COMPARATIVE ANTI-TRICHOMONAS VAGINALIS ACTIVITY EVALUATION OF SAMBUCUS NIGRA L. FLOWERS AND FRUITS EXTRACTS

Ecem Erdemir1image, Husniye Kayalar2*image, İbrahim Cavus3image, Ahmet Ozbilgin3image 

1Bozyaka Education Research Hospital, Izmir, Turkey.

2Ege University Faculty of Pharmacy  Department of Pharmacognosy, Bornova, Izmir, Turkey.

3Manisa Celal Bayar University, Faculty of Medicine, Department of Medical Parasitology, Manisa, Turkey.

 

Abstract

Background: Sambucus nigra or also known as Black elderberry or European elderberry is been used for the teatment and prevention of various diseases. The plant posseses health beneficial effects such as antimicrobial, antioxidant, antiulcerogenic, antidepressant, antidiabetic, antiviral and antiinflammatory.  However, there is limited report on the antitrichomoniasis activity of S. nigra. 

Objectives: The study aimed to comparatively analyze the in vitro trichomonicidal effects of various extracts prepared from both fruits and flowers of S. nigra. Half maximal inhibitory concentration (IC50) and minimum parasiticide (MPC) values of the extracts against metronidazole-resistant Trichomonas vaginalis 50143 strain were determined by the liquid microdilution method using 96-well microplates.

Results: After 48 hrs incubation with parasites, the highest activities were observed for 70% hydroethanolic extract, chloroform extract and methanol extract of fruits with IC50 values of 513, 531 and 566 µg/ml, respectively. Fruit extracts exhibited higher activity than flower extracts, and no activity was observed in methanol extract of flowers.

Conclusion: This study is the first comparative analysis of the anti-Trichomonas vaginalis activity of elderberry extracts. This study will serve as a reference source in isolating the components of elderberry fruits with antiparasitic activity.

Keywords: Metronidazole, plant extracts, Sambucus nigra, Trichomonas vaginalis, trophozoites.

 

 

INTRODUCTION

 

Sambucus nigra L., distributed in Europe, North Africa, West and Central Asia and North America, is one of the two species growing wildly in Turkey. This perennial shrub or 8-10 m tall tree with purplish fruit and creamy white flowers belongs to Adoxaaceae family1-3. S. nigra is also known as Black elderberry or European elderberry and believed to have been used since ancient times for the treatment and prevention of various diseases4,5. In traditional Chinese medicine S. ebulus or known as dwarf elder is widely used in bone and joint disorders6. In Europe and African countries, Sambucus species are traditionally used in the treatment of respiratory diseases such as asthma and bronchitis, throats, sinusitis, herpes, neuralgia, epilepsy and for dental problems for bronchitis and stomach ache7,8.

n Turkey, in addition to external use for the treatment of rheumatism and wounds, the decoction or infusion prepared from flowers and fruits of S. nigra are used as diuretic, diaphoretic, immune system booster and protective against cancer. The fruits are also used in the carpet and textile industry due to its colorful structure9-12.

Numerous pharmacological activities have been conducted on the fruits of S. nigra and proven to posses health beneficials such as antimicrobial, antioxidant, antiulcerogenic, anti-depressant, antidiabetic, antiviral and anti-inflammatory8,13. Due to various health benefits, the elderberry fruits are also included in the composition of jam, pie, ice cream, yoghurt, wine, tea and fruit juices6.

Trichomoniasis is one of the most common sexually transmitted diseases caused by T. vaginalis whose annual average prevalence is around 156 million in the world14. Trichomonas manifests itself with a number of symptoms, and different symptoms occur in men and women. This disease is also reported to be the causative agent of cervical cancer, pelvic inflammation and infertility15. Metronidazole is the most effective drug used in the treatment of trichomoniasis. Unfortunately, besides drug resistance, side effects such as gastrointestinal disorders, taste problems, nausea, vomiting, vertigo, neutropenia, skin reactions, cloudy urine and fatigue are reported for metronidazole16-19. Due to serious side effects of 5-nitroimidazole derivatives, a more accesible source of drugs with fewer side effects are still being investigated from natural sources20.

In a study conducted in Iran, plants registered in international databases were searched for their in vitro anti-Trichomonas activity. Garlic, lavender, eucalyptus, geranium, onion, yarrow, wormwood which are traditionally used for antiparasitic activities were found to be effective against T. vaginalis21. In other investigations conducted to find alternative sources for the treatment of trichomoniasis, rhubarb, turmeric and fennel were found to inhibit the growth and reproduction of parasites22,23. In a recent study, Niknam et al., reported the anti-T. vaginalis effect of methanolic extract prepared from S. nigra fruits from nothern Iran24.

