Gas Chromatography-Mass Spectrometry Characterization of Bioactive Compounds from Ziziphus nummularia (Burm. F.) Stem Bark with Promising In Vitro Antiplasmodial Activity.

OBJECTIVE: This study was carried out to evaluate the in vitro antiplasmodial activity of alcoholic and hydroalcoholic extract of Ziziphus nummularia stem bark as well as the determination of core structure fractionated from its crude extract.
MATERIALS AND METHODS: The antiplasmodial activity of stem bark extracts was investigated by in vitro schizont maturation inhibition assay against Plasmodium falciparum (both chloroquine sensitive and resistant). The crude extract was analyzed by gas chromatography-mass spectrophotometry (GC-MS). RESULT: Both alcoholic and hydroalcoholic extracts showed their potential toward in vitro antiplasmodial activity against P. falciparum strains MRC-02 (CQ sensitive) and RKL-09 (CQ resistant). Alcoholic extract showed more promising antiplasmodial activity with Inhibitory concentration (IC50) of 0.130 and 1.191 for MRC-02 and RKL-09 strains, respectively, as compared to hydroalcoholic extract with IC50 of 1.856 and 2.981 for MRC-02 and RKL-09 strains, respectively. No morphological changes in erythrocytes were observed when investigated for chemical injury to the erythrocytes. Chemical characterization of alcoholic extract was performed by GC-MS analysis to identify the bioactive compounds that are responsible for antiplasmodial activity. Alkyl esters, phenolics, and flavonoids were found as major compounds and also showed resemblance to existing antimalarial drugs.
CONCLUSION: The findings suggested that the investigated extracts will provide a foundation for combating the resistant strains of P. falciparum. Also, the compounds isolated from Z. nummularia crude extract are viable newer chemical antimalarial candidates requiring further investigation. Copyright:

INTRODUCTION

Malaria is an enormous health, social, and economic burden for over 40% of the world's population. It is one of the most devastating vector-borne parasitic diseases in humans with an incidence of almost 300–500 million clinical cases resulting in 1.5–2.7 million deaths. The incidence of malaria is high among the children, and every year causes one million deaths of children under the age of five years.[1] Among the four species of Plasmodium parasite responsible for disease in human beings, P. falciparum is the widest spread and the hazardous parasite. It shows resistance to almost every existing antimalarial drug and can lead to the fatal cerebral malaria, which often results in death.[2] Resistance is followed by reduction in the effectiveness of the existing antimalarial agents and concomitant increase in malaria-related morbidity and mortality. There is a widespread multidrug resistance to common antimalarial agents, one of the greatest challenges against malaria control.[34] The emergence and resistance to almost all antimalarial drugs remarks the crucial need for the development of newer antimalarial drugs, especially in the absence of antimalarial vaccine, when mankind has to rely on the use of chemical agents for treatment or vector control.[5] Natural products and medicinal plants have always been a potential resource for new drug development;[67] and may inspire the researchers to identify newer potent molecules of natural origin, their derivatives, and synthetic compounds designed from natural products or with natural product pharmacophores.[8]

Z. nummularia, locally known as chhoti ber, is a small, thorny bush distributed in northwestern India, and it majorly grows in dry and arid regions.[910] It belongs to the family Rhamnaceae, and ethnomedically, the fruit is used as astringent, tonic, digestible, laxative, aphrodisiac, antiemetic, to remove biliousness, thirst and burning sensation,[9] abdominal pain during pregnancy, as an antidote in aconite poisoning and externally applied for wounds, and bark decoction for the treatment of diarrhea, dysentery, and colic.[11] Z. nummularia is reported to possess the cyclopeptide alkaloids. These are macrocyclic compounds containing a 13-, 14-, or 15-membered ring. Traditionally, plants containing cyclopeptide alkaloids are used in malaria treatment.[12] Thus, keeping in the view, this study aimed to determine the possible antiplasmodial activity of alcoholic and hydroalcoholic extract of Z. nummularia stem bark against P. falciparum strains MRC-02 (CQ sensitive) and RKL-09 (CQ resistant).

