo antimalarial activity of the column-fractions of the hexane extract of Garcinia kola seedsagainst chloroquine-resistant P. berghei in mice, the probable mechanism of action and the phytochemical constituents of the active extract and its column-fractions using GC-MS. 

2 Materials and methods

2.1 Drugs and chemicals

All reagents/chemicals used in this study were of analytical grade. Sodium acetate and HEPES were purchased from Fluka. Pyridine 99%, glacial acetic acid and dimethylsulfoxide (DMSO; 99.7% purity) were purchased from Acros Organics. Hydrochloric acid 1M and sodium hydroxide 0.1M were purchased from Merck. All other chemicals used in this study were purchased from Sigma-Aldrich®, UK. The seeds of Garcinia kola were obtained from Bodija market, Ibadan, Nigeria.The proof of authentication was issued by Mr. A.A. Ekundayo, a plant taxonomist at Forestry Research Institute of Nigeria Ibadan (authentication number FHI 109896). 

2.2 Preparation of Plant extract

The seeds of Garcinia kola were air-dried and pulverised to a coarse powder using a table top blender. Five hundred grams (500 g) of the pulverised seeds was extracted exhaustively by cold maceration successively in hexane, dichloromethane and methanol at room temperature for 72 hours according to the method of Abiodun7. The extract was filtered, and the filtrate was concentrated at 40°C using a rotary evaporator.  Final solvent elimination was done using regulated water bath at 40°C. The extract was stored in a refrigerator (4°C) until required for assay. 

2.3 Animals

Inbred Swiss albino mice, weighing between 20-22g, aged 8-9 weeks, were used for all experiments. Animals were obtained from the animal house of the Malaria Research Laboratories, Institute for Advanced Medical Research and Training (IMRAT), University of Ibadan.  Animal care and management was carried out according to the NIH guide for the care and use of laboratory animals, NIH publication (2010)8. The mice were housed in groups of five in plastic cages, fed with mouse cubes and provided with water ad libitum.  The ethical approval for the study was obtained from the ethical review committee of the University of Ibadan.

2.3.1 Parasites

Chloroquine-resistant Plasmodium berghei (ANKA) strain used was obtained from the Malaria Research Laboratories, Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan. Parasitized red blood cells were obtained from donor-infected mouse by cardiac puncture in acid citrate dextrose (ACD) anticoagulant. The blood obtained by cardiac puncture was diluted to desired parasite density in 0.9 % NaCl solution by calculating the volume needed to infect the animals for the assay. Each mouse was inoculated intravenously with 1x106 parasitized red blood cell suspension in saline (0.2 mL). The day inoculation was done was designated as day zero (D0) and the subsequent days designated as D1, D2, D3….

2.4 Fractionation of the hexane extract of Garcinia kola seeds

Based on the observed in vivo antimalarial activity of the seed extracts of Garcinia kola, the hexane extract of GKS was fractionated on silica gel using column chromatography according to the method by Bajpai9. The hexane extract of GKS was chromatographed on silica gel with particle size of 230 - 400 mesh (Biopharma India) in a glass column (47cm x 3cm). Briefly, the column was packed with 200g of silica gel using hexane.  Fifteen grams (15 g) of the hexane extract was adsorbed with silica gel, dried packed into the column and chromatographed using a gradient solvent system consisting of hexane and ethyl acetate (100% hexane, 95:5 hexane/ETOAc, 90:10 hexane/ETOAc……) to elute the column. Eighty-five fractions were obtained and spotted on thin layer chromatographic (TLC) plates.

According to the method by Bajpai9, the plates were developed in a chamber containing the corresponding elution solvent of each fraction.  Plates were allowed to dry and thereafter introduced into concentrated sulphuric acid and then into a chamber containing iodine crystals. The plates were then viewed for similarities in retention times. Fractions with similar retention times were combined based on their TLC profile. A total of 31 pooled fractions were obtained and stored at 4ºC until when needed.

2.5 Evaluation of in vivo antimalarial activity of the fractions of the hexane extract of Garcinia kola seeds

The Peters’ 4-day suppressive test was used for the in vivo antimalarial assay10. Based on the percentage yield of the column-fractions, twelve (12) hexane column-fractions were evaluated.

Seventy (70) Swiss albino mice inoculated intravenously with 1×106 red blood cells infected with the CQ-resistant P. berghei ANKA strain.  Based on the fact that the crude hexane extract of GKS showed notable in vivo antimalarial activity at a dose of 200 mg/kg BW, the same dose (200 mg/kg BW) was used to evaluate the column fractions of the hexane extract of GKS. The infected animals were divided randomly into 14 groups of 5 animals each and were treated once daily via oral route for 4 days.  Two controls groups were used: one treated with chloroquine 10 mg/kg body weight given daily, while the second group of animals received corn oil (vehicle solution).  Response to treatment was monitored by preparing Thinbloodfilmsfrom tail snips of each animalonD4,D5,D6,D7,D9, D11 and D14 till D35 post inoculation or till the animal died and smearing on a glass slide. Each thin film was then air dried, fixed with methanol and stained with 10% Giemsa stain. 

