Evaluation of some plants used in Turkish folk medicine against parasitic infections for their in vivo anthelmintic activity

Journal of Ethnopharmacology 108 (2006) 211–216

Evaluation of some plants used in Turkish folk medicine against parasitic infections for their in vivo anthelmintic activity Esma Kozan a , Esra K¨upeli b , Erdem Yesilada c,∗ a

Department of Parasitology, Faculty of Veterinary Medicine, Afyon Kocatepe University, Afyonkarahisar, Turkey b Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 6330, Ankara, Turkey c Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, Kayisdagi 34755, Kadikoy, Istanbul, Turkey Received 2 March 2006; received in revised form 27 April 2006; accepted 8 May 2006 Available online 16 May 2006

Abstract Ethanolic and aqueous extracts obtained from nine plant species from seven families selected depending on their use in Turkish folk medicine, including Citrillus lanatus (Thunb.) Matsum. (seed), Jasminum fruticans L. (branches), Juniperus drupacea Labill. (fruits), Juniperus nana L. (fruit and leaves), Juniperus oxcycedrus L (fruit and leaves), Mentha longifolia L. (herba), Pinus nigra ssp. pallasiana (Lamb.) Richt. (fruits), Plantago lanceolata L. (leaves), and Zea mays L. (seed) were evaluated for their in vivo anthelmintic activity. Among the plant extracts studied, both ethanolic and aqueous extracts of Jasminum fruticans, Mentha longifolia and Pinus nigra ssp. pallasiana, the aqueous extracts of Zea mays, the ethanolic extracts of Citrillus lanatus, Juniperus drupacea (fruit), Juniperus oxcycedrus and Plantago lanceolata displayed significant anthelmintic activity against pinworms, Syphacia obvelata and Aspiculuris tetraptera, in mice. Rest of the extracts from plants did not show any remarkable anthelmintic activity. The results were considered significant at p < 0.05. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Anthelmintic activity; Aspiculuris tetraptera; Syphacia obvelata; Turkish folk medicine

1. Introduction Laboratory animals are suitable and necessary for the proper development of several biological assays. The utilization of these standard models is recommended aiming at the attainment of reliable and reproducible results. In spite of this approach, laboratory animals are seldom investigated for autochthonous ecto and endoparasites prior to their utilization in experiments. In conventional, semi-open facilities, rodent colonies are frequently infected with helminths. These parasites, if undetected, can interfere in development of protocols and alter the interpretation of final results (Pinto et al., 1994, 2001). Syphacia obvelata (Rudolphi, 1802) Seurat 1916 is the mouse pinworm occurring in the house mouse throughout the world. The mouse pinworm occurs in the large intestine of the host. Aspiculuris tetraptera (Nitzsch, 1821) Schulz, 1924 occurs in the large intestine of mice and other rodents (Soulsby, 1982).

∗

Corresponding author. Fax: +90 216 5780068. E-mail address: [email protected] (E. Yesilada).

0378-8741/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2006.05.003

Because of the frequent, worldwide transfer of mice between laboratories and researchers, pinworm parasites like Syphacia obvelata and Aspiculuris tetraptera are relatively common in laboratory mice. Although pinworm infection usually does not produce any clinical illness in normal mice, heavy worm burdens have been associated with rectal prolapse, intussusception, and intestinal impaction (Flynn et al., 1989). In addition, immunocomprimised mice, such as nude mice or SCID mice, are predisposed to large worm burdens, sequelae, and death (Zenner, 1998). Science parasitized mice are not desirable as experimental subjects, it is important to keep laboratory rodent colonies parasite free (Sueta et al., 2002). Syphacia obvelata and Aspiculuris tetraptera are important since they have been extensively used in determination of efficacy of several chemotherapeutic agents (Theodorides, 1976). For long time, mankind has developed through the world a traditional practice of medicine based on the knowledge of medicinal plants (Schillhorn van Veen, 1997). Anthelmintic plants offer a traditional alternative to manufactured anthelmintics that is both sustainable and environmentally acceptable. Such plants could have a more important role in the future

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Table 1 Traditional use of the plant materials in Turkish folk medicine Plant name and authors

Family

Local name

Part used

Traditional use (Lit.)

