Polysaccharide Peptide (PsP) of Ganoderma lucidum as vasa vasorum

anti-Angiogenesis agent in Dyslipidemic state by Measuring Lp-PLA2 and H2O2 Levels: In Vivo Study using wistar strain Rattus novergicus model of Atherosclerosis with Dyslipidemia


Titin Andri Wihastuti1, Fibe Yulinda Cesa2, Reyhan Amiruddin2, Meddy Setiawan3,

Danisa Namira Wijayanti2, Teuku Heriansyah4*

1Departement of Biomedicine Nursing, Faculty of Medicine, University of Brawijaya, Malang, Indonesia

2Departement of Biomedical Science, Faculty of Medicine, University of Brawijaya, Malang, Indonesia

3Faculty of Medicine, University of Muhammadiyah Malang.

4Departement Cardiology, Faculty of Medicine, University of Syiah Kuala, Aceh, Indonesia

*Corresponding Author E-mail: teuku_hery@unsyiah.ac.id



Dyslipidemia is one of risk factors for atherosclerosis induced by high fat diet. In atherosclerosis, an angiogenesis of immature and fragile vasa vasorum leads to atherosclerotic plaque destabilization. Angiogenesis of vasa vasorum is influence by oxidative stress and inflammation. Polysaccharide peptide (PsP) from Ganoderma lucidum (GL) contains bioactive compounds that possess antioxidant and anti-inflammatory properties which might become anti-angiogenesis agent in atherosclerosis. The aim of this study is to determine whether administration of PsP GL is able to decrease the number of vasa vasorum, H2O2 and Lp-PLA2 in atherosclerosis mouse model with dyslipidemia. True Experimental research design was conducted with Randomized Post Test Only Controlled Group Design. Twenty five Wistar strain Rattus novergicus were divided into 5 groups (n=5). Negative control group was administered with normal diet, positive control group was given high fat diet (HFD), and 3 treatment groups received HFD and PsP administration of 50mg/kgBW, 150 mg/kgBW, and 300mg/kgBW. Vasa vasorum measurement was performed using histopathological section of mouse aorta and measured using Scan Dot Slide Olyvia with 400x magnification. Serum Lp-PLA2 and H2O2 were measured using ELISA method. One way ANOVA analysis demonstrates that PsP GL significantly reduced vasa vasorum count (p<0,05) in mouse model of atherosclerosis with dyslipidemia. Duncan Post Hoc analysis indicates a significant difference in vasa vasorum count in 50mg/kgBW PsP treated group compared to other doses. The conclusions of this study is Polysaccharid peptide G. lucidum extract significantly reduces vasa vasorum count in Wistar strain Rattus novergicus with HFD administration.


KEYWORDS: Atherosclerosis, High Fat Diet, Oxidative stress, Polysaccharid peptide (PsP) extract, Vasa vasorum.




Cardiovascular diseases are the leading cause of death globally, contributing to around 31% of mortality worldwide, and more than 75% mortality in low-middle income countries1. It is estimated that 80% of CVD deaths are caused by heart attack and stroke. In Indonesia, the morbidity and mortality rate of CVD are predicted to affect 50% of the population2. Cardiovascular diseases are closely related to atherosclerosis. Risk factors that contribute to the high prevalence of CVD are hypertension, smoking, obesity, stress, lacks of physical activity, hypercholesterolemia, and dyslipidemia3


Dyslipidemia is a common cause of several organ dysfunctions. High serum Low Density Lipoprotein (LDL) and low serum High Density Lipoprotein (HDL) highly correspond to the incidence of coronary heart disease. LDL will initiate the development of atherosclerotic plaque. Oxidized LDL (OxLDL) is able to induce endothelial dysfunction which is heralded by the upregulation of adhesion molecules expression4. Dyslipidemia is a risk factor that stimulates ROS generation and OxLDL production. Reactive oxygen species (ROS) is a component of oxygen-derived molecules that, in the case of excessive activity, is considered to be harmful oxidant5. Some of these radical forms are free OxLDL is attached to endothelial wall. Attached oxLDL will demonstrate destructive figures in histopathological specimen of endothelial wall6.


