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

Authors: Jahangir Khan Achakzai; Muhammad Anwar Panezai; Abdul Manan Kakar; Basira Akhtar; Ali Akbar; Shahabuddin Kakar; Javed Khan; Nazima Yousaf Khan; Ghulam Mustafa Khan; Nizam Baloch; Muhammad Hashim Jan Khoso; Marina Panezai
Journal: Journal of Chemistry
 (2019)

---

## Abstract

In this study, GC-MS analysis has shown that whole plant hexane fraction ofAchillea 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.


---

## Body

## 1. Introduction

The genusAchillea 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 ofAchillea wilhelmsii was used.


### 2.2. Extraction

The plantAchillea 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 asn-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 againstLeishmania 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 180μL 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 ofAchillea 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].


## 2.1. Plant Material

In this research study, whole plant ofAchillea wilhelmsii was used.


## 2.2. Extraction

The plantAchillea 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 asn-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 againstLeishmania 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 180μL 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 ofAchillea 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 plantn-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 ofAchillea 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 ± 0.9Molecular formula, molecular mass, structure, and RT of compounds 1–66 of whole plantn-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 plantn-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
9.363Table 3
Molecular formula, molecular mass, structure, and RT of compounds 6–10 of whole plantn-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
10.622Table 4
Molecular formula, molecular mass, structure, and RT of compounds 11–15 of whole plantn-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
11.801Table 5
Molecular formula, molecular mass, structure, and RT of compounds 16–20 of whole plantn-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
17.124Table 6
Molecular formula, molecular mass, structure, and RT of compounds 21–25 of whole plantn-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
23.070Table 7
Molecular formula, molecular mass, structure, and RT of compounds 26–30 of whole plantn-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
27.184Table 8
Molecular formula, molecular mass, structure, and RT of compounds 31–35 of whole plantn-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
34.463Table 9
Molecular formula, molecular mass, structure, and RT of compounds 36–40 of whole plantn-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
40.349Table 10
Molecular formula, molecular mass, structure, and RT of compounds 41–45 of whole plantn-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
43.439Table 11
Molecular formula, molecular mass, structure, and RT of compounds 46–50 of whole plantn-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
48.360Table 12
Molecular formula, molecular mass, structure, and RT of compounds 51–55 of whole plantn-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
53.097Table 13
Molecular formula, molecular mass, structure, and RT of compounds 56–60 of whole plantn-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
58.962Table 14
Molecular formula, molecular mass, structure, and RT of compounds 61–66 of whole plantn-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
63.626Table 15
Mass spectra of compounds 1–5 of whole plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 plantn-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 ofAchillea wilhelmsii (WHFAW).Figure 2
Mass spectrum of compound 2(3H)-furanone, 5-ethenyldihydro-5-methyl- of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 6
Mass spectrum of compound butanoic acid, 2-methyl-, 2-methylbutyl ester of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 7
Mass spectrum of compoundn-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 ofAchillea wilhelmsii (WHFAW).Figure 9
Mass spectrum of compound isopinocarveol of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 11
Mass spectrum of compound 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 12
Mass spectrum of compound 2(10)-pinen-3-one of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 14
Mass spectrum of compound 3-cyclohexen-1-ol, 4-methyl-1-(1-methylethyl)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 15
Mass spectrum of compound 3,7-octadiene-2,6-diol, 2,6-dimethyl- of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 18
Mass spectrum of compound thymol of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 19
Mass spectrum of compound phenol, 2-methoxy-3-(2-propenyl)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 20
Mass spectrum of compound 7-hexadecene, (Z)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 21
Mass spectrum of compound caryophyllene of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 22
Mass spectrum of compound 1,6,10-dodecatrien-3-ol, 3,7,11-trimethyl-, (E)- of whole plant hexane fraction ofAchillea 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 ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 26
Mass spectrum of compound oxacyclotetradecan-2-one, 14-methyl of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 27
Mass spectrum of compound caryophyllene oxide of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 29
Mass spectrum of compound tetradecanoic acid of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 31
Mass spectrum of compound 3,7,11,15-tetramethyl-2-hexadecen-1-ol of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 32
Mass spectrum of compound cis-9-hexadecenoic acid of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 34
Mass spectrum of compoundn-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 ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 39
Mass spectrum of compound 9,12-octadecadienoic acid, methyl ester, (E,E)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 40
Mass spectrum of compound 9,12,15-octadecatrienoic acid, methyl ester, (Z,Z,Z)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 41
Mass spectrum of compound phytol of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 42
Mass spectrum of compound 9,12-octadecadienoic acid (Z,Z)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 43
Mass spectrum of compound 9,12,15-octadecatrienoic acid, (Z,Z,Z)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 44
Mass spectrum of compoundα-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 ofAchillea wilhelmsii (WHFAW).Figure 46
Mass spectrum of compound hexadecanoic acid, 1,1-dimethylethyl ester of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 47
Mass spectrum of compound 1-eicosanol of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 48
Mass spectrum of compound tetradecane, 2,6,10-trimethyl- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 49
Mass spectrum of compound butyl 9,12-octadecadienoate of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 50
Mass spectrum of compound butyl 9,12,15-octadecatrienoate of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 51
Mass spectrum of compound 1-docosene of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 52
Mass spectrum of compound heptacosane of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 53
Mass spectrum of compound hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl)ethyl ester of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 54
Mass spectrum of compound 1,2-benzenedicarboxylic acid, diisooctyl ester of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 56
Mass spectrum of compound 8,11,14-eicosatrienoic acid, (Z,Z,Z)- of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 57
Mass spectrum of compound 1-hexadecanol, 2-methyl- of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).Figure 61
Mass spectrum of compound stigmasterol of whole plant hexane fraction ofAchillea wilhelmsii (WHFAW).Figure 62
Mass spectrum of compoundβ-sitosterol of whole plant hexane fraction of Achillea wilhelmsii (WHFAW).Figure 63
Mass spectrum of compound quassin of whole plant hexane fraction ofAchillea 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 ofAchillea wilhelmsii (WHFAW).


## 4. Conclusion

In this research study, whole plantn-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.



---

*Source: 5734257-2019-10-01.xml*