Original Articles

Cytotoxic, Phytotoxic and Insecticidal Activities of Chrysophthalmum montanum (DC.) Boiss.

10.4274/tjps.07279

  • Fatma AYAZ
  • Nurgün KÜÇÜKBOYACI
  • Hayri DUMAN
  • Bilge ŞENER
  • Muhammad Iqbal CHOUDHARY

Received Date: 13.10.2016 Accepted Date: 13.02.2017 Turk J Pharm Sci 2017;14(3):290-293

Objectives: To investigate the in vitro cytotoxic, phytotoxic, and insecticidal activity of Chrysophthalmum montanum (DC.) Boiss.
Materials and Methods: The crude methanol (80%) extract of the aerial parts of C. montanum was fractionated to obtain n-hexane, chloroform, n-butanol, and remaining water fractions. The crude extract and subsequent solvent fractions of the plant were evaluated for their biological activities using screening bioassays such as cytotoxicity on brine shrimp lethality, phytotoxicity against Lemna minor L., and insecticidal activity against Rhyzopertha dominica and Tribolium castaneum.
Results: The cytotoxicity assay revealed that the crude extract, n-hexane, and chloroform fractions of the plant had positive lethality with LD50 values of 71.51, 126.62, and 75.95 µg/mL, respectively. The extract and its fractions, except for the remaining water fraction, showed phytotoxic activity, which was expressed as percentage growth regulation in a concentration-dependent manner. n-hexane and chloroform fractions in particular had 100% growth inhibition (GI) at 1000 μg/mL, followed by the n-butanol fraction (62.6% GI) and crude extract (40.0% GI) of the plant at the same concentration. Otherwise, all samples had no insecticidal activity against R. dominica and T. castaneum.
Conclusion: This study demonstrates that C. montanum contains bioactive compounds related to potential biological activities such as cytotoxic and phytotoxic.

Keywords: Chrysophthalmum montanum,Asteraceae,cytotoxic activity,phytotoxic activity,insecticidal activity

INTRODUCTION

Medicinal plants contain various chemical constituents that have potential to use for their biological activities. Natural resources that yield valuable phytochemical products are often used in the treatment serious diseases. Moreover, folk medicines can attribute to the discovery of a large number of clinically effective compounds.

Chrysophthalmum Schultz Bip., a member of the family Asteraceae, the tribus Inulaeae1, is represented by three species, namely Chrysophthalmum montanum (DC.) Boiss., C. dichotomum Boiss. & Heldr., and C. gueneri Aytac and Anderb2 in Turkey. C. montanum, known as “nezle otu and tutça”, is a herbaceous perennial plant mainly distributed in eastern parts of Turkey. Its aerial parts are traditionally used for the treatment of the common cold and sinusitis, as well as healing wounds on the body of human and animal in Turkey.3-5

To date, a few studies on the morphologic characteristics and preliminary evaluation of biologic antioxidant and antimicrobial activities have been reported on C. montanum.2,3,6-10 Only one recent phytochemical study has been conducted on the isolation of sesquiterpene lactones from C. montanum.11 However, there have been no other experimental studies for the scientific evaluation of phytotoxic, cytotoxic, and insecticidal effects of C. montanum.

The Asteraceae family has been intensively investigated in the treatment of various diseases in recent years. The family is well-known as a good source of sesquiterpene lactones, which are associated with antitumor, cytotoxic, antimicrobial, anti-inflammatory, and phytotoxic activities.12,13 In our ongoing research on C. montanum, we revealed that C. montanum had cytotoxicity against some cancer cell lines using sulforhodamine B assays.14 The aim of the present study was to investigate the therapeutic importance of C. montanum, which is relatively safe from toxic effects, for its phytotoxic, cytotoxic, and insecticidal activities by using screening bioassays.


EXPERIMENTAL

Chemicals

In the extraction and fractionation procedure, methanol, n-hexane, chloroform, and n-butanol were of analytical grade and purchased from Merck Co. (Darmstadt, Germany). Analytical thin-layer chromatography (TLC) was performed on precoated Kieselgel 60 F254 plates (Art. 5554, Merck). The plates were sprayed with anisaldehyde reagent [76% methanol (Merck) and 19% ortho-phosphoric acid (Riedel-De Haën, Buchs, SG Switzerland), 5% p-anisaldehyde (Merck)] and 20% H2SO4 (Merck) solution in MeOH (Merck).

