Journal of Chemistry
Volume 2019 (2019), Article ID 5734257, 26 pages
https://doi.org/10.1155/2019/5734257

Research Article

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

Jahangir Khan Achakzai,¹ Muhammad Anwar Panezai,¹ Abdul Manan Kakar,¹ Basira Akhtar,² Ali Akbar,³ Shahabuddin Kakar,⁴ Javed Khan,⁵ Nazima Yousaf Khan,¹ Ghulam Mustafa Khan,⁶ Nizam Baloch,⁶ Muhammad Hashim Jan Khoso,¹ and Marina Panezai¹

¹Institute of Biochemistry, University of Balochistan, Quetta 87300, Pakistan
²Department of Botany, University of Balochistan, Quetta 87300, Pakistan
³Department of Microbiology, University of Balochistan, Quetta 87300, Pakistan
⁴Department of Zoology, University of Balochistan, Quetta 87300, Pakistan
⁵Department of Microbiology, Quaid-i-Azam University, Islamabad 45320, Pakistan
⁶Department of Chemistry, University of Balochistan, Quetta 87300, Pakistan

Correspondence should be addressed to Muhammad Anwar Panezai; anwarpanezai@yahoo.com

Received 5 July 2019; Accepted 6 August 2019; Published 1 October 2019

Academic Editor: Gabriel Navarrete-Vazquez

Copyright © 2019 Jahangir Khan Achakzai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 6th 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.

S. no
Name of extracts/fractions/standard drugs
IC₅₀ (μg/mL) ± S.D.

1
WMEAW
>100

2
WHFAW
58.27 ± 0.52 moderate activity

3
WAFAW
>100

4
Amphotericin B
0.20 ± 0.5

5
Pentamidine
5.09 ± 0.9

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).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

1
Eucalyptol
C₁₀H₁₈O
154

7.606

2
2(3H)-Furanone, 5-ethenyldihydro-5-methyl-
C₇H₁₀O₂
126

7.833

3
Terpineol, cis-β-
C₁₀H₁₈O
154

8.528

4
2-Furanmethanol, 5-ethenyltetrahydro-α,α,5-trimethyl-, cis-
C₁₀H₁₈O₂
170

8.660

5
1,6-Octadien-3-ol, 3,7-dimethyl-
C₁₀H₁₈O
154

9.363

Table 3: Molecular formula, molecular mass, structure, and RT of compounds 6–10 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

6
Butanoic acid, 2-methyl-, 2-methylbutyl ester
C₁₀H₂₀O₂
172

9.458

7
n-Amyl isovalerate
C₁₀H₂₀O₂
172

9.568

8
2,2,6-Trimethyl-bicyclo[4.1.0]hept-1-yl-methanol
C₁₁H₂₀O
168

9.905

9
Isopinocarveol
C₁₀H₁₆O
152

10.476

10
Bicyclo[2.2.1]heptane-2,5-diol, 1,7,7-trimethyl-, (2-endo,5-exo)-
C₁₀H₁₈O₂
170

10.622

Table 4: Molecular formula, molecular mass, structure, and RT of compounds 11–15 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

11
3,6-Dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran
C₁₀H₁₆O
152

10.930

12
2(10)-Pinen-3-one
C₁₀H₁₄O
150

11.113

13
Bicyclo[2.2.1]heptan-2-ol, 1,7,7-trimethyl-, (1S-endo)-
C₁₀H₁₈O
154

11.215

14
3-Cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)-
C₁₀H₁₈O
154

11.508

15
3,7-Octadiene-2,6-diol, 2,6-dimethyl-
C₁₀H₁₈O₂
170

11.801

Table 5: Molecular formula, molecular mass, structure, and RT of compounds 16–20 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

16
3-Cyclohexene-1-methanol, α,α4-trimethyl-
C₁₀H₁₈O
170

11.867

17
1,7-Octadiene-3,6-diol, 2,6-dimethyl
C₁₀H₁₈O₂
170

14.071

18
Thymol
C₁₀H₁₄O
150

14.620

19
Phenol, 2-methoxy-3-(2-propenyl)-
C₁₀H₁₂O₂
164

16.319

20
7-Hexadecene, (Z)-
C₁₆H₃₂
224

17.124

Table 6: Molecular formula, molecular mass, structure, and RT of compounds 21–25 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

