




































 Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1 

 

CTMJ | traditionalmedicinejournals.com  

 

 

Impact of pycnogenol on oxidative and inflammatory damage in rat 

ovaries caused by ischemia/reperfusion 

Dr.Y.Sirisha 
1
, Dr.Aneela 

2
,Shivaprasad 

3
,Asraful hoque 

4,
 

Assistant professor 
1,2,3,4

, 

Department of Pharmacy, 

Samskruti College of Pharmacy, 

Kondapur (V), Ghatkesar (M) Medchal Dist, Telangana, India. 

 

Abstract 

The goal of this biochemical and histological study was to determine if pycnogenol (PYC) protected rats' ovaries 

from the ischemia/reperfusion (I/R) damage that resulted from experimental ovarian torsion.Methods: The six rats 

were divided into four equal groups and given the following names: SG (sham), PCG (pycnogenol 40 mg/kg), IRG 

(ovarian ischemia-reperfusion), and PIR (pycnogenol 40 mg/kg plus ovarian ischemia-reperfusion). The right ovary 

was made to undergo ischemia for two hours using vascular clips in the groups that received IRG and PYC 

treatments. Two hours after induction of ischemia, the ovary was reperfused. Next, the levels of MDA, tGSH, NF-κB, 

TNF-α, and IL-1β in the rat ovarian tissues were reviewed. Follicle counts were also conducted in addition to 

histological examinations of ovarian tissues.The outcomes are: The developing follicles in the ovary of the I/R-

induced group exhibited morphological and cellular deterioration as well as vascular disease, according to the 

histopathological investigation. Compared to the I/R-induced group, the PYC therapy group exhibited significantly 

reduced ovarian injury, edema, and vascular pathology (p < 0.05). Compared to the SG group, the I/R-induced 

group had considerably greater levels of MDA, NF-κB, TNF-α, and IL-1β, whereas the I/R damage group had 

significantly lower levels of tGSH (p < 0.05). Treatment with pycnogenol corrected the alterations in these 

biochemical indicators and the histological changes caused by I/R.Pycnogenol protects rat ovaries against I/R-

induced alterations in biochemical markers and histological disturbances, as shown in the conclusion. If we want to 

know how PYC affects ovarian damage, we need further research, preferably on people. 

Key terms: ischemia-reperfusion, ovarian injury, pycnogenol, malondialdehyde, tumor necrosis factor-alpha, 

interleukin-1β. 

 

INTRODUCTION  

Ovarian torsion occurs when the ovary rotates around 

its ligaments from which it receives support [1]. It is 

a gynecological emergency and affect women of all 

ages [2]. The most important risk factors are: being 

of reproductive age, presence of a mass in the ovaries 

exceeding 5 cm, pregnancy, ovulation induction and 

previous ovarian torsion [3]. However, ovarian 

torsion is seen even in normal ovaries [4]. Ovarian 

torsion is one of the causes of ovarian ischemia [2]. 

Ischemia is a condition in which the amount of 

oxygen in the tissue is reduced as a result of impaired 

blood flow in the vessels associated with the tissue, 

for whatever reason. On the other hand, reperfusion 

is the restoration of blood supply to the ischemic 

tissues [5]. Continuous reperfusion, after an ischemic 

attack, leads to a new physio-pathological process 

called “reperfusion injury”, which results in more 

severe tissue damage [6]. A delay in diagnosing and 

treating ovarian torsion result in severe ovarian 

damage and infertility [7]. Therefore, in the clinical 

setting, reperfusion of the ovaries by detorsion of the 

torsioned ovaries and preservation of their functions 

should be prioritized. Xanthine oxidase, the levels of 

which increase in tissue during ischemia, converts 

hypoxanthine to xanthine, using the abundant oxygen 

available as the tissue reperfuses [5]. As a result, a 

large proportion of the oxygen that reaches the tissue 

during reperfusion is converted into reactive oxygen 

species (ROS). Products with toxic properties, such 

as malondialdehyde (MDA), are formed due to the 

attack of cell membrane lipids by ROS [5]. In a study 

conducted by Ali and coworkers, it was reported that 



 Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1 

 

