<i>Physalis angulata</i> leaf extract ameliorates L-N<sup>G</sup>-nitroarginine methyl ester (L-NAME)-induced preeclampsia symptoms in rats through improved endothelial progenitor cells and endothelial cells due to reduced antiangiogenic factor and oxidative stress

Dian Nugrahenny; Achmad Rudijanto; Nur Permatasari; I Wayan Arsana Wiyasa; Mohammad Aris Widodo; Karyono Mintaroem; Edi Widjajanto; Mustofa Mustofa

doi:10.12688/f1000research.123006.1

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Research Article

Physalis angulata leaf extract ameliorates L-N^G-nitroarginine methyl ester (L-NAME)-induced preeclampsia symptoms in rats through improved endothelial progenitor cells and endothelial cells due to reduced antiangiogenic factor and oxidative stress

[version 1; peer review: 1 approved with reservations]

Dian Nugrahenny ^1,2, Achmad Rudijanto³, Nur Permatasari², [...] I Wayan Arsana Wiyasa⁴, Mohammad Aris Widodo², Karyono Mintaroem⁵, Edi Widjajanto⁶, Mustofa Mustofa⁷

Dian Nugrahenny ^1,2, Achmad Rudijanto³, [...] Nur Permatasari², I Wayan Arsana Wiyasa⁴, Mohammad Aris Widodo², Karyono Mintaroem⁵, Edi Widjajanto⁶, Mustofa Mustofa⁷

PUBLISHED 12 Jul 2022

Author details Author details

¹ Doctoral Program in Medical Sciences, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
² Department of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
³ Department of Internal Medicine, Faculty of Medicine, Universitas Brawijaya-dr. Saiful Anwar General Hospital, Malang, 65145, Indonesia
⁴ Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Brawijaya- dr. Saiful Anwar General Hospital, Malang, 65145, Indonesia
⁵ Department of Anatomical Pathology, Faculty of Medicine, Universitas Brawijaya- dr. Saiful Anwar General Hospital, Malang, 65145, Indonesia
⁶ Department of Clinical Pathology, Faculty of Medicine, Universitas Brawijaya- dr. Saiful Anwar General Hospital, Malang, 65145, Indonesia
⁷ Department of Pharmacology and Therapy, Faculty of Medicine, Public Health, and Nursing, Universitas Gadjah Mada, Yogyakarta, 65281, Indonesia

Dian Nugrahenny
Roles: Conceptualization, Formal Analysis, Funding Acquisition, Methodology, Project Administration, Software, Visualization, Writing – Original Draft Preparation

Achmad Rudijanto
Roles: Conceptualization, Data Curation, Supervision, Validation, Writing – Review & Editing

Nur Permatasari
Roles: Conceptualization, Data Curation, Supervision, Validation, Writing – Review & Editing

I Wayan Arsana Wiyasa
Roles: Conceptualization, Data Curation, Supervision, Validation, Writing – Review & Editing

Mohammad Aris Widodo
Roles: Conceptualization, Data Curation, Supervision, Validation, Writing – Review & Editing

Karyono Mintaroem
Roles: Conceptualization, Data Curation, Supervision, Validation, Writing – Review & Editing

Edi Widjajanto
Roles: Conceptualization, Data Curation, Supervision, Validation, Writing – Review & Editing

Mustofa Mustofa
Roles: Conceptualization, Data Curation, Supervision, Validation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Plant Science gateway.