21
Caryophyllene
C₁₅H₂₄
204

17.959

22
1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, (E)-
C₁₅H₂₆O
222

21.385

23
1H-Cycloprop[e]azulen-7-ol, decahydro-1,1,7-trimethyl-4-methylene-, [1ar-(1aα,4aα,7β,7aβ,7bα)]-
C₁₅H₂₄O
220

21.803

24
Caryophyllene oxide
C₁₅H₂₄O
220

21.942

25
Tetracyclo[6.3.2.0(2,5).0(1,8)]tridecan-9-ol, 4,4-dimethyl-
C₁₅H₂₄O
220

23.070

Table 7: Molecular formula, molecular mass, structure, and RT of compounds 26–30 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

26
Oxacyclotetradecan-2-one, 14-methyl
C₁₄H₂₆O₂
226

23.465

27
Caryophyllene oxide
C₁₅H₂₄O
220

23.890

28
9-(3,3-Dimethyloxiran-2-yl)-2,7-dimethylnona-2,6-dien-1-ol
C₁₅H₂₆O₂
238

25.493

29
Tetradecanoic acid
C₁₄H₂₈O₂
228

25.793

30
1,6,10-Dodecatrien-3-ol, 3,7,11-trimethyl-, [S-(Z)]-
C₁₅H₂₆O
222

27.184

Table 8: Molecular formula, molecular mass, structure, and RT of compounds 31–35 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

31
3,7,11,15-Tetramethyl-2-hexadecen-1-ol
C₂₀H₄₀O
296

27.814

32
cis-9-Hexadecenoic acid
C₁₆H₃₀O₂
254

28.305

33
2,6,10-Dodecatrien-1-ol, 12-acetoxy-2,6,10-trimethyl-(E,E,E)-
C₁₇H₂₈O₃
280

31.570

34
n-Hexadecanoic acid
C₁₆H₃₂O₂
256

32.486

35
Cholestan-3-ol, 2-methylene-, (3β,5α)-
C₂₈H₄₈O
400

34.463

Table 9: Molecular formula, molecular mass, structure, and RT of compounds 36–40 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

36
9-(3,3-Dimethyloxiran-2-yl)-2,7-dimethylnona-2,6-dien-1-ol
C₁₅H₂₆O₂
238

36.806

37
trans-Z-α-Bisabolene epoxide
C₁₅H₂₄O
220

38.475

38
1-Hexadecanol
C₁₆H₃₄O
242

39.127

39
9,12-Octadecadienoic acid, methyl ester, (E,E)-
C₁₉H₃₄O₂
294

39.903

40
9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)-
C₁₉H₃₂O₂
292

40.349

Table 10: Molecular formula, molecular mass, structure, and RT of compounds 41–45 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

41
Phytol
C₂₀H₄₀O
296

41.118

42
9,12-Octadecadienoic acid (Z,Z)-
C₁₈H₃₂O₂
280

42.297

43
9,12,15-Octadecatrienoic acid, (Z,Z,Z)-
C₁₈H₃₀O₂
278

42.700

44
α-Santonin
C₁₅H₁₈O₃
246

43.293

45
Limonen-6-ol, pivalate
C₁₅H₂₄O₂
236

43.439

Table 11: Molecular formula, molecular mass, structure, and RT of compounds 46–50 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

46
Hexadecanoic acid, 1,1-dimethylethyl ester
C₂₀H₄₀O₂
312

44.237

47
1-Eicosanol
C₂₀H₄₂O
298

46.851

48
Tetradecane, 2,6,10-trimethyl-
C₁₇H₃₆
240

47.137

49
Butyl 9,12-octadecadienoate
C₂₂H₄₀O₂
336

48.228

50
Butyl 9,12,15-octadecatrienoate
C₂₂H₃₈O₂
334

48.360

Table 12: Molecular formula, molecular mass, structure, and RT of compounds 51–55 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