CTMJ | traditionalmedicinejournals.com  

 

the ischemia/reperfusion (I/R) procedure also 

increased the levels of pro-inflammatory cytokines 

along with oxidants in ovarian tissue [8]. Pycnogenol 

(PYC), investigated for its protective effect against 

possible damage caused by the ovarian I/R 

procedure, is a standardized extract of Pinus maritime 

[9]. In many countries, PYC is used as a dietary 

supplement and as a phytochemical treatment for 

various diseases, from chronic inflammation to 

circulatory disorders. A large number of studies have 

determined the antioxidant and anti-inflammatory 

properties of PYC. It has been reported that the 

strong antioxidant activity of PYC is due to the 

synergistic interaction of its components [10]. Ozoner 

et al reported that PYC protects brain tissues by 

reversing the increase in MDA and pro-inflammatory 

cytokines and the decrease of total glutathione 

(tGSH) [13]. Turkler et al also showed that PYC had 

mitigating effects against cisplatin-induced uterine 

and ovarian tissue damage in rats [14]. Based on this 

information, it was thought that PYC might protect 

the ovaries from I/R injury. However, studies 

investigating the effect of PYC on I/R-induced 

ovarian injury were not found in the literature. In this 

study, the preventive effect of PYC treatment on 

ovarian injury induced by I/R procedure in rats was 

investigated using biochemical analysis and 

histopathological examination.  

EXPERIMENTAL  

Animals  

Eighteen female albino Wistar rats (245 – 258 g) 

were used in the study. Animals were provided from 

the Atatürk University Medical Experimental 

Application and Research Centre. The rats were 

housed in laboratory rooms at a temperature of 22 ± 2 

°C. The rooms were equipped with 12- hour 

automatic light–dark cycles. The rats were provided 

with food and water without restriction before and 

during the experiment. The study was approved by 

Erzurum Ataturk University Laboratory Animal 

Research Local Ethics Committee (Dated: 

04.30.2020, Meeting no. 4, Decision no. 60), and 

followed international guidelines for animal studies. 

Chemicals  

Thiopental sodium was purchased from I.E 

ULAGAY (Istanbul, Turkey) and PYC was 

purchased from Solgar (Leonia, America).  

Groups  

The rats used in this study were divided into three 

groups of six rats each and designated as follows – 

SG: sham operation group, PCG: pycnogenol (40 

mg/kg) group, IRG: ovarian ischemia-reperfusion 

group and PIR: pycnogenol (40 mg/kg) + ovarian 

ischemia-reperfusion group.  

Procedure  

Sterile conditions were provided for surgical 

procedures in the experimental design. One hour 

before anesthesia, PYC (40 mg/kg) was administered 

orally in the PCG and PIR groups [13]. SG and IRGs 

were also given normal saline orally. Thiopental 

sodium (25 mg/kg) was injected intraperitoneally (IP) 

one hour after administration of pycnogenol or saline. 

A vertical incision (2 – 3 cm) was made in the 

appropriate anatomical region of the rats to access the 

right ovaries in SG, PIR and IRGs (no application 

was made to the ovaries of SG animals). Two hours 

later, the clips were excised and the tissues 

reperfused for another two hours. The rats were 

sacrificed using sodium thiopental (50 mg/kg) and 

the right ovaries were excised. The ovarian tissues 

were subjected to biochemical and histopathological 

examination. 

Biochemical analyses 

 Preparation of samples  

Excised rat ovaries (0.2 g) were weighed, washed 

with NaCl (0.9 %) to remove blood and subsequently 

homogenized in liquid nitrogen. For MDA 

determination, 2 mL of the homogenate was 

transferred to a 1.15 % potassium chloride solution. 

To determine total glutathione (tGSH), phosphate 

buffer (pH = 7.4, 2 mL) was added to homogenate. 

The solution was subsequently centrifuged and 

filtrate was used for analysis.  

Determination of MDA levels  

For the determination of MDA, the pink-colored 

compound formed the reaction of thiobarbituric acid 

and MDA (µmol/g protein) at 100 °C was measured 

spectrophotometrically [13]. Homogenates were 

centrifuged (5000 rpm, 20 min) and 250 μL of the 

clear filtrate was added to 750 μL (0.08 %) 

thiobarbituric acid, 100 μL (8 %) sodium dodecyl 

sulphate, 750 μL (20 %) acetic acid and 150 μL 

purified water and allowed to mix. Then, this mixture 

was incubated at 100 °C for one hour. N-butanol (2.5 

mL) was added and the pink color produced was read 

at a wavelength of 532 nm. The standard calibration 

curve was prepared using an appropriate 



 Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

ISSN : 2693 - 6356 

2021 | Vol 4 | Issue 1 

 

CTMJ | traditionalmedicinejournals.com  

 

concentration range of 1,1,3,3,3-tetra-

methoxypropane [13].  

