In Vitro Antileishmanial Activity and GC-MS Analysis of Whole Plant Hexane Fraction of Achillea wilhelmsii (WHFAW)

Abstract

In this study, GC-MS analysis has shown that whole plant hexane fraction of Achillea wilhelmsii (WHFAW) consists of 66 compounds which exhibited antileishmanial activity. Antileishmanial bioassay was the method used for determining antileishmanial activity. The inhibitory concentration (IC₅₀) which was observed for whole plant hexane fraction of Achillea wilhelmsii (WHFAW) against parasitic and vector-borne disease, leishmaniasis, is 58.27 ± 0.52 μg/mL. For leishmanicidal assay, Leishmania major is the species used for analysis. Whole plant methanol extract of Achillea wilhelmsii (WMEAW) and whole plant aqueous fraction of Achillea wilhelmsii (WAFAW) exhibited no antileishmanial activity.

1. Introduction

The genus Achillea consists of more than 100 species and belongs to the family Asteraceae, innate to Western Asia and Europe [1]. Achillea means Achilles which in Greek language refers to hero who with the usage of yarrow treated the wounds of soldiers during battles [2]. High mountains of the Mediterranean are the location for the occurrence of Achillea species [3]. The extracts and fractions of Achillea exhibited analgesic, antiulcerogenic, anti-inflammatory, antipyretic, antioxidant, cytotoxic, antibacterial, hypoglycemic, and immunosuppressive properties [4–12]. Achillea wilhelmsii exhibited antimycobacterial, antihypertensive abdominal pain, flatulence, and antihyperlipidemic properties [13–15]. Chemically, Achillea wilhelmsii consist of sesquiterpenoids, cineol, flavonoids, borneol, alkaloids, thujene, carvacrol, caryophyllene, monoterpenoids, camphor, rutin, pinene, and linalool [16, 17]. The parasitic and vector-borne disease, leishmaniasis, is caused by 21 out of 30 species of Leishmania, and in the entire world, this is the 6^th infectious disease predominantly in tropical and subtropical countries [18]. Leishmaniasis is classified into three forms, for instance, visceral, cutaneous, and mucocutaneous [19, 20]. In this research study, the extract and fractions of Achillea wilhelmsii are examined against leishmaniasis.

2. Materials and Methods

2.1. Plant Material

In this research study, whole plant of Achillea wilhelmsii was used.

2.2. Extraction

The plant Achillea wilhelmsii was collected from Killi Murda Karez Chaman, Balochistan, Pakistan, and identified by Prof. Dr. Rasool Bakhsh Tareen, Department of Botany, University of Balochistan, Quetta, Pakistan. 0.5 kg whole plant of Achillea wilhelmsii was dried in shade and then grinded, soaked in 2 liters of methanol, kept for 7 days, and shaken daily. After 7-day period, methanol containing whole plant of Achillea wilhelmsii was filtered and vaporized with the help of rotary evaporator. Dried semisolid whole plant methanolic extract of Achillea wilhelmsii (WMEAW) was 53.2 g. This crude extract was examined for antileishmanial activity while the remaining extract was fractionated with solvents, for instance, n-hexane and aqueous.

2.3. Fractionation of Crude Extract

In a separatory funnel, with crude extract, two solvents were added such as n-hexane and aqueous. The separatory funnel was shaken thoroughly to create two layers such as n-hexane layer and aqueous layer. Both solvents were separately vaporized with the help of rotary evaporator to form whole plant hexane fraction of Achillea wilhelmsii (WHFAW) 8.1 g and whole plant aqueous fraction of Achillea wilhelmsii (WAFAW) 17.3 g. Both fractions were examined for antileishmanial activity [21, 22].

