# Essential Oil ofArtemisia annua L.: An Extraordinary Component with Numerous Antimicrobial Properties

Authors: Anna Rita Bilia; Francesca Santomauro; Cristiana Sacco; Maria Camilla Bergonzi; Rosa Donato
Journal: Evidence-Based Complementary and Alternative Medicine
 (2014)

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## Abstract

Artemisia annua L. (Asteraceae) is native to China, now naturalised in many other countries, well known as the source of the unique sesquiterpene endoperoxide lactone artemisinin, and used in the treatment of the chloroquine-resistant and cerebral malaria. The essential oil is rich in mono- and sesquiterpenes and represents a by-product with medicinal properties. Besides significant variations in its percentage and composition have been reported (major constituents can be camphor (up to 48%), germacrene D (up to 18.9%), artemisia ketone (up to 68%), and 1,8 cineole (up to 51.5%)), the oil has been subjected to numerous studies supporting exciting antibacterial and antifungal activities. Both gram-positive bacteria (Enterococcus, Streptococcus, Staphylococcus, Bacillus, and Listeria spp.), and gram-negative bacteria (Escherichia, Shigella, Salmonella, Haemophilus, Klebsiella, and Pseudomonas spp.) and other microorganisms (Candida, Saccharomyces, and Aspergillus spp.) have been investigated. However, the experimental studies performed to date used different methods and diverse microorganisms; as a consequence, a comparative analysis on a quantitative basis is very difficult. The aim of this review is to sum up data on antimicrobial activity of A. annua essential oil and its major components to facilitate future approach of microbiological studies in this field.


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## Body

## 1. Introduction

Artemisia annua L., a plant belonging to the Asteraceae family, is an annual herb native to China and it grows naturally as a part of steppe vegetation in northern parts of Chatar and Suiyan province in China at 1,000–1,500 m above sea level. This plant can grow up to 2.4 m tall. The stem is cylindrical and branched. Leaves are alternate, dark green, or brownish green. Odour is characteristic and aromatic while the taste is bitter. It is characterized by large panicles of small globulous capitulums (2-3 mm diameter), with whitish involucres, and by pinnatisect leaves which disappear after the blooming period, characterised by small (1-2 mm) pale yellow flowers having a pleasant odour (Figure 1). The Chinese name of the plant is Qinghao (or Qing Hao or Ching-hao which means green herb). Other names are wormwood, Chinese wormwood, sweet wormwood, annual wormwood, annual sagewort, annual mugwort, and sweet sagewort. In the USA, it is well known as sweet Annie because after its introduction in the nineteenth century it was used as a preservative and flavouring and its aromatic wreath made a nice addition to potpourris and sachets for linens and the essential oil obtained from the flowering tops is used in the flavouring of vermouth [1]. The plant is now naturalised in many other countries such as Australia, Argentina, Brazil, Bulgaria, France, Hungary, Italy, Spain, Romania, the United States, and the former Yugoslavia [2].Figure 1
Picture ofA. annua flowers (from http://upload.wikimedia.org/wikipedia/commons/5/59/Artemisia_annua_detail.jpeg).Due to the presence of the unique sesquiterpene endoperoxide lactone artemisinin (Qinghaosu), one of the most important plant-derived drug in the treatment of the chloroquine-resistant and cerebral malarias, the plant is cropped on a large scale in China, Vietnam, Turkey, Iran, Afghanistan, and Australia. In India, it is cultivated on an experimental basis in the Himalayan regions, as well as temperate and subtropical conditions [3].The essential oil which is rich in mono- and sesquiterpenes represents another source of potential commercial value [4]. Besides significant variations in its percentage and composition have been reported, it has been successfully subjected to numerous studies which mainly concern the antibacterial and antifungal activities. Diverse experimental studies have been reported to date using different methods and testing different microorganisms; therefore, a comparative analysis on a quantitative basis is very difficult. The aim of our review is to sum up data on antimicrobial activity ofA. annua volatiles and its major components to facilitate future approach of microbiological experimental in this field.