Plant material

The aerial parts of C. montanum (DC.) Boiss. were collected from the valley of Tohma River, Akçadağ, Malatya, Turkey at the flowering stage in July 2014. The plant material was identified by one of the authors (Professor, PhD Hayri Duman). An authenticated voucher specimen (Hayri Duman 10324) was deposited in the Herbarium of GAZI, Ankara, Turkey.

Preparation of extracts

The air-dried aerial parts of C. montanum (500 g) were extracted four times (4x3000 mL) with 80% methanol at 25°C by stirring for 2 days. Following filtration, the combined methanol extracts were evaporated in vacuo at 40°C to dryness. The concentrated MeOH extract (90.8 g, CM) were further fractionated through successive solvent extractions with n-hexane (11x250 mL), chloroform (8x250 mL), and n-butanol saturated with H2O (8x250 mL) in a seperatory funnel. Each extract, as well as its remaining aqueous phase (R-H2O) after solvent extractions were evaporated to dryness under reduced pressure to yield an “n-hexane fraction” (1.7 g, CMH), “CHCl3 fraction” (15.8 g, CMC), “n-BuOH fraction” (21.4 g, CMB), and “R-H2O fraction” (36.4 g, CMR), respectively.

Phytochemical analysis

The extracts of C. montanum (1 mg/mL) were applied to silica gel plates. The n-hexane and CHCl3 extracts were developed with the mixture of n-hexane:ethylacetate (65:35) and chloroform:acetone (80:20), respectively, as mobile phases. TLC plates were evaluated under UV light at 254 and 366 nm for the determination of fluorescent compounds. Anisaldehyde reagent and 20% H2SO4 were sprayed on the plates to visualize the separated compounds, and then the plates were heated for 5 min at 100°C. Sesquiterpenes appeared with pink and purple coloration.

Brine shrimp lethality assay

Brine shrimp (Artemia saline Leach) eggs (50 mg) were sprinkled in a hatching tank (a rectangular dish 22x32 cm) half-filled with filtered brine solution. The crude extract and subsequent solvent fractions of C. montanum (20 mg) were dissolved in 2 mL of methanol (stock solution). The stock solutions of the extracts were diluted to 10, 100, and 1000 µg/mL concentrations in three vials. The solvent was evaporated under a fume hood by keeping overnight. After hatching (2 days), 30 shrimps were added in each vial with the volume adjusted to 5 mL using sea water. The vials were incubated at 25-27°C for 24 hours under illumination. Other vials were supplemented with solvent and reference cytotoxic drug (Etoposide: 7.46 µg/mL), which served as negative and positive controls, respectively. The number of brine shrimps that survived was counted in each vial and LD50 values with 95% confidence intervals were determined using Finney computer software.15,16

Pytotoxicity assay

The phytotoxicity assay was performed for the crude extract and subsequent solvent fractions of C. montanum against Lemna minor L.17 The medium was prepared by mixing various constituents in 1000 mL distilled water. KOH pellets were added for the adjustment of pH at 6.0-7.0. The extracts (30.0 mg) were dissolved in 1.5 mL of methanol, which served as a stock solution. The stock solutions of the extracts were diluted to get final concentrations as 10, 100, and 1000 µg/mL (nine flasks, three for each dilution). After evaporating the solvent overnight under sterile conditions, 20 mL medium and 10 plants were added to each flask, each one containing a rosette of two fronds of L. minor. Other flasks were supplemented with medium and reference plant growth inhibitor (Paraquate) as negative and positive controls, respectively. All flasks were incubated in a growth cabinet for seven days at 30°C. The number of fronds per flasks was counted and recorded at the end of the incubation period. Growth regulation (GR) as a percentage (%) was determined using the formula given below:

The criteria indicate that the GR (%) of 0-39 for low activity,

                          10-Number of the fronds

                              in the test samples

GR (%) -------------------------------------------------x100

                           Number of the fronds

                           in the negative control

40-59 for moderate activity, 60-69 for good activity, and >70 for significant activity were detected.