Abstract

Background: This study aimed to determine the ameliorative effects of Physalis angulata leaf extract on L-N^G-nitroarginine methyl ester (L-NAME)-induced preeclampsia symptoms in rats.
Methods: Phytochemical analysis of the extract was performed with liquid chromatography-high resolution mass spectrometry (LC-HRMS). Pregnant Wistar rats were randomly divided into five groups (n=6). Preeclampsia rats were injected with L-NAME on gestation days 9 to 18 (G9–G18), while sham rats were injected with the same vehicle volume. Three groups of preeclampsia rats were orally supplemented with the extract at doses of 500, 1500, and 2500 mg/kg body weight/day on G12–G18. The tail-cuff method measured blood pressures. Spectrophotometry determined urine protein levels. Serum soluble fms-like tyrosine kinase (sFlt)-1 levels were evaluated using an enzyme-linked immunosorbent assay (ELISA). Serum and placental malondialdehyde (MDA) levels, superoxide dismutase (SOD) activities, and nitric oxide (NO) levels were measured by colorimetry. Immunohistochemistry was used to determine tail artery endothelial nitric oxide synthase (eNOS), placental eNOS, and placental hypoxia-inducible factor (HIF)-1α expressions. Circulating angiogenic cells (CACs) and endothelial colony-forming cells (ECFCs) were counted using flow cytometry.
Results: L-NAME injection increased blood pressures, 24-h total urine protein level, serum sFlt-1 level, serum and placental MDA levels, percentages of CACs and ECFCs, and placental HIF-1α expression. It also decreased serum and placental SOD activities, serum NO level, tail artery and placental eNOS expressions compared to the sham group. Physalis angulata leaf extract administration lowered blood pressures, urine protein level, sFlt-1 level, MDA levels, percentages of CACs and ECFCs, and placental HIF-1α expression. The extract increased SOD activities, NO level, tail artery and placental eNOS expressions compared to the preeclampsia group.
Conclusions: Physalis angulata leaf extract reduces antiangiogenic factor and oxidative stress. It also enhances eNOS/NO signaling. Thus, it improves EPC and endothelial cell function and reverses L-NAME-induced hypertension and proteinuria in preeclampsia rats.

Keywords

Antiangiogenic, Antioxidant, Cutleaf groundcherry, Endothelial cells, Hypertension, Hypoxia, Nitric oxide, Physalis, Preeclampsia, Pregnancy, Stem cells

Corresponding author: Dian Nugrahenny

Competing interests: No competing interests were disclosed.

Grant information: This work was supported by the Research and Community Service Institution (LPPM) Universitas Brawijaya: HPP UB No. 696.150/UN10.C10/PN/2019 and the Research and Community Service Unit (BPPM) Faculty of Medicine-Universitas Brawijaya: Competitive Grant PNBP FKUB No. 32/SK/UN10.F08.06/KS/2019.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Nugrahenny D et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Nugrahenny D, Rudijanto A, Permatasari N et al. Physalis angulata leaf extract ameliorates L-N^G-nitroarginine methyl ester (L-NAME)-induced preeclampsia symptoms in rats through improved endothelial progenitor cells and endothelial cells due to reduced antiangiogenic factor and oxidative stress [version 1; peer review: 1 approved with reservations]. F1000Research 2022, 11:780 (https://doi.org/10.12688/f1000research.123006.1) First published: 12 Jul 2022, 11:780 (https://doi.org/10.12688/f1000research.123006.1) Latest published: 12 Jul 2022, 11:780 (https://doi.org/10.12688/f1000research.123006.1)

Introduction

Preeclampsia is one of the most severe hypertension conditions during pregnancy. It is distinguished by new-onset hypertension and proteinuria after more than 20 weeks of gestation.¹ Preeclampsia affects 3% of all pregnancies globally.² Preeclampsia is one of the leading causes of maternal mortality in underdeveloped nations, where access to prenatal and intrapartum care is generally limited.³ Preeclampsia also dramatically raises the cost of healthcare services.⁴

Endothelial dysfunction contributes to preeclampsia’s pathogenesis, resulting in peripheral vasoconstriction and multiorgan damage.⁵ Endothelial dysfunction persists after birth, increasing the risk of future cardiovascular and cerebrovascular disorders in the mother⁶^,⁷ and the child.⁷^,⁸ This condition underlines the importance of alleviating systemic endothelial dysfunction in preeclampsia.

One essential factor in improving endothelial dysfunction is endothelial progenitor cells (EPCs).⁹ Previous studies showed that EPC function decreases in preeclampsia.¹⁰^,¹¹ Thus, it results in inadequate endothelial cell regeneration (reendothelialization) and impacts various clinical manifestations of preeclampsia.⁹^,¹²

Although the pathophysiology of preeclampsia is becoming more understood, finding a definitive treatment has been difficult.¹³ Increasing data suggest that treatment strategies to restore the underlying endothelium dysfunction may alleviate the broad systemic manifestations in preeclampsia.¹⁴ A central common metabolite in vascular dysfunction associated with preeclampsia is nitric oxide (NO).¹⁵