To the best of our knowledge this is the first comparative research in scientific literature on the anti-Trichomonas activity of elderberry fruits and flowers. In this study, the extracts were prepared from fruits and flowers of Sambucus by using solvents with different polarities and are analyzed for their in vitro antiparasitic activity against T. vaginalis. While there have been recent studies on the antioxidant, antimicrobial, cytotoxic and antiviral activities of S. nigra6,8, it is surprising that only one study has been conducted on the activiy of S. nigra fruits against the common T. vaginalis parasite24. This in vitro anti-T. vaginalis activity research will provide a basis for future in vivo trichomonocidal activity studies on S. nigra.

 

MATERIALS AND METHODS

 

Plant materials and preparation of extracts

S. nigra flowers and fruits were collected in June and August 2023, respectively. The plant species was authenticated by pharmacist Muammer Şen from Konya. The plant materials were dried at room temperature. The dried fruits and flowers were purchased from Pharmacist Muammer Şen, whose the owner of Temmuz Organic Farm, Selçuklu, Konya, Turkey. An amount of 5 g of fruits and flowers were grounded and macerated with solvent (25% hydroethanolic solution, 70% hydroethanolic solution, methanol and chloroform) with a plant/solvent ratio of 1:10 at room temperature under stirring for 48 hrs. The extracts were filtered through Whatman no.1 paper and solvent was evaporated to dryness by rotary evaporator under reduced pressure. The extracts were lyophilized and stored at -20°C until analysis25.

Preparation of Tyrypticase Yeast Extract Maltose (TYM) Medium

Metronidazole resistant reference strain T. vaginalis ATCC 50143 was obtained from Celal Bayar University, Faculty of Medicine, Parasitology Bank, Manisa. In order to revive T. vaginalis parasites and to bring to logarithmic phase, tyrpticase yeast, maltose (TYM) medium was used. TYM medium is mainly composed of 20 g trypticase, 5 g maltose, 10 g yeast extract, 0.2 g L-ascorbic acid, 1 g L-cysteine, 0.5 g agar and 0.8 g pH regulators (K2HPO4-KH2PO4). Materials other than agar were weighed in the required amounts and dissolved in 900 ml distilled water using magnetic stirrer. The pH was adjusted to 6, followed by addition of agar and the mixture was sterilized for 20 minutes at 121oC under 15 psi. At the end of sterilization process, TYM medium was divided into 4 ml screw cap glass tubes. To make a total volume of 5 ml, before use, 100 IU/ml streptomycin, 100 IU/ml penicillin and commercially obtained inactive horse serum were added to TYM medium26,27.

Revitalizing the parasites and bring into logarithmic phase

T. vaginalis strain was quickly thawed in a 37oC water bath within 1.5-2 minutes and then centrifuged  and washed twice with fresh medium to remove remaining DMSO. After washing, the parasites accumulated at the bottom were added to TYM medium with 16% horse serum and incubated in the oven at 37oC.The growth of parasites was observed under a microscope. Then, the strains were then transferred to fresh medium to ensure average adaptation. Parasites that receive sufficient and necessary substances and pass the latent phase are expressed as production by multiplying their numbers in a generation period specific to their species is defined as logarithmic phase. Strains entering logarithmic phase were transferred to cell flask and incubated for the growth of parasites in high volumes27,28.

Investigation of the antitrichomonial activity of extracts

Half maximal inhibitory concentration (IC50) and minimum parasiticide concentration (MPC) values of the extracts against metronidazole-resistant T. vaginalis 50143 strain were determined in vitro by the liquid microdilution method in 96-well microplates28. After the trophozoites entered the logarithmic phase, they were counted on the Thoma chamber using trypan blue stain and their viability was determined. In the experiments, trophozoites with a survival rate of over 95% were used and the amount of parasites was adjusted to 5x103 parasites/ml using a Thoma chamber. 5x10³ parasites/ml T. vaginalis trophozoites were added to each well except for the negative control and the microplates were incubated at 37°C for 48 hrs. At the end of incubation, the motility of T. vaginalis trophozoites was evaluated by direct examination under an inverted microscope, and parasite viability was determined using trypan blue. For growth control (positive control), two wells containing T. vaginalis without tested extracts were used, and for contamination control (negative control), two wells containing the extracts without  T. vaginalis were used. Then, the plates were incubated at 37°C under aerobic conditions. At the 24th and 48th hrs of incubation, the motility of T. vaginalis trophozoites was evaluated under an inverted microscope, and their viability was evaluated on a Thoma counting chamber using trypan blue stain. The extract concentration in the last well, where approximately half of the T. vaginalis trophozoites were determined to be alive using trypan blue stain, was accepted as the IC50 value and the MPC value was determined by taking trophozoites from the wells containing immobile parasites, passage them into a new TYM medium, and checking for growth at the 24th and 48th hrs29.