Thereafter the Giemsa stained thin blood films on glass slides were examined under a high-power microscope objective lens (x100) to quantify theparasitaemia by counting the number of parasitized blood cells among at least 1000 red blood cells. The difference between the mean value of the control groupandthoseofthetreatmentgroupswascomputed.Thepercent suppression (% suppression) of parasite growth was calculated using this formula by Li11:

S = Suppression

2.6 Beta-Hematin assay

A colorimetric assay to determine the probable mechanism of action of the hexane extract of Garcinia kola seeds and its fractions with in vivo antimalarial activity was performed using the beta hematin assay as described by Vargas12.  Briefly, stock solutions of extracts and fractions were dissolved in 0.1M HCL/ MeOH / DMSO (5:3:2). The quantitative determination of the β-hematin synthesis inhibition of the extracts and fractions was done at 1.39 and 0.69 mg/mL. Ten microliter (10 µL) of the seed extract or fraction was dispensed into wells of a 24-well microtiter plate, thereafter, 10 µL of 1M HCL was added. One hundred microliter (100 µL) of freshly prepared hematin solution was added into the wells in row A and B of the microtiter plate. The plate was shaken at 450rpm for 10 minutes. Sixty microliter (60 µL) of saturated pre-warmed acetate solution was then added to all the wells and incubated at 60ºC for 90 minutes. 

Seven hundred and fifty microliter (750 µL) of 15% pyridine was then added to the first and third rows (rows A and C) while 750 µL of HEPES (Sigma) was added to the second and fourth wells (rows B and D) of the microtiter plate.  The plate was shaken for 10 minutes at 450rpm and then allowed to settle for 15minutes.  One hundred microliter (100 µL) from the test plate was transferred in duplicates into a 96- well plate.  The absorbance was read at 405nm on a Spectramax Gemini XS microplate reader (Paul Bucher Analytik und Biotechnologie Schutzen graben 74051 Basel).  The residual absorbance for each sample (ΔAAnalysis) designated (X) due to inhibition of the β-hematin was calculated using the formula

X = (Absorbance value in row A) - (Absorbance value in row B)

The residual absorbance (ΔACLT;Blank) designated (Y) of the sample independent from the inhibition of the β- hematin complex was calculated using the formula

Y = (Absorbance value in row C) - (Absorbance value in row D)

The resulting inhibition of the β-hematin synthesis induced by the analyzed sample was calculated using the formula: -

 IAnalysis = ΔAAnalysis (X) – ΔACLT:Blank (Y)  

IAnalysis values ≥ 0 are indicative of the ability of the sample to inhibit the conversion of heme to heamozoin.

2.7 Gas chromatograph/mass spectrometry of the hexane extract and fractions of Garcinia kola seeds

The constituents of the hexane extract of Garcinia kola seeds and its active fractions (GKHF4 GKHF5, GKHF15 and GKHF95) were determined using a gas chromatographic system (Agilent Technologies, Model 7890) fused to a silica capillary column (30m x 0.32mm) (column model HP5 MS) and equipped with a mass spectrophotometric detector (Agilent technologies, model 5975). The mobile phase was helium (99.99% purity) and was set at a flow rate of 1.2 mL /min. One microliter of the sample was injected into the column after the sample was derivatized.  The oven temperature was held at 80º C for one-minute holding time and raised from 80ºC to 240ºC at the rate of 10ºC/min and then held at 240ºC for 6 minutes, while the detector temperature was 250º C.  The MS scanning range was (50-2000m/z).

Compounds were identified by comparing the retention indices of peaks on HP5 MS column with literature values. Interpretation on the mass spectrum of individual compound was compared with the known compounds stored in the software database library (insPECT), while the relative percentage of each compound was calculated by comparing the average peak area obtained for each compound to the total areas obtained.

2.8 Statistical analysis

Graph Pad prism version 5.0 (Graph-Pad Software, San Diego, CA, USA) was used for all statistical analyses. Chemosuppression of parasite growth and survival of animals were expressed as mean ± SEM. Statistical significance of means of different variables were analysed using Student’s t test and one- way analysis of variance between groups (ANOVA) was used to compare difference in percentage inhibition of parasite growth. For all statistical tests, P≤0.05 was considered significant.

