ANTIFUNGAL, CYTOTOXIC AND PHYTOTOXICITY OF AERIAL PART OF RANUNCULUS MURICATUS

Muhammad Shahzad Aslam ¹, Rubina Rehman², Bashir A. Choudhary², Abdul Subhan Ijaz ², Muhammad Uzair², Muhammad Syarhabil Ahmad¹.

¹ School of Bioprocess Engineering, University Malaysia Perlis, Kompleks Pusat Pengajian, Jejawi 3, 02600 Arau, Perlis, Malaysia.

²  Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan.

Corresponding author:  Email: aslammuhammadshahzad@gmail.com

DOI: http://doi.org/10.22270/ujpr.v1i1.R2

ABSTRACT

The present research is preliminary biological screening of aerial plant of Ranunculus muricatus (Ranunculaceae). Dichloromethane and methanol extracts of the aerial plant were investigated for their antifungal, phytotoxic and cytotoxic activities.  Anti-fungal, cytotoxicity and phytotoxicity activities were performed by agar tube dilution assay, brine shrimp lethality bioassay and lemna bioassay respectively.

Dichloromethane and methanolic extracts exhibited significant phytotoxicity against Lemna minor having Paraquat as standard drug and incubation condition (28±1ºC). None of extracts presented any significant cytotoxic activity having Imipenum and Etoposide as standard drug respectively. Both extract had non-significant antifungal activity but it has been noted that methanol extract showed 30% inhibition with linear growth at 70 mm, when compared with control; only against Microsporum canis.  Ranunculus muricatus showed significant phytotoxicity. The phytotoxicty assay is a valuable major screen for weedicide investigation. Additionally, modern studies are currently carried out to identify the allelopathic constituents by isolation, purification and structure elucidation to find out as effective herbicidal.

Keywords: Biological screening; cytotoxicity, phytotoxicity, Ranunculus muricatus.

INTRODUCTION

Ranunculus muricatus also known as Ranunculus pseudo-muricatus Baltter and Hallb is indigenous to Atlantic, S. Europe, W. and S. W. Asia, Crimea, Caucasus, S. Siberia, Pakistan and India. Flowering period is between March and April¹. It is recognised with different names in different part of world such as spiny buttercup (English); Chambul, jaghagha, Latokari, Korgandal (Folk). This plant is slightly poisonous. Whole plant is traditionally used as decoction for periodic fever and asthma². The plant reported to contain stigmasterol-4-ene-3, 6-dione, stigmasterol, Anemonin, Aescin lactone dimethyl ether, beta-valley sterol, protocatechuic aldehyde, protocatechuic acid, and luteolin factors³. The findings of anemonin as chemical constituents in Ranunculus muricatus may justify the uses of these species against fever, rheumatism and rubefacient in Asian traditional medicines⁴.  Present study is conducted to document the in vitro biological activities of Ranunculculus muricatus.

conducted to document the in vitro biological activities of Ranunculculus muricatus. Antifungal, phytotoxic and cytotoxic bioassay has studied.

EXPERIMENTAL

Plant material

Ranunculus muricatus was collected from Jallo pind, Lahore. Total weight of wet plant collected was 15kg while 5kg of dried plant was obtained after drying. The plant was identified by Dr. Altaf Hussain Dasti, Professor, Institute of pure and applied Biology, Bahauddin Zakariya University, Multan. 

Extraction

The air-dried aerial part of plant material was grounded and extracted successively with dichloromethane and methanol (thrice with each solvent) at room temperature occasional shaking for 24hrs. Extracts were concentrated by Rotavapor-R20 at 35ºC.  

Anti-fungal bioassay

Test fungi such as Candida albicans, Aspergillus flavus, Microsporum canis Fusarium solani, Candida glabrata were employed for preliminary screening. Extracts were dissolved in sterile DMSO to serve as stock solution. Sabouraud dextrose agar was prepared by mixing Sabouraud 4% glucose agar and agar in distilled water. Known amount of media was dispensed into screw capped test tubes. Test tubes containing media were autoclaved 121°C for 15 minutes. Tubes were allowed to cool to 50°C and the desired concentration of extract was added into non-solidified media. The tubes were allowed to solidify at room temperature.

Each tube was inoculated with a 4mm diameter piece of in culture of fungi. All culture-containing tubes were inoculated at optimum temperature of 28-30°C for growth for 7 to 10 days. Culture was examined at least twice a weekly during the incubation. With no visible growth of microorganism is taken to represent the MIC of the test sample which is expressed in µg⁵. 

