Open Access

Presence of phthalate derivatives in the essential oils of a medicinal plant Achillea tenuifolia

  • Azadeh Manayi1,
  • Mahdieh Kurepaz-mahmoodabadi1,
  • Ahmad R Gohari1,
  • Yousef Ajani2 and
  • Soodabeh Saeidnia1Email author
DARU Journal of Pharmaceutical Sciences201422:78

https://doi.org/10.1186/s40199-014-0078-1

Received: 8 October 2014

Accepted: 18 November 2014

Published: 28 November 2014

Abstract

Background

Phthalate, esters of phthalic acid, are mainly applied as plasticizers and cause several human health and environment hazards. The essential oils of Achillea species have attracted a great concern, since several biological activities have been reported from varieties of these medicinal species. On the other side, due to the problems regarding the waste disposal in developing countries, phthalate derivatives can easily release from waste disposal to the water and soil resulting in probable absorption and accumulation by medicinal and dietary plants. As a matter of fact, although the toxicity of phthalate derivatives in human is well-known, food crops and medicinal plants have been exposing to phthalates that can be detected in their extracts and essential oils. Achillea tenuifolia (Compositea) is one of these herbaceous plants with traditional applications which widely growing in Iran.

Finding

The plant root was subjected to hydro-distillation for 4 h using Clevenger type apparatus to obtain its essential oil before and after acid treatment. Both of the hydro-distilled essential oils were analysed by GC-MS method resulted in recognition of their constituent. Phthalate contamination as (1, 2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (5.4%) and phthalic acid (4.5%), were identified in the first and second extracted oils, respectively.

Conclusion

As a warning, due to the potential role of phthalates to cause reproductive toxicity, disturb of endocrine system and causing cancers, medicinal plants have to be considered through quality control for detection of these compounds.

Keywords

Achillea tenuifolia Compositae Phthalate contamination Acid treatment

Findings

Regarding the recent published articles on probable pollution of medicinal plants and other natural medicines like marine algae to phthalate [1],[2], finding a detection and even quantification method for phthalates, which can be accurate, fast and cost effective, is a considerable challenge particularly in standardization of herbal extracts and phytopharmaceuticals.

In fact, phthalates are the esters of phthalic acid and mainly used as plasticizers. They are manufactured by reacting phthalic anhydride with alcohols (ranged from methanol (C1) to tridecyl alcohol (C13)) in both straight and branching chains. Due to the toxicity concerns related to lower molecular weight phthalates (3–6 C), they are now being slowly replaced in the US, Canada, and European Union by high molecular weight phthalates (>6 C). The reason might be behind their higher permanency and durability in nature [2]. It is assumed that six million tonnes plasticizers are consumed every year, of which phthalates used in a large number of products including enteric coated pharmaceutical pills and supplements (as viscosity control agents), gelling agents, film formers, stabilizers, dispersants, lubricants, binders, emulsifying agents, and suspending agents [3]. These compounds interfere with endocrine systems in humans specially sex hormones and thyroids [4]. In addition, induction of inflammation, early puberty in girls, oxidative stress, asthma, and allergic symptoms were reported because of these compounds [5]-[7]. Literature review showed that these compounds could exhibit toxicity in liver, kidney, lung and testis in both animal and human [2],[5]. Accumulation of phthalates may occur in a variety of herbal medicines especially those are growing up in water and rivers due to the exposure of plants’ roots to the polluted wastewater. Consequent exposure of animals and humans to phthalate by using polluted herbs, crops and vegetables is possible, since phthalates accumulate in plants [1].

Achillea tenuifolia is distributed in the north and north-west of Iran with small yellow flowers, woody based and several stems [8]. This plant has been used as traditional herbal remedies against sweating and bleeding along with regulation of menstrual cycle and reduction of heavy bleeding and pain [9]. The previous study revealed that the oil of the plant compromised of several monoterpenes and sesquiterpenes [9]-[12]. There is also a report on the phytochemical content of the root extract demonstrating the presence of tannins, sterols and terpenoids [13].

Recently, we reported high percentage of phthalate in a medicinal plant, Lythrum salicaria[14]. In continuing our research on detection of phthalate in medicinal and food plants, here we focused on detection of these compounds in the root oil of A. tenuifolia.

Methods

Plant material and isolation of essential oils

The roots of A. tenuifolia were collected from Qazvin province (1500 m above the sea level) in June 2011(No. 1624) deposited at the Herbarium of Institute of Medicinal Plants, Jahade-Daneshgahi (ACECR), Karaj, Iran.

Air-dried roots (200 g) were submitted to hydro-distillation in a Clevenger-type apparatus for 4 h, subsequently, 10 mL hydrochloric acid (Merck, Darmstadt, Germany) (1 N) was added to the residue of the root over night at room temperature and hydro-distilled again for 4 h. As a result of acid attendance in the mixture, hydrolysing procedure of glycosidic components was successfully facilitated. The oils after extraction were separately collected in screw capped glass vials and dried over anhydrous sodium sulphate (Merck, Darmstadt, Germany) and stored at 4°C until analyses.