## 2. Plant Distribution and Yield of the Volatiles

Essential (volatile) oil ofA. annua can reach yields of 85 kg/ha. It is synthesised by secretory cells, especially of the uppermost foliar portion of the plant (top 1/3 of growth at maturity) which contains almost double number if compared with the lower leaves. It is reported that 35% of the mature leaf surface is covered with capitate glands which contain the terpenoidic volatile constituents. Essential oil fromA. annua is distributed, with 36% of the total from the upper third of the foliage, 47% from the middle third, and 17% from the lower third, with only trace amounts in the main stem side shoots and roots. The yield of the oil generally ranges between 0.3 and 0.4% but it can reach 4.0% (V/W) from selected genotypes. Several studies have permitted the conclusion thatA. annua crop could be harvested much before onset of flowering for obtaining high yields of artemisinin and the crop must be allowed to attain maturity to obtain high yields of the essential oil [5, 6].Yield (herbage and essential oil content) can be increased with added nitrogen and the greatest growth was obtained with 67 kg N/ha. Increasing density of plants tended to increase essential oil production on an area basis, but the highest essential oil yields (85 kg oil/ha) were achieved by the intermediate density at 55,555 plants/ha receiving 67 kg N/ha. Finally the planting date and harvest time can influence the maximum concentration of the produced essential oil [6].


## 3. Chemical Profile of the Essential Oil

The essential oil, generally obtained by hydrodistillation of the flowering tops, analysed with GC-MS, revealed a great variability both in the qualitative and quantitative composition.Chemical profile is generally influenced by the harvesting season, fertilizer and the pH of soils, the choice and stage of drying conditions, the geographic location, chemotype or subspecies, and choice of part plant or genotype or extraction method. In Table1, the main constituents (>4%) of the investigated samples are reported.Table 1
Compounds (>4%) isolated from essential oil ofArtemisia annua L.
Compound
Country
%
Reference
Artemisia alcohol
China (Cult)
7.5
[20]
USA-CA
5.2
[21]
Serbia
4.8
[10]
Artemisia ketone
Not stated
38.0
[22]
France
52.5
[23]
Serbia
35.7
[10]
Egypt
13.9
[15]
China
2.21
[11]
Bosnia
30.7
[9]
USA-CA
35.7
[24]
China (Cult)
63.9
[20]
USA-IN
68.5
[25]
England
61.0
[26]
Vietnam
0.1–4.4
[27]
Indian (Cult)
58.8
[28]
India (Cult)
11.5
[29]
Turkey
22
[19]
Borneol
Not stated
20.0
[22]
England
7.0
[26]
China (Cult)
15.9
[30]
Camphene
Iran
7
[13]
Camphene hydrate
USA-IN
12.0
[25]
Camphor
Vietnam
21.8
[3]
Serbia
4.2
[10]
Egypt
5.08
[15]
France
27.5
[23]
China (Cult)
21.8
[20]
Vietnam (Cult)
3.3
[20]
Bosnia
15.8
[9]
Iran
1.92
[14]
Italy
17.6
[5]
Indian (Cult)
15.75
[28]
India (Cult)
8.4
[29]
France
43.5
[12]
Iran
48
[13]
Turkey
31
[19]
Trans-Cariophyllene
Egypt
7.73
[15]
β-Caryo phyllene
Italy
9.0
[5]