51
1-Docosene
C₂₂H₄₄
308

50.476

52
Heptacosane
C₂₇H₅₆
380

50.607

53
Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester
C₁₉H₃₈O₄
330

50.739

54
1,2-Benzenedicarboxylic acid, diisooctyl ester
C₂₄H₃₈O₄
390

51.318

55
9,12-Octadecadienoic acid (Z,Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester
C₂₁H₃₈O₄
354

53.097

Table 13: Molecular formula, molecular mass, structure, and RT of compounds 56–60 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

56
8,11,14-Eicosatrienoic acid, (Z,Z,Z)-
C₂₀H₃₄O₂
306

53.207

57
1-Hexadecanol, 2-methyl-
C₁₇H₃₆O
256

55.396

58
dl-α-Tocopherol
C₂₉H₅₀O₂
430

57.842

59
Pregnan-20-one, 3-(acetyloxy)-5,6 : 16,17-diepoxy-, (3β,5α,6α,16α)-
C₂₃H₃₂O₅
388

57.937

60
Campesterol
C₂₈H₄₈O
400

58.962

Table 14: Molecular formula, molecular mass, structure, and RT of compounds 61–66 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
Name
Molecular formula
Molecular mass
Structure
RT

61
Stigmasterol
C₂₉H₄₈O
412

59.357

62
β-Sitosterol
C₂₉H₅₀O
414

60.126

63
Quassin
C₂₂H₂₈O₆
388

60.338

64
9,19-Cyclolanost-24-en-3-ol, (3β)-
C₃₀H₅₀O
426

61.195

65
9,19-Cyclolanost-24-en-3-ol, acetate, (3β)-
C₃₂H₅₂O₂
468

62.908

66
Ethanol, 2-(9-octadecenyloxy)-, (Z)-
C₂₀H₄₀O₂
312

63.626

Table 15: Mass spectra of compounds 1–5 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

1
154(M⁺), 111(389), 108(482), 93(503), 84(443), 81(727), 71(506), 69(455), 55(403), 43(999), 41(320)

2
126(M⁺), 111(999), 99(255), 98(210), 83(220), 71(410), 67(320), 56(310), 55(620), 43(510), 27(255)

3
154(M⁺), 111(183), 93(316), 81(230), 79(171), 71(534), 69(240), 67(142), 55(335), 43(999), 41(500)

4
170(M⁺), 94(224), 69(201), 68(330), 67(266), 59(999), 55(346), 43(668), 41(523), 39(233), 27(270)

5
154(M⁺), 121(232), 93(760), 80(321), 71(999), 69(426), 67(206), 55(637), 43(598), 41(564), 39(179)

Table 16: Mass spectra of compounds 6–10 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

6
172(M⁺), 103(133), 85(705), 71(224), 70(999), 57(818), 55(227), 43(427), 42(123), 41(348), 29(311)

7
172(M⁺), 85(999), 71(263), 70(884), 57(626), 55(262), 43(589), 42(175), 41(349), 29(234), 27(154)

8
168(M⁺), 95(700), 85(749), 84(849), 83(509), 81(794), 79(577), 69(842), 55(728), 43(550), 41(999)

9
152(M⁺), 92(708), 91(543), 83(624), 79(350), 70(797), 69(561), 55(859), 41(999), 39(396), 27(336)

10
170(M⁺), 108(722), 95(379), 93(669), 49(467), 67(561), 55(627), 43(450), 41(999), 39(596), 27(509)

Table 17: Mass spectra of compounds 11–15 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

11
152(M⁺), 137(999), 109(311), 95(129), 93(155), 79(156), 69(342), 67(131), 55(159), 41(239), 29(118)

12
150(M⁺, 299), 108(759), 107(398), 81(999), 79(335), 69(424), 53(970), 41(673), 39(319), 27(307)

13
154(M⁺), 110(196), 95(999), 93(111), 67(92), 55(150), 43(176), 41(323), 39(198), 29(160), 27(213)

14
154(M⁺), 111(494), 93(505), 86(247), 71(999), 69(226), 67(184), 55(265), 43(544), 41(356), 27(175)

15
170(M⁺), 85(80), 83(82), 82(999), 71(634), 67(365), 55(113), 43(572), 41(144), 39(60), 27(102)

Table 18: Mass spectra of compounds 16–20 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