One of the natural ingredients to improve nitric oxide bioavailability is Physalis leaf extract. Previous study found that Physalis leaf extract increases EPCs number and enhances reendothelialization in rats given deoxycorticosterone acetate (DOCA)-salt.¹⁶ The species of Physalis found in abundance in Indonesia is Physalis angulata L. It is easy to grow and is often only considered a weed.¹⁷ This study aimed to evaluate whether Physalis angulata leaf ethanol extract might alleviate L-N^G-nitroarginine methyl ester (L-NAME)-induced preeclampsia symptoms in rats by improving EPC and endothelial cell function. L-NAME is a nonselective NOS inhibitor, thereby decreasing NO production.¹⁸

Methods

Ethical considerations

The institutional research ethics council of Universitas Brawijaya authorized all experimental protocols (No.1191-KEP-UB, December 10, 2019). The reports are compliant with Animal Research Reporting of In Vivo Experiments (ARRIVE) guidelines 2.0.¹⁹ All efforts were made to alleviate any suffering of animals. Trained researchers carried out all procedures, including injections and administration of extract through an oral gavage feeding tube.

Plant materials

Physalis angulata leaves were collected from the Research Institute of Spices and Medicinal Plants (BALITTRO) Bogor, Indonesia. The plant was identified as Physalis angulata L. (Solanaceae family) by the School of Life Sciences and Technology (SITH) at Institut Teknologi Bandung (ITB), Indonesia (No. 3538/I1.CO2.2/PL/2016, October 13, 2016). The fresh leaves were thoroughly washed to remove soil residues with distilled water and then dried at room temperature (RT) for a week. The dried leaves were then ground using a grinder into a fine powder. The powder was stored in an aluminum foil-wrapped airtight container at −20°C before being used.

Plant extraction

The crude ethanol extract was prepared by maceration. Physalis angulata leaf powder (100 g) was stirred mechanically with 900 mL of 96% (v/v) ethanol for 24 h at RT. The residue was extracted thrice. The extract obtained was then filtered through filter papers (Whatman No. 1, Cat# 1001-150). The filtrate was evaporated using vacuum distillation and a rotary evaporator (Janke & Kunkel, IKA-Labortechnik, Germany) at a temperature of 70–80°C. The crude ethanol extract was then stored in an aluminum foil-wrapped airtight container at −20°C before being used.

Phytochemical analysis with LC-HRMS

Phytochemical analysis of the Physalis angulata leaf extract was performed with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The LC-HRMS system was composed of a Dionex™ UltiMate™ 3000 RSLCnano System (Cat# ULTIM3000RSLCNANO) with a micro-flow meter (Cat# 6041.7903A) from Thermo Scientific™. The analytical column used was Thermo Scientific™ Hypersil GOLD™ aQ (1.0 mm × 50 mm × 1.9 μm particle size, Cat# 25302-051030). The LC system was linked to a Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap™ MS System (Cat# IQLAAEGAAPFALGMBDK) with heated electrospray ionization (HESI) with positive or negative ionization at 300°C, a nitrogen gas sheath flow of 20 arbitrary units, and an auxiliary nitrogen gas flow of 10 arbitrary units.

The temperature of the analytical column was kept constant at 30°C. (A) MS-grade water (Cat# 900682) with 0.1% (v/v) formic acid and (B) MS-grade acetonitrile with 0.1% (v/v) formic acid (Cat# 900686) were employed as mobile phases (all from Sigma-Aldrich). Separation was accomplished using the following mobile phase gradient: (i) elution with 5% B from the injection time until 2 minutes, (ii) a linear gradient of 5–60% B for the next 13 minutes, (iii) a further linear gradient 60–95% B for 10 minutes, (iv) a further linear gradient 5–95% A for 0.1 minutes, and finally (vi) elution with 95% A for 4.9 minutes (total chromatographic run time was 30 minutes). The injection volume was 5 μl, and the flow rate was 40 μl per minute.

Electrospray ionization (ESI)⁺ was carried out with a spray voltage of 3.80 kV, a capillary temperature of 300°C, and a capillary voltage of 3.80 kV. It had a resolution of 70,000 full widths at half maximum peak height (FWHM) at m/z 200. A total ion chromatogram (TIC) was obtained using a mass range of 50–750 m/z, a target of 3,000,000 charges, and a maximum ionization-time of 250 ms. A complete scan for positive ions (ESI⁺) was performed, and data-dependent MS/MS employed normalized collision energy.

