IN-VITRO THROMBOLYTIC ACTIVITY OF PHENOLIC COMPOUND DEGRADING PRODUCT FROM LIGNIN
SUGARCANE BAGGASE USING Ochrobactrum sp.
Arie Srihardyastutie1*, M. Farid R1, Tri Ardyati2, Anna Roosdiana1, Indah Prihartini3
1Chemistry departement, Faculty of Mathematics and Natural Science, University of Brawijaya,
2Biology departement Faculty of Mathematics and Natural Science, University of Brawijaya,
3Biotechnology Departement, Univeritas Muhammadiyah Malang
*Corresponding email : [email protected]
Received day month year; Accepted day month year (will be given)
ABSTRACT
Sugarcane bagasse (Saccharum officinarum L) is a readily available waste product of cane sugar processing. The content of lignocelluloses in sugarcane bagasse is approximately 52.7% cellulose, 20% hemicelluloses, and 24.2% lignin. Lignin can be degraded enzymatically by using microorganisms, such as Ochrobactrum sp. Monomer derived from lignin degradation using these bacteria in the optimum condition of sugarcane fermentation (pH 6, temperature 40 °C, for 5 days of incubation, the concentrations of starter 29%) by GC-MS yielded phenolic compounds such as 4-methyl-2, 6-di-tert-butylphenol; 2,6-di-tert- butylquinone; phenol, and p-hydroxybenzaldehide. The thrombolytic activity of these lignin monomers can be tested in-vitro by measuring the ability of clot lysis. Lignin did not have in-vitro clot lysis activity, whereas the lignin monomers in filtrat of fermented sugarcane bagasse have an in-vitro clot lysis activity, although its capacity is not as high as streptokinase.
Key word: degradation, lignin, Ochrobactrum sp., in-vitro clot lysis activity
INTRODUCTION
Sugarcane bagasse (Saccharum officinarum L) is an available waste product of cane- sugar processing, which has limited in the cellulose utilization such as an alternative energy or animal feed sources. The content of lignocelluloses in sugarcane bagasse was approximately 52.7% cellulose, 20% hemicelluloses and 24.2% lignin. Lignin is a polymer, which is mainly composed of phenyl propane or C9 unit. There are three kinds of C9 units contained in the lignin, the unit p-hydroxyphenylpropane, guaiacylpropane and syringylpropane [1].
Degradation of lignin can be either chemically or enzymatically. The enzymatically lignin degradation can be done by using lignocellulolytic microorganisms, such as fungi and some bacteria, one of which is Ochrobactrum sp. Lignin degrading bacteria contains several enzymes that are specific in its ability to catalyze the production of several useful components. Therefore, the bacterial enzymatic system was expected to be used in the bioconversion of lignin into intermediate metabolites. Monomers derived from lignin degradation come from a class of alkaloid, phenyl propanoid, terpenoids, and flavonoids, which are phenolic derivative compounds [2]. The structure and composition of phenolic
monomers as lignin degradation products can give an indication of the active compounds contained in the degradation of lignin. In general, phenol derivatives possess antioxidant and anti-platelet [3].
The ability of phenol derivatives or phenolic compounds as anti-platelet are widely used in the medical world, such as for the treatment of stroke. Currently, stroke still becomes a commonly problem found in the world and requires very expensive treatment. Research in recent decades suggests that thrombosis has a very closely to the pathogenesis of ischemic stroke. The drugs that may interfere the formation of a blood clot or thrombus are often used in the treatment of this disease [4]. One method to study the ability of a material to interfere the thrombus formation is to measure the in-vitro activity of a compound in clot lysis.
Therefore, there should be an effort to find out the results of lignin degradation using Ochrobactrum sp and the ability of clot lysis of the phenolic monomers from lignin fermentation process.
EXPERIMENT
Chemicals and instrumentation
Chemicals used for research should include sugarcane bagasse, toluene, ethanol, 5%
NaOH, HCl 6M, chloroform, 3.1% sodium citrate, 2% calcium chloride, 100 µL Streptokinase (SK), distilled water, MSM medium. The composition of the MSM used in this study was follow (g/L): NaNO3 4.0, NaCl 1.0, KCl 1.0, CaCl2.2H2O 0.1, KH2PO4.12H2O 3.0, MgSO4 0.2, FeSO4.7H2O 0.001; 2 mL trace element stock solution composed of (g/L):
FeCl3.6H2O 0.08, ZnSO4.7H2O 0.75, CoCl2.6H2O 0.08, CuSO4.5H2O 0.075, MnSO4.H2O 0.75, H3BO3 0.15, Na2MoO4.2H2O 0.05.