16
170(M⁺), 136(462), 121(487), 95(125), 93(602), 92(165), 81(331), 67(166), 59(999), 43(314), 41(155)

17
170(M⁺), 82(526), 71(851), 69(172), 68(375), 67(999), 55(410), 43(864), 41(406), 39(176), 27(246)

18
150(M⁺, 248), 136(89), 135(999), 117(87), 115(91), 91(157), 77(80), 51(56), 41(55), 39(94)

19
164(M⁺, 999), 149(415), 137(210), 131(279), 121(245), 104(223), 103(344), 91(280), 77(378), 55(271)

20
224(M⁺), 97(360), 83(517), 70(446), 69(628), 57(572), 56(547), 55(999), 43(664), 41(569), 29(242)

Table 19: Mass spectra of compounds 21–25 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

21
204(M⁺), 133(646), 107(389), 105(372), 93(937), 91(551), 81(389), 79(614), 69(976), 55(432), 41(999)

22
222(M⁺), 107(275), 93(560), 81(241), 79(192), 71(370), 69(999), 67(249), 55(272), 43(410), 41(586)

23
220(M⁺), 205(440),159(365), 119(524), 105(555), 93(449), 91(647), 79(379), 55(343), 43(999), 41(709)

24
220(M⁺), 95(420), 93(661), 91(573), 81(373), 79(885), 69(407), 67(377), 55(393), 43(999), 41(927)

25
220(M⁺), 136(999), 135(148), 105(132), 93(130), 91(260), 79(217), 69(190), 67(163), 55(174), 41(508)

Table 20: Mass spectra of compounds 26–30 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

26
226(M⁺), 98(479), 97(375), 84(327), 83(448), 69(601), 56(376), 55(999), 43(491), 41(908), 29(354)

27
220(M⁺), 95(420), 93(661), 91(573), 81(373), 79(885), 69(407), 67(377), 55(393), 43(999), 41(927)

28
238(M⁺), 93(372), 85(316), 81(999), 71(623), 69(366), 67(292), 55(375), 43(919), 41(727), 29(314)

29
228(M⁺), 185(292), 129(512), 73(999), 71(336), 69(353), 60(904), 57(652), 55(707), 43(721), 41(677)

30
222(M⁺), 107(221), 93(402), 81(207), 71(384), 69(798), 67(242), 55(283), 53(153), 43(669), 41(999)

Table 21: Mass spectra of compounds 31–35 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

31
296(M⁺), 123(892), 95(962), 82(986), 81(999), 71(748), 68(728), 57(811), 55(852), 43(965), 41(811)

32
254(M⁺), 97(376), 96(251), 82(292), 83(461), 69(648), 67(290), 56(305), 55(999), 43(374), 41(587)

33
280(M⁺), 107(176), 95(228), 94(138), 93(341), 81(186), 69(259), 68(314), 67(154), 43(999), 41(213)

34
256(M⁺), 129(435), 83(267), 73(980), 71(373), 69(351), 60(999), 57(840), 55(767), 43(817), 41(574)

35
400(M⁺), 105(344), 97(344), 95(666), 93(390), 83(528), 81(735), 79(413), 71(689), 69(999), 67(632),

Table 22: Mass spectra of compounds 36–40 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

36
238(M⁺), 93(372), 85(316), 81(999), 71(623), 69(366), 67(292), 55(375), 43(919), 41(727), 29(314)

37
220(M⁺), 109(320), 107(263), 93(291), 91(247), 79(234), 67(295), 57(311), 55(431), 43(999), 41(901)

38
242(M⁺), 97(573), 83(760), 82(394), 70(425), 69(822), 57(653), 56(410), 55(999), 43(710), 41(758),

39
294(M⁺), 95(450), 82(450), 81(670), 79(330), 69(350), 68(400), 67(999), 55(720), 54(380), 41(910)

40
292(M⁺), 180(270), 95(400), 93(470), 81(430), 80(420), 79(999), 67(780), 55(570), 43(310), 41(610)

Table 23: Mass spectra of compounds 41–45 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

41
296(M⁺), 123(184), 81(223), 71(999), 69(239), 68(199), 57(334), 56(169), 55(259), 43(381), 41(260)

