ORIGINAL ARTICLE

Extraction-based blood coagulation activity of marigold leaf:a comparative study

Nandita Dasgupta & Shivendu Ranjan & Saleh M. A. Arabi Mohammed &

Pradeep Singh Jadon & Samuel S. Melvin & Annie Deborrah Harris &Arkadyuti Roy Chakraborty & Chidambaram Ramalingama

Received: 25 September 2013 /Accepted: 20 May 2014# Springer-Verlag London 2014

Abstract Marigold (Tagetes erecta), besides being an orna-mental plant, has various medicinal properties—it is nematoci-dal, fungicidal, antibacterial and insecticidal and aids in woundhealing. Our work is focused on the blood clotting activity of itsleaf extracts. Extraction was done by conventional as well as theSoxhlet method, which was found to be much more efficientusing a 1:1 ratio of ethanol to water as solvent. Blood clottingactivity of the leaf extract was examined using prothrombin timetest using the Owren method. For both extraction methods, theyield percentage and coagulation activity in terms of coagulationtime were analysed. Marigold leaf extract obtained using theSoxhlet method has shown very good blood coagulation prop-erties in lower quantities—in the range of microlitres. Furtherresearch is needed for identification and quantification of itsbioactive compounds, which could be purified further and en-capsulated. Since marigold leaf has antibacterial properties too,therefore, it might be possible in the future to develop an anti-septic with blood coagulation activity.

Keywords Bloodclotting .Soxhletextraction .Tageteserecta(Marigold) . Prothrombin time test

Introduction

Tagetes erecta, also known as marigold, is a popular orna-mental plant and grows in various agroclimates. Beside its useas an ornamental plant, it is also cultivated as a crop. Thespecies Tagetes minuta, T. erecta, Tagetes patula and Tagetestenuifolia are the most common in tropical countries. Bioac-tive extracts from different parts of this plant exhibit nemato-cidal, fungicidal and insecticidal activities (Padma et al.1997). Marigold leaf extracts have also shown antibacterialproperty against several gram-positive and gram-negativebacteria (Nandita et al. 2012). It also has antioxidant(Gordana et al. 2004) and wound healing properties (Bashirand Gilani 2008). Research is still in progress to find thepossible mechanism of action for the wound healing propertyof marigold leaves.

Coagulation of blood after vascular spasm and plateletaggregation involves formation of prothrombinase and theconversion of prothrombin into thrombin, which helps inconversion of fibrinogen into fibrin (clot). Prothrombin time(PT) test is the most commonly used coagulation test in healthcare (Horsti 2002). In the present study, marigold leaf extractswere obtained from two different extraction methods (conven-tional and Soxhlet) and comparative analysis of yield percent-ages, as well as blood coagulation activity (in terms of coag-ulation time), was performed.

Material and methods

Marigold (T. erecta) leaves were collected from the localmarket of Vellore, India. All standard chemicals were obtainedfrom the various chemical units E. Merck India Ltd., HiMediaPrivate Ltd. and S. D. Fine-Chem. Ltd. (India).

Nandita Dasgupta and Shivendu Ranjan contributed equally to this work.

N. Dasgupta : S. Ranjan : S. M. A. Arabi Mohammed : P. S. Jadon :S. S. Melvin :A. D. Harris :A. R. Chakraborty :C. RamalingamaInstrumental and Food Analysis Laboratory, IndustrialBiotechnology Division, School of Bio Sciences and Technology,VIT University, Vellore 63014, India

S. RanjanDepartment of Zoology, Jai Prakash University, Chapra, Bihar, India

S. M. A. Arabi Mohammed (*)School of Bio Sciences and Technology, VIT University, Vellore,Tamil Nadu, Indiae-mail: amsaleh@vit.ac.in

Comp Clin PatholDOI 10.1007/s00580-014-1943-5

Processing of marigold leaves

Fresh marigold leaves were chopped and subjected to dryingin a hot air oven in a static environment at a temperature of 40±5 °C for 2 days. The leaves were then crushed to a powderedform using an electric grinder and stored in an airtight glassbottle at 4 °C for further analysis.

Conventional extraction

Of the powdered leaves, 5.00±0.05 g was taken in threeconical flasks, and 50 ml of different solvents was addedseparately and kept in an orbital shaker at 150 rpm at 50 °Covernight (Daniel et al. 2007). This extraction process wascarried out using three different types of solvents—water,ethanol and ethanol/water (1:1).

Soxhlet extraction

An amount of 5.00±0.05 g of powdered marigold leaves wasplaced in a Whatman cellulose thimble (25 mm×100 mm).Samples were extracted in a Soxhlet extraction system using150 ml of different solvents. The heating power was set to twocycles per hour so that six cycles of extraction was achievedwithin 3 h (Andri et al. 2009). As with the conventionalextraction, this process was carried out using three differentsolvents—water, ethanol and water/ethanol (1:1).

Exclusion of solvent

The crude extract solutions obtained were then filtered usingWhatman filter paper and concentrated using a vacuum rotaryevaporator (BUCHI Rotavapor Model R-210) at 60 °C orlower to remove the ethanol, lyophilized to remove waterand dried using a freeze dryer (LT5S, Lyophilization Systems,Inc., USA, with five shelves of 0.9 m2). The lyophilizer wasused to reduce the temperature down to −30 °C, and thecoolants used were R404A (44 % w/w pentafluoroethane,52 % w/w 1,1,1-trifluoroethane, 4 % w/w 1,1,1,2-tetrafluoroethane) and R508R (54 % w/w hexafluoroethane,46 % w/w trifluoroethane) at 150 and 200 psi, respectively.

