Endothelial Protective and Antioxidant Effect of Polysaccharide

Peptide of Ganoderma Lucidum in STEMI and NSTEMI Patients

with Diabetes as a Risk Factor

Djanggan Sargowo

, Nurul Aini Rahmawati

2,3

*, Faris Wahyu Nugroho

, Putri Annisa Kamila

, Dedy

Irawan

, Monika Sitio

, Liemena Harold Adrian

, Aditya Reza Pratama

, Yusuf Arifin

, Rizki

Fadlan

, Diah Ivana Sari

, Chamelia Rohadatul ‘Aissy

, Evita Febriyanti Purwanto

, Firyal Nadiah

Rahmah

, Merkuri Puspasari Sukatman

, Rakhmawati Fahmiy

Department of Cardiology and Vascular Medicine, Faculty of Medicine, Brawijaya University/Saiful Anwar General

Hospital, Malang, Indonesia

Master Program of Biomedical Science, Faculty of Medicine, Brawijaya University, Malang, Indonesia

Department of Physiotherapy, Faculty of Health Science, University of Muhammadiyah Malang, Indonesia

Jalan Bendungan Sutami 188A Malang 65145

Keywords: STEMI, NSTEMI, Diabetes, Atherosclerosis, Endothelial dysfunction, Antioxidant, Ganoderma Lucidum.

Abstract: Diabetes induced increases of Reactive Oxygens Species (ROS) Production. Oxidative stress and endothelial

dysfunction are induced by high free radical. In Previous studies has been found that PsP of Ganoderma

Lucidum has no toxicity in animal model and can be act as an antioxidants and antiendothelial dysfunction.

This study was purposed to prove the effect of PsP of Ganoderma Lucidum as antioxidant and protective

endothelial cell in STEMI and NSTEMI patients with diabetes. This clinical trial study conducted to 50

patients that diagnosed STEMI, NSTEMI and Diabetes, with pre- and post-test control group design. The

parameters are MDA, SOD, CEC, and EPC counts. Patients divided into two group randomized, one group

were given PsP Ganoderma Lucidum 750mg/day in 3 divided dose, and another group were given placebo

for 90days. Normally distributed data will be analyzed by Dependent T-test and abnormally distributed data

will be analyze by Wilcoxon test. The result are in PsP group patiens MDA level significantly decreased with

P=0,030, SOD significantly increased with P=0,000, EPC count significantly increased with P=0,016, CEC

count significantly decreased with P= 0,020. It concluded PsP of Ganoderma Lucidum act as antioxidant and

protective endothelial cell against pathogenesis of atherosclerosis in STEMI and NSTEMI patients with

diabetes.

1 INTRODUCTION

The pathophysiology of vascular damage in diabetes

is complex and involves abnormalities in endothelial

cells, vascular smooth muscle cells, and platelet

function. Hyperglycemia reduces the availability of

endothelial nitric oxide (NO) and vascular function

through several mechanisms, especially an increase in

the number of reactive oxygen species (ROS) derived

from mitochondria and cytoplasm. ROS is not only

the final effector but also the triggering factors of

each of the following pathways involved in blood

vessel damage caused by hyperglycemia: polyol

pathway, formation of glycation end products

(AGEs), de-novosynthesis of diacylglycerol (DAG),

which leads to the activation of isoform protein-

kinase C (PKC) and the hexosamine pathway (Bonora

& DeFronzo, 2018).

Knowledge of the factors involved in the process

of atherosclerosis, the main markers of antioxidants

such as SOD or markers for oxidative stress such as

MDA will help in the treatment of this disease.

Pathological processes in endothelial due to oxidative

stress using CEC (circular endothelial cells) which

prove the existence of endothelial injury and EPC

(endothelial progenitor cells) that improve vascular

repair. The second marker representing vascular and

endothelial integrity is important in the

pathophysiology of cardiovascular disease

(Rajendran et al., 2013).

Sargowo, D., Rahmawati, N., Nugroho, F., Kamila, P., Irawan, D., Sitio, M., Adrian, L., Pratama, A., Ariﬁn, Y., Fadlan, R., Sari, D., ’Aissy, C., Purwanto, E., Rahmah, F., Sukatman, M. and

Fahmiy, R.