Citrillus lanatus (Thunb.) Matsum.

Cucurbitaceae

Karpuz

Seeds

Jasminum fruticans L

Oleaceae

¨ uzg¨oz¨u Ok¨

Branches

Juniperus drupacea Labill.

Cupressaceae

Andiz

Leaves, fruits

Juniperus nana L.

Cupressaceae

Ardic

Leaves, fruits

Juniperus oxycedrus L.

Cupressaceae

Dikenli ardic, Cirti

Leaves, fruits

Mentha longifolia L.

Lamiaceae

Yarpuz

Aerial parts

Pinus nigra ssp. pallasiana (Lamb.) Richt.

Pinaceae

Cones

Plantago lanceolata L.

Plantaginaceae

Zea mays L.

Poaceae

Karac¸amsakizi, Camkozalagi Mambel, Ilandili, Zimanugmari, Belhevzar Misir

1 kg of seed is ingested on an empty stomach against worms or pericarp is cut into small pieces and swallowed (Sezik et al., 2001) Juice from cut end of a freshly cut branch is heated over fire and applied, against a parasitic disease in animals (Honda et al., 1996) Crushed and boiled, decoction is used internally as anthelmintic (Yesilada et al., 1993) In spite of use of two other Juniperus species (drupacea and oxycedrus) in traditional medicine, this species has not been reported to possess a relevant utilization. This plant was tested to reveal whether or not possesses a relevant effect not discovered in folk medicine Applied externally against parasitic disease (Sezik et al., 1997) Decoction is used as tea or fresh plant is eaten against worms (Yesilada et al., 1993) Decoction is used as tea against worm (Fujita et al., 1995) Fresh leaves are pounded to obtain juice and applied externally on skin against parasites in animals (Tabata et al., 1994) Seeds of corn and dried white beans are cooked together and taken orally against intestinal worms (Yesilada et al., 1999)

control of helminth infections in the tropics (Hammond et al., 1997). In our ongoing project on the medicinal plants used in Turkish traditional medicine for the treatment of parasitic diseases, we undertook the present screening study in order to elucidate traditional use of the selected plants from the scientific view point. Nine plant species whose selections were based on ethnomedical information gathered from Turkish folk medicine which were used either for humans or domestic animals have been evaluated for their in vivo anthelmintic activity. The selected plants as the subject of this study are listed in Table 1 with their vernacular names and recorded utilizations in Turkish folk medicine. Two kinds of extracts with ethanol and water were prepared from each plant and their inhibitory effects on Syphacia obvelata and Aspiculuris tetraptera infections for the assessment of anthelmintic activity in mice were examined.

Laves

Seeds

2. Materials and methods 2.1. Plant materials Plant materials were collected from different localities in Turkey. Voucher specimens were authenticated by Prof. Dr. Hayri Duman of Department of Biology, Faculty of Science & Art, Gazi University, and were deposited in the Herbarium of Faculty of Pharmacy, Gazi University, Ankara, Turkey. Collection sites, parts used and herbarium numbers for each plant material is given in Table 2. 2.2. Preparation of plant extracts Each plant material was dried under shade and powdered to a fine grade by using a laboratory scale mill. The plant parts and

Table 2 The collected plant parts, collection sites during collection and percentage yields of EtOH and H2 O extracts Plant name

Collected parts

Collection sites

Herbarium numbers

EtOH extract (w/w, %)

H2 O extract (w/w, %)

Citrillus lanatus Jasminum fruticans

Seed Branch

Purchased from a bazaar Ankara, vicinity of Kurtbogazi dam

– GUE 2357

23.74 19.95

33.86 12.92

Juniperus drupacea

Leaf Fruit

Ankara, Kizilcahamam, G¨uvem village

GUE 2358

18.46 26.05

16.99 33.77

Juniperus nana Juniperus oxycedrus Mentha longifolia Pinus nigra ssp. pallasiana Plantago lanceolata Zea mays