Pressure was lowered to 3.3×10−4 Pa for primary drying and3.3×10−5 Pa for secondary drying. Heat was supplied andmaintained at 25 °C to help the ice sublimate into vapour.After 16 h of drying, the samples were collected and stored inairtight bottles at −4 °C for further experimentation.

Prothrombin time measurement by the Owren method

Coagulation was analysed by measuring PT by the Owrenmethod (Tietz 1995) with a simple modification, i.e. insteadof thromboplastin reagent, we used marigold leaf extractsfor our analysis. Human blood samples were collected byvenepuncture. The blood was decalcified by EDTA to pre-vent blood clotting (Horsti 2001). The blood cells (RBCs,WBCs, platelets, etc.) were separated from the plasma bycentrifugation of 13,000 rpm at 4 °C for 10 min (Guderet al. 1997). Blood plasma was collected in test tubes andkept in a water bath at 37 °C for 2 min. According to theexperimental design, 80 μl of marigold leaf extracts (bothconventional and Soxhlet extracts) were added to 1 ml ofblood plasma and one plasma sample was kept as control.The extract started to counteract the effect of EDTA andclotting was observed. The coagulation time was then tab-ulated. Prothrombin time should be analysed within 2 h fora sample at room temperature or within 4 h for a sample at4 °C from the time of sample collection (Tietz 1995; Dacieand Lewis 1995; Ravel 1995).

Statistical analysis

All statistical analysis was performed and expressed as meanof parallel duplicates of ANOVA correlation. P<0.05 wasconsidered as statistically significant.

Results

The yield percentage of the marigold leaf using the conven-tional extractionmethodwith different solvent systems (water,ethanol and ethanol/water in 1:1 ratio) was 3.45±0.08, 4.35±

Table 1 Comparative yield percentage of marigold leaf by conventional and Soxhlet extraction methods with different solvents and the respectivecoagulation times

Solution number Solvents Conventional Soxhlet

Yield (%) Coagulation time (s) Yield (%) Coagulation time (s)

1 Water 3.45±0.08 23.79±0.07 4.65±0.05 22.59±0.08

2 Ethanol 4.35±0.06 22.42±0.06 5.51±0.07 18. 26±0.09

3 Ethanol/water (1:1) 5.46±0.09 21.54±0.06 6.79±0.08 13.14±0.06

Comp Clin Pathol

0.06 and 5.46±0.09, respectively. Comparatively, the Soxhletmethod yield percentage was 4.65±0.05, 5.51±0.07 and 6.79±0.08, respectively (Table 1). Based on these data, it can beconcluded that the Soxhlet extraction is suitable for bioac-tivity analysis since it has greater yield than the conven-tional method.

When extracts were mixed with blood plasma, the coagu-lation time was analysed. The coagulation time using extractsobtained using the conventional technique was 23.79±0.07,22.42±0.06 and 21.54±0.06 s with the solvents water, ethanoland water/ethanol (1:1), respectively Using extracts obtainedvia the Soxhlet technique, the coagulation times were 22.59±0.08, 18. 26±0.09 and 13.14±0.06 s with the respectivesolvent, i.e. water, ethanol and water/ethanol (1:1). From thisresearch, it can be concluded that the bioactive compoundsresponsible for the blood coagulation are extracted more effi-ciently using the Soxhlet method compared with the conven-tional method of extraction. Also, it can be concluded thatwater and ethanol together as a solvent in the ratio of 1:1shows better bioactivity in terms of coagulation time.

Discussion

Marigold leaves were subjected to two different extractionmethods, conventional and Soxhlet, using different solvents(water, ethanol, water and ethanol in equal ratio) separately. Itwas observed that the extract yield obtained by the Soxhletextraction method with water/ethanol (1:1) as the solvent washighest and therefore is a suitable solvent for investigation.The decrease in coagulation time dependent upon the extrac-tion procedure and the solvent used can be attributed to thefact that marigold leaf extract may act as thromboplastinreagent in the Owren method for PT test and the thromboplas-tin agents are more efficiently extracted from Soxhlet usingwater and ethanol as a solvent in the ratio of 1:1 (Fig. 1).

In conclusion, marigold leaf extract shows very good bloodcoagulation properties in the microlitre range; therefore, furtheridentification and quantification of its bioactive compoundsshould be carried out in order to enable further purification andeven encapsulation. Microwave-assisted extraction, a relativelynovel extracting approach using a microwave applicator as an

energy source, has received increasing attention recently(Kaufmann and Christen 2002) and may achieve even betterresults for marigold leaf extraction. There are several parameterswhich are important for the extraction of active ingredient com-pound, i.e. time of microwave radiation, intensity of microwave,solvent concentration, solid solvent ratio and the temperature towhich the sample is subjected (Kosar et al. 2005).

As marigold leaves have many medicinal properties, e.g.antibiotic, antifungal, nematocidal, wound healing, etc., theymay have a future role as an antiseptic with blood coagulationactivity. The leaf extract could also be emulsified and used as acoagulant in tissue engineering (Danie et al. 2013).

Acknowledgments Dr.Aaron Southgate, Department of SurgicalResearch,Northwick Park Institute for Medical Research, Harrow, Mid-dlesex, UK is acknowledged for technical support and Englishcorrection. Chancellor and Vice Chancellor, VIT University, Vellore,TN, India are acknowledged for providing financial help for the projectunder the category of RGEMS-VC Fund, VIT.

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