Endothelial Protective and Antioxidant Effect of Polysaccharide Peptide of Ganoderma Lucidum in STEMI and NSTEMI Patients with Diabetes as a Risk Factor.

DOI: 10.5220/0009121700600065

In Proceedings of the 2nd Health Science International Conference (HSIC 2019), pages 60-65

ISBN: 978-989-758-462-6

Treatment using natural ingredients has long been

used, one of which is the administration of

polysaccharide peptides (PSP) derived from

Ganoderma Lucidum extract. Ganoderma Lucidum is

an oriental mushroom, has a history in China, Japan

and other Asian countries as an ingredient that adds

to health and longevity. This fungus is dark and large.

In China, Ganoderma Lucidum is called Lingzhi,

while in Japan it is called Reishi or Mannentake.

Lingzhi contain bioactive compound, namely β-

glucan has been proven to have an effect on

controlling blood sugar, immune system modulation,

hepatoprotective and many other effects

Molecular weight, conformation, and degree of

branching are influenced β-glucan properties. The

different structure of β-glucan can affect its

antioxidant activity. This antioxidant activity can also

be influenced by sources and extraction methods. The

molecular weight of β-glucan extracted from barley

with warm water is 40,000-100,000 Da, while

oligomers made from β-glucan macromolecules with

enzymatic degradation by lichenase have a molecular

weight of around 2,000 Da. The smaller molecular

size, inferred, can result in a reduction in hydroxyl

radical uptake activity or a reduction in activity of

antioxidant. The reduced molecular weight of about

1/20 - 1/50 can cause a reduction in antioxidant

activity by about half from before. This finding shows

that β-glucan still carries out hydroxyl radicals at

various molecular sizes, even with different potentials

(Kofuji et al., 2012).

β-1,3 / 1,6-D-Glucan contained in PsP has been

analyzed by the Complex Carbohydrate Research

Center-University of Georgia, USA and the analysis

shows that PsP has a large molecular weight (>

3,755,000 Da). β-1,3 / 1,6-D-Glucan in PsP is also

known to have complex triple helix solution

conformation and branching. Large molecular

weight, triple helix formation and complex branching

shows the potential of β-glucan in PsP as a powerful

immunomodulator and antioxidant. The molecular

weight of β-glucan in PsP extracted from Ganoderma

Lucidum can be compared with the molecular weight

of β-glucan taken from other sources. The molecular

weight greater than the molecular weight of β-glucan

from other sources shows the potential of β-glucan in

PsP as a stronger antioxidant compared to other

sources (HE et al., 2010).

Research on Peptide Polysaccharide (PsP) of

Ganoderma Lucidum has been started since 2013

which aims to find out the effect of PsP

administration on Rattus novergicus strain Wistar rats

with a high-fat diet and diabetes mellitus. The results

showed that PsP is a chronic anti-inflammatory and

antioxidant in Rattus novergicus strain Wistar rats

(Sargowo et al., 2018). In 2014, an acute and sub-

chronic PsP toxicity study was conducted. In this test,

observations of blood chemical parameters and

histopathology of organs including kidney, lung,

heart, aorta, and liver in Rattus novergicus strain

Wistar rats.

The results showed that 5 variations of PsP dose

did not have a toxic effect with a certain dose in

Rattus novergicus strain Wistar rats (Wihastuti

et al.,

2016). In 2015 a study was conducted to determine

the effect of giving PSP as a chronic anti-

inflammatory, antioxidant, antilipid, and endothelial

antidisfunction in stable Angina Pectoris patients and

at high risk of heart disease based on Framingham

scores. The results showed that PsP has an anti-

inflammatory, antioxidant, antilipid, and endothelial

anti-inflammatory function so that it can play a role

as a secondary preventive for Angina Pectoris

patients. Stable and primary preventive for patients at

high risk of heart disease based on Fraingham scores

(Sargowo et al., 2018).

Based on the results of the study, the

administration of PsP is thought to be beneficial for

patients with STEMI and NSTEMI. Therefore,

further research is needed on the potential of β-1,3 /

1,6-D-glucans from mycelia Ganoderma Lucidum

extract as an antioxidant and endothelial protection in

STEMI and NSTEMI patients with diabetes.

