Anti-Trichomonas vaginalis properties of the oil of Amomum tsao-ko and its major component, geraniol

Abstract

Context: Trichomonosis, caused by the flagellate protozoan Trichomonas vaginalis, is the most common non-viral sexually transmitted disease (STD) and 5-nitroimidazole drugs are used for the treatment. However, a growing number of T. vaginalis isolates are resistant to these drugs, which make it becomes an urgent issue.

Objective: The current study was designed to evaluate the anti-T. vaginalis activity of the essential oil from A. tsao-ko used in traditional Chinese medicine and as a spice and its main component, geraniol.

Materials and methods: The anti-T. vaginalis activities of A. tsao-ko essential oil and geraniol were evaluated by the minimum lethal concentration (MLC) and 50% inhibitory concentration (IC₅₀) in vitro. The morphological changes of T. vaginalis were observed by transmission electron microscopy (TEM). Additionally, sub-MLC concentration treatment with sub-MLC A. tsao-ko essential oil and geraniol was also performed.

Results: This study shows that MLC/IC₅₀ of A. tsao-ko essential oil was 44.97 µg/ml/22.49 µg/ml for T. vaginalis isolate Tv1, and 89.93 µg/ml/44.97 µg/ml for T. vaginalis isolate Tv2. Those of geraniol were 342.96 µg/ml/171.48 µg/ml, respectively. After A. tsao-ko essential oil or geraniol treatment, obvious similar morphological changes of T. vaginalis were observed by TEM: the nuclear membrane was damaged, nuclei were dissolved, and the chromatin was accumulated; in the cytoplasm, numerous vacuoles appeared, rough endoplasmic reticulum dilated, the number of ribosomes were reduced, organelles disintegrated, the cell membrane was partially damaged, with cytoplasmic leakage, and cell disintegration was observed. The action time did not increase the effect of A. tsao-ko essential oil or geraniol against T. vaginalis, as no significant difference was observed after sub-MLC concentration treatment for 1, 3, and 5 h with A. tsao-ko essential oil and geraniol.

Discussion and conclusion: The study describes the first report on the activity and morphological changes of A. tsao-ko essential oil and geraniol against T. vaginalis. The results obtained herein presented new opportunities for antitrichomonal drugs.

• In vitro activity
• morphological changes
• transmission electron microscopy

Introduction

Trichomonosis, caused by the parasitic protozoan Trichomonas vaginalis, is the most prevalent non-viral sexually transmitted disease (STD) worldwide with 248 million new cases annually [World Health Organization (WHO), 2010)]. The infection has been associated with serious health consequences including adverse pregnancy outcomes (Cotch et al., 1997), acute infections associated with pelvic inflammatory disease (Cherpes et al., 2006), increased risks of infertility (Goldstein et al., 1993), and facilitation of HIV infection acquisition (Van Der Pol et al., 2008). So far, 5-nitroimidazole drugs have been used in the treatment of trichomonosis. Metronidazole and tinidazole were the two drugs of choice recommended by the United States Food and Drug Administration for the treatment of this STD (Helms et al., 2008). Although the cure rate was high, these drugs demonstrated treatment failure which was normally related to non-compliance because of side effects such as headache, dry mouth, metallic taste, glossitis, and urticaria caused by lengthy treatment or high doses (Garduño-Espinosa et al., 1992; Kapoor et al., 1999; Oxberry et al., 1994), or reinfection (Rocha et al., 2012). Moreover, an increasing number of metronidazole-resistant T. vaginalis isolates have been reported since metronidazole was introduced for the treatment of trichomoniasis in 1959 (Blaha et al., 2006; Butler et al., 2010; Durel et al., 1967; Narcisi & Secor, 1996; Schmid et al., 2001; Upcroft et al., 2006). In addition, the metronidazole-resistant isolates were cross-resistant to other members of the 5-nitrothiazole compounds including ornidazole, tinidazole, nitazoxanide, and furazolidone (Wright et al., 2010). Therefore, to improve the current chemotherapy against T. vaginalis infection, the natural products with high effectiveness and low toxicity may be an alternative therapeutic source.

