In vivo antimalarial activity, toxicity, and phytochemical composition of total extracts from securidaca longepedunculata Fresen. (polygalaceae)

Joseph Mwanzia Nguta

doi:10.4103/bbrj.bbrj_82_19

ORIGINAL ARTICLE

Year : 2019 | Volume : 3 | Issue : 3 | Page : 196-201

In vivo antimalarial activity, toxicity, and phytochemical composition of total extracts from securidaca longepedunculata Fresen. (polygalaceae)

Joseph Mwanzia Nguta
Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, College of Agriculture and Veterinary Sciences, University of Nairobi, Nairobi, Kenya

Date of Submission	23-Mar-2019
Date of Decision	10-May-2019
Date of Acceptance	11-Jun-2019
Date of Web Publication	10-Sep-2019

Correspondence Address:
Dr. Joseph Mwanzia Nguta
Department of Public Health, Pharmacology and Toxicology, Faculty of Veterinary Medicine, College of Agriculture and Veterinary Sciences, University of Nairobi, P. O. Box 29053-00625, Nairobi
Kenya

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_82_19

Abstract

Introduction: Alternative antimalarial drugs are urgently required. Securidaca longepedunculata Fresen. (Polygalaceae) is a medicinal plant with a long history of use in African ethnomedicine to treat malaria and other illnesses. The efficacy, safety, and chemical composition of chloroform: methanol (1:1) and aqueous total extracts from the leaves, stem, and roots of S. longepedunculata were investigated. Methods: Adult Swiss female mice were infected with 10⁷ erythrocytes parasitized with Plasmodium berghei (strain ANKA) on day 0 as a model of malaria. Effects of crude extracts at a dosage rate of 100 mg/kg of body weight on parasitemia were measured over a 4-day period. To evaluate acute toxicity, the mice were administered crude extracts by oral gavage at 50, 300, and 2000 mg/kg of body weight and observed over a 24-h period. Cytotoxic effects of crude extracts were measured using human epithelial-2 cells in a 96-well microtiter plate over a 24-h period. Results: Chloroform: methanol (1:1) and aqueous root extracts demonstrated significant chemosuppressive activities of 91.46% and 87.64%, respectively (P < 0.05). CC₅₀values ranged from 115 to 140 μg/mL and an LD₅₀>2000 mg/kg body weight. Crude extracts contained alkaloids, anthraquinones, flavonoids, saponins, steroids, tannins, and triterpenoids. Conclusion: The findings from the current study validate ethnopharmacological use of the plant, while demonstrating its potential as a possible source of new lead molecules against malaria.

Keywords: Antimalarial, drug discovery, efficacy, medicinal plants, safety, secondary metabolites

How to cite this article:
Nguta JM. In vivo antimalarial activity, toxicity, and phytochemical composition of total extracts from securidaca longepedunculata Fresen. (polygalaceae). Biomed Biotechnol Res J 2019;3:196-201

How to cite this URL:
Nguta JM. In vivo antimalarial activity, toxicity, and phytochemical composition of total extracts from securidaca longepedunculata Fresen. (polygalaceae). Biomed Biotechnol Res J [serial online] 2019 [cited 2023 Mar 26];3:196-201. Available from: https://www.bmbtrj.org/text.asp?2019/3/3/196/266565

Introduction

Malaria is a major cause of death globally, with approximately 3.3 billion people worldwide being at risk of infection.^[1] The burden is heaviest in the African region, where an estimated 90% of all malaria deaths occur in children under 5 years, who account for 78% of all deaths.^[2] The infectious disease is of global concern and of national interest in Kenya because of development of resistant strains of Plasmodium falciparum to majority of the existing drugs including chloroquine (CQ).^[3] This has made malaria chemotherapy a major problem.^[4] In Kenya, of the 22 million people at risk of malaria infection, 70% of them live in rural areas, while about 34,000 Kenyan children under the age of 5 years die every year from malaria compared with a total estimate of 42,000 deaths arising from all diseases combined.^[5]