Insecticidal activity

The crude extract and subsequent solvent fractions of C. montanum were tested against Rhyzopertha dominica and Tribolium castaneum using impregnated filter paper.18 The samples (200 mg) were dissolved in 3 mL of methanol and served as stock solution. The samples (1019.10 µg/cm2) were applied to filter paper of appropriate size (9 cm or 90 mm) on petri plates using a micropipette. The plates were left for 24 hours to evaporate the solvent. The next day, 10 insects of each species were placed in each plate (test and control) using a clean brush. Permethrin (239.5 µg/cm2) was used as positive control; methanol was used as negative control. The plates were incubated at 27°C for 24 hours with 50% relative humidity in the growth chamber. For the calculation, the number of survivals of each species was counted and mortality (M) (%) was determined using the following formula:

                                  100 - Number of insects alive

                                          in the test samples

M (%)= ------------------------------------------------------------------ X100

                                      Number of insects alive

                                              in the control


RESULTS AND DISCUSSION

In this study, we investigated the crude (80% methanol) extract and its fractions of C. montanum for their primary screening bioassays including cytotoxic, phytotoxic, and insecticidal activities. The cytotoxic properties of C. montanum were investigated at concentrations of 10, 100, and 1000 µg/mL, using etoposide as a standard. The methanol extract, n-hexane, and chloroform fractions of the plant had positive lethality with LD50 values of 71.52, 126.62, and 75.95 µg/mL against the brine shrimps, respectively (Table 1).

The phytotoxicity of the investigated samples on L. minor was observed to have dose-dependent activity because low activity was detected in the n-hexane fraction with 12.5 and 18.7% inhibition at 10 and 100 µg/mL, respectively. Moderate phytotoxic activity was found in the methanol extract (40.0% inhibition) at 1000 µg/mL. Good phytotoxic activity was found in the chloroform fraction (68.7% inhibition) at 100 µg/mL and n-butanol fraction (62.6% inhibition) at 1000 µg/mL. Significant phytotoxic activity was shown in the n-hexane and chloroform fractions of the plant; 100.0% inhibition for each fraction at 1000 µg/mL (Table 2).

The methanol extract and fractions of C. montanum were also screened for their insecticidal effects against R. dominica and T. castaneum using permethrin as a standard drug. There were no insecticidal effects on all samples against T. castaneum and R. dominica (Table 3).

The brine shrimp lethality assay is not specific for any particular physiologic effects. However, the cytotoxic effect of the natural constituents on the shrimp larvae was especially correlated with their anticancer potentials. This preliminary method, which has been developed for screening, fractionation, and monitoring of physiologically active natural products, is clearly a more rapid, inexpensive, and general bioassay.16 Moreover, phytotoxic and insecticidal constituents are mostly important to develop natural herbicides and insecticides that are safe, cost effective, and user-friendly for the environment.19

According to our results, the n-hexane and chloroform fractions of C. montanum were found as promising samples due to having cytotoxicity on brine shrimp. In our recent study, n-hexane and chloroform fractions of the plant also exhibited cytotoxicity on selected cancer cell lines.14 In addition, our findings demonstrate that n-hexane and chloroform fractions of C. montanum possess significant phytotoxicity against L. minor. Our preliminary phytochemical detection using TLC showed that sesquiterpenes were as prominent components in the bioactive chloroform fraction of the plant.


CONCLUSION

In summary, the present study firstly depicts the potential of the extracts of C. montanum on biologic activities such as cytotoxicity against brine shrimp and phytotoxic effects, which indicate that the plant might be considered as a new potential source in the research of new drugs. Accordingly, further investigations to identify the responsible bioactive compound(s), principally sesquiterpenes, are ongoing on C. montanum.


ACKNOWLEDGEMENTS

This study was supported by TUBİTAK-2214/A, TUBİTAK-2211/A and ICCBS-HEJ.

Conflict of Interest: No conflict of interest was declared by the authors.


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