GC-MS analysis

The essential oil was analysed by GC-MS method on a Thermoquest-Finnigan Trace GC-MS instrument (ThermoQuest, Manchester, UK) equipped with a DB-5 fused silica column (60 m × 0.25 mm i.d., film thickness 0.25 μm). The oven temperature was raised from 60°C to 250°C at a rate of 5°C/min and held for 10 min; transfer line temperature was 250°C. Helium was used as a carrier gas at a flow rate of 1.1 mL/min with a split ratio equal to 1/50. The quadrupole mass spectrometer was scanned over the 35–465 amu with an ionizing voltage of 70 eV and an ionization current of 150 μA. The compounds were identified by comparison of retention indices (RI, DB-5) with those reported in the literature and libraries [15]-[23].

Results and discussion

The hydro-distillation of the root of A. tenuifolia resulted in extraction of the essential oils before and after acidic hydrolysis in extremely scarce amounts of colourless oils. In order to make sure about the sources of phthalate compounds in this study, no plastic container was used all through the procedure, and no solvent was used during extraction process except for hydrochloric acid that was purchased by analytical grade with no phthalate pollution. In addition, the solvents, used for injection of the samples to GC-MS, were injected alone to the chromatograph just before sample injection in order to detect probable contamination peaks. Taking together, any phthalate peaks detected in this study would highly unlikely be originated from storage, extraction and analysis procedure. GC-MS analysis of the volatile oils revealed the presence of 24 and 29 volatile components in the oils before and after acid treatment, representing 95.3% and 94.2% of the total oils, respectively (Table 1). Palmitic acid (36.9%), 5-dodecyldihydro-2(3H)-furanone (14.9%) and pentadecanoic acid (5.7%) were detected as the major constituents of the untreated essential oil, while the major volatile aglycones were identified as iso-valeric acid (24.9%), palmitic acid (15.8%), cyclohexane (13.3%), cyclohexadecanolide (7.2%) and 5-dodecyldihydro-2(3H)-furanone (6.1%) in the hydrolysed oil. Chemical structures of the identified compounds are illustrated in the Figure 1. However, in the previous study on the aerial parts of this plant, monoterpenes were characterized as the major constituents of the oil [5],[6]. Regarding the present results, palmitic acid and 5-dodecyldihydro-2(3H)-furanone were dominant in both volatile oils. The most considerable point found among the identified compounds is the presence of phthalate contaminations (compounds 31 and 32 in Figure 1) in both oils identifying as 1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester (5.4%) in the oil before acid treatment and phthalic acid (4.5%) in the oil after acid treatment. Presence of phthalic acid in the oil after acid treatment probably attributed to the hydrolysis of its derivatives during acid treatment.
Table 1

Percentage composition of the essential oils obtained from A. tenuifolia root before and after acidic hydrolysis

No.

Identified compounds

KI

RT

Percentage (%)

Contenta

Contentb

1

Cyclohexane

752

5.12

-

13.3

2

n-octane

900

6.68

-

0.4

3

Iso-valeric acid

976

8.15

-

24.9

4

2-methyl butanoic acid

978

8.19

-

0.6

5

n-decane

1098

11.16

0.6

1.2

6

Benzene-acetaldehyde

1146

12.43

-

0.2

7

Linalool oxide (cis) furanoid

1176

13.3

-

0.4

8

Linalool oxide (trans) pyranoid

1191

13.73

-

0.3

9

Camphor

1245

15.23

0.3

-

10

Terpinene-4-ol

1273

16.15

0.5

-

11

Alpha-terpineal

1289

16.54

0.6

-

12

Dodecane

1297

16.8

-

0.5

13

Eugenol

1453

21.28

1

2.7

14

Methyl eugenol

1490

22.07

0.4

-

15

n-dodecanol

1493

22.15

0.4

-

16

n-tetradecane

1497

22.27

-

0.3

17

Pentadecane

1597

24.81

-

0.1

18

Dodecanoic acid

1649

26.04

2.6

-

19

Spatulenol

1687

26.99

2

-

20

Caryophyllene oxide

1690

27.07

2

-

21

Hexadecane

1697

27.23

-

0.4

22

Tetradecanal (myristaldehyde)

1763

27.54

3.9

1.8

23

Dill apiol

1771

28.91

-

0.1

24

Apiol

1788

29.3

0.5

-

25

Tetradecanoic acid (myristic acid)

1842

30.95

4

1.9

26

Cyclocolorenone

1855

30.8

0.5

-

27

Octadecanal

1895

31.3

-

0.3

28

Hexadecanal

1897

31.7

0.3

0.4

29

Pentadecanoic acid

1902

32.28

5.7

4.5

30

6,10,14-trimethyl, 2-pentadecanone

1928

32.38

0.9

-

31c

Phthalic acid

1944

32.7

-

4.5

32c

1,2-benzenedicarboxylic acid, bis (2-methylpropyl) ester

1955

32.93

5.4

-

33

Hexadecanoic acid (palmitic acid)