Vietnam (Cult)
5.6
[20]
Vietnam
3.3–8.6
[27]
China (Cult)
5.98
[30]
India (Cult)
12.2
[29]
France
8.9
[12]
Caryophyllene oxide
China
5.13
[11]
Chrysanthenone
Vietnam
1.1–7.3
[27]
India (Cult)
10.19
[28]
1,8-Cineol
France
11.66
[23]
Serbia
5.5
[10]
Egypt
8.13
[15]
Bosnia
4.8
[9]
USA-IN
22.8
[25]
USA-CA
31.5
[21]
Iran
9.4
[13]
Iran
11.4
[14]
Turkey
10
[19]
β-Farnesene
Italy
10.2
[5]
Vietnam
1.1–12.8
[27]
Egypt
5.32
[15]
China (Cult)
12.9
[30]
Germacrene D
Vietnam (Cult)
18.3
[20]
Italy
21.2
[5]
Vietnam
0.3–18.9
[27]
China (Cult)
10.9
[30]
France
15.6
[12]
α-Guaiene
China (Cult)
4.7
[20]
Linalool
Vietnam
0.1–4.2
[27]
Iran
8.1
[14]
Linalool acetate
England
10.0
[22]
Myrcene
China (Cult)
5.1
[20]
USA-CA
4.6
[21]
Vietnam
0.1–8.5
[27]
α-Pinene
USA-CA
11.2
[21]
USA-IN
16.0
[25]
Serbia
16.5
[10]
(Trans)-Pinocarveol
France
10.9
[12]
Serbia
4.8
[10]
Sabinene
France
9.4
[12]
Spathulenol
Iran
4.97
[14]
Iran
4.9
[13]Analysis ofA. annua essential oils revealed the presence of mainly monoterpenoids and sesquiterpenes and the profiles showed great differences in the three main components, artemisia ketone, 1,8-cineole, and camphor, depending on the global phytogeographic origin. Oils can be grouped into the following:(i)
Vietnamese oil with 3.3–21.8% camphor and 0.3–18.9% germacrene D,(ii)
Chinese oil with high content of artemisia ketone (64%),(iii)
Indian oil with 11.5–58.8% of artemisia ketone,(iv)
French oil with 2.8–55% artemisia ketone, 1.2–11.6% 1,8-cineole, and 15% germacrene D,(v)
North American oil with 35.7–68% artemisia ketone and 22.8–31.5% 1,8-cineole,(vi)
Iranian oil with 48% camphor and 9.4% 1,8-cineole.The presence of volatile oil is also reported in fruits and roots. Sesquiterpenes are the most abundant chemicals identified in the essential oil of the fruits; in particular, caryophyllene oxide (9.0%), caryophyllene (6.9%), (E)-β-farnesene (8.2%), and germacrene D (4.0%) are identified. However, only 52% of the total components were identified [7].Upon hydrodistillation, the dried roots ofArtemisia annua L. cultivar Jwarharti, a pleasantly fragrant essential oil, have been obtained with a yield of 0.25%. The oil was rich in sesquiterpenes and oxygenated sesquiterpenes and hadcis-arteannuic alcohol (25.9%), (E)-β-farnesene (6.7%), β-maaliene (6.3%), β-caryophyllene (5.5%), caryophyllene oxide (4.4%), and 2-phenylbenzaldehyde (3.5%) as its major components [8].Recently, the analysis of aromatic waters, obtained from plants collected at full blooming, showed the presence, among others, of camphor (27.7%), 1,8-cineole (14%), artemisia ketone (10.1%),α-terpineol (6.1%),trans-pinocarveol (5.4%), and artemisia alcohol (2%). From plants at the preflowering stage, aromatic waters gave camphor (30.7%), 1,8-cineole (12.8%), artemisia alcohol (11.4%), artemisia ketone (9.5%), alpha-terpineol (5.8%), andtrans-pinocarveol (3.0%) as the main constituents. The qualitative and quantitative profiles of the two aromatic waters were similar [5].