3 Results

In vivo antimalarial activity of the column-fractions of the hexane extract of Garcinia kola seeds

The antimalarial activity of the column-fractions from the hexane extract, the most active of the three extracts evaluated is presented in table 1.  Parasitaemia on day 4 ranged from 4.4% to 22.8% in animals treated with 200 mg/kg of the fractions, while it was 0.00% and 15.5% in the treated and untreated control animals.  Four fractions, GKHF4, GKHF5, GKHF15 and GKHF95 of hexane extract of GKS produced a chemosuppression of 71.7%, 67.3%, 70.9% and 59.6% respectively compared to the untreated control (P ≤ 0.05), while fractions GKHF1921, GKHF2427, GKHF4346 and GKHF4750 produced chemosuppression ranging between 13.1% and 30.5%. Fractions GKHF13, GKHF14, GKHF3942 and GKHF6668 produced no chemosuppression at 200 mg/kg. 

The survival time of animals treated with the fractions ranged from 8 -15 days, while animals in the untreated control group survived till day 8. The mean survival time of the mice treated with the fraction (GKHF4) that produced the highest chemosuppression was 18 days. In all cases, remarkable suppression of parasiteamia by extracts/fractions translated into a longer mouse survival.

3.1 Quantitative β-hematin assay on the hexane seed extract of Garcinia kola and its active antimalarial fractions

The IAnalysis value obtained for chloroquine a standard drug, the hexane extract of Garcinia kola seeds and its active fractions was ˃ 0 (Table 2), indicative of the ability of chloroquine, the hexane extract of Garcinia kola seeds and its fractions to inhibit the conversion of heme to heamozoin. The IAnalysis value obtained for chloroquine, a standard drug used as a control was 0.22±0.03 at 1.39 µg/mL, while the IAnalysis values obtained for the extracts and fractions ranged between 0.13±0.02 µg/mL and 0.23±0.04 µg/mL, with GKHF4 producing the highest IAnalysis value of 0.23±0.04 µg/mL, which was comparable to chloroquine (Table 2). At 0.69 µg/mL, chloroquine produced an IAnalysis value of 0.51±0.02 µg/mL, while the extracts and fractions evaluated produced IAnalysis values ranging between 0.12±0.01 µg/mL and 0.23±0.02 µg/mL (Table 2).  These values obtained are indicative of the probable mechanism of action of the extract and its active antimalarial fractions.

3.2 Gas chromatography of the active fractions of the hexane extract of Garcinia kola seeds

Gas chromatography-mass spectrometry revealed the presence of alkyl arenes, cycloalkanes and acylic alkenes in GKHF4, GKHF5, GKHF15 and GKHF95, (fractions of the hexane extract of GKS with good in vivo antimalarial activity) (Tables 3, 4, 5 and 6).

The percentage of heptacosane, an acyclic alkane, tricosane, an acyclic alkane, benzene (pentylheptyl), benzene (propylnonyl), benzene (butylnonyl), benzene (butylhepyl) and Kaur-16-ene, a diterpene in GKHF4 was 13.83%, 8.75%, 3.81%, 3.13%, 3.33%, 2.93% and 3.83% respectively (Table 3).

The percentage of arenes like benzene,(3,3-dimethyldecyl)(14.12%), benzene,(1-pentylocyl) (benzene, (1,butylpentyl) and benzene, (1,butylheptyl) in GKHF5 was 14.12%, 10.55%, 8.76% and 8.17% respectively ( Table 4).  The percentage of 4-oxatricyclo [20.8.0.0] triconta-1(20)7(16)-diene, a halogenated oxacycloalkane, 1-phenathrene, a carboxylic acid and octadecenoic acid in GKHF15 was 62.40%, 11.81% and 4.99% respectively ( Table 5), while the percentage of 13-tetradece-11-yn-1-ol, an alcohol, tridecanedial, an ester, Oct-5-en-2-ol, an arene,  5-phenylvaleric, a monocarboxylic acid, allylionone, a ketone and 9,19-cycloanost-24-en-3-ol, an alcohol was 27.14%, 26.43%,22.20%, 8.68% and 2.95% respectively (Table 6).

4 Discussion

In the absence of an efficient and readily available vaccine for the prevention of malaria, chemotherapy still remains the main stay for the treatment of malaria. The emergence and spread of Artemisinin resistant parasites across Southeast Asia in a major setback in the progress made on a global basis in malaria chemotherapy1,2.  

This development poses a serious health care issue for malaria endemic regions and for the world at large13. One possible source for a replacement of the artemisinins might lie in nature based on the antecedence of antimalarial drug discovery. In this study, efforts were directed at evaluating the antimalarial potential of the column fractions the hexane extracts of Garcinia kola seeds, their constituents and their probable mechanism of action.