Finally, results were processed for qualitative screening using Thermo Scientific™ Compound Discoverer™ 3.0 (Cat# OPTON-31061) with Thermo Scientific™ High-Resolution Accurate-Mass MS/MS Spectral Library (Cat# IQLAAEGABSFAPYMBHK). The analyte filter requirements were isotopic pattern matching score >70%, area (max.) >10⁶, and mass deviation for each ion less than 2.5 ppm between observed and theoretical mass. [(Theoretical mass–observed mass)/observed mass] × 10⁶ was defined as mass deviation ∆m.

The sample was prepared in the following manner. Ethanol (900 μl) was used to dilute 100 μl of the extract (a dark green paste). The material was vortexed for 1 minute using Thermo Scientific™ Basic Vortex Mixers (Cat# 88882012) before being spun down (6000 rpm, RT, 2 minutes) with Thermo Scientific™ Sorvall™ ST 8 Small Benchtop Centrifuge (Cat# 75007205). The supernatant was collected and placed into a vial after being filtered using a 0.22 μm syringe filter. The sample was placed in the autosampler before it was injected into the LC-HRMS system.

Experimental animals

Healthy virgin female Wistar rats aged 10–12 weeks (160–200 g) and male Wistar rats (200–250 g) were acquired from the Faculty of Pharmacy, Universitas Gadjah Mada, Indonesia. For a week, rats were subjected to laboratory settings. Rats were kept in clean plastic cages in groups, with ad libitum access to standard feed and tap water and a 12-hour light/dark cycle at a temperature of 25±2°C.

The sample size was determined a priori based on the formula from Steel and Torrie (1995): (t-1) (r-1)≥15, where t is the number of groups and r is the number of replications in each group. The minimum number of replications was 4.75 (rounded to 5). In each group, 1 rat was added as a backup in case a rat died or gave birth before the termination time. So, the number of pregnant rats needed for the 5 groups was 30. In this study, no rats died or gave birth before termination time.

The inclusion and exclusion criteria were established a priori. The inclusion criteria were healthy pregnant Wistar rats aged 10–12 weeks (160–200 g). Physically healthy rats were characterized by active movement, clear eyes, shiny white fur, no hair loss, and no diarrhea. While the exclusion criteria were rats who died or gave birth before termination time.

Timed pregnant rats were obtained by estrus synchronization with Sardjono et al. (2019) methods with modifications. The females were housed together and separated from males for two weeks to become anestrus (the Lee-Boot effect). Females were then exposed to male urine (dirty bedding from the male’s cage) for 72 h to induce estrus synchronization (the Whitten effect). Vaginal swab and histological analysis with Giemsa staining were conducted to determine the estrus phase. An estrus female was mated overnight with a male at 1:1. Females with a vaginal plug in the morning after cohabitation were considered pregnant on day 0 (G0; term, G21–23) and were used in this study. The timed pregnancy rate was 70%.²⁰

The randomization was performed on a web-based Research Randomizer. A total of 30 pregnant rats were randomly divided into five groups (n=6). A sham group (sham) received an abdominal subcutaneous injection of phosphate-buffered saline (PBS, bioWORLD Cat# 41620016-2) on G9–G18. A preeclampsia group (PE) received an abdominal subcutaneous injection of L-NAME (Cayman Chemical Cat# 80210) at a dose of 90 mg/kg body weight (BW)/day on G9–G11, then 75 mg/kg BW/day on G12–G18. Treatment groups were preeclampsia rats orally administered with Physalis angulata leaf ethanol extract at doses of 500 (D1), 1500 (D2), or 2500 mg/kg BW/day (D3) on G12–G18. The doses of the extract were determined according to our previous study.¹⁶

Blood pressure measurement

Conscious rats were held in a heated plastic chamber, and their systolic and diastolic blood pressures were measured non-invasively using rat tail-cuff blood pressure device. The CODA^® High Throughput System with 2 activated channels (Kent Scientific Corp. Cat# CODA-HT2) was employed.²¹ Blood pressures were monitored in the morning on G7 (before L-NAME injection), G12, G15, and G19.

