Introduction

Inflammation is a normal part of the body’s response to injury or infection; it occurs when the body releases chemical signals that trigger an immune response to fight off pathogens or heal damaged tissue (National Cancer Research Institute), usually caused by physical injury,  harmful chemicals, and protein denaturation, a process in which  proteins lose their quaternary, tertiary, and secondary structures (Yani  et al.). One of the affecting factors includes the increased temperature,  causing enzyme to denature.

Protein Denaturation Assay

• Path of victory: the plant extract that significantly reduces the denaturation of BSA in this assay may be considered to have potential anti-inflammatory properties.

• IC50 (half maximal inhibitory concentration) values - a representation of the concentration of the plant extracts required to reduce the denaturation of BSA proten by 50% - are used to indicate the potency of the extracts in inhibiting BSA protein.

Castanopsis tunggurut

• An IUCN endangered species of 20-25 metres  tall evergreen tree mainly found in West  Sumatera, Malay Peninsula, Simalur, Banka  Island, and West Java.  

• Has highly durable bark, used as black ink in  rattan work, trunk for construction works, and  fruits for consumption.  

• Medicinal properties are yet to be discovered.

UV/VIS Spectrophotometry

UV/VIS spectroscopy is used to measure the amount of wavelengths of UV light absorbed by a substance in a sample in comparison to a blank sample, providing information on the concentration of that particular substance contained in the  sample.  Test samples are measured at 660 nm for their turbidity, indicating the amount  of suspended particles (due to BSA denaturation) in each sample; the greater the result value, the more protein denatured, and hence, the lower the inhibition rate of the plant extract. Acceptable values are at a range of 0.1-1.2. Values beyond  that are considered of high inaccuracy, and therefore are ineligible to be used as  data.

Inhibition rate is calculated with the equation:

% inhibition of denaturation = 100 x (1 - A2/A1), where A1 is absorption of the control sample, A2 is absorption of the test sample.

Sample Preparation and Extraction with 70% Ethanol

• Two extracts are used in this experiment: C. tungurrut ethanol extracts.  

• In this investigation, the samples of C. tungurrut were harvested from Kebun  Raya Cibodas. 235g of dried simplicia C. tungurrut leaves (natural ingredients in  the form of dried materials used for medicine) was completely submerged with  70% ethanol with a volume of 1.175 L and macerated with occasional stirring for  24 hours.  

• The pulp was then filtered and further macerated with a new solvent of the same  amount of 70% ethanol. This process was repeated twice.  

• The volume of thickened 70% ethanol extract obtained was evaporated with  rotary vacuum evaporator and dried in the oven at 40℃.  

• 10.000 ppm of C. tungurrut ethanol extract was dissolved in DMSO, and was  then diluted into concentrations of 400, 500, 600, 700, and 800 ppm.

Sample Preparation and Extraction with Ethyl Acetate

• The same samples of C. tungurrut harvested from Kebun Raya Cibodas were  used to create the extract with ethyl acetate. Simplified leaves of C. tungurrut  (Blume) A.DC. were pulverised using a blender into fine powder.  

• 260g of the powder was dissolved in 1.3 L of ethyl acetate and poured into a  closed jar, allowing the solution to sit for 24 hours away from the Sun. The simplicia that had been soaked for 24 hours was then filtered. The pulp was once  again submerged into 1.3 L of ethyl acetate as a solvent, allowed to sit for  another 24 hours. This process was done twice with the pulp of simplicia.  

• The filtrate obtained was then concentrated using a rotary evaporator at 40℃ and  thickened using an oven at 50℃.  

• 10 000 ppm of C. tungurrut ethyl acetate extract was dissolved in DMSO, and  was then diluted into concentrations of 1000, 1500, 2000, 2500, and 3000 ppm.

Result and Discussion

Ethanol Extract

Graphic result can be seen in the image section.

• As can be seen in Fig. 2., there is a linear correlation between the  concentrations of C. tungurrut ethanol extract (400, 500, 600, 700,  800 ppm), showing that C. tungurrut ethanol extract is able to  inhibit inflammation caused by protein denaturation.

• The IC₅₀ of C.  tungurrut ethanol extract is found to be 618.841 ppm, with the  equation of y = 0.138x - 35.4.  The IC₅₀ of C. tungurrut ethanol extract is found to be 239.789  ppm, with the equation of y = 0.1329x + 18.132.  

• Compared with the IC₅₀ of diclofenac potassium, the IC₅₀ of C.  tungurrut ethanol extract is much higher. The lower IC₅₀ value of  diclofenac potassium indicates that it is a better inhibitor of protein  denaturation, and thus, a more potent anti-inflammatory drug.

• Fig. 4. shows identification and quantification of 78 compounds  detected in C. tungurrut ethanol extract. Seeing from the high  number of peaks, it can be inferred that C. tungurrut ethanol  extract contains an abundant source of bioactive compounds  which may possibly give rise to its anti-inflammatory property. 

• Considering that a previous study done confirms the anti-oxidant  property of this extract which also helps in reducing inflammation  by suppressing pro-inflammatory cytokines, and inhibiting key  signaling pathways and enzymes involved in immune processes  (Mucha et al.), it is verified that C. tungurrut ethanol extract does  possess the ability to inhibit inflammation via protein denaturation inhibition.

Ethyl Acetate Extract

• Fig. 5. shows a negative correlation between the concentrations of C.  tungurrut ethyl ethanol extract (400, 500, 600, 700, 800 ppm), indicating its  inability to inhibit inflammation caused by protein denaturation. IC₅₀ of the  plant extract is unable to be calculated due to its negative result.  

• The flat line and unidentifiable peaks of LC-MS (Fig. 6.) show absence of  anti-inflammatory metabolites. Considering that ethyl acetate has the ability to  extract Omega-6 fatty acids which are pro-inflammatory, there is a possibility  that the mentioned metabolite is what is detected in the extract, therefore  contributing to inflammation instead of reducing it, confirming the negative  trend of Fig. 5.

Conclusion

• While Castanopsis tungurrut may not be as effective as the well-known non steroidal anti-inflammatory drug diclofenac potassium, it does contain anti inflammatory bioactive compounds that inhibit protein denaturation, as  displayed by the ethanol extract data. This does not seem to be the case with  C. tungurrut ethyl acetate extract, however.  

• As this investigation only examines one anti-inflammatory pathway with one  methodology and two kinds of extracts, the results of this investigation is  unable to fully represent and determine the anti-inflammatory properties and  efficacy of C. tungurrut as an inflammatory drug. Thus, there is a need to  conduct further investigations on the plant’s contents of bioactive compounds  and metabolites, as well as examinations on its efficacy in reducing  inflammation in several other inflammatory pathways with different kinds of  extracts.