The strain of Ochrobactrum sp. used in this study was obtain from culture collection of Indah Prihartini (Biotechnology Laboratory of UMM). These strain was identified by sequencing in 16 SrRNA.
Instrumentation applied for analysis include Soxhlet apparatus, incubator, rotary evaporator, Buchner funnel, UV spectrophotometer (Shimadzu 1601), and GC-MS (Shimadzu QP-2010S).
Procedure
Fermentation of Lignin from Sugarcane Bagasse using Ochrobactrum sp.
Sugarcane bagasse were first dried in sunlight and cut into small pieces (± 1.5 cm).
The cut bagasse was ground to pass 1 mm size screen. The dried powder were first extracted with toluene: ethanol (2:1, v/v) in Soxhlet apparatus for 6 hours and the dewaxed meal was allowed to dry in oven for 16 hours at 60 ⁰C. The dried powder was soaked in NaOH 5% for 6 hours at 50 ⁰ C for desilication. The pulp was then washed and acidified into pH 5.5 by using HCl 6 M. The solution was evaporated until 20% volume left and then added with three times volume of ethanol. Then it was filtered using a Buchner funnel. The filtrate of lignin was evaporated in a vacuum rotary evaporator, and then was acidified into pH 2 with HCl 6 M. The residue of lignin was filtered using a Buchner funnel and dried at room temperature.
After that, 1% (v/v) of this lignin’s residue was added with 72 h-aged-Ochrobactrum sp.
cultures in the MSM pH 6 and kept in shaker at 150 rpm at 40 oC for 5 days. The degradation product of lignin from this fermentation process was kept for further analysis.
Identification of monomers from lignin degradation
The monomers of lignin degradation product were isolated from culture filtrate of the organism grown on lignin by extraction with chloroform. The chloroform was evaporated with rotary evaporator apparatus, then the monomer residue was solved in distilled water.
Then, the filtrate was analyzed further by using UV spectrophotometer and GC-MS to predict the concentration of lignin monomers and the structure of the lignin degradation products, respectively. After that, 100 µL aqueous monomer lignins were added to the microcentrifuge tube containing the clots to test the thrombolytic activity.
Clot Lysis Activity
Cow blood was collected from Dau-Malang Slaughterhouse, Indonesia and immediatedly citrated using 3.1% sodium citrate solution. Thousand microliters of blood was added to preweighed microcentrifuse tubes. Two hundred microlitres of 2% calcium chloride then added to each of these tubes, mixed well and incubated at 37 oC for 45 min for clot formation. The serum from formed clot was aspirated out without disturbing the clot and each of these tubes was weighed again to determined the weight of the clot (clot weight = weight of clot containing tube – weight of tube alone). Then, 100 µL of aqueous monomer lignin was added separately into each microcentrifuge tubes containing pre-weighed clot. In addition, 100 µL Streptokinase (SK) (as a positive control) and 100 µL of distilled water (as a negative control) were separately added to the control tubes. All tubes were then incubated at 37 oC for 90 minutes and observed for clot lysis. After incubation, the fluid released was removed and tubes were again weighed to observe the weight difference after clot disruption.
The weight difference obtained taken before and after clot lysis was expressed as percentage of clot lysis.
RESULT AND DISCUSSION
The Identification of Lignin Degradation Products
Generally, GC-MS analysis showed that lignin degradation during sugar bagasse fermentation at the optimum conditions produces phenolic compounds, namely 4-methyl-2, 6-di-tert-butylphenol (10.65%) and 2, 6-di-tert-butylquinone (0.84 %) as major products. In addition this fermentation proccess also produced phenol and p-hydroxybenzaldehide in minor proportion. The MS spectra of the major products shown in Figure 1. Meanwhile, the effect of concentration of Ochrobactrum sp starter on the concentration of the major products can be seen in Table 1.