42
280(M⁺), 96(388), 95(617), 82(516), 81(827), 79(430), 68(437), 67(999), 55(680), 54(429), 41(523)

43
278(M⁺), 108(391), 95(478), 93(503), 91(299), 81(373), 80(460), 79(999), 67(624), 55(463), 41(408)

44
246(M⁺, 667), 173(999), 172(429), 135(559), 91(793), 77(544), 69(491), 55(499), 45(351), 41(461)

45
236(M⁺), 119(175), 109(214), 107(236), 93(398), 91(209), 85(206), 57(999), 55(287), 43(403), 41(493)

Table 24: Mass spectra of compounds 46–50 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

46
312(M⁺), 73(303), 69(191), 61(153), 60(278), 57(622), 56(999), 55(317), 43(526), 41(444), 29(316)

47
298(M⁺), 97(542), 83(620), 82(350), 71(330), 69(617), 57(743), 56(358), 55(893), 43(999), 41(799)

48
240(M⁺), 155(134), 85(500), 84(179), 71(641), 69(147), 57(999), 56(256), 55(282), 43(782), 41(384)

49
336(M⁺), 109(379), 96(439), 95(699), 82(489), 81(949), 79(409), 69(349), 68(389), 67(999), 55(669)

50
334(M⁺), 121(299), 108(469), 95(679), 93(559), 91(309), 81(499), 80(409), 79(999), 67(689), 55(509)

Table 25: Mass spectra of compounds 51–55 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

51
308(M⁺), 97(622), 83(617), 71(406), 70(362), 69(687), 57(902), 56(437), 55(999), 43(967), 41(859)

52
380(M⁺), 99(140), 85(416), 71(622), 69(176), 57(999), 56(139), 55(283), 43(798), 41(275), 29(135)

53
330(M⁺), 239(380), 98(710), 84(480), 74(630), 69(390), 57(850), 55(690), 43(999), 41(740), 29(480)

54
390(M⁺), 167(350), 150(107), 149(999), 83(100), 71(224), 70(264), 57(341), 55(218), 43(200), 41(225)

55
354(M⁺), 262(390), 95(490), 82(370), 81(840), 79(420), 69(390), 67(999), 55(780), 54(380), 41(690)

Table 26: Mass spectra of compounds 56–60 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

56
306(M⁺), 93(470), 91(380), 81(630), 80(750), 79(780), 67(960), 57(400), 55(810), 43(810), 41(999)

57
256(M⁺), 97(386), 83(460), 71(514), 70(341), 69(580), 57(999), 56(572), 55(670), 43(888), 41(633)

58
431(M + 1], 84), 430(M⁺], 261), 205(98), 166(114), 165(999), 164(322), 57(95), 55(77), 43(165), 41(84)

59
388(M⁺), 328(999), 105(500), 95(378), 93(469), 91(730), 81(348), 79(545), 77(505), 55(580), 53(405)

60
400(M⁺), 145(285), 107(403), 105(306), 95(414), 81(436), 71(312), 57(452), 55(617), 43(999), 41(470)

Table 27: Mass spectra of compounds 61–66 of whole plant n-hexane fraction of Achillea wilhelmsii (WHFAW).

Compound
m/z (% relative abundance)

61
412(M⁺), 159(418), 133(444), 105(427), 95(398), 91(427), 83(692), 81(638), 69(636), 55(999), 43(431)

62
414(M⁺), 107(378), 105(392), 95(387), 91(377), 81(427), 69(348), 57(447), 55(639), 43(999), 41(561)

63
388(M⁺), 302(346), 165(310), 152(642), 127(369), 91(399), 77(298), 69(806), 55(510), 43(999), 41(735)

64
426(M⁺), 135(320), 121(350), 109(440), 107(400), 95(430), 93(370), 81(440), 69(999), 55(549), 41(579)

65
468(M⁺), 121(278), 109(324), 107(311), 95(456), 93(260), 81(316), 69(752), 55(485), 43(999), 41(572)

66
312(M⁺), 96(708), 95(546), 83(670), 82(968), 81(627), 69(770), 67(664), 55(999), 43(577), 41(621)

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.

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