MATERIALS AND METHODS

Collection of plant material

Stem bark of Z. nummularia was collected from Sikandrabad, Uttar Pradesh, India, in the month of July, and authenticated by Principal Scientist, National Bureau of Plant Genetic Resources (NBPGR), Pusa, Delhi, India. The voucher specimen (NHCP/NBPGR/2016-4/5633) was preserved in NBPGR.

Preparation of crude extracts

Plant material was subjected to dry in shade, coarsely powdered, and extracts processed by cold percolation method with ethanol (95%) and ethanol and water (1:1). The extracts were decanted, filtered with Whatman no. 1 filter paper, and concentrated at reduced pressure below 40°C using rotary evaporator to obtain dry extract. The alcoholic and hydroalcoholic extract were designated as AZN and HAZN, respectively, and taken up for biological screening. The extracts were also investigated by chemical identification method for the presence of major phytochemical constituents.[13]

In vitro antimalarial assay

Parasite cultivation

In vitro antiplasmodial activity of AZN and HAZN was assessed against P. falciparum strains, MRC-02 (CQ sensitive) and RKL-09 (CQ resistant), obtained from the National Institute of Malaria Research, New Delhi, India, and maintained in a continuous culture using the standard method described by Trager and Jensen.[14] Parasites were cultured in human B (+ve) erythrocytes in RPMI-1640 media supplemented with 25mM HEPES buffer, 10% human B (+ve) serum, 0.2% sodium bicarbonate (Sigma-Aldrich, St. Louis, Missouri), 40 µg/mL of gentamicin sulfate, and maintained at 5% CO2.[15] Cultures containing predominantly early ring stages were synchronized by the addition of 5% d-sorbitol (Sigma-Aldrich) lysis, used for testing. Hematocrits were adjusted at 10%, and parasite cultures were used when they showed 2% parasitemia.[16]

Preparation of tested extracts

The plant extracts were dissolved in DMSO (dimethyl sulfoxide), (Sigma-Aldrich), and filtered through Millipore sterile filters (mesh 0.22 µm, Merck Millipore, Molsheim, France). The filtrate was used for testing at different concentrations of 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL.[17]

In vitro antiplasmodial assay

Filtered sterilized extracts (100, 50, 25, 12.5, 6.25, and 3.125 μg/mL) were incorporated into 96-well tissue culture plate containing 200 μL of P. falciparum culture with fresh red blood cells (RBCs) diluted to 2% hematocrit. Negative control (fresh RBCs without Plasmodium diluted to 2% hematocrit) and positive control (parasitized blood cells culture treated with chloroquine) were added to each set of experiments.[18] Growth of the parasites from duplicate wells of each concentration was monitored in Giemsa-stained blood smears by counting the number of schizont per 100 asexual parasites. Percent schizont maturation inhibition was calculated by the formula:

where, Nt and Nc represent the number of schizont in the test and control well, respectively. The consequences of the antimalarial screening are expressed as the drug concentration resulting in 50% inhibition (IC50) of parasite growth using HN-NonLin V1.1.[19]

Chemical injury to erythrocytes

To assess any chemical injury to erythrocytes that might be attributed to the extract, 100 μL of erythrocytes were incubated with 100 μg/mL of the extract at a dose equal to the highest used in the antiplasmodial assay. The conditions of the experiment were maintained as in the case of antiplasmodial assay. After 48h of incubation, thin blood smears were stained with Giemsa stain and observed for morphological changes under high-power light microscopy. The morphological findings were compared with those in the erythrocytes that were uninfected and not exposed to extract.[20]

Gas chromatography–mass spectrophotometry

Of the two tested extracts, AZN was found to be more potent than HAZN. Therefore, AZN was subjected for the separation and characterization of bioactive compounds using GC–MS.