2027

34.32

36.6

15.8

34

9-octadecanoic acid (oleic acid)

2048

34.38

9.7

-

35

Cyclohexadecanolide

2053

34.9

-

7.2

36

Ethyl stearate

2079

35.41

0.9

-

37

Docosane

2087

35.57

0.5

0.2

38

Ethylhexadecanoate

2091

35.65

-

0.4

39

Heneicosane

2145

37.72

-

0.5

40

5-dodecyldihydro-2(3H)-furanone

2150

37.92

14.9

6

41

Ethyl linoleate

2177

38.95

1.3

2.5

42

Nonadecanal

2226

40.3

-

2

43

Tricosane

2322

42.63

-

0.8

Hemiterpenoids

 

-

24.9

Monoterpenes

 

2.8

3.4

Sesquiterpenes

 

4.5

-

Phenylpropanoids

 

0.5

0.1

CXHy

 

1.3

17.8

CxHyOz

 

86.4

48

Phthalate contamination

 

5.4

4.5

Total

 

95.5

94.2

KI: Kovats Index on DB-5 with reference to n-alkanes injected after the oil at the same chromatographic conditions, RT: Retention Time, a: values of the percentages before acidic hydrolysis, b: values of percentage after acidic hydrolysis, c: phthalate derivatives contaminations.

Figure 1

Chemical structures of some identified components and phthalates (31 and 32) from the essential oils of A. tenuifolia root.

Detection of the mentioned compounds in the oils revealed that these contaminations are able to absorb from water and soil into the plant root. The plant, employed in this study, was gathered from a mountainous region near a seasonal river, which was surrounded by lots of disposed plastics and water bottles. Therefore, the source of contamination would most probably be polluted water particularly, regarding the point that we reduced the probable external contamination during storage, extraction and analysis procedure. Actually, these phthalate derivatives are widely used in plastic items, medical and pharmaceutical products, health care products, food containers, toys and paints. It seems that in Iran, the major sources of these compounds might be disposal plastics and chemical factories. Phthalate contaminations have previously been reported from the essential oils of the plants in several studies reported phthalate contaminations in the plants oils [14],[24]-[27]. Exposure to phthalates during pregnancy produced serious adverse effects like miscarriage, low birth weight, and preterm birth trough induction of inflammation and oxidative stress [6]. Moreover, fetal exposure to phthalate is associated with behavioral and mental ability; for instance in the third trimester of pregnancy they caused neurogical problems in children even until 4–9 years [28]. Although finding such a toxic manmade group of compounds is not a new concern and they are now replaced in the USA, Canada, and European Union by other plasticizers, but there is a complicated situation in developing countries. In fact, U.S. Environmental Protection Agency (EPA) has current management plan that includes the following eight phthalates: dibutyl phthalate (DBP), diisobutyl phthalate (DIBP), butyl benzyl phthalate (BBP), di-n-pentyl phthalate (DnPP), di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DnOP), diisononyl phthalate (DINP), and diisodecyl phthalate (DIDP), of which, BBP, DEHP, and DBP cause the most toxicity to terrestrial organisms, fish, and aquatic invertebrates. Medical device assessments for DEHP have been developed by Food and Drug Administration (FDA), Health Canada Medical Devices Bureau and the European Union Scientific Committee on Medicinal Products and Medical Devices. They concluded that premature infants are the population most highly exposed to phthalates via these uses. Furthermore, The European Commission (2005) banned DEHP, DBP and BBP in all toys and childcare articles. Encouraging industry to move away from phthalates is future plan of EPA [29].

Conclusion

Finding the phthalate esters in the essential oil of A. tenuifolia indicated that these toxic compounds, which have been used as the plasticizers in chemical and pharmaceutical industries, are able to be simply released into the water and soil and accumulate in the plants even in the medicinal species that are growing wildly in mountainous areas surrounded by lots of municipal solid wastes, disposed plastics and water bottles. Derivatives of phthalate esters are able to cause reproductive and developmental toxicity [1],[26] regarding their chemical structures. The toxicity of phthalate esters have been well-documented demonstrating that different organisms and tissues of the human and animal bodies could be affected by them including kidney, liver, thyroid and testes [1],[2],[30]. Besides, they could sensitize eye, skin and mucus membranes in human [2]. Taken together, pollution of medicinal plants to phthalate esters in developing countries seems cause a major problem in human health area, which needs more attention in both quality control and standardization of herbal medicines as well as Food and Drug policies or strategies by Ministry of Health.

Declarations

Acknowledgements

This paper is the result of an in-house study and no grants or funds have been received.

Authors’ Affiliations

(1)
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences
(2)
Institute of Medicinal Plants (IMP), Iranian Academic Centre for Education, Culture and Research (ACECR)

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