The Effect of Andrographis paniculata Nees on Oxidative Stress and Parasitemia Levels of Plasmodium berghei Infected Rats

Anita Lidesna Shinta Amat¹, Hilkatul Ilmi², Lidya Tumewu², Harianto Notopuro³,

Indah Setyawati Tantular³, Achmad Fuad Hafid^2,4, Aty Widyawaruyanti^2,4*

¹Faculty of Medicine, University of Nusa Cendana, East Nusa Tenggara 85228, Indonesia.

²Natural Product Medicine Research and Development, Institute of Tropical Disease,

Universitas Airlangga, Surabaya 60115, Indonesia.

³Department of Parasitology, Faculty of Medicine, Universitas Airlangga, Surabaya 60286, Indonesia.

⁴Department of Pharmaceutical Sciences, Faculty of Pharmacy,

Universitas Airlangga, Surabaya 60115, Indonesia.

*Corresponding Author E-mail: aty-w@ff.unair.ac.id

ABSTRACT:

Background: During malaria infection, oxidative stress arises due to the high metabolic rate of the multiplying parasite within the erythrocyte. Malondialdehyde (MDA), a product of lipid peroxidation and glutathione (GSH) has been suggested as a biomarker of oxidative stress. The Ethyl acetate (EA) fraction from the ethanol extract of Andrographis paniculata was shown to inhibit Plasmodium berghei in vivo. However, the antimalarial mechanism of the EA fraction, specifically on oxidative stress has not been investigated previously. Therefore, this study aimed to investigate the effects of the EA fraction on parasitemia levels, GSH and MDA levels of P. berghei infected rats. Methods: Female Wistar rats infected with P. berghei were divided into three groups. Group one received no treatment (negative control), group two was treated with 1.4 mg/200 g body weight of chloroquine diphosphate as positive control, and group three was treated with the EA fraction at a dose equal to andrographolide 3.5 mg/200 g body weight. The treatments lasted for four days (day 0 to day 3) and parasitemia was observed from day 0 to day 4. Rats were sacrificed and blood taken intracardially on day 4 after parasitemia observation. GSH was measured using an ELISA reader at a wavelength of 415 nm. MDA was observed via spectrophotometry at a wavelength of 532 nm. Results: The EA fraction at a dose equal to andrographolide 3.5 mg/200 g body weight was able to inhibit parasite growth by 81.97±9.14%. The GSH levels of the negative control, positive control and EA fraction treated group were 139.30±75.93 μMol/mL, 81.06±53.26 μMol/mL and 105.71±76.00 μMol/mL, respectively. Furthermore, the MDA level of negative control, positive control and EA fraction treated group were 11.18±0.70 nMol mL, 8.81±1.26 nMol/mL and 9.40±0.74 nMol/mL, respectively. No significant differences were detected between treatment groups regarding their GSH levels. Additionally, there was a significant difference in MDA levels between the negative control and positive control groups; as well as a significant difference between the negative control and the EA treated group. However, no significant difference in MDA levels between the EA fraction treated group and positive control group. Interestingly, a correlation was found between parasite growth inhibition and MDA levels among groups (p<0.05). Conclusion: The EA fraction of A. paniculata significantly decreased MDA levels which correlated significantly with parasitemia levels of P. berghei infected rats. The antimalarial activity of the EA fraction may have been correlated with oxidative stress mechanisms and this correlation could be explained in part by the decreased production of MDA.

KEYWORDS: Ethyl Acetate Fraction, Andrographis paniculata, Plasmodium berghei, Oxidative stress, Rattus novergicus.

INTRODUCTION:

Andrographis paniculata is a popular medicinal plants abundant in South Eastern Asia. This herb is belonging to Acanthaceae family and contain a large number of chemical constituents, mainly lactones, diterpenoids, diterpene glycosides, flavonoids and flavonoids glycosides. It has a wide range of medical and pharmacological applications^1-4. It has been reported to exhibit anti-inflammatory⁵, antibacterial^6-9, antioxidant^10,11, antimalarial^12,13and various other activities. The aqueous extract was reported had no significant toxicity on brine shrimp lethality test¹⁴.