Leaf + fruit Leaf + fruit Herb Fruit Leaf Seed

Ankara, Kizilcahamam, G¨uvem village Ankara, Kizilcahamam, G¨uvem village Amasya, Kurnaz village Samsun, Vezirkopru Ankara, Kurtbogazi dam Amasya, Kurnaz village

GUE 2359 GUE 2360 GUE 2361 GUE 2362 GUE 2363 –

17.79 18.99 8.69 39.04 10.95 23.15

10.52 10.23 14.75 10.4 7.22 39.46

E. Kozan et al. / Journal of Ethnopharmacology 108 (2006) 211–216

the extract yields (w/w) are given in Table 2. The extracts were prepared as described below. Ethanolic (EtOH) extract: dried plant material (10 g) was extracted with 90% EtOH at room temperature two times (×300 ml). The combined ethanolic extract was evaporated to dryness in vacuo to give crude EtOH extract. Aqueous (H2 O) extract: Dried plant material (10 g) was extracted with distilled water at room temperature two times (×300 ml). The combined aqueous extract was lyophilized to give the crude H2 O extract. 2.3. Pharmacological procedures 2.3.1. Animals Male Swiss albino mice (20–25 g) of 8–10 weeks old were purchased from the animal breeding laboratories of Refik Saydam Central Institute of Health (Ankara, Turkey). The animals were left for 2 days to acclimatize to animal room conditions and maintained on a standard pellet diet and water ad libitum. The food was withdrawn on the day before the experiment, but the animals were allowed free access to water. All mice were kept at 20–25 ◦ C, with 45% relative humidity, a light/dark cycle of 12 h and 10–15 changes of fresh air per hour in cycle. A

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minimum of six animals was used in each group. Throughout the experiments, animals were processed according to the suggested European ethical guidelines for the care of laboratory animals. 2.3.2. Parasitology Natural infections were identified by the use of cellophane impression of perianal region and by the technique of ZnCl2 + NaCl centrifugal flotation of feces. Cellophane tape tests were used for diagnosis of Syphacia obvelata and ZnCl2 + NaCl centrifugal flotation were used for diagnosis of Aspiculuris tetraptera (Sueta et al., 2002). Egg counts showed a mean of 178 Syphacia obvelata eggs per anal tape per mouse and 400 Aspiculuris tetraptera eggs per mouse before treatment. Infected mice were divided into 22 groups by random selection, 21 treated (n = 6) and one control (n = 6) group. Test samples were given orally at a close of 100 mg/kg body weight to test animals after suspending in 0.5% sodium carboxymethyl cellulose (CMC) suspension in distilled water. The control group animals received the same experimental handling as those of the test groups except that the drug treatment was replaced with appropriate volumes of the dosing vehicle. Doramectin (0.2 mg/kg) in 0.5% CMC was used as reference drug.

Table 3 The efficacy of some plants in naturally acquired Syphacia obvelata in mice Material

Parts used Extract type Dosage (mg/kg)

Control

Anal tape results before treatment (days 13 and 0)

Anal tape results after treatment (days 4 and 7)

Parasite counts recovered at necropsy (day 7) Total

Min–max

GO

S.E.

+

+

786

45–210

114.5

15.8

Efficacy (%)