2.4. Antileishmanial Bioassay

The extracts and fractions of medicinal plants were examined against Leishmania major (promastigotes) in culture by means of microplates. Using normal physiological saline, in bulk, promastigotes of Leishmania major (MHOM/Pk/88/DESTO) were grown in NNN biphasic medium. Then, for the culturing of promastigotes, RPMI 1640 medium (Sigma, St Louis, USA) supplemented with 10% heat-inactivated FBS (PAA Laboratory GmbH, Austria) was used. At log phase, the promastigotes were harvested, and then for 10 min at 2000 rpm, the leishmanial parasites were centrifuged. At same speed and time, the Leishmania major was washed three times with saline. Lastly, under microscope, by using Neubauer chamber, the promastigotes were counted and diluted with the addition of fresh culture medium to develop the leishmanial parasites with a final density of 10⁶ cells/mL.

In a 96-well microtitre plate, culture medium with 180 μL was added in different wells. PBS with pH 7.4 having 0.5% DMSO and 0.5% MeOH was used for the dissolution of extracts and fractions of medicinal plants to make 1000 mg/mL stock solution. The extracts and fractions with concentration of 20 μL were introduced in wells, and this was further serially diluted to develop working solution with 1–100 μg/mL range. The parasite culture with 100 μL was introduced in all wells. During this protocol, two rows were left; one row was for negative control which received medium while the other for positive control that received the standard antileishmanial drugs such as amphotericin B (Fluka) and pentamidine (ICN). The 96-well microtitre plate was incubated for 72 h between 21 and –22°C. With the help of microscope, the culture was observed for cell viability; for instance, the number of motile cells was counted on an improved Neubauer counting chamber, and with the help of software Ezfit 5.03 (Perella Scientific, USA), the IC₅₀ values of extracts and fractions with antileishmanial activities were calculated [23].

2.5. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis Triple Quadrupole Acquisition Method MS Parameters

For identification and quantification of Achillea wilhelmsii compounds: 2 μL of Achillea wilhelmsii extract or fraction was directly injected into the gas chromatograph mod. 6890N Network GC System (Agilent Technologies, Palo Alto, CA) together in the presence of mass spectrometer mod. 5973 Network Mass Selective Detector (Agilent Technologies, Palo Alto, CA) and furnished in the presence of a column HP-5MS (30 m length, 0.25 mm interior diameter, 0.25 μm film width; Agilent Technologies, Palo Alto, CA). Helium gas was off. Injection was made into a split-splitless injector (split ratio of 30 : 1) at 250°C. The oven program was the following: 70°C for 3 min then 6°C/min to 180 for 5 min, then 6°C/min to 280°C for 10 min, and then 8°C/min to 290°C for 20 min. The MSD transfer line was set at a temperature of 250°C; MSD temperature quadrupole was of 150°C and ionization temperature was 230°C. Mass spectra were seventy electrovolts, and scan achievement was accomplished in the series between 35 and 300 m/z. The identification of the components of the Achillea wilhelmsii extract or fraction was assigned by matching their mass spectra with those available in the libraries NIST 02 and WILEY [24].

3. Results and Discussion

Antileishmanial results have shown that whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW) exhibited antileishmanial activity. The inhibitory concentration (IC₅₀) which is observed for whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW) against parasitic and vector-borne disease, leishmaniasis, is 58.27 ± 0.52 μg/mL. For leishmanicidal assay, Leishmania major is the species used for analysis. In this bioassay, amphotericin B (IC₅₀: 0.29 ± 0.05 μg/mL) and pentamidine (IC₅₀: 5.09 ± 0.09 μg/mL) were used as positive control drugs to compare the parasite inhibition with that whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW). This activity was perceived under an incubation period of 72 h and an incubation temperature of 22°C. Whole plant methanolic extract of Achillea wilhelmsii (WMEAW) and whole plant aqueous fraction of Achillea wilhelmsii (WAFAW) exhibited no antileishmanial activity. The antileishmanial activities of extract and fractions of Achillea wilhelmsii are shown in Table 1.

Table 1 

Antileishmanial activities of extracts and fractions of whole plant of Achillea wilhelmsii.

Molecular formula, molecular mass, structure, and RT of compounds 1–66 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW) are shown in Tables 2–14 while mass spectra interpretation of compounds 1–66 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW) are presented in Tables 15–27. Mass spectra of compounds 1–66 are shown in Figures 1–66.