Urine protein measurement

Total protein level was measured in 24-h urine samples on G12 and G18. Urine sampling was performed non-invasively by placing the rats in metabolic cages with urine and stool separators. Rats were fasted to avoid contaminating the collected urine. The collected urine was centrifuged (3500 rpm, 4°C, 10 min). Before analysis, the supernatant was decanted, labeled, and stored at −40°C. The total urine protein content was evaluated by measuring absorbance at 280 nm with a Thermo Scientific™ NanoDrop™ 1000 spectrophotometer (Cat# ND-1000).²²

Specimen collection

Rats were sacrificed on G19 by terminal anesthetization with ketamine at 40 mg/kg BW intraperitoneally. Maternal blood was collected by cardiac puncture. The blood (2 mL) was put into a tube containing ethylenediaminetetraacetic acid (EDTA). The residual blood volume was centrifuged (4500 rpm, RT, 10 min) to obtain the serum, then labeled and stored at −40°C until analysis.

Laparotomy was performed. The uterus was extracted in toto and opened at the anti-mesometrial side. Thus, the placenta remained attached to the uterus. The tail arteries were also collected. The tissues were then fixed in a 10% neutral-buffered formalin solution (pH 7.2, Sigma-Aldrich Cat# HT501128) for 24 h and paraffin-embedded as per the standard procedure.²³ Some placentas were gently perfused with cold PBS before being kept at −40°C until analysis.

Enzyme-linked immunosorbent assay

Serum sFlt-1 levels were determined in duplicate using a rat VEGFR1/Flt-1 ELISA kit (Elabscience Biotechnology Cat# E-EL-R0911). The protocol was carried out following the manufacturer’s instructions.

Colorimetry

Serum NO levels (Cat# E-BC-K035-M), serum and placental malondialdehyde (MDA) levels (Cat# E-BC-K025-M), and serum and placental superoxide dismutase (SOD) activities (Cat# E-BC-K020-M) were measured in duplicate using colorimetric assay kits from Elabscience Biotechnology. The protocol was performed according to the manufacturer’s instructions.

Immunohistochemistry

The formalin-fixed, paraffin-embedded (FPPE) placenta was sectioned (3 μm thick) parallel to the mesometrial-fetal axis. The FPPE tail artery was also sectioned. After dewaxing, hydrating, and inhibiting endogenous peroxidase activity, sections were treated with the primary antibody overnight at 4°C. Mouse monoclonal IgG_2a κ nitric oxide synthase 3 (NOS3) antibody (A-9) (Cat# sc-376751, RRID: AB_2832203) and IgG₁ κ hypoxia-inducible factor (HIF)-1α antibody (28b) (Cat# sc-13515, RRID: AB_627723) were employed as primary antibodies. All from Santa Cruz Biotechnology. According to the manufacturer’s instructions, immunoreactivity was detected using N-Histofin^® Simple Stain MAX PO (MULTI) (Nichirei Biosciences Cat# 414151F).

Stained sections were examined under the microscope (Olympus BX51 Microscope). The sections were then photographed in brightfield imaging mode using Olympus whole-slide scanner (Olympus dotSlide Virtual Microscopy System with Canon Powershot SX10IS Digital Camera) operated with OlyVIA V2.4 (Build 9003) software (Olympus Soft Imaging Solutions GmbH).²⁴

Tail artery eNOS expression was observed throughout the tunica intima, while placental eNOS and HIF-1α expressions were observed in five high-power fields in the basal zone. The analysis was carried out with ImageJ for Windows software (an open-source Java image processing program). The percentage (%) area of the tail artery tunica intima expressing eNOS (brown-stained cells) was calculated compared to the total area of tunica intima. The optical density of eNOS expression in the placental basal zone was measured in arbitrary units. The number of trophoblast giant cells in the placental basal zone expressing HIF-1α was also counted.

Circulating EPC isolation and flow cytometry

According to our previous study, circulating EPCs were isolated with modifications.¹⁶ Briefly, mononuclear cells (MNCs) were isolated by Lymphoprep™ 1.077 g/mL (Alere Technologies AS Cat# AXS-1114545) density gradient centrifugation (1600 rpm, RT, 30 min), then washed three times with PBS. Isolated MNCs were then stained with mouse monoclonal IgG₁ κ CD34 antibody (ICO115) conjugated to phycoerythrin (Cat# sc-7324, RRID: AB_2291280), IgG₁ λ Flk-1/KDR/VEGFR2 antibody (D-8) conjugated to fluorescein isothiocyanate (Cat# sc-393163, RRID: AB_2920761), and IgG₁ κ CD45 antibody (35-Z6) conjugated to phycoerythrin-cyanine5 (Cat# sc-1178, RRID: AB_627074) (all from Santa Cruz Biotechnology). EPCs numbers were analyzed using BD FACSCalibur™ Flow Cytometer from BD Biosciences (Cat# 342973).