## 4. Antimicrobial Activities of the Essential Oils

The essential oil ofArtemisia annua has been the subject of numerous studies to test the antibacterial and antifungal activity. Tests were carried out both on the whole oil (Table 2) and on its principal components such as camphor, 1,8-cineol, α-pinene, and artemisia ketone (Table 3). The main gram-positive bacteria tested withA. annua volatiles obtained by hydrodistillation wereStaphylococcus aureus [9–14],Enterococcus hirae [12],Enterococcus faecalis [14],Streptococcus pneumoniae,Micrococcus luteus [9],Bacillus cereus [14],Sarcina lutea [10],Bacillus subtilis [9, 11],Bacillus thuringiensis [11],Bacillus spp. [14], andListeria innocua [15]. The gram-negativeEscherichia coli [9, 11–14],Escherichia coli UPEC-Uropathogenic [14],Escherichia coli ETEC-Enterotoxigenic [16],Escherichia coli EPEC-Enteropathogenic [16],Escherichia coli EIEC-Enteroinvasive [16],Escherichia coli STEC-Shiga-toxin producer [16],Shigella sp. [10],Salmonella enteritidis [10],Klebsiella pneumoniae [10],Haemophilus influenzae [9], andPseudomonas aeruginosa [9, 13, 14] were tested. Some strains of yeasts includingCandida albicans [10, 12, 13],Candida krusei [9], andSaccharomyces cerevisiae [12, 13] and molds likeAspergillus fumigatus [10] were also tested (Table 2).Table 2
Tests carried out on the whole oil.
Bacterial strains
Agar disk diffusion
Concentration
Reference
MIC
mg/mL
Reference
MBC
mg/mL
Reference
Gram-positive
S. aureus
Not active
[12]
Active
32
[13]
Active
5.00%
[11]
Active
0.0156–0.0313
[11]
Active
10 mg/mL
[9]
Active
5.0–10.0
[10]
Low activity
>20.0–10.0
[10]
Active
10 mg/mL
[14]
Active
0.031
[14]
Active
0.031
[14]
E. hirae
Active
0.1 mg/mL
[12]
E. faecalis
Active
[9]
Active
10 mg/mL
[14]
Active
0.026
[14]
Active
0.031
[14]
S. pneumoniae
Active
10 mg/mL
[9]
M. luteus
Not active
[9]
B. cereus
Active
10 mg/mL
[14]
Active
0.053
[14]
Active
0.055
[14]
Low  activity
20
[10]
Low activity
20
[10]
B. subtilis
Active
5.00%
[11]
Active
0.00781-0.00781
[11]
Active
10 mg/mL
[9]
B.  thuringensis
Active
5.00%
[11]
Active
0.0313–0.0156
[11]
B.sp.
Active
10 mg/mL
[14]
Active
0.026
[14]
Active
0.053
[14]
L. innocua
Not active
[15]
Sarcina lutea
Active
2.5
[10]
Active
2.5
[10]
Gram-negative
E. coli
Not active
[12]
Active
64
[13]
Active
5.00%
[11]
Active
0.0313-0.0313
[11]
Active
10 mg/mL
[9]
Low  activity
20
[10]
Low activity
20
[10]
Active
5 mg/mL
[14]
Active
0.017
[14]
Active
0.024
[14]
UPEC
Active
5 mg/mL
[14]
Active
0.026
[14]
Active
0.031
[14]
Shigellasp.
Low  activity
20
[10]
Low activity
20
[10]
S. enteritidis
Active
5
[10]
Low activity
20
[10]
K. pneumoniae
Low  activity
20
[10]
Low activity
20
[10]
H. influenzae
Active
10 mg/mL
[9]
P. aeruginosa
Not active
[13]
Active
10 mg/mL
[9]
Active
10 mg/mL
[14]
Active
0.025
[14]
Active
0.053
[14]
Fungal strains
Agar disk diffusion
Concentration
Reference
MIC
mg/mL
Reference
MFC
mg/mL
Reference
C. albicans
Active
0.2 mg/mL
[12]
Active
2
[13]
Low  activity
20
[10]
Not active
>20
[10]
C. krusei
Active
10 mg/mL
[9]
S. cerevisiae
Active
0.2 mg/mL
[12]
Active
2
[13]
A. fumigatus
Active
5
[10]
Active
5
[10]Table 3
Tests on the main components ofA. annua essential oil.
Artemisia ketone
α-Pinene
1,8-Cineole
Camphor
Reference
α-Terpineol
Reference
MIC(mg/mL)
MBC(mg/mL)
MIC(mg/mL)
MBC(mg/mL)
MIC(mg/mL)
MBC(mg/mL)
MIC(mg/mL)
MBC(mg/mL)
MIC(%, v/v)
MBC(%, v/v)
Bacterial strains
Gram- positive
S. aureus
0.07–0.15
0.3–0.6
>10
>10
2.5–5
2.5–5
2.5–5
2.5–>10
[10]
Sarcina lutea
2.5
10
1.25
2.5
0.6
1.25
2.5
2.5
[10]
B. cereus
0.6
0.6
>10
>10
20
20
10
10
[10]
Gram- negative
E. coli
10
10
>10
>10
20
20
>10
>10
[10]
Shigellasp.
0.6
0.6
>10
>10
10
20
>10
>10
[10]
S.  enteritidis
0.6
10
0.6
5
5
10
>10
>10
[10]
K. pneumoniae
2.5
2.5
>10
>10
5
5
1.25
5
[10]
Fungal strains
C. albicans
10
10
>10
>10
5
20
5
5
[10]
0.25
0.5
[18]
C. glabrata
0.12
0.5
[18]
C. dubliniensis
0.12
0.25
[18]
C. krusei
0.12
0.5
[18]
C. guillermondii
0.12
0.25
[18]
C. parapsilosis
0.06
0.5
[18]
C. tropicalis
0.5
0.5
[18]
A. fumigatus
2.5
2.5
5
5
1.25
2.5
2.5
2.5