While synthetic pharmaceutical agents continue to dominate research, attention has increasingly been directed to natural products in a view to combat drug resistance.^[6] Since natural products are a proven template for the development of new scaffolds of drugs, plant species still serve as a rich source of many novel biologically active compounds, yet very few plant species have been thoroughly investigated for their medicinal properties, and thus, there is renewed interest in phytomedicine research.^[7] Hence, a pharmacognostic investigation of plants for the establishment of complementary medicine for malaria within traditional plants is necessary.^[8] In many African communities, local herbs are used for the treatment of malaria. These herbs have helped to reduce mortality and morbidity rates, especially in the rural areas of the developing world where antimalarial drugs are not readily available.^[9]

Securidaca longepedunculata is locally known as Mzigi by the Digo community of Kwale County in Kenya. It is a multipurpose medicinal plant with a long history of use in African traditional medicine. In Zimbabwe, the roots are used against fever, pains, epilepsy, pneumonia, tuberculosis, venereal diseases, and syphilis,^[10] while in Botswana, they are used against coughs and also as an aphrodisiac.^[11] In the neighboring Uganda, the roots are used against malaria, fever, and ascariasis.^[12] In Nigeria, the roots are used to treat fever, toothache, tuberculosis, sexual boost, abortion, constipation, coughs, and rheumatism.^[10] The roots are also used as a blood purifier, aphrodisiac, and for psychoactive purposes in South Africa.^[13] The root extracts are used to treat menstrual pains and gonorrhea in Nigeria.^[14] The leaves are used to treat headaches, skin infections, skin cancer, and as a contraceptive in Nigeria.^[10] In Burkina Faso, the stem bark is used to treat skin diseases,^[15] while in Nigeria, it is used to treat malaria, dysentery, typhoid fever, and frequent stomachache.^[10] In Kenya, the whole plant is used against malaria.^[16]

These ethnomedicinal uses suggest that the most commonly used plant part is the root and that the plant species is used to treat many diseases, including malaria, fever, pains, tuberculosis, coughs, and sexually transmitted infections in different geographical zones. Despite its wide usage, little has been done to evaluate its safety and efficacy as an antimalarial phytomedicine. The current study was therefore designed to investigate the antimalarial activity, acute toxicity, cytotoxicity, and to analyze the phytochemical composition of the aqueous and organic crude extracts from the leaves, stem, and the roots of S. longepedunculata in an effort to authenticate the anecdotal efficacy and safety of this medicinal plant against malaria.

Methods

Plant materials

The plant materials were collected following study approval by the Faculty of Veterinary Medicine Biosafety, Animal Care and Use Committee. The three plant parts, namely leaves, stem, and roots, from S. longepedunculata used in this study were collected from their natural habitat in Shimoni, Msambweni Sub-county, and Kwale County in Kenya during June 2018 based on earlier ethnopharmacological studies.^[16] The three plant parts are traditionally used by the Msambweni community against malaria. The study area in South coast centered around 04° 28' 59.2“S latitude and 039° 33' 36.2”E longitude in and around Shimoni. The plant was identified by Mr. Kimeu Musembi, a taxonomist at the University of Nairobi herbarium, where a voucher specimen (No. JM 06/2018) was deposited. The study species was validated using the Medicinal Plant Names Services database of the Royal Botanic Gardens, Kew (http://mnps.kew.org/mnps-porta). The plant parts were chopped into small pieces; air-dried at room temperature (25°C) under shade and pulverized using a laboratory mill (Christy and Norris Ltd., England).

Preparation of extracts

Considering that the Msambweni community usually uses hot water to prepare their herbal remedies as decoctions, and sometimes concoctions, aqueous hot infusions of each plant part were prepared (50 g of powdered material in 500 mL of distilled water) in a water bath at 60°C for 1 h. The extracts that were obtained were filtered and then freeze-dried. Chloroform: methanol (1:1) extracts (50 g powder in 500 mL of solvent) were prepared by maceration of the plant material with chloroform: methanol (1:1) at room temperature for 48 h. The mixture was filtered and the filtrate concentrated to dryness in vacuo. The dry solid extracts were stored at −20°C in airtight containers until used.