Statistical analysis

The results of three parallel studies carried out at two different times were averaged and the viability percentages corresponding to all concentrations in which two-fold serial dilutions were analyzed with the Graphpad program and IC50 and MPC values were expressed as µg/ml of the extracts.

RESULTS AND DISCUSSION

 

In this study, the anti-T. vaginalis activity of S. nigra fruits and flowers extracts prepared with solvents of different polarities was comparatively analyzed. The parasite viability percentages of the fruit and flower extracts at 24 and 48 hrs are demonstrated in Table 1, Table 2, and Table 3, Table 4 respectively. The extracts of S. nigra reduced the number of live T. vaginalis parasites in a time and concentration dependent manner. The fruit extracts exhibited higher trichomonocidal activity then the flower extracts. As shown in Table 5, after 24 hrs of incubation the chloroform and 70% hydroethanolic extracts of fruits showed the highest inhibition of parasites with IC50 values of 1136 and 1138 µg/ml respectively.

S. nigra fruit methanolic extracts’ IC50 were calculated as 1373 and 566 µg/ml for 24 hrs and 48 hrs respectively whereas no activity was observed at 48 hrs for the methanolic extracts of flowers even at concentration 25000 µg/ml. Among the flower extracts, the highest activity was observed in the chloroform extract with IC50 value at 605 µg/ml. The 70% hydroethanolic extract of fruits exhibited the highest trichomonocidal activity with 513 µg/ml IC50, while it was 8 µg/ml for metronidazole. In a study conducted on trichomonocidal activities of herbs used in Korea, the methanolic extracts of Torilidis fructus, Sophorae radix and Agrimonae herba were reported to show complete trichomonocidal activity at the concentration of 400 g/ml where as metronidazole used as positive control showed 0.94% viability at the concentration of 5 g/ml33.

In another survey on the anti-T. vaginalis activitiy, hydroalcoholic extracts of Eugenia caryophyllata, Camellia sinensis and Terminalia chebula were reported to have the best anti-trichomonal activity with IC50 values of 1.21, 1.62 and 1.66 mg/ml, respectively34. In the present study, the 70% hydroethanolic fruits extract which had the highest anti-T. vaginalis activity among the investigated fruit and flower extracts, inhibited the 50% of parasites at the concentration of  0.513 mg/ml which is  much lower concentration than those extracts which were reported to have significant activity. In a previous report, methanolic extract of S. nigra fruits were evaluated for in vitro anti-Trichomonas activity at concentrations ranging from 100-800 µg/ml and at 800 µg/ml the extract was reported to have 100% efficacy after 48 hrs24. In contrast, in this study fruit methanolic extract inhibited the 50% of parasites at 566 µg/ml at 48 hrs. 95% parasite inhibition was observed in methanol extract at 1562.6 µg/ml. Obtained results showed that S. nigra has acceptable efficacy in vitro and according to minimum parasiticide concentrations fruit extracts were significantly better than flower extracts.

However in vivo  trichomonocidal activity search is required for further studies. Among the phytochemicals in S. nigra, flavonoids, anthocyanins, phenolic acids, cyanogenic glycoside and vitamin A and vitamin C have been reported8. Research has been conducted mostly on the phenolic components of the elderberry plant30-32, but as observed in this study, chloroform extract, which is known to contain more lipophilic compounds, also showed strong activity.   In addition to hydroethanolic and methanolic extracts of fruits, the chloroform extracts of fruits and flowers are also worth for investigating antitrichomonocidal candidates.

 

CONCLUSIONS

 

There are limited studies on the activity of elderberry against trichomoniasis. This study is the first comparative analysis of the anti-T. vaginalis activity of S. nigra extracts. This study will serve as a reference source in isolating the components of elderberry fruits with antiparasitic activity. Anti-T. vaginalis bioactivity guided assay is planned for further studies for the isolation of active constituents of fruits and flowers of S. nigra.

 

ACKNOWLEDGEMENTS

 

We would like to render our thanks to Parasitology Bank of Faculty of Medicine, Celal Bayar University for supplying the parasite strains.

 

AUTHOR’S CONTRIBUTION

 

Erdemir E and Çavus I: investigation, data curation, writing and editing. Kayalar H: plant material supply, methodology. Kayalar H and Ozbilgin A: supervision

Final manuscript was read and approved by all authors.

 

DATA AVAILABILITY

 

The data will be available to anyone upon request from the corresponding author.

 

CONFLICT OF INTEREST

 

None to declare.

 

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