OxLDL could initiate chemoattractants release, which will further induce macrophage migration to the lesion site, and reactive oxygen species (ROS) generation, and produce cascades of oxidative stress. In other words, OxLDL is capable of inducing chronic inflammatory response OxLDL is degraded into lysophosphatidylcholie and oxidized nonesterified fatty acid by lipoprotein associated phospholipase (Lp-PLA2) which has proinflammatory properties, and hypothesized by several researchers as a marker of atherosclerotic plaque development7. Lp-PLA2 is produced in proinflammatory cells such as monocytes, macrophages, T lymphocytes, and mast cell8.


Atherosclerosis is a condition characterized by plaque deposition in the vascular wall as a result of endothelial lesion formation due to inflammatory process and the formation of thrombi (static plaque) as well as emboli (dynamic plaque)1. It begins as inflammatory reaction against the endothelial cells and artery wall components 9. The process of atherogenesis closely corresponds to oxidative stress within the vessels10. ROS that is produced includes OH-, lipid radicals, RO and hydrogen peroxide (H2O2). The enhanced generation of ROS dominates the intrinsic antioxidant defenses resulting in the cells' status known as oxidative stress11. This process occurs as a compensatory mechanism against endogenous antioxidant production. Oxidative stress is defined as imbalance of ROS generation and cellular endogenous antioxidant production12,13. It is also a condition in which generation of free radicals and other reactive oxygen species overwhelms the endogenous antioxidant defense of the body and also damage cells14,15.16 It includes molecular oxygen, since ground-state oxygen has two unpaired electrons17. Oxidative stress that occurs in the vascular wall induces the angiogenesis of vasa vasorum in hypercholesterol-fed animals2.


Atherosclerosis will induce adventitial vasa vasorum growth, producing irregular pattern of neovascularization diameter enlargement. Derivative from adventitia is vasa vasorum neovascularization, which plays an important role in the atherosclerotic plaque development and plaque destabilization. Recent research observed that the increase of density and permeability of adventitial vasa vasorum occurred prior to the increased intima-media thickness and subsequent endothelial dysfunction. Inflammatory activity within the plaque causes plaque instability which increased the vulnerability of the plaque and development of clinical features of atherosclerosis, induced by the impairment and leakage of immature neovascularization originating from adventitial vasa vasorum18.


Novel study using Ganoderma lucidum peptide is shown to possess strong antioxidant activity with minimal or negligible side effects19. Antioxidant properties of polysaccharide peptide of G. lucidum include free radicals scavenging activity20. Antioxidant is a compound capable of neutralizing reactive free radicals into relatively stable nonreactive forms to protect the cells from harmful effects of free radicals and can be used for the treatment of liver injury and other diseases21,22.



Animal Preparation:

2-3 months old Wistar strain Rattus novergicus weighing 150-200gr were kept in Central Sciences Laboratory, Brawijaya University, Malang. Prior to treatment, all animals were acclimatized for 2 weeks, followed by division of rats into 5 groups (n=5), including negative group (normal), positive group (HFD), HFD+PSP dose of 50mg/kgBW group, HFD+PSP dose of 150mg/kgBW group, and HFD+PSP dose of 300mg/kgBW group.


Administration of Normal and High Fat Diet:

Negative group was fed with normal diet which consisted of PARS 66.6% and wheat flour 33.4%. Other groups were given high fat diet which consisted of PARS 57.3%, wheat flour 31.8%, cholesterol 1.9%, cholic acid 0.1%, and lard 8.9%. Diet was administered in each group for 12 weeks.