Phytochemical screening

Phytochemical analyses were conducted on aqueous and chloroform: methanol (1:1) crude extracts from the leaves, stem bark, and the roots of S. longepedunculata for the presence of steroids, alkaloids, tannins, anthraquinones, flavonoids, and saponins using standard methods.^[17],[18]

In vivo determination of antimalarial activity

CQ-sensitive P. berghei strain ANKA was used to assess thein vivo antimalarial activity. The assay protocol was based on 4-day suppressive test.^[19] The parasite strain was maintained by serial passage of blood from an infected mouse to a naive mouse. Forty female Swiss mice (6–7 weeks old; 20–22 g) were randomly infected by intraperitoneal inoculation of 10⁷ erythrocytes parasitized with P. berghei in a saline suspension of 0.2 mL on day 0 (D0) and allocated to eight groups of five mice in each cage. They were fed on standard pellets and water ad libitum. The animals were housed in the Animal House at Kenya Medical Research Institute (KEMRI) and the Institute's Animal Care and Use Committee gave approval for the study. Plant extracts were solubilized in 10% Tween 80 (chloroform: methanol [1:1] extracts) or in physiological saline (water extracts) and administered once daily orally by oral gavage (D0 to D3) at a concentration of 100 mg/kg/day in a dose volume of 0.2 mL. Two groups (five mice each) served as negative and positive controls, respectively. The negative group received physiological saline/Tween 80, while the positive group was treated with reference drug CQ diphosphate at a dose of 5 mg/kg/day orally. Each day from D1 to D4, thin blood smears were made from the tail of each mouse, stained with 10% Giemsa in phosphate buffer, pH 7.2, and examined microscopically for assessment of parasitemia. The mean parasitemia in each group of mice on D4 was used to calculate the percentage chemosuppression for each extract using the formula:

(A − B) ÷ (A) ×100

where A was the mean parasitemia in the negative control and B was the parasitemia in the test group.^[20] Extract activity was determined by percentage reduction of parasitemia in treated groups compared with untreated infected mice. For all the groups of experimental mice used, survival time in days was recorded and the mean for each group calculated.

Acute toxicity

Fifty-seven healthy Swiss female mice weighing 20–22 g were divided into 17 treatment groups and 1 control group. Each group comprised three mice in each cage and had access to tap water and food, except for a short-fasting period (12 h) before oral administration of a single dose of the extract. The water extracts were dissolved/suspended in distilled water while chloroform: methanol (1:1) extracts were solubilized in Tween 80 and administered by oral gavage at 50, 300, and 2000 mg/kg.^[21] The general behavior of mice was observed continuously for 1 h after the treatment and then intermittently for 4 h, and thereafter, over a period of 24 h.^[22] The mice were further observed for up to 14 days following treatment for any signs of toxicity and the latency of death.

Cell cytotoxicity assay

Thein vitro cytotoxicity properties of the extracts were evaluated using a tetrazolium salt 3-[4.5-dimethylthiazol- 2-yl]-2.5-diphenyltetrazolium bromide (MTT) colorimetric method. Human epithelial-2 (HEp-2) cells obtained from KEMRI center for traditional medicine and drug research were cultured in Minimum Eagle Essential Medium. Trypsinized cells were seeded in a 96 microtiter well plates at 2 × 10⁴ cells per well in 100 μL. Crude extracts were suspended in culture media, to give a final stock solution concentration of 2500 μg/mL, and serially diluted across 96-well microtiter plates (100 μL), and incubated at 37°C with 5% CO₂ for 24 h in a humidified carbon dioxide incubator. Four hours prior to the end of each exposure period, an MTT mixture (20 μL/well) was added. After the completion of exposure period, the plates were then placed on a microplate reader shaken for 10 s and the absorbance of the formazan product was read at 562 nm. Each experiment was repeated on three separate occasions. Two internal controls were set up for each experiment: (1) an IC₀ consisting of cells only and (2) IC₁₀₀ consisting of medium only. Background absorbance due to the nonspecific reaction between crude extracts and the MTT reagent was deducted from exposed cell values.^[23] Dose–response curves were plotted for the crude extracts after correction by subtracting the background absorbance from the controls. The percentage inhibition was determined using the formula:

%I = (100× [1 − At/Ac])

where (At) is the absorbance of treated well and (Ac) is the absorbance of control well. CC₅₀ value is the concentration of sample required to inhibit 50% of the cell proliferation and was calculated from a calibration curve by a linear regression^[24] using Microsoft Excel.