Phytotoxicity bioassay

Prepared inorganic medium of 5.5-6.0 pH attained with KOH pellets. 10 vials per dose 500, 50, 5 and control were prepared. 15mg of the extract was dissolved in 15ml of the solvent. 1000, 100 and 10µl of solution to vials for testing allow the solvent to evaporate overnight. 2ml of medium was added in each vial containing a single plant a rosette of three fronds. The vials were placed in a glass dish filled with 2cm of water, sealed the container with stopcock grease and glass plate. Placed the dish along with vials in growth chamber for seven days at 25°C under fluorescent and incandescent light. Count the number of fronds per vials on day 3 and 7. Analyzed the data as percent of control with ED 50 computer program⁵.

Brine shrinp lethality bioassay

Brine shrimp cytotoxicity assay was accomplished according to the standard procedure described by McLaughlin⁶. Three concentrations (1000, 100, and 10 ppm) of the plant extracts were used in

this assay. Brine shrimp larvae were hatched in a small partitioned tank in artificial seawater. Illumination was provided on one side to attract newly hatched larvae. Brine shrimp larvae with second in star stage were used in this assay.

Plant extracts of respective concentrations were added to dram vials. To each dram vial ten brine shrimp larvae were added. Negative control was prepared by evaporating 0.5ml of methanol in dram vials and then by adding sea salt solution to it. Following 24 hrs of incubation, survivors were counted by using magnifying glass. The experiment was repeated three times. Mortality data was transformed by probit analysis in finny computer program to estimate ED50 value. Percentage of mortality was also calculated at all concentrations⁶.

RESULTS AND DISCUSSION

Dichloromethane and methanol extracts of the aerial plant of Ranunculus muricatus were studied for their antifungal, phytotoxic and Brine Shrimp lethality bioassay. Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata were employed for fugitoxic effect of the extracts. It has been noted that MeOH extract of Ranunculus muricatus showed 30% inhibition with linear growth at 70mm, as compared with control; only against Microsporum canis at the concentration of 400µg/ml for incubation period of seven days at 27ºC with reference to Miconazole as standard. While dichloromethane extract does not showed any activity as shown in Table 1.

Dichloromethane and methanolic extracts of the aerial part of Ranunculus muricatus showed significant phytotoxicity at concentrations of 1000 µg/ml, 100 µg/ml and 10 µg/ml against Lemna minor.  Dichloromethane and methanolic extract of Ranunculus muricatus does not showed

cytotoxicity even at highest level having Etoposide as standard drug containing 28±1ºC as incubation condition.

CONCLUSION

The phytotoxicity assay is a valuable major screen for weedicide investigation. Weeds are one of the main issues of poor agronomic efficiency in the developing countries. Expensive, lethal and non-specific synthetic pesticides are used now. Using natural sources pesticides will not only reducers the adverse effect of pesticides but also help us to return to natural flora. Natural herbicides destroy specific targets, while leaving the wanted crop comparatively undamaged. Additionally, modern studies are currently carried out to discover the phytotoxic constituents of the plant by isolation, purification and structure elucidation to find out as effective herbicidal.

CONFLICT OF INTERESTS

Declared none.

ACKNOWLEDGEMENTS

The authors are grateful for the support provided by the department of Pharmacy Bahauddin Zakariya University, Multan and School of Bioprocess Engineering, University Malaysia Perlis, Malaysia. We also wish to acknowledge the technical support of HEJ Research institute of Chemistry, University of Karachi, Karachi, Pakistan.

REFERENCES

1. Nasir E, Ali SI. Flora of Pakistan 1986: No.172.
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3. Lingjie W, Xiao-Zhong G. The chemical composition of the fruit butter cup. China Modern Applied Pharmacy. 2009; 6:460-462.
4. Aslam MS, Choudhary BA, Uzair M, Ijaz AS, The genus Ranunculus: A Phytochemical and Ethnopharmacological Review. Int J Pharm Pharmaceu Sci. 2012; 4(5): 15-22.
5. Rehman A, Choudhary MI and Thomsen WJ. Bioassay technique for drug development Harwood Academic Publishers. 2001: 16-17, 23-24, 65-66.
6. McLaughlin JL. Crown gall tumors on potato discs and brine shrimp lethality: two single bioassays for plant screening and fraction. In: Hostetsmann, K, ed., Methods in Plant Biochemistry. Academic Press, London. 1991. 1-31.

Table 1:  Results of in-vitro antifungal bioassay of Ranunculus muricatus

Table 2:  Results of in vitro phytotoxic bioassay of Ranunculus muricatus

Table 3: Results of in-vitro cytotoxic bioassay of Ranunculus muricatus