0

Citrillus lanatus

SD

EtOH H2 O

100 100

+ +

+ +

248 329

28–62 43–66

39.7 74.1

5.3 4.2

65.3** 35.3

Jasminum fruticans

BR

EtOH H2 O

100 100

+ +

+ +

238 361

36–45 44–77

30.5 58.9

4.5 5.5

73.4*** 48.5**

Juniperus drupacea

FR

EtOH H2 O EtOH H2 O

100 100 100 100

+ + + +

+ + + +

286 428 321 454

35–56 45–109 29–87 55–132

47.0 94.1 79.4 110.2

4.4 13.3 9.5 13.9

59.0** 17.8 30.7 3.8

LF Juniperus nana

FR + LF

EtOH H2 O

100 100

+ +

+ +

537 510

54–134 80–127

109.2 111.8

10.7 14.9

4.6 2.4

Juniperus oxycedrus

FR + LF

EtOH H2 O

100 100

+ +

+ +

381 452

46–78 65–109

52.5 94.9

5.7 10.7

54.1** 17.1

Mentha longifolia

AE

EtOH H2 O

100 100

+ +

+ +

238 294

29–51 15–65

37.6 42.2

5,1 67,1** 6.9 63,1**

Pinus nigra ssp. pallasiana FR

EtOH H2 O

100 100

+ +

+ +

282 389

31–67 44–86

45.1 62.2

5.9 6.1

60.6** 45.7*

Plantago lanceolata

LF

EtOH H2 O

100 100

+ +

+ +

393 463

33–77 46–87

66.9 79.4

6.6 5.1

41.6* 30.7

Zea mays

SD

EtOH H2 O

100 100

+ +

+ +

546 405

78–115 60–91

98.2 48.5

9.9 5.6

14.2 57.6**

+

−

0

Doramectin

0.2

0

0

0

100

AE, aerial parts; BR, branch; EtOH, ethanol extract; FR, fruits; GO, geometric mean; H2 O, water extract; LF, leaves; P, pericarp; SD, seeds; S.E., standard error. * p < 0.05. ** p < 0.01. *** p < 0.001.

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Table 4 The efficacy of some plants in naturally acquired Aspiculuris tetraptera in mice Material

Parts used

Extract type

Dosage (mg/kg)

Control

Faecal egg results before treatment (days 4 and 0)

Faecal egg results after treatment (days 4 and 7)

Parasite counts recovered at necropsy (day 7)

Efficacy (%)

Total

Min-Max

GO

SE

+

+

263

15–97

35.3

13.2

0

Citrillus lanatus

SD

EtOH H2 O

100 100

+ +

+ +

130 135

6–29 7–36

13.4 21.2

3.5 5.9

62.0*** 39.9

Jasminum fruticans

BR

EtOH H2 O

100 100

+ +

+ +

152 134

2–20 10–34

11.1 20.3

5.6 4.9

68.6*** 42.0*

Juniperus drupacea

FR

EtOH H2 O EtOH H2 O

100 100 100 100

+ + + +

+ + + +

148 164 114 148

3–27 10–45 2–36 5–50

16.8 31.5 27.4 39.8

5.8 8.9 9.9 9.3

52.4** 11.3 22..9 –

LF Juniperus nana

FR + LF

EtOH H2 O

100 100

+ +

+ +

86 97

29–85 23–78

35.9 38.8

8.2 9.1

Juniperus oxycedrus

FR + LF

EtOH H2 O

100 100

+ +

+ +

146 94

2–28 12–56

17.9 31.2

5.7 5.9

48.9* 11.6

Mentha longifolia

AE

EtOH H2 O

100 100

+ +

+ +

133 119

3–21 4–28

13.9 14.1

4.3 6.2

60.6*** 60.1**

Pinus nigra ssp. pallasiana

FR

EtOH H2 O

100 100

+ +

+ +

148 169

11–36 9–38

15.1 21.5

5.2 6.1

57.2** 39.1

Plantago lanceolata

LF

EtOH H2 O

100 100

+ +

+ +

120 123

8–29 12–39

19.6 22.6

5.6 5.5

44.5* 35.9

Zea mays

SD

EtOH H2 O

100 100

+ +

+ +

146 163

7–44 11–36

31.2 18.2

8.8 4.4

11.6 48.4**

+

−

0

Doramectin

0.2

0

0

0

– –

100

AE, aerial parts; BR, branch; EtOH, ethanol extract; FR, fruits; GO, geometric mean; H2 O, water extract; LF, leaves; P, pericarp; SD, seeds; S.E., standard error. * p < 0.05. ** p < 0.01. *** p < 0.001.