Evaluation of tGSH levels  

GSH (nmol/g protein) is a compound containing a 

sulphydryl group in its structure. Sulfhydryl groups 

reduce 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), 

and a yellow color is formed [14]. Homogenates were 

centrifuged (12,000 rpm, 10 min) and 500 μL of the 

supernatant was added into test tubes containing 1500 

μL of measuring buffer, 100 μL DTNB and 7,900 μL 

of methanol and the solution was allowed to mix. 

This mixture was incubated at 37 °C for 30 minutes. 

The absorbance of the yellow compound was read at 

412 nm and the sample concentrations were 

calculated using a standard curve prepared according 

to GSH standard [14].  

Assessment of NF-κB, TNF-α and IL-1β levels  

Rat-specific enzyme-linked immunosorbent assay 

(ELISA) kits were used to determine NFκB (µg/g 

protein), TNF-α (pg/mL) and IL-1β (pg/mL) 

concentrations in ovarian tissue homogenates. Kits 

for rat NF-κB (Catalog no. 201-11-0288) were 

procured from SunRed (China) while those for TNF-

α (Catalog no. YHB1098Ra) and IL-1β (Catalog no. 

YHB0616Ra) were purchased from Shanghai 

(China). Analyses were carried out following the 

manufacturer’s manual.  

Histopathological analysis  

For light microscopy evaluation, the samples were 

placed in a 10 % formaldehyde solution to prevent 

any deterioration in the histological structures of the 

tissues. The tissues were then washed and gradually 

treated with (70 – 100 %) alcohol for dehydration. 

They were made transparent with xylol and blocks 

were obtained using paraffin wax. Sections (4 – 5 

µm) obtained from these blocks were stained with 

hematoxylineosin. Tissues were examined and 

photographed (Olympus DP2-SAL, Olympus® Inc. 

Tokyo, Japan). The histopathologist who performed 

the evaluation was not aware of the groups. Tissue 

damage was graded between 0 and 3 (0: absent, 1: 

mild, 2: moderate, 3: severe). Follicle classification 

and counting were performed at 100x magnification.  

Statistical analysis  

The IBM SPSS 22 software was used for all 

statistical analyses. The biochemical and follicle 

count data were presented as mean ± standard 

deviation (SD). The data were subjected to the 

Shapiro-Wilk test, and the results showed that they 

were normally distributed. A one-way ANOVA was 

used for analysis. Post-hoc test selection was made 

based on Levene’s test. The Games-Howell test was 

used in cases where variance homogeneity was not 

provided (MDA), and Tukey’s HSD test was used 

when it was provided (tGSH, NF-κB, TNF-α, IL-1β 

and follicle count). For histopathological statistics, 

the Kruskal Wallis test - Mann Whitney U test was 

used. P < 0.05 was considered statistically 

significant.  

RESULTS  

MDA and tGSH levels  

Malondialdehyde (MDA) levels were significantly 

elevated in ovarian tissues obtained from the IRG 

(5.37 ± 0.23) compared to the sham operation (2.23 ± 

0.05) and PYC alone (1.47 ± 0.23) groups (p < 

0.001). MDA levels in rats treated with PYC (2.92 ± 

0.37) prior to I/R were significantly lower than in the 

IRG (p < 0.001, Figure 1). On the other hand, tGSH 

levels were found to be decreased in the IRG (1.71 ± 

0.76) compared to the sham operation (4.60 ± 0.17) 

and PYC alone (5.86 ± 0.07) groups (p < 0.001). This 

decrease in tGSH levels was reversed by PYC (3.73 ± 

0.20) administration prior to I/R (p < 0.001, Figure 

1).  