Chemicals

All the chemicals and reagents used in the study were obtained from Sigma-Aldrich (St. Louis, Mo., USA).

Statistical analysis

Multiple group comparisons were made using ANOVA one-parameter tests. The Welch's t-test was used to test the significance of differences between mean results obtained for different samples, and Dunnett's test was used for multiple comparisons of significance between results of the same sample means against controls (GraphPad Instat™ V2.04, GraphPad Software, San Diego, CA). Values with P < 0.05 were considered statistically significant.

Results

Parasitemia ranged from 1.93% to 19.07% among the treated mice [Table 1]. Chemosuppression activities ranging from 18.42% to 91.46% were observed for the 100 mg/kg/day dose during the 4 days of crude extract administration. The highest chemosuppression of 91.46% was observed in the chloroform: methanol (1:1) crude root extract, while the lowest chemosuppression of 18.42% was observed in the aqueous crude stem extract. In general, crude extracts from both the stem and the leaves of S. longepedunculata did not exhibit significant antimalarial activities compared to the positive control (P > 0.05). Among the aqueous crude extracts, the stem and the leaves had chemosuppressive rates of 18.42% and 38.05%, respectively, while among the chloroform: methanol (1:1) crude extracts, both the stem and the leaves had chemosuppressive rates of 19.09% and 54.04%, respectively [Table 1].

Table 1: Antimalarial activities of the aqueous and organic extracts of Securidaca longipedunculata

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The chemosuppression of both aqueous and chloroform: methanol (1:1) crude root extracts from S. longepedunculata of 87.64% and 91.46%, respectively, were not significantly different from that of CQ (P< 0.05), which showed the highest chemosuppressive activity of 96.99% [Table 1]. In terms of survival time, mice treated with both aqueous and chloroform: methanol (1:1) crude root extracts of S. longepedunculata and those treated with CQ at a dose of 10 mg/kg/day, survived for the entire 14 days of test period [Table 1]. Up to 60% and 40%, respectively, of the mice treated with the chloroform: methanol (1:1) and aqueous crude leave extracts survived the test period, while only 20% of the test mice treated with aqueous and chloroform: methanol (1:1) crude stem extracts, survived the test period [Table 1]. None of the mice in the negative control group was alive by D14 of the test period as shown [Table 1].

It was observed that none of the plant extracts produced mortality at a dosage rate of 50 and 300 mg/kg body weight. At 2000 mg/kg body weight, one mortality was observed in the aqueous and chloroform: methanol (1:1) total root extract-treated groups. However, the rest of the test mice survived the 14 days observation period. All the six total extracts studied were found to be nontoxic to HEp-2 cells. Median cytotoxic concentrations (CC₅₀) values ranged from 115 to 140 μg/mL. All the crude extracts were evaluated for the presence of major phytochemical compounds. The aqueous root extracts were found to contain steroids, tannins, anthraquinones, and saponins, while the stem total extracts contained steroids, tannins, and saponins. Crude leaf extracts were found to contain steroids, tannins, flavonoids, and saponins [Table 2].

Table 2: Phytochemical constituents from Securidaca longipedunculata aqueous crude extracts

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The organic crude extracts from the root, stem, and the leaves were found to contain low-to-high concentrations of steroids, alkaloids, tannins, anthraquinones, flavonoids, saponins, and cardiac glycosides [Table 3].

Table 3: Phytochemical constituents from Securidaca longipedunculata organic crude extracts

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The chloroform: methanol (1:1) total extracts from the root, stem, and the leaves were found to contain low-to-high concentrations of steroids, alkaloids, tannins, anthraquinones, flavonoids, and saponins [Table 3].