Method adopted on GC column, RXi-5-Sil MS (30m × 0.25mm with 0.25 µ) with column flow, 1mL/min with split ratio, 1/25. Oven temperature started at 70°C hold for 1min rising with a rate of 10°C/min till 180°C and finally with rate of 15°C/min till 280°C and hold for 8min. Injector port temperature was 250°C, interface temperature was 280°C, and ion source temperature was 280°C. Mass programming—scanning from 3–40min at scan speed 2000 μ/second with starting m/z 40.0–550.0 with solvent cut time of 2.5min. Samples were prepared by dissolving 10mg of extract in 10mL of methanol (ultrapure high performance liquid chromatography grade). Take 1mL of the dilution and again dilute it to 10mL. Make a program with injection volume, 1 µL to obtain GC–MS spectra and match the peaks with NIST 11 and Willy database. The MS for each peak was compared with those documented in library data for their chemical structure [Table 1], [Figure 1].

Table 1

Gas chromatography–mass spectrometry analysis of alcoholic extract of Ziziphus nummularia

S. no.	Name of component	RT	Area (%)
1.	8-Methyl-8-azabicyclo[3.2.1]octane-3-carbonitrile	6.34	25.19
2.	2-Methoxy-4-vinylphenol	6.49	3.71
3.	Pyrogallol 1,3-dimethyl ether	6.80	3.04
4.	Ethyl 1H-1,2,4-triazol-1-yl methyl carbonate	6.90	2.58
5.	Melezitose	7.21	3.83
6.	6-Methoxy-8-methyl-8-azabicyclo[3.2.1]octan-3-ol	7.52	8.92
7.	Melezitose	7.74	10.68
8.	(2-Phenyl-1,3-dioxolan-4-yl)methyl 9-octadecenoate, cis-	7.85	1.97
9.	-allose	8.28	3.91
10.	2,4-Di-tert-butylphenol	8.37	4.65
11.	á-d-glucopyranoside, methyl	9.09	6.78
12.	(E)-2,6-dimethoxy-4-(prop-1-en-1-yl) phenol	10.42	1.99
13.	(E)-4-(3-hydroxyprop-1-en-1-yl)-2-methoxyphenol	10.87	5.25
14.	Hexadecanoic acid, methyl ester	13.17	3.74
15.	n-Hexadecanoic acid	13.61	4.32
16.	trans-Sinapyl alcohol	14.03	1.84
17.	Propionic acid, 3-amino-3-(4-isopropylphenyl)-	19.03	1.97
18.	Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester	21.10	2.00
19.	3’-Hydroxy-5,6,7,4’-tetramethoxyflavone	22.54	1.91
20.	9,19-Cyclolanostan-3-ol, 24,24-epoxymethano-, acetate	27.58	1.74

Figure 1

Gas chromatography–mass spectrometry spectrum of alcoholic extract of Ziziphus nummularia stem bark

RESULTS

In vitro antiplasmodial assay

This assay was used for the assessment of inhibition of growth of P. falciparum strains MRC-02 (CQ sensitive) and RKL-09 (CQ resistant) by prepared extracts. AZN was found to possess comparable activity with chloroquine diphosphate (IC50, 0.13 µg/mL) against chloroquine-sensitive strain (MRC-02) of P. falciparum and active against both chloroquine-sensitive (MRC-02) as well as chloroquine-resistant strain (RKL-09) with resistance index (RI) 0.109. In vitro antiplasmodial activity of AZN was followed by HAZN with RI, 0.623, indicating the moderate activity of HAZN against the chloroquine-resistant strain [Table 2].

Table 2

In vitro antimalarial activity of Ziziphus nummularia stem bark against MRC-01 and RKL-09 strains of Plasmodium falciparum

Plant name	Extract	IC50 (µg/mL)a		RId
		MRC-02b	RKL-09c
Z. nummularia	HAZN	1.856	2.981	0.623
	AZN	0.130	1.191	0.109
Chloroquine	CQ	0.097	0.995	0.0975

aConcentration corresponding to 50% growth inhibition of the parasite bChloroquine-sensitive strain of P. falciparum, IC50,μg/mL cChloroquine-resistant strain of P. falciparum, IC50, μg/mL dRI = Resistance index (IC50 values of MRC-02 strain/IC50 values against RKL-09)

On microscopical examination, no chemical injury to erythrocytes was observed as indicated by no morphological differences in uninfected erythrocytes incubated with test extracts after 48h of incubation.

The phytochemical studies revealed that the test extracts have variety of phytochemical constituents, namely alkaloids, flavonoids, tannins, steroids, and carbohydrates [Table 3].