The mice were isolated in individual cages for faecal collection on day four pretreatment and on days 0, 4 and 7 posttreatment. Faecal pellets were collected, weighed and 60 ml of ZnCl2 + NaCl added for each gram of faeces. The pellets were broken up and then strained. The strained solution was used for determination of faecal egg count using a two chamber McMaster slide. Examination cellophane tape preparation on day 13 pretreatment and on days 0, 4 and 7 post-treatment. On day 7 post-treatment the mice were euthanised using diethyl ether, gastrointestinal tract removed and washed with normal saline. The contents were examined under a steromicroscope to count and identify any parasite presence. Efficacy was calculated from the geometric means of S. obvelata and A. tetraptera counts as follows: efficacy (%) =

a−b × 100 a

where a is the geometric mean number of parasites in control group and b is the geometric mean number of parasites in treated group.

2.3.3. Statistical analysis of data Data obtained from animal experiments was expressed as mean standard error (±S.E.M.). Statistical differences between the treatments and the control were evaluated by ANOVA and Students–Newman–Keuls post hoc tests. p < 0.05 was considered to be significant (* p < 0.05; ** p < 0.01; *** p < 0.001). 3. Results and discussion In order to select plant materials with possible antihelmintic activity, the database of Turkish folk medicines (TUHIB), and ethnobotanical studies which were published on Turkish traditional medicine so far were reviewed. Eight materials were determined to be used against parasitic infections of various types, which are listed in Table 1. Inhibitory effects of the extracts from selected materials on Syphacia obvelata and Aspiculuris tetraptera infections were studied for the assessment of anthelmintic activity. Results of assays are given in Tables 3 and 4. Citrillus lanatus seeds are used against worms in Turkish folk medicine (Sezik et al., 2001). Our experiments revealed

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that EtOH-extract of Citrillus lanatus seeds possess a significant anthelmintic activity, but the activity of aqueous extract was weak (Tables 3 and 4). Previously it was experimentally shown that Cucurbita pepo seeds, another plant from the same family, possesses anthelmintic activity against Ascaris, Taenia ve Oxiuris parasites (Chung and Ko, 1976; Younis et al., 2000). Aspiculuris tetraptera and Syphacia obvelata, parasites were studied in the present study, are also classified under Oxiurid group and cause parasitic infection in mice. Since, Citrillus lanatus and Cucurbita pepo are effective against Oxiuris, the seeds of these plants can show anthelmintic effect on Aspiculuris tetraptera and Syphacia obvelata with similar mechanisms. The stem juice of Jasminum fruticans is used to treat parasitic infections in domestic animals (Honda et al., 1996). Both aqueous and EtOH extracts from the branches of Jasminum fruticans showed significant anthelmintic activity which proved the folkloric utilization, but that of EtOH extract was more pronounced as shown in Tables 3 and 4. Different parts or products, i.e., fruits, leaves, wood or tar, obtained from Juniperus species are used to treat several parasitic diseases in Turkish folk medicine. Crushed and boiled fruit of Juniperus drupacea are used internally as anthelmintic in Turkish folk medicine (Yesilada et al., 1993). Leaves and fruit of another Juniperus species, Juniperus oxcycedrus, are also applied externally against parasitic diseases (Sezik et al., 1997). As shown in Tables 3 and 4, EtOH extracts from the fruits of both species showed potent anthelmintic activity, while the effect of aqueous extracts were not noteworthy. These results revealed that principles found in the resin of these plants might possess the relevant activity. As a matter of fact, tar of these species has been used widespread since centuries. Although such an utilization was not described in folk medicine, we also studied the effects of another species, Juniperus nana leaves and fruit using the same experimental models. Neither ethanol nor aqueous extracts of Juniperus nana showed anthelmintic activity, which proves why this species has not been used in folk medicine. Decoction of the dried aerial as tea or fresh leaves of Mentha longifolia are taken orally to get rid of worms (Yesilada et al., 1993). Both aqueous and EtOH extracts showed significant anthelmintic activity as shown in Tables 3 and 4. According to a previous research, tannins, which are naturally occuring secondary metabolites in Lamiaceae, might reduce worm burden. Results has shown that dietary inclusion of condensed tannin in quebracho exctract reduces egg output and worm burden in sheep that are infected by Trichostrongylus colubriformis (Butter et al., 2000). These results were confirmed by others researchers in their studies (Anthanasiadou et al., 2000). Villasenor et al. (2002), isolated beta-sitosterol from Mentha cordifolia as the analgesic principle and reported the anthelmintic effect against Ascaris suum. The same compound was also reported to be effective against a nematode, Caenorhabditis elegans, in vitro (Deepak et al., 2002). Decoction of Pinus nigra ssp. pallasiana cones is applied internally against worms (Fujita et al., 1995). Both aqueous and EtOH extracts from P. nigra ssp. pallasiana cones showed potent anthelmintic activity as shown in Tables 3 and 4. Sev-