2 METHODS

The study was true experimental using double blinds

randomized perspective with pre-test and post-test

design. All patients who participated in this study

were patients who came from the cardiology

outpatient unit of the Saiful Anwar Malang Hospital

and Lavalette Malang Hospital which had agreed to

participate in this study and had signed informed

consent. Following are the inclusion, exclusion, and

drop out criteria in this study.

2.1 Study Subject

The study group consisted of 50 patients with STEMI

and NSTEMI. They are admitted to CVCU or heart

poly of Saiful Anwar Hospital Malang and signed the

informed consent. Patients who drinking alcohol a

day more than 50g of ethanol, has acute hepatitis,

kidney failure, hematological abnormalities and

bleeding, anaphylactic disorder, using steroid, have

transplant of grafting organ, sepsis, drug abuse in

previous year, have low moderate risk according

Endothelial Protective and Antioxidant Effect of Polysaccharide Peptide of Ganoderma Lucidum in STEMI and NSTEMI Patients with

Diabetes as a Risk Factor

Framingham cardiovascular risk score criteria

according to ATP III, rheumatological abnormalities,

neoplasm less than 5 years, severe disease and severe

comorbidity were excluded from the study. All

protocols in this study were agreed with informed

consent and the Ethics Committee of the Medical

Faculty of Brawijaya University, Malang Indonesia

(171 / EC / KEPK / 07/2018)

2.1 Collection of Specimens and

Biochemical Analysis.

Venous blood sample were collected from all the

patient, 20 ml of blood was collected from each

patient. The blood sample was centrifuges at 1000

rpm for 15 minutes at 2-8 ° C. Measurement of SOD

and MDA in humans by taking pretest and posttest

blood preparations then using the ELISA kit method.

SOD uses an ELISA kit that is applied in quantitative

in vitro determination of SOD concentrations in

humans in serum, plasma, and other body fluids. A

detectable dose of SOD of at least 37.5 pg / mL. MDA

is measured using ELISA with the ELISA

competitive method. MDA in the sample will

compete with MDA on the plate, with the aim that the

two compete to bind antibodies that are specific to

MDA. The amount of CEC was measured by the

flowcytometry method of blood plasma samples with

anti-CD146 antibody markers labeled Phycoerythrin

and anti-CD45 labeled FITC. EPC counts were

measured by flowcytometry from blood plasma

samples with Phycoerythrin labeled anti-CD133

antibody markers and FITC labeled anti-CD34

All data are expressed of mean ± SD. Paired T test

was used to estimate the differences between groups.

If the data are abnormally distributed, Wilcoxon test

will be used to analyze. SPSS 25.0 was used for

statistical analyze and the statistically significant was

set at p<0,05.

3 RESULTS AND DISCUSSION

In this study there were five patient who dropped out

caused by allergic reactions. In the extract group there

was significant decrease in MDA and CEC and there

was significant increase in SOD and EPC.

Anthropometric results and characteristics of

research subjects can be seen in table 1.

Table 1: Characteristics of research subjects and anthropometric examination results.

Variabel Mean pretest

Mean Posttest

p- value

PsP Placebo

SOD 2,8878±0,9315 3,4099± 0,1171 2,6807± 0,1930 0,000

MDA 20,8239±1,5969 16,7858± 2,1828 23,6827 ± 2,26851 0,03

CEC 88,1119±13,928 61,618± 9,5877 48,3465 ± 7,48505 0,02

EPC 101,4794±8, 116 93,7319±9,2121 101,1762±11,088 0,16

Table 2: The result of SOD, MDA, CEC, EPC.