Amomum tsao-ko Crevost et Lemaire (tsao-ko), called “Caoguo” or “tsao-ko”, which belongs to the family Zingiberaceae, was used as spice in Chinese cuisine and Chinese Traditional Medicine for the treatment of stomach disorders and throat infection (Yu et al., 2010). A. tsao-ko exhibited a wide range of biological activities, such as antimicrobial, antioxidant, and cytotoxic activities, as well as plasma and liver triacylglycerol decreasing activity (Martin et al., 2000; Moon et al., 2004, 2005; Yang et al., 2008, 2009; Yu et al., 2010). In a previous study, we found that A. tsao-ko essential oil from the dried fruit of tsao-ko cultured in Yunnan, China, had antibacterial effects (Peng et al., 2010). Geraniol shown to be one of the main components of A. tsao-ko essential oil as determined by GC-MS with a content of 13.69%. Geraniol is a widely used fragrance ingredient (Lapczynski et al., 2008). It also demonstrated pharmaceutical activities such as antimicrobial activity (Mendonça et al., 2009), restoring antibiotic activity against multidrug-resistant isolates (Lorenzi et al., 2009), insecticidal (Barros et al., 2009; Khallaayoune et al., 2009), and antitumor activity (Kim et al., 2011). In this study, the anti-T. vaginalis activities of A. tsao-ko essential oil and geraniol were assessed. The morphological changes of T. vaginalis after pretreatment with A. tsao-ko essential oil or geraniol were also studied by transmission electron microscopy (TEM).

Materials and methods

Drugs, media, and reagents

Geraniol was obtained from Sigma (St. Louis, MO) (Lot: MKBH7931V). Metronidazole was purchased from Sichuan Kelun Pharmaceutical Co., Ltd (Chengdu, China) (Lot: M11120602). Jieeryin was purchased from Sichuan Enwei Pharmaceutical Co., Ltd (Chengdu, China) (Lot: 1107102). Trichomonas medium No. 2 (each liter nutrient solution containing 13.6 g pancreatic digest of casein, 2.4 g papaic digest of soybean meal, 4.0 g sodium chloride, 2.0 g di-basic potassium phosphate, 2.0 g glucose, 14.4 g liver peptone, 16.0 g dextrose, 0.05 g calcium d-pantothenic acid, 0.125 g chloramphenicol, 200 ml horse serum) was purchased from OXOID Co., Ltd (Adelaide, South Australia). Polysorbate 80 and phosphate buffer were purchased from Chengdu Changzheng Huabo Co., Ltd (Chengdu, China). Other reagents were purchased from Sigma, Shanghai, China.

Origin and preparation of T. vaginalis

Two isolates of T. vaginalis (Tv1, Tv2) were obtained from vaginal secretions of out-patients at Sichuan Provincial Maternal and Child Health Hospital, Chengdu, China in 2012, and identified by optical microscopy and TEM.

Trichomonas vaginalis suspensions were prepared as described by Moon et al. (2006) with modifications. T. vaginalis was axenically cultured at 37 °C in trichomonas medium No. 2. The growth of T. vaginalis was monitored using the trypan blue exclusion method (Mishell & Shiigi, 1980). Trichomonas vaginalis was harvested at the mid-logarithmic phase with more than 95% viable cells by centrifugation at 500 rpm for 10 min, and was adjusted to 2 × 10⁵ cells/ml by pre-warmed fresh medium at 37 °C. The survival rate was more than 95% as observed by microscopy.

Preparation of essential oil from A. tsao-ko

Amomum tsao-ko cultivated in Yunnan province, China, was purchased from Beijing Tongren Drug Store (Beijing, China) in 2012 and identified by Prof. Min Li at Chengdu University of Traditional Chinese Medicine. The dried fruits were ground and distilled for 4 h using a Clevenger-type apparatus (Yang et al., 2008). The essential oil was dried by anhydrous sodium sulfate and then stored at 4 °C. The density of the oil was 920.90 mg/ml.