PsP preparation and administration:

Preparation of PSP extract consisted of 2 stages, including upstream, which comprised G. lucidum fungi selection, followed by inoculation in Potato Dextrose Agar (PDA) medium, until miselin growth was observed. Next, miselin was inoculated back into fluid medium with incubation temperature of 24-26oC for 40-50 days in dark place. Downstream process constituted harvesting of G. lucidum miselia and homogenization process, then followed by extraction and centrifugation. Afterward, the miselia underwent precipitation, evaporation, and lioilization to produce solid and dry extract. Subsequently, measurement of β-1,3/1,6-D-Glukan concentration was performed. PsP was administered in 3 doses groups, which were 50 mg/kgBW, 150mg/kgBW, and 300mg/kgBW. In PsP treated group, animals were given HFD for 8 weeks, followed by PSP extract administration 4 weeks afterward.



Prior to surgery, euthanasia using ether was performed, followed by surgery to obtain samples from the aorta. Afterward, the samples were collected inside 10% formaldehyde solution for fixation prior to histological slides preparation.


Lipid Profile Measurements:

Lipid profile measurements were performed using serum analysis by Cobas Mira Analyzer (PT Roche Indonesia, Jakarta, Indonesia).


H2O2 Analysis:

H2O2 serum measurement was performed using colorimetric hydrogen peroxide kit (Assay Design, Cambridge, MA, USA) and was observed in 570nm by enzyme linked immune-absorbent assay reader (ELISA kit, Boehringer-Mannheim GMBH, Mannheim, Germany).


Lp-PLA2 level measurement:

Plasma Lp-PLA2 analysis was performed using observation in 570nm by enzyme linked immune-absorbent assay reader (ELISA kit, Boehringer-Mannheim GMBH, Mannheim, Jerman).


Vasa Vasorum count measurement:

Detailed histopathological observation using HE stain and 400x magnification was performed to count the number of Vasa Vasorum. Measurement was performed using software Olyvia. Vasa vasorum was measured through all magnification field. Further, measurement was performed twice to minimize bias, followed by Intraclass Correlation Coefficien analysis using SPSS software.


Statistical Analysis:

One Way ANOVA test was performed to determine the effect of PsP in vasa vasorum count in 5 treatment groups. Further, Post Hoc Analysis (Duncan Test) was performed to determine the difference among groups.



Histopatological section of vasa vasorum:


Fig. 1: Identification of Vasa Vasorum with HE stain. (A) Negative control group (Normal). (B) Positive control group (HFD). (C) PsP 1 group is group that receives HFD and PSP 50 mg/kgBW. (D) PSP 2 group receives HFD and PSP 150 mg/kgBW. (E) PsP 3 receives HFD and PsP 300 mg/kgBW. (HE, 400x)


Table 1: Parameters measurements


Normal Diet

High Fat Diet

High Fat Diet + PSP (mg/kgBW/Day)







14,25 ± 3,30

9,25 ± 18,00

84,29 ± 35,20

62,13 ± 145,50

64,67 ± 40,62

10,38 ± 119,00

40,92 ± 23,05

26,50 ± 80,13

28,47 ± 8,01

21,75 ± 41,88


Total Cholesterol


47,20 ± 9,96


139,65 ± 59,68


115,75 ± 20,46

(81,00- 132,00)

103,05 ± 34,71


89,50 ± 21,73

(66,00- 115,00)




76,20 ± 24,68

(42,00- 106,00)

661,60 ± 217,13

(358,00 - 882,00)

537,60 ± 231,18

(204,00 - 762,00)

347,80 ± 198,67


274,80 ± 183,63





232,81 ± 6,58

(226,25- 240,00)

258,75 ± 67,19

(190,00 - 370,00)

249,75 ± 17,79

(223,75 - 265,00)

240,62 ± 32,66

(207,50 - 282,50)

239,00 ± 19,87

(218,75 - 262,50)


Vasa Vasorum (Cell/ Cross section)

4,60 ± 2,40

9,20 ± 2,68

4,20 ± 2,58

6,60 ± 1,94

9,60 ± 3,71




Ganoderma lucidum (GL) constitutes as a basidiomycete white rot fungus which has been widely used as traditional Chinese medicine for thousands of years. GL is known to possess bioactive materials that possess anti-tumor, antioxidant, anti-virus, anti-inflammation, antidiabetic, and neuroprotective activity23.