Sample pretest mean Sample posttest mean

Value

PsP Placebo PsP Placebo

Gender M: 53,78%

Age (Years) 59,26

Weight (kg) 58,35 63,8

58,12 63,84 0,743

Height (m) 1,55±0,07

BMI 24.24 26.27 24.14 26.29 0.706

WC (cm) 87.5 93,92 88.85 94.08 0.482

Pulse 81.85 74.36 75.2 77.24 0.000

SBP (mmHg) 148.35 148,48 134.5 147,6 0.021

DBP (mmHg) 86.4 85.64 80 83.44 0.317

GDP 139,81 151,5 109,937 129,312 0,041

HBA1C 7,934 8,125 7,156 8,121 0,025

HSIC 2019 - The Health Science International Conference

This study purposed to prove the potential of β-

1,3 / 1,6-D-Glukan (Polysaccharide Peptide) from

Miselia Ganoderma Lucidum as an antioxidant and

endothelial protective in STEMI and NSTEMI

patients with Diabetes as a risk factor. Data analysis

showed that extract of Ganoderma Lucidum could

reduce MDA levels significantly (p = 0.03), it can

significantly increase SOD (p = 0.00), decrease in

CEC levels significantly (p= 0,02), and significantly

increase in SOD level (p = 0.016). This data shows

that β-1,3 / 1,6-D-glucan (Polysaccharide Peptide)

has an antioxidant effect and increases the potential

of endothelial cells in carrying out angiogenesis even

in oxidative stress conditions and it is associated with

SOD improvement, especially MnSOD. β-1,3 / 1,6-

D-Glucan (Polysaccharide Peptide) increases the

expression of MnSOD and increases its antioxidant

activity. This increase can then reduce free radicals in

endothelial cells and increase cell resistance to

oxidative stress.

SOD has enzymatic activity to scavenge on

superoxide anion molecules (O2), through the

mechanism of redox reactions. Under oxidized

conditions, SOD will release excess electrons in

superoxid anions, and release O2 molecules. Under

reduced conditions, SOD will catalyze the reaction of

superoxid anions with hydrogen cations (2H +) to

form hydrogen peroxide (H2O2) which is more

unreactive. Determination of the form of reduced or

oxidized SOD, is determined by the amount of redox

from metal ions that are conjugated with SOD.

Furthermore, H2O2 will be catalyzed further by

enzymes catalase and glutathione peroxidase (Fukai

and Ushio-Fukai, 2011).

At high conditions of free radicals, there is an

initial response to an increase in SOD production,

specifically Cu / ZnSOD through the PI3K / Akt

transmission pathway and NF-κB transcription factor

in cells exposed to oxidants. However, in conditions

of high free radicals that continue, causing problems

in the process of SOD formation, especially in

MnSOD and EC SOD. MnSOD is made on

mitochondria, which require "mitochondrial import

machines" to be used in the cytosol and carry out their

functions. In high conditions of free radicals, there is

a disturbance in the process of important

mitochondrial, which can reduce the level of

cytosolic MnSOD (Candas and Li, 2014). In addition,

SOD scavenge metabolites, namely H2O2 can also be

the amount of functional SOD2 protein, also can be

displayed mRNA from SOD2. It can act as H2O2

affecting the amount of SOD2 protein in the post-

translational process (Candas and Li, 2014).

Malondialdehyde (MDA) is a product of lipid

peroxidation and one of the oxidant markers most

often studied. MDA is involved in many disease such

as cancer, Alzheimer’s, diabetes and heart disease

observed an increase in MDA which contribute to

heart disease. Other research states that agents that

decrease MDA and increase SOD provide benefits

and are even considered as therapeutic agents for

heart disease because they can reduce oxidative stress

in the body (Kim, Yun and Kwon, 2016).

Endothelial dysfunction is characterized by a state

of endothelial stiffness, pro inflammation, and pro

thrombi nature. This occurs in most cardiovascular

diseases, such as coronary disease, peripheral

vascular disease, hypertension, chronic heart failure,

diabetes, chronic kidney failure, and severe viral

infections. High free radicals cause oxidative stress,

and can cause endothelial dysfunction.

Cardiovascular risk factors are associated with an

increase in ROS sources such as NADPH oxidase,

xanthine oxidase, cyclooxygenase and mitochondria.

Free radicals can disrupt the balance of nitric oxide,

damage the endothelium, and if it occurs in a long

time, will affect the permeability of blood vessels,

causing toxic substances easily enter the tissues.

Oxidative stress that occurs can also cause increased

proliferation of vascular smooth muscle cells,

increase in metalloproteinase matrix and be involved

in the formation of atherosclerotic plaque (Alessio et

al., 2013).