Determination of minimum lethal concentration (MLC) and 50% inhibitory concentration (IC₅₀)

Amomum tsao-ko essential oil (or geraniol) mixed with polysorbate 80 (v/v, 10:1) was emulsified by adding physiological saline while stirring. The emulsion containing 10% essential oil (or geraniol) was diluted with trichomonas medium No. 2. This assay was performed according to Wright et al. (2010) and Delmas et al. (2002) with modifications. Amomum tsao-ko essential oil and geraniol were diluted by geometric dilutions in 96-well plates, ranging from 22.48 µg/ml to 46.05 mg/ml of A. tsao-ko essential oil, and from 21.43 µg/ml to 43.90 mg/ml of geraniol. In addition, three positive drug controls (trichomonas medium + metronidazole, or ornidazole, or Jieeryin + T. vaginalis), one solvent control (trichomonas medium + polysorbate 80 + T. vaginalis), and one growth control (trichomonas medium + T. vaginalis) were set up and investigated at the same time. After 48 h of incubation at 37 °C, viable T. vaginalis cells were identified and counted microscopically on the basis of the morphology and motility. The minimum lethal concentration (MLC) was defined as the lowest drug concentration in which no motile organism was observed. The 50% inhibitory concentration (IC₅₀) value was determined as the drug concentration in which 50% T. vaginalis cells were alive and 50% died (Delmas et al., 2002). All experiments were carried out in triplicate.

Inspectation of ultrastructural changes of A. tsao-ko essential oil-treated or geraniol-treated T. vaginalis by TEM

The ultrastructural changes of T. vaginalis isolate (Tv2) treated with A. tsao-ko essential oil (or geraniol) were observed by TEM. Tv2 were incubated at 37 °C, and 2.0 × 10⁵ T. vaginalis/ml were treated in trichomonas medium No. 2 containing sub-MLC A. tsao-ko essential oil or geraniol for 1, 3, and 5 h. The cells were centrifuged at 1200g at 4 °C for 10 min, and were added to PBS (pH 7.4) containing 3% glutaraldehyde at 4 °C for 1 h. The cells were then postfixed in 1% buffered osmium tetroxide for 1 h, stained with 1% uranyl acetate and dehydrated in graded series of ethanol. The samples were cryofixed with a pressure of about 2100 bars and a reduction in temperature of 8 °C/s. The cryofixed cells were kept in liquid nitrogen and cryosubstituted in pure acetone containing 2% of osmium tetroxide and 0.1% of uranyl acetate for 72 h at −90 °C. The temperature was gradually increased to 4 °C (5 °C/h). Samples were kept at this temperature for 2 h, followed by 2 h at room temperature, and were then washed twice with acetone. The fixed cells were then embedded in Epon812. Ultra-thin sections were prepared and stained with uranyl acetate and lead citrate. Microscopy was performed with a HITACHI, H-600IV TEM at the West China Medical School, Sichuan University, China. The control samples were treated with physiological saline instead of A. tsao-ko essential oil or geraniol.

Results

In vitro activity of A. tsao-ko essential oil and geraniol against T. vaginalis

As shown in Table 1, A. tsao-ko essential oil and geraniol showed obvious anti-T. vaginalis activity in vitro. The activity of A. tsao-ko essential oil was stronger than that of geraniol. MLCs and IC₅₀ values of A. tsao-ko essential oil were 44.97 µg/ml/22.49 µg/ml for T. vaginalis isolate Tv1, and 89.93 µg/ml/44.97 µg/ml for T. vaginalis isolate Tv2. Those of geraniol were 342.96 µg/ml/171.48 µg/ml. When the activity of A. tsao-ko essential oil was compared with two standard drugs (metronidazole and ornidazole) and geraniol, the essential oil was about 10-fold less active than metronidazole, 20-fold less than ornidazole, and 4-fold to 8-fold more active than geraniol. However, in contrast to the Traditional Chinese Medicine Jieeryin, a well-known therapeutic agent for killing Trichomonas vaginitis in China, A. tsao-ko essential oil and geraniol were 80-fold and 20-fold to 40-fold more active, respectively.

Table 1. In vitro anti-T. vaginalis activity of essential oil and geraniol from A. tsao-ko.

	Tv1				Tv2
	IC50		MLC		IC50		MLC
Compounds	v/v	Value (µg/ml)	v/v	Value (µg/ml)	v/v	Value (µg/ml)	v/v	Value (µg/ml)
Metronidazole		2.44		4.88		4.88		9.77
Ornidazole		1.22		2.44		2.44		4.88
Essential oil	1:20480	22.49	1:10240	44.97	1:10240	44.97	1:5120	89.93
Geraniol	1:5120	171.48	1:2560	342.96	1:5120	171.48	1:2560	342.96
Jieeryin	1:256		1:128		1:128		1:64

The IC₅₀ value was defined as the concentration of a compound at which it inhibits 50% T. vaginalis cells. MLC means minimal lethal concentration of the drug against T. vaginalis. MLC and IC₅₀ values represent the mean of three independent experiments.