The potential antimalarial activity of A. paniculata has been widely reported. Importantly, the extract was shown to be active in vitro against Plasmodium falciparum and in vivo against Plasmodium berghei^12,13. In addition to this, the fractions and isolated compound also demonstrated antimalarial activitiy. This activity was likely due to its phytochemical constituents, specifically diterpene lactone compounds. Andrographolide is one of the major diterpene lactone compounds found in Andrographis paniculata and exhibits antimalarial activity with an IC₅₀ value of 9.1 μM¹⁵. Previously, it was reported that the ethyl acetate (EA) fractions of A. paniculata is an andrographolide rich fraction. Its antimalarial activity was demonstrated on Plasmodium berghei infected mice with an ED₅₀ value of 6.75 mg/kg BW and this classified it as a highly active antimalarial substance. The EA fraction was able to significantly increase survival time compared to the negative control¹⁶.

During malaria infection, oxidative stress arises due to the high metabolic rate of the multiplying parasite within the erythrocyte¹⁷. Malondialdehyde (MDA), a product of lipid peroxidation and glutathione (GSH) has been suggested as a useful biomarker of oxidative stress¹⁸. The lactone of the andrographolide group is capable of binding to the thiol and amine groups of reduced glutathione (GSH), hence influencing the oxidative stress condition in the parasite with elevated levels of H₂O₂¹⁹.

Malaria parasites attack red blood cells, which contain hemoglobin, and the hemoglobin has a heme composed of four porphyrine rings bound to iron (Fe). When hemoglobin is degraded within the parasite, heme degradation causes the release of this iron (Fe) and the degraded heme into the food vacuole of the parasite. Here it is converted to hemozoin, which is itself a nutrient for the parasite^20,21.

The iron (Fe) causes Fenton and Haber-Weiss reactions, forming Reactive Oxygen Species (ROS) such as H₂O₂ (hydrogen peroxide) and ∙O₂ (superoxide anion). The parasite increases the production of GSH to neutralize ROS and assure its survival within the host body. This the same process as is used by the human body as a defense mechanism against excessive oxidants with the catalysis of several enzymes including superoxide dismutase, gluthation peroxidase and thiol peroxidase. The decreased enzyme activity can lead to a decrease in the production of malondialdehyde (MDA) and prevent the lysis of host cell membranes²¹.

Based on previous reports, we know that A. paniculata possesses antimalarial activity due to andrographolide, a diterpene lactone compound, which is the most relevant component of this plant. Andrographolide has also been shown to have the capability to affect the oxidative stress condition of parasites. Here, we studied the correlation between the antimalarial activity of the EA fraction, which contains andrographolide with GSH and MDA levels as biomarkers of oxidative stress on P. berghei infected rats. The correlation of these factors indicated the antimalarial mechanism of the EA fraction of A. paniculata on oxidative stress.

MATERIALS AND METHODS:

Material:

The EA fractions of A. paniculata which consists of 20.9% andrographolide was obtained from the Institute of Tropical Disease, Universitas Airlangga. Chloroquine diphosphate salt was used as a positive control and obtained from Sigma (C6628). Glutathione assay kit was obtained from Sigma (CS0260). QuantiChrom^TM TBARS Assay kit DTBA-100 was used for MDA quantification.

Parasite:

P. berghei ANKA strain was obtained from the Eijkman Institute for Molecular Biology, Jakarta. This parasite was further maintained at the Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia.

Animals:

Female Wistar rat (Rattus novergicus) aged 1-2 months and ±80-120 g of weight were obtained from the Animal Experimental Development Unit-Gajah Mada University, Yogyakarta, Indonesia. They were maintained at the Animal Laboratory of the Institute of Tropical Disease, Universitas Airlangga. Permission and approval for animal studies were obtained from the Faculty of Veterinary Medicine, Universitas Airlangga, with ethical clearance No: 427-KE.

Determination parasitemia growth and inhibition:

Female Wistar rats were divided into three groups (seven rats in each group) and infected intraperitonelly with 400 µl, 1x10⁵ of P. berghei. The growth of the parasitemia was observed each day for four days until the parasitemia reached 1%. Group one was an untreated group (negative control), group two was treated with 1.4 mg/200 g body weight of chloroquine diphosphate (positive control), and group three was treated with the EA fractions at a dose equal to andrographolide 3.5 mg/200 g body weight. Treatment was conducted for four days (day 0 to day 3). The parasitemia was observed from day 0 to day 4. On day 4, rats were sacrificed and blood taken intracardially.