Factors that can induce endothelial cell release

from the vascular wall include mechanical injury to

the vascular wall, after arterial or venous blood

sampling, and acute plaque rupture. ROS activity

together with prolonged inflammation that disrupts

the integrity of integrins and cadherins can also cause

endothelial cell release. In this study, the results

obtained by giving PsP of Ganoderma Lucidum, a

significant decrease in the number of CEC compared

with placebo. This indicates that PsP Ganoderma

Lucidum can improve endothelial dysfunction that

occurs in the process of atherosclerosis. CEC itself is

known that can be induced through various processes

that occur in atherosclerosis, such as persistent

inflammation and high free radical conditions

(Rajendran et al., 2013).

Protease enzymes and pro-inflammatory

cytokines produced will disrupt the integrity of

integrin and cadherin proteins that function for

endothelial cell adhesion (Schmidt, Manca and

Hoefer, 2015). The number of CECs in addition to

indicating endothelial dysfunction, also gives an idea

of the severity of the atherosclerotic process.

Sugimoto et al. reported that there was a positive

Endothelial Protective and Antioxidant Effect of Polysaccharide Peptide of Ganoderma Lucidum in STEMI and NSTEMI Patients with

Diabetes as a Risk Factor

correlation between the number of CECs and Carotid

Plaque Score indicating the severity of

atherosclerosis(Sugimoto et al., 2015).

Inversely related to CEC, EPC has a negative

correlation with the progression of atherosclerosis by

having a role in regenerating damaged vascular

endothelium. EPC is a derivative of stem cells in the

bone marrow, which are able to stimulate

proliferating and migrating. Proliferation and

migration of EPC depend on cytokines and growth

factors that result from vascular endothelial damage

processes, such as VEGF, IGF-1, SDF-1α, to

erythropoietin. EPC in circulation is divided into 2

pathways of differentiation, namely early EPC (also

called angiogenic EPC) and late EPC (also called out-

growth EPC). Early EPC plays a role in the process

of angiogenesis, whereas late EPC, which can

differentiate to form mature endothelial cells, plays a

role in the process of vascular endothelial

regeneration. This difference is supported by the

expression of adhesion molecule markers in late

EPC, such as VE-cadherin and von Willebrand

Factor (Shantsila, Watson and Lip, 2007). In this

study, the results obtained by giving PsP of

Ganoderma Lucidum increased the number of EPC

compared to the placebo group significantly.

These results indicate that PsP of Ganoderma

Lucidum can increase the process of endothelial cell

regeneration, which is damaged in the development

of the atherosclerotic process. This can be thought to

be caused by the anti-oxidant properties of PsP

Ganoderma Lucidum. Lin et al. reported that

increased free radical production correlates with a

decrease in the amount of EPC through the induction

of apoptosis from EPC. Similar results were obtained

in studies from Fiorito et al. which illustrates the

increase in the amount of EPC in the administration

of vitamins C and E as anti-oxidants. In addition, an

increase in the amount of EPC due to the

administration of this vitamin is also induced by a

decrease in TNF-α levels which indicates the

presence of anti-inflammatory factors that play a role

in increasing the amount of EPC (Fiorito et al., 2008).

TNF-α is a marker of inflammation, and various

studies have described its relationship with EPC.

Research by Chen et al. gave the results that the

incubation of EPC with TNF-α which described the

pro-inflammatory environmental conditions caused a

decrease in EPC proliferation and migration and

induced apoptosis of EPC. In addition, exposure with

TNF-α reduces the ability of EPC adhesion by

decreasing the expression of VEGFR and SDF-1

proteins that play a role in adhesion with sub

endothelium (Chen et al., 2011). Other studies from

Koshikawa et al. reported that hsCRP levels were

negatively correlated with the amount of EPC in

circulation. The hsCRP protein is one of the

inflammatory markers produced more in chronic

diseases such as diabetes and atherosclerosis

(Koshikawa et al., 2010). From these studies it can be

concluded that inflammatory conditions reduce EPC

activity in endothelial regeneration.

4 CONCLUSIONS

From this study, it can be concluded that PsP of

Ganoderma Lucidum can be act as antioxidant and

endothelial protection in STEMI and NTEMI patients

with diabetes as a risk factor.

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HSIC 2019 - The Health Science International Conference

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Endothelial Protective and Antioxidant Effect of Polysaccharide Peptide of Ganoderma Lucidum in STEMI and NSTEMI Patients with

Diabetes as a Risk Factor