Discussion

The current study was designed to investigatein vivo antimalarial activity,in vitro cytotoxicity, acute toxicity, and also to analyze the phytochemical composition of aqueous and chloroform: methanol (1:1) crude extracts from the leaves, stem, and roots of S. longepedunculata. In addition, the study sought to authenticate the claims on anecdotal antimalarial efficacy and safety made against this plant by traditional healers from Msamweni Sub-county, South Coast Kenya. The results of thein vivo antimalarial study demonstrate chemosuppression of the multiplication of P. berghei in mice in the treated groups. Usage of effective antimalarial drugs in correct dosage causes a decrease in parasitemia levels that is essential for recovery. Plant extracts exhibiting high chemosuppressive values reduced parasitemia levels enabling mice to survive up to the 14^th-day postinfection. The experimental results indicated that out of the six crude extracts of S. longepedunculata tested, the chloroform: methanol (1:1) root extract showed the lowest parasitemia (1.93%) compared to distilled water (22.58%) and the highest chemosuppression of 91.46% compared to that of CQ of 96.99%. There was no significant difference (P< 0.05) between the observed chemosuppression caused by CQ and the aqueous/chloroform: methanol (1:1) root extracts of S. longepedunculata. The highest parasitemia in all the treated groups was observed with distilled water (22.58%), demonstrating that all the tested extracts had activity against P. berghei malaria parasite in Swiss albino mice.

The mice survival time in days was inversely related to the percentage parasitemia. The survival time of mice treated with CQ with parasitemia of 0.68% and the aqueous/chloroform: methanol (1:1) root crude extracts of S. longepedunculata with parasitemia of 2.78% and 1.93%, respectively, survived for the whole observation period of 14 days. Mice treated with distilled water with parasitemia levels of 22.58% did not survive the 14-day observation period. The results from this study showed that aqueous and chloroform: methanol (1:1) root extracts of S. longepedunculata had high chemosuppressive activities against P. berghei malaria parasite in Swiss albino mice thus, in part, concurring with the traditional use of this plant for antimalarial therapy. This demonstrates that the plant can offer a potential source or an important lead in antimalarial drug discovery and development. Data generated from the current study marks the first step in screening the plant for novel molecules against P. falciparum malaria. The current findings support earlier observations of a chemosuppresssive value of 82.6% exhibited by methanolic extracts from the roots of S. longepedunculata,^[25] further validating the ethnomedicinal utilization of the study plant part against illnesses with malaria symptoms by the Msambweni community. It will be plausible to hypothesize that the observed antimalarial activity caused by chloroform: methanol (1:1) root extract was partly associated with secondary metabolites extracted by methanol. This could explain earlier antimalarial observations.^[25]

The results observed in the present study are also in agreement with earlier reports^[26] which observed significant antiplasmodial activity from dichloromethane leaf extracts of S. longepedunculata. S. longepedunculata have been used against malaria in traditional medicine, but roots are the most widely used part against various diseases.^[27] These reports are validated by observations from the current study. In the current findings, chemosuppressive activity of 91.46% was observed from the chloroform: methanol (1:1) root extract at a dose of 100 mg/kg/day. In accordance with the World Health Organization (WHO),^[28] crude plant extracts displaying high levels of chemosuppression (>90%) at 250 mg/kg/day may be recommended for further development into herbal remedies, providing an affordable source for malaria treatment in endemic areas.