Determination of GSH level:

The level of GSH was determined using Glutathione assay kit Sigma (CS0260). The determination method was based on the manufacturer’s instructions. Brieftly, the blood sample was first deproteinized with 5% 5-Sulfosalicylic Acid Solution (5% SSA), centrifuged to remove the precipitated protein and then assayed for glutathione. Following this, 5 µl of sample was mixed in a 96-well plate with 5 µl of 5% SSA and 150 µl of working solution, which contained assay buffer, diluted glutathione reductase enzyme solution and 5,5’-Dithiobis (2-nitrobenzoic acid) [DTNB] solution. This mixture was then incubated for 5 minutes at room temperature and 50 µl of the diluted NADPH solution was added. The absorbance was read at 415 nm with a BioRad iMark^TM Microplate absorbance reader.

Determination of MDA level:

The level of MDA was determined using QuantiChrom^TM TBARS Assay kit DTBA-100. For this assay, 500 µl of blood sample was mixed with 4.5 ml of PBS solution to obtain 4 ml of supernatant. The supernatant was then added to 1 ml of 15% TCA solution and 0.37% of TBA solution (in 0.25 N HCl). The mixture was then heated in a 80^oC waterbath for 15 min and cooled at room temperature for 60 min. Following this, the mixture was centrifugated at 3,000 rpm for 15 min. The absorbance was read at 532 nm with a spectrophotometer (Spectroquant Merck Pharo 300).

Statistical analysis:

The average (mean) and standard deviation (SD) of the number of parasitemia, GSH and MDA plasma levels were calculated. Data analysis was conducted by Kolmogorov-Smirnov normality analysis, homogeneity analysis and Post Hoc tests. Data with a normal mean distribution was analyzed by analysis of variance (ANOVA) while data with abnormal mean distribution was analyzed by the Kruskal-Wallis test. If there were significant differences in mean between groups then these steps were followed by a Least Significant Different (LSD) Test. A p value <0.05 was considered significant.

RESULTS:

Parasitemia growth in the group treated with the EA fraction was lower compared to the negative control group. The parasitemia growth was significantly different among groups (P<0.05). The parasitemia growth and inhibition can be seen in Figure 1 and Table 1 below.

Figure 1. Parasitemia growth of infected rats on day 0 to day 4

Table 1. Parasitemia growth, percentage growth inhibition, GSH and MDA level of P. berghei infected rats treated with EA fraction of A. paniculata

Groups	Parasitemia growth (%)*	Inhibition (%)*	GSH (µMol/mL)*	MDA (nMol/mL)*
1 Negative control	0.72±0.03^a	-	139.29 ± 75.92	11.18 ± 0.71^ab
2 Positive control	0.00±0.00^a	100.00 ± 0.00	81.06 ± 53.26	8.81 ± 1.26^a
3 Ethyl acetate fraction	0.13±0.06^a	81.97 ± 9.14	105.71 ± 76.01	9.39 ± 0.74^b

*Data represent Mean±SD (n=7); a,b: there was significant different between groups.

The positive control group exhibited the lowest GSH and MDA levels among all three groups. Nevertheless, the EA fraction treated group had lower GSH and MDA levels compared to the negative control group. We observed no significant difference in the GSH levels of the three groups. Interestingly, there was a detectable and significant difference in MDA levels between the negative control and positive control groups; as well as a significant difference between the negative control and the EA treated group. However, we did not find any significant difference in MDA levels between the EA fraction treated group and positive control group. This suggests that the effects of the EA fraction treated group and positive group on MDA levels was similar.

There was no statistically significant correlation between GSH levels and the inhibition of parasitemia growth but there was a negative (linear) correlation between MDA levels and the inhibition of parasitemia growth. Hence, lower MDA levels are associated with greater inhibition of parasitemia growth. Futhermore, there was no correlation between GSH levels and MDA levels.

DISCUSSION:

The EA fraction, applied at a dose equal to andrographolide 3.5 mg/200 g body weight, inhibited parasite growth by 81.97 ± 9.14%. Hence, the antimalarial activity of EA fraction of A. paniculata has been demonstrated in vivo both on P. berghei infected mice and rats.