In our study, 4 rat models were given atherogenic diet for 12 weeks because it was sufficient for development of hypercholesterolemia compared to rat with 5 and 8 weeks atherogenic diet administration24.


High fat diet administration caused dyslipidemia, which manifested as the increase of body LDL concentration 25,26. Development of vasa vasorum angiogenesis in rat with High Fed Diet administration corresponds to vascular oxidative stress process. Oxidative stress results from the imbalance between oxidant and antioxidant compounds. The increased oxidative stress in vascular wall will contribute to the development of atherogenesis2. Our study observed the number of angiogenesis in vasa vasorum, and several parameters related to oxidative stress including Lp-PLA2.


Lp-PLA2 through oxLDL will produce bioactive products such as lyso PC and oc=x NEFA which stimulate pro-inflammatory cytokines production (IL-6, IL1b, dan TNF-a)27. Our study indicates that, Lp-PLA2 expression, marker of inflammation process, was not significantly reduced (P=0,802); however, in PsP treatment group of PsP 50, 150 and 300mg/kgBW which underwent Lp-PLA2 reduction, reduction of inflammatory process was identified. This result aligns with previous research conducted by Wihastuti et al, which indicated that 150mg PsP administration resulted in reduction of IL-6 level. Research conducted by Fan Xu et al (2017) concluded that PsP is capable of inhibiting DOX-induced pro-inflammatory cytokine production2,23.


Anti-oxidant administration significantly reduced H2O2 level, in which stress oxidative state was dangerous for biological body condition28. The level measurement in H2O2 level measurement was significant H2O2 level reduction (P<0,003), which indicates that PsP administration in rat model directly inhibited oxidative stress process. The composition of H2O2 into water may occur according to the antioxidant compounds as the antioxidant component present are good electron donors, they may accelerate the conversion of H2O2 to H2O29. PsP enhances antioxidant defense mechanism by upregulating body SOD activity, an antioxidant enzyme that contributes as a nutrient antioxidant to protect from oxidative stress. However, previous research conducted by Wihastuti et al indicated that there were no significant difference in SOD level in PsP treated rats. However, PsP can potentially be used as antioxidant therapy to lower oxidative stress.


Our study observed marked reduction in vasa vasorum number in 50 and 150 PsP treated groups. However, administration of 300mg of PSP shifted towards positive rat model. Inhibition of vasa vasorum angiogenesis by PSP was supported by the decline in H2O2 level and down regulation of LP-PLA2 expression in dyslipidemic rats. Our findings are consistent with a study conducted by Fan Xu et al which concluded that PsP in GL is shown to hinder anti-inflammatory mediator which subsequently impairs the development of IL-6 and TNF-a and impairs angiogenesis processes of immature vasa vasorum23,24



Administration of Polysaccharide Peptide of Ganoderma lucidum down regulates the expression of Lp-PLA2, H2O2 and vasa vasorum angiogenesis in animal model compared to high fat diet group. The dose of 50 mg/kgBW has been proven to reduce the LP-PLA2, H2O2, and vasa vasorum angiogenesis comparable to normal or physiologic condition in animal model. The results of this study are consistent with previous studies, suggesting that antioxidant administration will reduce oxidative stress and inflammatory processes to inhibit atherogenesis processes6



We acknowledge the Sahabat Lingkungan Hidup Surabaya for preparing the PsP extract of Ganoderma lucidum. And this study was funded by Directorat of High Education through Brawijaya University Indonesia.



The authors declare no conflict of interest.



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Received on 29.10.2019           Modified on 25.12.2019

Accepted on 19.01.2020         © RJPT All right reserved

Research J. Pharm. and Tech. 2020; 13(7): 3241-3245.

DOI: 10.5958/0974-360X.2020.00574.0