Morphological changes of T. vaginalis after A. tsao-ko essential oil treatment

Figure 1 shows the internal structure of untreated T. vaginalis Tv2 cell. The cell in normal condition without environment disturbance shows an integrated membrane (6 in Figure 1) and nucleus (1 in Figure 1), hydrogenosomes (3 in Figure 1), and the base of the flagellum (5 in Figure 1). Significant ultrastructural changes in A. tsao-ko essential oil-treated Tv2 cell were observed by TEM. The morphological changes of Tv2 with sub-MLC essential oil (44.97 μg/ml) for 1 h are shown in Figure 2(A–F). In the nucleus, chromatin accumulation (e in Figure 2D and E), a damaged nuclear membrane or disappearing nuclear envelope (i in Figure 2C and E), and dissolving nuclei (j in Figure 2F) were observed. In the cytoplasm, numerous vacuoles (a in Figure 2B and C), autophagic vacuoles (b in Figure 2C and D), dilation of the rough endoplasmic reticulum (d in Figure 2B), reduction or disappearance of ribosomes (f in Figure 2D), organelles disintegration (g in Figure 2E and F), partial cell membrane damaging (h in Figure 2F), cytoplasmic leakage (k in Figure 2F), cell disintegration, and necrosis (Figure 2F) were observed. There was no obvious difference among 1, 3, and 5 h treatments. These results indicate that the treatment time is not the main factor on the effect of A. tsao-ko essential oil against T. vaginalis, and the A. tsao-ko essential oil takes action in a relatively short period of time.

Figure 1. Transmission electron micrographs of untreated T. vaginalis. In untreated-T. vaginalis cell, nucleus (1), rough surfaced endoplasmic reticulum (2), free hydrogenosomes (3), ribosome (4), flagella basal body, (5) and cytoplasmic membrane (6) were observed by TEM in this figure.

Figure 2. Transmission electron micrographs of A. tsao-ko essential oil-treated T. vaginalis. Transmission electron micrographs of T. vaginalis cell treated with sub-MLC A. tsao-ko essential oil (44.97 µg/ml) for 1 h were shown in (A–F). Vacuoles (a), autophagic vacuoles (b), perinuclear space widening (c), swollen rough surfaced endoplasmic reticulum (d), chromatin accumulation (e), ribosomes disappearing (f), organelles disintegration (g), damaged cytoplasmic membrane (h), nuclear envelope disappearing (i), nuclei dissolving (j), and cytoplasmic leakage (k) were observed.

Morphological changes of T. vaginalis after geraniol treatment

Similar morphological changes were observed in Tv2 cells treated with geraniol at sub-MLC (171.48 µg/ml) for 1, 3, and 5 h, with the observation in 1 h represented in Figure 3(A–F). Compared with the changes in A. tsao-ko essential oil-treated Tv2 cells, similar morphological changes were observed, such as numerous vacuoles (a in Figure 3A), autophagic vacuoles (b in Figure 3A, B, and E), rough endoplasmic reticulum dilation (d in Figure 3C), perinuclear space widening (c in Figure 3A and C), chromatin accumulation (e in Figure 3C), disappearing nuclear envelope (i in Figure 3E), dissolving nuclei (j in Figure 3E), ribosome reduction or disappearing (f in Figure 3D and F), organelles disintegration (g in Figure 3D, E, and F), partial cell membrane damaging (h in Figure 3E and F), and cytoplasmic leakage (k in Figure 3F). Disintegration and necrosis of Tv2 cells were observed in Figure 3(E) and (F). These results indicated that the anti-T. vaginalis mechanism of geraniol might be similar to that of A. tsao-ko essential oil.

Figure 3. Transmission electron micrographs of gernaiol-treated T. vaginalis. Transmission electron micrographs of T. vaginalis cell treated with sub-MLC gernaiol (171.48 µg/ml) for 1 h were observed in (A–F). Vacuoles (a), autophagic vacuoles (b), perinuclear space widening (c), swollen rough surfaced endoplasmic reticulum (d), chromatin accumulation (e), ribosomes disappearing (f), organelles disintegration (g), damaged cytoplasmic membrane (h), nuclear envelope disappearing (i), nuclei dissolving (j), and cytoplasmic leakage (k) were observed.