In vitro cytotoxicity was used as a starting point in evaluating the potential toxicity of S. longepedunculata crude extracts. None of the extract tested was found to be toxic to HEp-2 cell lines with CC₅₀>100 μg/mL. According to earlier reports,^[29]S. longepedunculata aqueous root bark extract was cytotoxic to Ehrlich ascites cancer cells, an observation that was not reported in the current study. This could be attributed to the fact that a whole root extract was used in the current study, while a root bark was utilized in an earlier study.^[29] Further, it was observed that some compounds that do not showin vitro cytotoxicity may possessin vivo toxicity due to pharmacokinetic and immunological factors.^[30] The current study was extended to evaluatein vivo toxicity of the total extracts from the leaves, stem, and root of S. longepedunculata, which were found to be safe. However, the current study recommends further subchronic and chronic toxicological studies on this plant parts. Acute toxicity studies revealed that mice given a dose of 2000 mg/kg from the aqueous total extracts derived from the leaves and stem bark of the study plant did not show any change in behavior following administration of the crude extracts as compared to the control. There was no noticeable change in food and water intake of the test animals. This study is also in concurrence with earlier studies^[25] which recorded the absence of death or any sign of toxic manifestations in rats at a dose of 2000 mg/kg and registered LD₅₀ values >2000 mg/kg following oral administration of aqueous crude extracts from the leaves and stem bark of S. longepedunculata. Similar findings were observed with oral LD₅₀ values of 3162 mg/kg after oral administration of S. longepedunculata aqueous leaf extracts.^[31] It has been also reported that there was no observed toxicity following a 28-day oral administration of S. longepedunculata aqueous crude root extract in mice with the LD₅₀ being above 2700 mg/kg body weight.^[32] However, earlier studies reported that S. longepedunculata organic crude root extracts exhibited liver and kidney injury in the test mice.^[33] This observation is in agreement with data generated from the current study where death was reported following oral administration of chloroform: methanol (1:1) crude root extracts. This calls for dose regulation in preparation of herbal concoctions from the roots of S. longepeducnculata. The local community of Msambweni uses the root extract from S. longepedunculata in combination with other medicinal plants in malaria treatment. This could be the plausible explanation for the absence of toxicity reports from the study community in earlier studies.^[16] The current study, therefore, demonstrates that S. longepedunculata aqueous and chloroform: methanol (1:1) crude extracts are safe and have no significant toxic effects in normal doses in Swiss albino mice. Hence, they could be developed as a standardized safe and efficacious herbal medicine against malaria for use by the rural and urban populace, locally and internationally.

The current study has demonstrated the presence of numerous phytochemical constituents useful in the treatment of numerous diseases. This justifies the traditional use of this plant to treat various ailments. The chloroform: methanol (1:1) crude extracts revealed the presence of alkaloids, anthraquinones, flavonoids, saponins, steroids, tannins, and triterpenoids. This study agrees with previous studies reporting the presence of the observed secondary metabolites.^[31] Alkaloids, flavonoids, and anthraquinones observed in the current study have documented antimalarial properties and perhaps responsible for the observed antimalarial activity. Phytochemical screening of S. longepedunculata crude extracts has demonstrated the presence of bioactive principles that can be used to explain the observed antimalarial activity of this plant.

It would be plausible to hypothesize that the diverse pharmacological activities associated with phytoconstituents present in S. longepedunculata leaf, root, and stem crude extracts are responsible for the observed biological activity. The current findings validate, in part, the ethnopharmacological use of the roots of S. longepeducnculata in traditional treatment of malaria, thus recommending domestication and propagation of the study plant, and development of an antimalarial herbal product according to the WHO guidelines. The observed phytochemical compounds can be used as biomarkers in quality assurance of antimalarial herbal products developed from the roots of S. longepedunculata. To the best of our knowledge, we validate for the first time, the ethnopharmacological use of S. longepedunculata against malaria by the Msambweni community of South Coast, Kenya.

Conclusion

The current study investigated the antimalarial activity, safety, and phytochemical composition of the leaves, stem, and roots of S. longepedunculata. Results from the current study demonstrate that the aqueous and chloroform: methanol (1:1) crude root extracts of S. longepedunculata were efficacious against P. berghei parasites in vivo, lending support, in part, the traditional use of this plant against malaria. The conservation status of this plant is also of great concern since the roots are the most widely used part, exposing the plant to the risk of eradication as the roots get harvested. Sustainable root harvesting techniques by the local communities is encouraged.

Acknowledgments

The author is greatly indebted to the Msambweni community, for sharing their valued ethnopharmacological knowledge. The University of Nairobi, Department of Pharmacology and Toxicology is acknowledged for valuable technical support.

Financial support and sponsorship

The author is grateful to Bill and Melinda Gates Foundation, through Grant No. #OPP52155 for funding the current study.

Conflicts of interest

There are no conflicts of interest.

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