Table 2 

Molecular formula, molecular mass, structure, and RT of compounds 1–5 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 3 

Molecular formula, molecular mass, structure, and RT of compounds 6–10 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 4 

Molecular formula, molecular mass, structure, and RT of compounds 11–15 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 5 

Molecular formula, molecular mass, structure, and RT of compounds 16–20 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 6 

Molecular formula, molecular mass, structure, and RT of compounds 21–25 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 7 

Molecular formula, molecular mass, structure, and RT of compounds 26–30 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 8 

Molecular formula, molecular mass, structure, and RT of compounds 31–35 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 9 

Molecular formula, molecular mass, structure, and RT of compounds 36–40 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 10 

Molecular formula, molecular mass, structure, and RT of compounds 41–45 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 11 

Molecular formula, molecular mass, structure, and RT of compounds 46–50 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 12 

Molecular formula, molecular mass, structure, and RT of compounds 51–55 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 13 

Molecular formula, molecular mass, structure, and RT of compounds 56–60 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 14 

Molecular formula, molecular mass, structure, and RT of compounds 61–66 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 15 

Mass spectra of compounds 1–5 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 16 

Mass spectra of compounds 6–10 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 17 

Mass spectra of compounds 11–15 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 18 

Mass spectra of compounds 16–20 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 19 

Mass spectra of compounds 21–25 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 20 

Mass spectra of compounds 26–30 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 21 

Mass spectra of compounds 31–35 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 22 

Mass spectra of compounds 36–40 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 23 

Mass spectra of compounds 41–45 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 24 

Mass spectra of compounds 46–50 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 25 

Mass spectra of compounds 51–55 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 26 

Mass spectra of compounds 56–60 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Table 27 

Mass spectra of compounds 61–66 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 1 

Mass spectrum of compound eucalyptol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 2 

Mass spectrum of compound 2(3H)-furanone, 5-ethenyldihydro-5-methyl- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 3 

Mass spectrum of compound terpineol, cis-β- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 4 

Mass spectrum of compound 2-furanmethanol, 5-ethenyltetrahydro-α,α,5 trimethyl-, cis- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 5 

Mass spectrum of compound 1,6-octadien-3-ol, 3,7-dimethyl- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 6 

Mass spectrum of compound butanoic acid, 2-methyl-, 2-methylbutyl ester of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 7 

Mass spectrum of compound n-amyl isovalerate of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 8 

Mass spectrum of compound 2,2,6-trimethyl-bicyclo[4.1.0]hept-1-yl-methanol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 9 

Mass spectrum of compound isopinocarveol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 10 

Mass spectrum of compound bicyclo[2.2.1]heptane-2,5-diol, 1,7,7-trimethyl-, (2-endo,5-exo)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 11 

Mass spectrum of compound 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 12 

Mass spectrum of compound 2(10)-pinen-3-one of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 13 

Mass spectrum of compound bicyclo[2.2.1]heptan-2-ol, 1,7,7-trimethyl-, (1S-endo)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 14 

Mass spectrum of compound 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 15 

Mass spectrum of compound 3,7-octadiene-2,6-diol, 2,6-dimethyl- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 16 

Mass spectrum of compound 3-cyclohexene-1-methanol, α,α4-trimethyl- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 17 

Mass spectrum of compound 1,7-octadiene-3,6-diol, 2,6-dimethyl of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 18 

Mass spectrum of compound thymol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 19 

Mass spectrum of compound phenol, 2-methoxy-3-(2-propenyl)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 20 

Mass spectrum of compound 7-hexadecene, (Z)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 21 

Mass spectrum of compound caryophyllene of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 22 

Mass spectrum of compound 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, (E)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 23 

Mass spectrum of compound 1H-cycloprop[e]azulen-7-ol, decahydro-1,1,7-trimethyl-4-methylene-, [1ar-(1aα,4aα,7β,7aβ,7bα)]- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 24 

Mass spectrum of compound caryophyllene oxide of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 25 

Mass spectrum of compound tetracyclo[6.3.2.0(2,5).0(1,8)]tridecan-9-ol, 4,4-dimethyl- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 26 

Mass spectrum of compound oxacyclotetradecan-2-one, 14-methyl of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 27 