Discussion

Anti-T. vaginalis activity of A. tsao-ko essential oil and its main component, geraniol

Interest has increased in recent decades in natural products with medicinal activity from plants which are still the major sources of innovative therapeutic agents for infectious diseases, cancer, lipid disorders, and immunomodulation (Altmann, 2001). Plant extracts (Frasson et al., 2012), essential oils (Moon et al., 2006), and isolated substances such as saponins (Rocha et al., 2012), β-glycosides (Arthan et al., 2008), and alkaloids (Giordani et al., 2011) have shown anti-T. vaginalis activity, but until now, no study on the action of A. tsao-ko essential oil and geraniol against T. vaginalis or other protozoa is available. In this study, the anti-T. vaginalis activity of A. tsao-ko essential oil and geraniol against two isolates of T. vaginalis was investigated, showing obvious anti-T. vaginalis activity. Although the activity of A. tsao-ko essential oil and geraniol less active than metronidazole and ornidazole, it is stronger than Jieeryin, a well-known therapeutic agent for killing T. vaginitis in China. These results indicated that a rich variety of A. tsao-ko can be used as potential therapeutic natural resource for the development of antitrichomonal drugs. As far as we know, this is the first demonstration of the potential anti-T. vaginalis activity of A. tsao-ko essential oil and geraniol.

We have identified 24 compounds in A. tsao-ko essential oil by GC-MS, representing 97.00% of the total oil (unpublished data), while geraniol represents only about 13.69% of total oil volume. It is unknown whether A. tsao-ko essential oil contains other anti-T. vaginitis constitute or if interactions exist between geraniol and other constitute. Consequently, further studies are necessary to determine the relationship between chemical composition and anti-T. vaginalis activity, as well as to detail the mechanism of action of those bioactive molecules in A. tsao-ko essential oil. Besides that, the risk of cell toxicity due to essential oil ingestion or skin contact, the role of the delivery systems, and the potential application of A. tsao-ko essential oil and geraniol for the treatment of parasitical infections require further investigation.

Morphological changes of T. vaginalis after A. tsao-ko essential oil or geraniol treatment

At present, limited studies have suggested that essential oils could interfere with biochemical pathways (Lopes et al., 1999; Valentin et al., 1995). The mechanism of A. tsao-ko essential oil and geraniol against T. vaginalis was preliminarily investigated by TEM after the susceptibility assay in this study. Morphological changes of A. tsao-ko essential oil-treated and geraniol-treated Tv2 cell are shown in Figures 2 and 3, respectively. Compared with untreated Tv2 cell, severe morphological changes were observed by TEM after A. tsao-ko essential oil treatment for 1 h (Figure 2A–F), such as a large number of vacuoles, decreased or disappeared ribosome, rough endoplasmic reticulum dilated, chromatin margination, perinuclear space widening, the nuclear envelope disappearing, and nuclei dissolving. Similar morphological changes can be observed in geraniol-treated Tv2 cell (Figure 3A–F). Partially damaged cytoplasmic membranes and leakage of cytoplasmic contents were also observed. Eventually, the damaged cytoplasmic membrane and organelles might cause Tv2 cells to die. Therefore, we presume that A. tsao-ko essential oil and geraniol induce reduction in the trophozoite viability probably by damaging membrane and organelles.

Besides that, the influence of action time on T. vaginalis of A. tsao-ko essential oil or geraniol was analyzed by TEM among 1 h, 3 h, and 5 h treatments, but no obvious differences were observed. Therefore, the action time was not an important factor in T. vaginalis morphological changes caused by sub-MLC essential oil or geraniol. The drug concentration might rather be the most influential factor, which requires further investigation.

Conclusions

In conclusion, A. tsao-ko essential oil and geraniol demonstrated anti-T. vaginalis activities. Their anti-T. vaginalis activity might be resulted from damaged membrane and organelles. Therefore, A. tsao-ko essential oil and geraniol might be potentially useful as alternative lead compounds for development of antitrichomonal drugs.

Declaration of interest

The authors declare that there are no conflicts of interest. This study was financially supported by the Funds of the Department of Science and Technology of Sichuan Province (No. 2011JYZ004), the Sichuan Provincial Department of Education (No. 11ZB206, 15ZB0238), Research Project of Sichuan Provincial Health Department (130307) and the General Program of Chengdu Medical College (No. CYZ11-002).