The GSH level in group three (EA fraction) was 105.71 μMol/mL, lower than in the negative control group (139.29 μMol/mL). This suggests that the EA fraction of A. paniculata was able to bind reduced glutathione so that total GSH level decreased. The binding of andrographolide with GSH causes GSH depletion. This depletion as well as elevated levels of ROS (H₂O₂) can affect the existing systems in the cell. Glutathione is regulated by the enzyme γ-glutamilsistein synthetase (γ-GCS) and glutathione synthetase (GS) to synthesize GSH and glutathione reductase (GR). This reaction occurs because of the chemical structure of andrographolide. This structure contains unsaturated lactone at the α, β position, which is easily attacked by the amines and thiols of GSH²². The plasma glutathione level in the positive control group was 81.05 μMol/mL, which was lower than the negative control group at 139.22 μMol/mL. This showed that chloroquine was able to bind reduced glutathione.

When compared to the positive control group, the EA fraction group exhibited a higher plasma GSH level. This may have been due to the toxic effect of chloroquine on P. berghei which was a consequence of chloroquine bonding with ferriprotoporphyrine IX (FPP) in Plasmodium food vacuoles, hence, preventing heme polymerization and degradation of FPP depending on GSH. This mechanism plays an important role in preventing fatal damage to the parasitic cell membrane²³.

The EA fraction group showed MDA level of 9.2911 mMol/mL. These were lower than those observed in the negative control group (11.2278 mMol/mL). This indicated that andrographolide able to inhibit lipid peroxidation formation so that MDA formation was inhibited.

Lipid peroxidation events cause damage to lipids via ROS. If these events occur on the membrane, they can cause fluidity loss, decreased membrane potential, increased permeability and membrane damage. The decrease in MDA levels after exposure to the EA fraction was thought to be related to the antiplasmodium effect of andrographolide on the ring stage, inhibiting parasite development into tropozoites or schizons and subsequently deteriorating erythrocytes¹³. The ability of A. paniculata Nees to protect erythrocyte membranes against decreased lipid peroxidation may occur due to its activity as an antioxidant.

The positive control group (chloroquine) displayed MDA levels of 9.06 mMol/mL, it was lower than the 11.27 mMol/mL detected in the negative control group. This indicated that chloroquine was able to inhibit the formation of lipid peroxidation so that MDA formation was inhibited. When compared to the positive control group, the EA fraction group had higher MDA levels but these differences were not statistically significant (p<0.05). The ability of chloroquine to inhibit lipid peroxidation is due to its role in preventing heme polymerization and degradation of FPP thus preventing fatal damage to parasitic cell membranes²⁴.

Based on the results of our study, the inhibition of parasitemia was not correlated to GSH levels but was correlated with MDA levels. Chloroquine and the EA fraction decreased GSH and MDA levels compared to the negative control. It is possible that chloroquine and the EA fraction were able to bind GSH so that ROS neutralization was disturbed. Nevertheless, chloroquine, a standard antimalarial drug, and andrographolide, the major compounds of the EA fraction, inhibited lipid peroxidation thereby decreasing MDA levels.

CONCLUSION:

The EA fraction of A. paniculata significantly decreased MDA levels which were negatively correlated with parasitemia levels of P. berghei infected rats. The antimalarial activity of the EA fraction may be correlated with oxidative stress mechanisms and could be explained, at least in part, by the decreased production of MDA.

ACKNOWLEDGEMENT:

This research was funding in part of Natural Product Medicine Research and Development (NPMRD), Institute of Tropical Disease, Universitas Airlangga and Directorate General of Research Technology and Higher Education through Penelitian Unggulan Strategis Nasional contract No.266/UN3.14/LT/2015.

REFERENCES:

1. Niranjan A, Tewari SK, Lehri A. Biological activities of Kalmegh (Andrographis paniculata Nees) and its active principles-A review. Indian Journal of Natural Products and Resources 2010;1(2):125-135.

2. Madhavi S, Prakash Rao S. Review literature: Andrographis paniculata. J Pharmacology and Pharmacodynamics 2018; 10(4):166-170.

3. Chauhan ES, Sharma K, Bist R. Andrographis paniculata: A review of its phytochemistry and pharmacological activities. Research J Pharm and Tech 2019; 12(2):891-900.