Mass spectrum of compound caryophyllene oxide of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 28 

Mass spectrum of compound 9-(3,3-dimethyloxiran-2-yl)-2,7-dimethylnona-2,6-dien-1-ol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 29 

Mass spectrum of compound tetradecanoic acid of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 30 

Mass spectrum of compound 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, [S-(Z)]- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 31 

Mass spectrum of compound 3,7,11,15-tetramethyl-2-hexadecen-1-ol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 32 

Mass spectrum of compound cis-9-hexadecenoic acid of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 33 

Mass spectrum of compound 2,6,10-dodecatrien-1-ol, 12-acetoxy-2,6,10-trimethyl-(E,E,E)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 34 

Mass spectrum of compound n-hexadecanoic acid of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 35 

Mass spectrum of compound cholestan-3-ol, 2-methylene-, (3β,5α)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 36 

Mass spectrum of compound 9-(3,3-dimethyloxiran-2-yl)-2,7-dimethylnona-2,6-dien-1-ol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 37 

Mass spectrum of compound trans-Z-α-bisabolene epoxide of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 38 

Mass spectrum of compound 1-hexadecanol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 39 

Mass spectrum of compound 9,12-octadecadienoic acid, methyl ester, (E,E)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 40 

Mass spectrum of compound 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 41 

Mass spectrum of compound phytol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 42 

Mass spectrum of compound 9,12-octadecadienoic acid (Z,Z)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 43 

Mass spectrum of compound 9,12,15-octadecatrienoic acid, (Z,Z,Z)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 44 

Mass spectrum of compound α-santonin of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 45 

Mass spectrum of compound limonen-6-ol, pivalate of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 46 

Mass spectrum of compound hexadecanoic acid, 1,1-dimethylethyl ester of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 47 

Mass spectrum of compound 1-eicosanol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 48 

Mass spectrum of compound tetradecane, 2,6,10-trimethyl- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 49 

Mass spectrum of compound butyl 9,12-octadecadienoate of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 50 

Mass spectrum of compound butyl 9,12,15-octadecatrienoate of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 51 

Mass spectrum of compound 1-docosene of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 52 

Mass spectrum of compound heptacosane of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 53 

Mass spectrum of compound hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 54 

Mass spectrum of compound 1,2-benzenedicarboxylic acid, diisooctyl ester of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 55 

Mass spectrum of compound 9,12-octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 56 

Mass spectrum of compound 8,11,14-eicosatrienoic acid, (Z,Z,Z)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 57 

Mass spectrum of compound 1-hexadecanol, 2-methyl- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 58 

Mass spectrum of compound dl-α-tocopherol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 59 

Mass spectrum of compound pregnan-20-one, 3-(acetyloxy)-5,6 : 16,17-diepoxy-, (3β,5α,6α,16α)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 60 

Mass spectrum of compound campesterol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 61 

Mass spectrum of compound stigmasterol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 62 

Mass spectrum of compound β-sitosterol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 63 

Mass spectrum of compound quassin of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 64 

Mass spectrum of compound 9,19-cyclolanost-24-en-3-ol, (3β)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 65 

Mass spectrum of compound 9,19-cyclolanost-24-en-3-ol, acetate, (3β)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

Figure 66 

Mass spectrum of compound ethanol, 2-(9-octadecenyloxy)-, (Z)- of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).

4. Conclusion

In this research study, whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW) exhibited antileishmanial activity and consists of 66 compounds which was analyzed by GC-MS. Whole plant methanol extract of Achillea wilhelmsii (WMEAW) and whole plant aqueous fraction of Achillea wilhelmsii (WAFAW) exhibited no antileishmanial activity. Therefore, the compounds present in the whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW) which has shown antileishmanial activity will be the drugs for the treatment of leishmaniasis with least side effects and toxicity.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

The authors are thankful to Hussain Ebrahim Jamal (HEJ), Research Institute of Chemistry, University of Karachi, Karachi, Pakistan, for providing antileishmanial, GC-MS analysis, and Institute of Biochemistry, University of Balochistan, Quetta, Pakistan, for providing lab facilities for extraction and fractionation.