Table 3

Qualitative phytochemical investigations of Ziziphus nummularia stem bark

Chemical group	Name of extract
	AZN	HAZN
Alkaloids	+	+
Flavonoids	+	+
Tannins	+	+
Glycosides	−	−
Steroids	+	−
Carbohydrates	+	+
Proteins	−	−

+ = Present, − = Absent

Gas chromatography–mass spectrophotometry analysis

Of the two tested extracts, AZN was found to be more potent than HAZN. Therefore, AZN was subjected to separation of bioactive compounds using GC–MS, leading to identification of 20 compounds. The MS for each peak was compared with those documented in library data for their chemical structure [Table 1], [Figure 1].

DISCUSSION

Test extracts (AZN and HAZN) prepared from Z. nummularia stem bark were assessed for in vitro antiplasmodial activity and were reported to possess the same against both MRC-02 (CQ sensitive) and RKL-09 (CQ resistant) strains of P. falciparum. Activity of a test sample may be expressed as IC50, that is, concentration required to inhibit 50% of parasite's activity. Lower IC50 and hence higher activity of AZN also indicate the accumulation of active metabolites into the ethanol extract. The activity of the extracts can be predicated on the basis of chemical constituents present in the extract. Therefore, tested extracts were subjected to the phytochemical screening and GC–MS analysis to reveal the nature of phytoconstituents present in the extract.

Various researchers had concluded that secondary metabolites of plants such as alkaloids, flavonoids, and triterpenoids have been reported to possess antiplasmodial activity.[212223] Preliminary phytochemical studies also revealed the presence of alkaloids, flavonoids, tannins, and steroids, which are accountable for observed antiplasmodial activity of crude extracts. Also, GC–MS analysis of AZN revealed the presence of some pharmacologically active compounds such as phenolics, N-containing moieties, steroids, polyunsaturated fatty acids, and esters. Some antimalarial drugs possess alkyl esters as their main functional group. The series of n-alkyl or aryl ester groups have great potential as in vitro antimalarial drugs.[24] Fosmamide, a newly developed inhibitor of malarial isoprenoid enzyme, also consists of acyloxy alkyl ester.[25] Ethyl 1H-1,2,4-triazol-1-ylmethyl carbonate, hexadecanoic acid, methyl ester, and hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester are derivatives of alkyl esters and may be attributed for the activity under study.

In the same way, various authors also showed the efficacy of phenolic compounds in inhibiting the growth of P. falciparum parasite.[2627] GC–MS analysis also revealed the presence of various phenolic compounds, namely trans-sinapyl alcohol, pyrogallol 1,3-dimethyl ether, 2,4-di-tert-butylphenol, and 2-methoxy-4-vinylphenol. 3′-Hydroxy-5,6,7,4′-tetramethoxyflavone, flavonoid moiety, was also identified as one of the component of AZN. Flavonoids were reported to possess the antiplasmodial activity either by modulating the cellular signaling pathway promoting schizonticidal activity[28] or chelating with nucleic acid base pairing of the parasite.[29] Phenolics and flavonoids possess antioxidant potential and may help in improving the pathological condition by lowering the elevated levels of free radicals formed during severe malaria.[30] From the above discussion, it can also be said that no single compound is responsible for the antiplasmodial activity. Synergistic or enhanced activity may be a result of mixture of substances contained in a crude plant extract, for example, some flavonoids may cause significant reduction in IC50 value of artemisinin but they themselves do not possess any antiplasmodial activity.[31] The findings were influencing and suggested that identified bioactive compounds may be responsible for the biological activity of Z. nummularia stem bark and these compounds may further act as a basis for developing newer chemical entities with targeted pharmacological activities.

CONCLUSION

The present investigation revealed the in vitro inhibition of growth of P. falciparum parasite by Z. nummularia stem bark extract, and it may assist as a foundation for developing alternative antimalarial drugs and drug combinations to combat with the drug-resistant parasites. Thus, summing up the biological and chemical affirmations, it can be said that the study will provide a platform for further exploration of the drug for in vivo studies, toxicity studies, and identification of newer, safer compounds with much greater antimalarial potential.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.