In Vitro Antileishmanial Activity and GC-MS Analysis of Whole Plant Hexane Fraction of Achillea wilhelmsii (WHFAW)
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.
The genus Achillea consists of more than 100 species and belongs to the family Asteraceae, innate to Western Asia and Europe . Achillea means Achilles which in Greek language refers to hero who with the usage of yarrow treated the wounds of soldiers during battles . High mountains of the Mediterranean are the location for the occurrence of Achillea species . 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 6th infectious disease predominantly in tropical and subtropical countries . 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.
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 .
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 .
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.
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.
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.
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.
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.
S. Saeidnia, A. R. Gohari, N. Mokhber-Dezfuli, and F. Kiuchi, “A review on phytochemistry and medicinal properties of the genus Achillea,” DARU, vol. 19, no. 3, pp. 173–186, 2011.View at: Google Scholar
R. Kotan, A. Cakir, F. Dadasoglu et al., “Antibacterial activities of essential oils and extracts of Turkish Achillea, Satureja and Thymus species against plant pathogenic bacteria,” Journal of the Science of Food and Agriculture, vol. 90, no. 1, pp. 145–160, 2009.View at: Publisher Site | Google Scholar
K. Javidnia, R. Miri, and H. Sadeghpour, “Composition of the volatile oil of Achillea wilhelmsii C. koch from Iran,” DARU, vol. 12, no. 2, pp. 63–66, 2004.View at: Google Scholar
World Health Organization, Leishmaniasis, WHO, Geneva, Switzerland, 2015.
S. Kaur, H. Patel, V. Sharma, P. Garg, and Roy. N, “Leishmania major structural database,” International Journal of Integrative Biology, vol. 7, no. 2, pp. 63–68, 2009.View at: Google Scholar
B. Jehan, K. Shehla, and S. Mohammad, “Antimicrobial potentials of fresh Allium cepa against gram negative bacteria and fungi,” Pakistan Journal of Botany, vol. 45, pp. 1–6, 2013.View at: Google Scholar
J. K. Achakzai, M. A. Panezai, M. A. Kakar et al., “In vitro anticancer MCF-7, anti-inflammatory, and brine shrimp lethality assay (BSLA) and GC-MS analysis of whole plant butanol fraction of Rheum ribes (WBFRR),” BioMed Research International, vol. 2019, Article ID 3264846, 8 pages, 2019.View at: Publisher Site | Google Scholar
R. Atta-ur, M. I. Chaudhary, and W. Thompson, Bioassay Techniques for Drug, vol. 60, Harwood Academic publishers, Amsterdam, The Netherlands, 2001.
E. Ahmed El-Wakil, M. Mohamed El-Sayed, and E. E. Abdel-Lateef, “GC-MS investigation of essential oil and antioxidant activity of Egyptian white onion (Allium cepa L.),” International Journal of Pharmaceutical Sciences and Research IJPSR, vol. 6, no. 3, 2015.View at: Google Scholar