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eral groups of natural compounds mainly flavonoids (Niemann, 1988), tannins (Harborne and Baxter, 2001) and piperidine alkaloids (Stermitz et al., 1994) have been recognized in some Pinus species. Tannins are applied as astringents, anti-hemorrhagics and anti-abortives in veterinary medicine. Recently, studies showed that tannins can be used as an alternative to anthelmintics (Carrai et al., 2003; Paolini et al., 2003). Hence it may be speculated that the anthelmintic activity of the plant might be due to its tannin content. On the other hand, Abe et al. (1989) reported the inhibitory effect of two lignin derivatives from the cone of Pinus parviflora against Hymenolepis nana (Cestoda) infection in infant mice. Pounded leaf extract or juice obtained from the fresh leaves of Plantago lanceolata is used externally against parasites in animals (Tabata et al., 1994). Both aqueous and EtOH extracts showed significant anthelmintic activity, although EtOH extract was more pronounced. Seeds of corn (Zea mays) and dried white beans are cooked together and taken orally, against intestinal worms (Yesilada et al., 1999). Although the EtOH extract did not show any activity, aqueous extract showed potent anthelmintic activity against both parasites. In conclusion, all plants which are reported to treat parasitic disorders in Turkish folk medicine have shown to possess in vivo anthelmintic activity against both parasites. Particularly, Mentha longifolia and Jasminum fruticans showed the highest inhibitory rates against pinworms. Both ethanol and aqueous extract of Mentha longifolia and Jasminum fruticans were found to be significantly active. In overall evaluation, ethanol extracts were found generally highly effective, except Zea mays seeds, while only the aqueous extract was active. Although the aqueous extract of Pinus nigra ssp. pallasiana cones was found effective against both pinworms, the value against Syphacia obvelata infections was only found statistically significant. This study has clearly demonstrated that all the selected materials which were reported to be used against parasitic infections in Turkish folk medicine, as recorded in the TUHIB, possess statistically significant relevant activity. Further studies may yield the discovery of effective and safe new anthelmintic agents. References Abe, M., Okamoto, K., Konno, K., Sakagami, H., 1989. Induction of antiparasite activity by pine cone lignin-related substances. In Vivo 3, 359–362. Anthanasiadou, S., Kyriazakis, I., Jackson, F., Coop, R.L., 2000. Effects of shortterm exposure to condensed tannins on adult Trichostrongylus colubriformis. Veterinary Record 146, 728–732. Butter, N.L., Dawson, J.M., Wakelin, D., Buttery, P.J., 2000. Effect of dietary tannin and protein concentration on nematode infection Trichostrongyus colubriformis in lambs. Journal of Agricultural Science (Cambridge) 134, 89–99. Carrai, V., Borgognini-Tarli, S.M., Huffman, M.A., Bardi, M., 2003. Increase in tannin consumption by sifaka (Propithecus verreauxi verreauxi) females during the birth season: a case for self-medication in prosimians. Primates 44, 61–66. Chung, W.C., Ko, B.C., 1976. Treatment of Taenia saginata infection with mixture of areca nuts and pumpkin seeds. Zhonghua Min Guo Wei Sheng Wu Xue Za Zh 9, 31–35. Deepak, M., Dipankar, G., Prashanth, D., Asha, M.K., Amit, A., Venkataraman, B.V., 2002. Tribulosin and ␤-sitosterol-d-glucoside, the

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