4. Firdous J, Latif NA, Mona R, Mansor R, Muhamad N. Andrographis paniculata and its endophytes: A review on their pharmacological activities. Research J Pharm and Tech 2020; 13(4):2027-2030.

5. Tajuddin SA, Tariq M. Antiinflammatory activity of Andrographis paniculata Nees (Chirayata). Nagarjun 1983; 27:13-14.

6. Iwari Rk, Pandey R, Shukla SS, Tiwari P, Shah T. Antibacterial activity of aerial part of Andrographis paniculata. Res J Pharmacognosy and Phytochem 2014; 6(3):122-125.

7. Mary RNI, Banu N. Inhibition of antibiofilm mediated virulence factors in Pseudomonas aeruginosa by Andrographis paniculata. Research J Pharm and Tech 2017;10(1):141-144.

8. Geetha S, Rajeswari S. A preliminary study on Phytochemical screening, proximate and antibacterial activities of Andrographis paniculata seed extract. Research J Pharm and Tech 2019;12(5):2083-2088.

9. Mary RNI, Banu N. Antiquorum sensing potential of andrographolide from Andrographis paniculata in Vibrio harveyi. Research J Pharm and Tech 2017; 10(2):449-452.

10. Verma N, Vinayak M, Antioxidant action of Andrographis paniculata on lymphoma, Mol Biol Rep 2008: 35:535-540.

11. Kaur N, Gupta J. Comparison of phytochemical extraction solvents for Andrographis paniculata. Research J Pharm and Tech 2017:10(5):1271-1276.

12. Rahman NNNA, Furuta T, Kojima S, Takane K, Ali Mohd M. Antimalarial Activity of extracts of Malaysian medicinal plants. J Ethnopharmacol 1999; 64 (3), 249–254.

13. Mishra K, Dash AP, Swain BK, Dey N. Antimalarial activities of Andrographis paniculata and Hedyotis corymbosa extracts and their combination with curcumin. Malaria J 2009; 8 (26), 1-9.

14. Mamatha A. Brine shrimp lethality test of Andrographis paniculata. Research J Pharm and Tech 2014; 7(7):743-745.

15. Mishra K, Dash AP, Dey N. Andrographolide: A novel antimalarial diterpene lactone compound from Andrographis paniculata and its interaction with curcumin and artesunate. J Trop Med 2011; 1-6.

16. Widyawaruyanti A, Astrianto D, Ilmi H, Tumewu L, Setyawan D, Widiastuti E et al, Antimalarial activity and survival time of Andrographis paniculata fraction (AS202-01) on Plasmodium berghei infected mice. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2017; 8(1S): 49-54.

17. Kavishe RA, Koenderink JB, Alifrangis M. Oxidative stress in malaria and artemisinin combination therapy: Pros and Cons. FEBS J 2017; 284:2579-2591.

18. Bilgin R, Yalcin MS, Yucebilgic G, Koltas IS, Yazar S. Oxidative stress in vivax malaria. Korean J Parasitol 2012; 50(4):375-377.

19. Zhang Z, Chan GK, Li J, Fong WF, Cheung HY. Molecular Interaction between Andrographolide and Glutathione Follows Second Order Kinetics. Chem Pharm Bull 2008; 56(9): 1229-1233.

20. Pandey AV, Chauhan VS. Heme polymerization by malarial parasite: a potential target for antimalarial drug development. Current Science 1998; 75(9): 911-918.

21. Duran-Bedolla J, Rodriguez MH, Saldana-Navor V, Cerbon M. Oxidative Stress: Production in Several Processes and Organelles During Plasmodium sp Development. Oxidants and Antioxidants in Medical Science 2013; 2(2): 93-100.

22. Zhang Z. Reactions and Computational Studies of Andrographolide Analogues with Glutathione and Biological Nucleophiles. 2007. Thesis: University of Hongkong.

23. Meierjohann S, Walter RD, Muller S. Regulation of Intracellular Glutathione Levels in Erythrocytes Infected with Chloroquine-sensitive and Chloroquine-resistant Plasmodium falciparum. Biochem J 2002; 368(Pt3):761-8.

24. Ngaha EO. Some biochemical changes in the rat during repeated chloroquine administration. Toxicology Letters 1982; 10: 145-149.