NF-κB, TNF-α and IL-1β production  

As shown in Figure 2, NF-κB production in ovarian 

tissues subjected to I/R (IRG) was higher (5.70 ± 

0.33) than in the sham operation (2.39 ± 0.36) and 

PYC alone (1.65 ± 0.15) groups (p < 0.001). The 

PYC significantly reversed I/Rinduced increase in 

NF-κB levels (p < 0.001). There was no significant 

difference in the NF-κB levels between PIR group 

and SG (p = 0.220). Furthermore, TNF-α levels were 

higher in the IRG (4.62 ± 0.19) than in the sham 

operation (1.72 ± 0.16) and PYC alone (1.21 ± 0.09) 

groups (p < 0.001). PYC significantly reduced the 

increase in TNF-α amounts in ovaries subjected to 

I/R (p < 0.001; Figure 2). In addition, the IRG 

exhibited a significantly higher IL-1β (6.28 ± 0.20) 

than those of the sham operation (2.58 ± 0.34) and 

PYC alone (1.35 ± 0.21) groups (p < 0.001). 

Pretreatment with PYC significantly reversed the 

increase in IL-1β levels in ovaries subjected to I/R (p 

< 0.001, Figure 2).  

Histopathological findings  

As shown in Figure 3 A and B and Table 1, 

microscopic analyses of the ovarian sections of the 

sham operation and PYC groups show normal 



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2021 | Vol 4 | Issue 1 

 

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histological structure with developing follicle 

structures, corpus luteum, interstitial area and vessels. 

In the ovarian sections of I/R group (IRG) however, 

there was pronounced morphological and cellular 

degeneration of the developing follicles, abnormal 

bleeding foci in the corpus luteum and edema in the 

interstitial region were observed. 

 

Figure 1: MDA and tGSH levels in ovarian tissues of experimental groups. Note: *P < 0.001 vs. IRG. MDA: 

malondialdehyde; tGSH: total glutathione; SG: sham operation; PCG: pycnogenol (40 mg/kg); IRG: ovarian 

ischemia-reperfusion; PIR: pycnogenol (40 mg/kg) + ovarian ischemia-reperfusion 

 

Figure 2: NF-κB, TNF-α and IL-1β levels in ovarian tissues of experimental groups. Note: *P < 0.001 vs. IRG. NF-

κB: nuclear factor kappa B; TNF-α: tumor necrosis factor-alpha; IL-1β: interleukin one beta. SG: sham operation; 

PCG: pycnogenol (40 mg/kg); IRG: ovarian ischemia-reperfusion; PIR: pycnogenol (40 mg/kg) + ovarian ischemia-

reperfusion 

 



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Figure 3 (A – E): Hematoxylin-eosin staining of the 

ovary section of the experimental groups; A. Healthy 

control group. DF: developing follicle. Int: interstitial 

: blood vessel, x100. B.area. CL: corpus luteum.  

PYC group. DF: developing follicle. Int: interstitial 

area. : blood vessel, x100. C.CL: corpus luteum.  

Ischemia/reperfusion group. DF: degeneration in 

developing follicles, Int: edematous interstitial area, : 

dilated andCL: degenerated corpus luteum,  

congested blood vessel, x100. D. 

Ischemia/reperfusion group. DF: degeneration in 

developing follicles, Int: dense edematous interstitial 

: dilated andarea, CL: degenerated corpus luteum,  

congested blood vessel, x100. E. I/R + PYC group. 

DF: developing follicle with normal morphology, Int: 

interstitial area with normal appearance, CL: corpus : 

normal blood vessel, x100luteum,   

Severe dilatation and congestion were also noted in 

the blood vessels (Figure 3 C and Table 1). 

Furthermore, degeneration was also observed in the 

oocytes of the developing follicles, and areas of 

intense edema containing vacuolized cells were 

found in some places in large interstitial areas (Figure 

3 D and Table 1). In contrast, histopathological 

examination of the treatment group (PIR) detected a 

normal appearance of the developing follicles and 

corpus luteum, elimination of edema in the interstitial 

field and absence of dilatation and congestion of the 

blood vessels (Figure 3 E and Table 1). As shown in 

Table 2, I/R procedure decreased the number of 

developing and primordial follicles compared to rats 

in the sham operation and PYC-only groups (p < 

0.05). The number of developing and primordial 

follicles counted in PYC + I/R-treated rats was higher 

than in I/R group (p < 0.05). Atretic follicle and 

corpus luteum counts were the same in all groups (p 

> 0.05). 