Garcinia kola is an angiosperm belonging to the family Guttifereae/Clusiaceae. It is a highly valued ingredient in African ethno-medicine because of its varied uses which include medicinal and social uses. The presence of organic compounds within the seeds confers some medicinal properties on Garcinia kola seeds (GKS). These properties include antibacterial, antifungal, hepatoprotective and cardio protective4,5.  The search for newer antimalarial drugs/templates which will serve as alternatives to the artemisinins and the use of Garcinia kola seeds for the treatment of several illnesses according to folklore has necessitated the evaluation of the antimalarial activity of the column fractions of the hexane extract of Garcinia kola seeds, their probable mechanism of action and their chemical constituents.

Results from a previous study indicate that the hexane extracts of Garcinia kola seeds possess good in vitro and in vivo antimalarial activity and were non cytotoxic to Vero cells. The ability of the hexane extract of Garcinia kola seeds to destroy P.berghei parasites in Swiss albino mice and cause no harm to the Vero cells, which are normal epithelial cells of the African green monkey is an its indicator of selective toxicity, and as such can be considered as potential lead for clinical management of malaria.  In this present study, the hexane extract of Garcinia kola seeds which demonstrated the highest activity in vivo and four of its fractions were active against the asexual erythrocytic stages of P. berghei.  A key requirement for antimalarial agents is their ability to eliminate asexual blood stage parasites13,14.  This is an important process targeted by multiple antimalarials in asexual blood stage parasites. It involves the degradation and detoxification of host heamoglobin. Asexual blood stage parasites after establishing themselves within a parasitophorus vacuole, import heamoglobin into their acidic digestive vacuole for the eventual synthesis of parasite proteins necessary for the survival of the parasite13.

Heamoglobin is thereafter digested by parasite proteases, liberating alpha and beta chains which are cleaved into peptides and then into amino acids. These amino acids are required for the synthesis of parasite proteins13,15.  This process causes the release of Fe2+iron-containing reactive heam moieties that are oxidized inside the digestive vacuole to Fe3+ triggering oxidative stress. Within the vacuoles, Fe3+ is detoxified into heamozoin13

Several 4-aminoquinolines, notably chloroquine and amodiaquine are thought to act by inhibiting the detoxification of toxic heam into haemozoin.  Other parasite specific biological processes targeted by antimalarial drugs include the inhibition of folate synthesis by the antifolates like sulphadoxine/pyrimethamine and the inhibition of mitochondrial electron transport chain by naphthoquinones like atovaquone16. The cleavage of the endoperoxide bridge with the toxic heme which is a byproduct of heamoglobin degradation, leading to a production of oxidative stress and cellular damage is also a parasite specific biological process targeted by antimalarials such as the artemisinins 14,15.

The mechanism of action of the hexane extract of Garcinia kola seeds and the corresponding active fractions appears similar to that of the aminoquinolines.  The findings from the beta-hematin inhibition assay shows that the hexane extract of Garcinia kola seeds and the active fractions act by the inhibition of the conversion of heam to heamozoin. This inhibition produced by the hexane extracts and active fractions results a buildup of toxic heme within the food vacuole of the parasite and a production of oxidative stress leading to the death of the parasites14. This buildup of toxic heme produced by the hexane extract of Garcinia kola seeds and its active fractions may partly explain the observed in vitro and in vivo antimalarial activity.

Efforts in this study were also devoted to identifying the constituents of the active column-fractions from the hexane extract of Garcinia kola seeds using Gas chromatography/mass spectrometry.  An abundance of arenes in the fractions GKHF4, GKHF5, GKHF15 and GKHF95 of hexane extract of Garcinia kola seeds was observed in this study.  Arenes are a group of compounds that possess antimalarial properties17.  These compounds interact with free heme, inhibit heamozoin formation, and prevent Plasmodium falciparum growth in vitro in a concentration-dependent manner 17. Arenes dependently promote oxidative stress in Plasmodium falciparum by the generation of intraparasitic oxidants, protein carbonyls, and lipid peroxidation products. Furthermore, they deplete intraparasite GSH levels, which is essential for antioxidant defense and survival during intraerythrocytic stages17. The production of oxidative stress by the arenes within the active fractions combined with the inhibition of the conversion of toxic heme to non-toxic haemozoin might be the probable mechanism of action of the active fractions of the hexane extract of Garcinia kola seeds.

5 Conclusion

The observed antimalarial activity of the hexane extract of Garcinia kola seeds and four of its column-fractions justify its use in folklore for the treatment of febrile illnesses, thus further studies on the column-fractions in order to develop a potential antimalarial drug is necessary. 

6 Conflict of interest

The authors declared no conflict of interest

7 Author’s contributions

GOG made substantial contribution to the conception, the design of the study, gave final approval of the version to be published. GOA made substantial contribution to the conception of the study. OOA made substantial contribution to the analysis and interpretation of data. TSU made substantial contribution to the collection, analysis and interpretation of data, and drafting of manuscript.

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