[10]The main gram-positive bacteria tested with methanol, chloroform, ethanol, hexane, and petroleum ether extracts ofA. annua wereStaphylococcus aureus [14, 17],Enterococcus faecalis [14],Micrococcus luteus [17],Bacillus cereus [14, 17],Bacillus subtilis [17],Bacillus pumilus [17], andBacillus sp. [14]. The gram-negativeEscherichia coli [14, 17],Escherichia coli UPEC [14],Salmonella typhi [14, 17], andPseudomonas aeruginosa [14, 17] were tested.In addition, several single main components were investigated (Table3), including α-terpineol [18] tested onC. albicans,C. glabrata,C. dubliniensis,C. guilliermondii,C. krusei,C. parapsilosis, andC. tropicalis; artemisia ketone, α-pinene, 1,8-cineole, and camphor [10] tested onC. albicans,B. cereus,S. aureus,S. lutea,E. coli,K. pneumoniae,Ps. aeruginosa,S. enteritidis,Shigella sp., andA. fumigatus.The antifungal activity of the essential oil was also evaluated against economically important foliar and soil-borne fungal pathogens of tomato. The essential oil was active againstSclerotinia sclerotiorum,Botrytis cinerea,Phytophthora infestans, andVerticillim dahliae [19].Different methods were used to evaluate the antibacterial and antifungal properties and included agar disk diffusion method [9, 11, 14, 17], minimal inhibition concentration (MIC) [9, 10, 12–14, 16–18], minimal bacterial concentration (MBC) [10, 14], and minimal fungicidal concentration (MFC) [10, 18] as reported in Table 2.The results related to agar disk diffusion method (Table2) show that some important pathogens are sensitive toA. annua essential oil obtained by hydrodistillation.S. aureus,S. pneumoniae,E. coli, UPEC,H. influenzae,P. aeruginosa,C. albicans, andC. krusei were inhibited by the action of the oil.H. influenzae,S. pneumoniae, andC. krusei were more sensitive; their inhibition zones diameters were >60, 50, and 30 mm, respectively. Satisfactory results were also achieved with genusBacillus. On the contrary,M. luteus andL. innocua were resistant to this essential oil. Since the use of agar disk diffusion method is limited by the hydrophobic nature of most essential oils and plant extracts components that prevents their uniform diffusion through the agar medium, the most authors report the results obtained with MIC and MBC methods.However, from the literature it is observed that the results obtained by agar disk diffusion method were confirmed by the liquid medium methods (MBC and MIC). At present there is no complete agreement on the concentration of the extracts to be considered active or inactive. Duarte and coworkers [16] proposed a classification to be applied to the extracts based on MIC values; this author considers MIC up to 500 μg/mL as strong inhibitors, MIC between 600 and 1500 μg/mL as moderate inhibitors, and MIC above 1600 μg/mL as weak inhibitors. In recent years, many different microbial species of medical interest have been tested from which emerged encouraging results except in the case ofE. coli with special pathogenic characters (ETEC, EPEC, EIEC, and STEC) sensitive only at high concentrations of the extracts.As concerns the results obtained against fungal strains, the data are rather limited. The results are contrasted againstC. albicans but have to be more explored, while data related toA. fumigatus andC. krusei are encouraging.Further studies have been performed with the main components present inA. annua essential oil (see Table 3). These studies show that artemisia ketone is the component of the oil that has the greatest antimicrobial activity; in fact, it always turns out to be effective against bacteria and some fungi (C. albicans andA. fumigatus) at very low concentrations (range 0.07–10 mg/mL). The other compounds tested in the studies have produced variable results; however, it should be emphasized the fact that all the compounds tested by liquid methods were active (range 1.25–5 mg/mL) againstA. fumigatus, a dangerous microorganism frequently responsible for nosocomial infections in immunosuppressed subjects.


## 5. Concluding Remarks

During the last decade several authors have evaluated the antimicrobial activity ofArtemisia annua and some of its main components. The composition of the essential oil shows great differences in the three main characteristic components, namely, artemisia ketone, 1,8-cineole, and camphor, depending on the global phytogeographic origin. Besides the different chemical profiles, artemisia essential oil has revealed strong antimicrobial properties towards numerous bacterial strains, both gram-positive and gram-negative, and diverse fungal strains, including many pathogens. Biological effects are the result of a synergism of all molecules contained in an essential oil, even if it is possible that the activity of the main components is modulated by other minor molecules, but the activity of the isolated constituents is also remarkable.Artemisia annua volatile constituents appear to be a resource of many biologically active compounds which will hopefully give new economically important by-product. The good results obtained encourage further researches aiming at a possible application of these substances in food and pharmaceutical and cosmetology fields.



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