Table 1 showed that the concentration starter used has a different effect on the both products’s concentration. The higher concentration starter used up to 29% will lead to the higher concentration of 4-methyl-2,6-di-tert-butylphenol produced. But, the using of concentration starter above 29% can reduce the compound’s concentration. Meanwhile, the using of of 17% to 23% concentration starter increases the concentration of 2,6-di-tert- butylquinone. However, the concentration of that compounds decreases during the using of 29% to 33% of concentration starter. Even the using of the 7% starter did not produce 2,6-di- tert-butylquinone. This indicated that both products are further degraded into more simple compounds during fermentation. Based on the data in Table 1, the optimum concentration of
Figure 1. The spectra MS of the major products of lignin degradation from sugarcane bagasse fermentation using Ochrobactrum sp. at the optimum conditions (5 days, pH 6 and
temperature of 40 ° C). a) The spectrum MS of 4-methyl-2,6-di-tert-butylphenol b) The spectrum MS of 2, 6-di-tert-butylquinone
Table 1. Effect of starter concentration on the monomer lignin concentration during fermentation at the optimum conditions (5 days, pH 6 and temperature of 40 ° C)
No Monomer Lignin
Starter concentratio
n
Lignin monomers concentration (%)
1 4-methyl-2,6-di-tert-butylphenol
9 % 1,31 %
17 % 8,49 %
23 % 8,99 %
29 % 10,65 %
33 % 7,93%
2 2,6-di-tert-butylquinone 9 % -
17 % 0,77 %
23 % 0,62 %
29 % 0,84 %
33 % 1,47 %
The ThrombolyticticActivity of Phenolic Compounds of Lignin Degradation
The thrombolytic activity was studied by comparing % clot cow blood samples treated with lignin, lignin monomers produced by fermentation, streptokinase (control +) and distilled water (control -). The results were shown in Table 2. The data showed that monomer lignin has thrombolytic ability, although its ability was not as high as streptokinase.
Lignin monomers in the filtrate of fermented sugarcane bagasse have higher thrombolytic activity than lignin. The thrombolytic activity of thus filtrate could be attributed to the presence of some identified phenolic compounds, such 4-methyl-2, 6-di-tert- butylphenol, 2, 6-di-tert-butylquinone, phenol, and p-hydroxybenzaldehide According to Rabiner and Molinas [6], phenolic compounds such as phenol can shorten the life time of PF3 accelerating platelet aggregation. The interaction mechanism of phenolic compounds in PF3 is still unknown, but it has been indicated that phenolic compounds act as enzyme inhibitors [6]. Acceleration of time aggregation will causes smaller aggregates formation which facilitates its decomposition.
Table 2. % Clot lysis cow blood samples were treated with lignin, monomer lignin, streptokinase (control +) and distilled water (control -)
Drugs % clot lysis
Streptokinase 30.000 IU (control +) 79,44 ± 4,19
Lignin 18,45 ± 8,81
Lignin monomers 1,25 mg/mL 34,50 ± 3,23 Destilled water (control -) 13,43 ± 2,89
CONCLUSION
In conclusion, lignin didn’t have the clot lysis activity, while the lignin monomers have thrombolytic capability, although its ability is not as high as streptokinase.
ACKNOWLEDGMENT
Thanks to the Ministry of Education through the Directorate General of Higher Education that has funded this research through funding schemes DIPA UB Research
REFERENCES
[1] Graber, J.H., Ralphan, J., Lapiere, C., and Berriere, Y, Genetic and Molecular Basis of Grasses Cell-Wall Degradability, I. Lignin-Cell Wall matrix Interaction Plant Biology and Pathology, http://www.dfrc.wisc.edu/DFRCWebPDFs/2004-Grabber-CR-327-455, Retrieved on May, 26, 2017.
[2] Bhat, S.V., B.A. Nagasampagi and M. Sivakhumar, Chemistry of Natural Products, 2006, Springer Berlin Heidelberg, New York.
[3] Luceri, C., L. Giannini, M. Lodovici, E. Antonucci, R. Abbate, E. Masini and P. Dolara..
p-Coumaric Acid, A Common Dietary Phenol, Inhibits Platelet Activity in Vitro and in Vivo. British J. Nutrition, (2007), 97, 458-463.
[4] Coull, B.M., L.S. Williams, L.B. Goldstein, J.F. Meschia, D. Heitzman, S. Chaturvedi, K.C. Johnston, S. Starkman, L.B. Morgenstern, J.L. Wilterdink, S.R. Levine and J.L.
Saver, Anticoagulants and Antiplatelet Agents in Acute Ischemic Stroke, J. The American Heart Association, (2002), 33, 1934-1942.
[5] Rabiner and Molinas, The role of phenol and phenolic acids on the thrombocytopathy and defective platelet aggregation of patients with renal failure, The American Journal of Medicine, (1970), 49, 346-351.