DISCUSSION  

Ovarian ischemia may occur for various reasons, 

including surgical interventions, ovarian masses or 

ovarian torsion. It is an emergency gynecological 

situation that cause serious complications like 

infertility [15]. Reperfusion of ischemic ovaries after 

the detorsion procedure causes reperfusion damage. 

Therefore, it is increasingly recommended to add a 

conservative treatment to detorsion therapy [1]. The 

lipid peroxidation process that occurs in the cell due 

to oxidative stress leads to the formation of free 

radicals [9]. Malondialdehyde (MDA), the end 

product of lipid peroxidation, rises in I/R state, 

indicating the development of oxidative stress [9]. 

 

Agents like GSH, which interact with free radicals 

and are reduced to more stable molecules, also have 

the ability to repair lipid peroxides [11]. In a study 

where I/R was performed in rat ovaries, there was an 

increase in MDA amounts and a depletion in total 

antioxidant capacity in ovarian tissues [8]. Oxidative 

damage was detected in I/R group rats’ ovaries, with 

an increase in MDA levels and a decrease in tGSH 

levels compared to the sham group rats. Furthermore, 

in the PYC + I/R group, PYC protected the ovaries 



 Chinese Traditional Medicine Journal | 2021 | Vol 4 | Issue 1 

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2021 | Vol 4 | Issue 1 

 

CTMJ | traditionalmedicinejournals.com  

 

against I/R injury by limiting MDA increase and 

tGSH decrease. This result is in agreement with 

Ozoner et al who showed that PYC protects brain 

tissues against I/R injury by inhibiting the rise of 

MDA and pro-inflammatory cytokines while 

increasing tGSH levels as well [11]. Also, Ozer 

Sehirli et al established a model of renal I/R damage 

and found that MDA levels were significantly 

reduced in rats receiving a single dose of 10 mg/kg 

PYC intraperitoneally [9]. NF-κB serves as a 

transcription factor for the production of several pro-

inflammatory cytokines that play important roles in 

regulating inflammation and immune responses [16]. 

Previous studies established that ROS increases NF-

κB production [17].  

In addition, Kocaturk et al found an increase in NF-

κB levels in kidney tissues following I/R application 

[16]. In the current study, I/R application led to an 

elevation of tissue NF-κB concentrations. The fact 

that NFκB levels in I/R + PYC group did not increase 

as in I/R group but were close to those in the sham 

group suggests that PYC inhibits NF-κB. NF-κB 

specifically triggers the production of TNF-α, IL1, 

IL-6, lymphotoxin, and IFN-γ [17]. The results from 

this study showed that TNF-α and IL-1β levels 

increased at the tissue level in I/R group. According 

to the biochemical results, PYC given before I/R 

significantly suppressed the increase of TNF-α and 

IL-1β. Ali et al also demonstrated that ovarian I/R 

increased tissue TNF-α expression in their study [9]. 

Pycnogenol had a similar effect in kidney tissues 

exposed to I/R and reversed the increase in tissue 

TNF-α and IL-1β [16]. The data indicate that PYC 

suppresses inflammation by reducing I/Rinduced 

oxidative stress and pro-inflammatory cytokine 

increase in the ovary. In this study, the protective 

effect of PYC on ovarian reserve was also evaluated 

by determining the number of follicle loss. 

Previously. Turkler et al showed that PYC 

ameliorated cisplatin-induced histopathological 

changes in the uterus and ovaries [14]. Additionally, 

another study showed that pycnogenol reduced liver 

damage in an I/R rat model [6]. In this study, it was 

observed that the amount of primordial and 

developing follicles was lower in the PYC-treated 

group. Congestion, bleeding, edema and follicular 

degeneration, which are changes that occur due to I/R 

damage in ovarian tissue, were also observed 

histopathologically. Conversely, PYC pretreatment 

significantly ameliorated I/R-induced ovarian 

damage.  

CONCLUSION  

The results of this study confirm that I/R procedure 

causes serious damage to ovarian tissues. Pycnogenol 

protects the ovaries against I/R damage and possesses 

antioxidant activities, including the stabilization of 

intracellular antioxidant defense systems as well as 

reduction of ROS and lipid peroxidation products. 

This study has shown that PYC is beneficial in the 

treatment of I/R-induced ovarian injury. Further in 

vivo studies on the effect of PYC on ovarian injuries, 

including in humans, would be required. 

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