Statistical analysis

GraphPad Prism 9.3.1 for Windows (GraphPad Software Inc., San Diego, CA, USA) was used to perform all statistical analyses. Free alternatives such as SOFA or JASP Statistics could be used. The data is displayed as mean±standard deviation (SD). The differences in blood pressures and total urine protein levels between groups were examined using a two-way analysis of variance (ANOVA) and Tukey’s multiple comparisons test. One-way ANOVA followed by Tukey’s multiple comparisons test was used to compare group differences in the other variables. A p-value <0.05 was considered statistically significant. Before we looked at the data, we had determined the analytical stages.

Results

Phytochemical analysis

As shown in Table 1, compounds observed in Physalis angulata leaf ethanol extract were various amino acids, peptides and derivatives, alkaloids, phenolics, nucleobases and derivatives, fatty acids and derivatives, organic acids, steroids, and miscellaneous. Among the identified alkaloids were trigonelline and DL-stachydrine. While among the identified phenolics were kaempferol, rutin, quercetin, and chlorogenic acid. The steroid detected was withanolide A.¹⁹

Table 1. Compounds detected in Physalis angulata leaf ethanol extracted by LC-HRMS.

Chemical Family		Analyte	RT [min]	Formula	Theoretical Mass [M + H]⁺	Observed Mass [M + H]⁺	∆m (ppm)	Area (Max.)
Amino acids, peptides, and derivatives		Choline	1.053	C₅H₁₃NO	103.099714	103.09974	-0.3	616,797,316.57
		Betaine	0.948	C₅H₁₁NO₂	117.078979	117.07889	0.8	226,806,761.10
		Valine	1.328	C₅H₁₁NO₂	117.078979	117.07892	0.5	81,526,589.52
		L-Norleucine	1.345	C₆H₁₃NO₂	131.094629	131.09438	1.9	48,881,450.64
		2-Hydroxyphenyl-alanine	1.476	C₉H₁₁NO₃	181.073894	181.07367	1.2	25,542,530.11
		L-Phenylalanine	1.483	C₉H₁₁NO₂	165.078979	165.07871	1.6	22,476,555.69
		N6,N6,N6-Trimethyl-L-lysine	1.233	C₉H₂₀N₂O₂	188.152478	188.15220	1.5	22,389,817.55
		L-Pyroglutamic acid	1.452	C₅H₇NO₃	129.042594	129.04247	1.0	15,771,178.50
		Proline	1.669	C₅H₉NO₂	115.063329	115.06328	0.4	14,605,229.00
		Nicotinic acid	1.430	C₆H₅NO₂	123.032029	123.03194	0.7	10,913,391.35
		Pipecolic acid	1.338	C₆H₁₁NO₂	129.078979	129.07879	1.5	8,610,852.51
		L-Glutamic acid	0.842	C₅H₉NO₄	147.053159	147.05296	1.4	8,428,291.33
		Isoleucine	0.999	C₆H₁₃NO₂	131.094629	131.09438	1.9	7,726,199.80
		Leucylproline	1.517	C₁₁H₂₀N₂O₃	228.147393	228.14699	1.8	5,540,961.06
		N-Acetyl-L-leucine	5.707	C₈H₁₅NO₃	173.105193	173.10506	0.8	3,489,258.34
		Kynurenic acid	4.454	C₁₀H₇NO₃	189.042594	189.04242	0.9	1,275,019.10
Alkaloids	β-Carbolines	2,3,4,9-Tetrahydro-1H-β-carboline-3-carboxylic acid	5.272	C₁₂H₁₂N₂O₂	216.089878	216.08953	1.6	69,503,822.51
	β-Carbolines	Norharman	6.348	C₁₁H₈N₂	168.068748	168.06854	1.2	2,383,323.13
	Tropanes	Tropine	1.476	C₈H₁₅NO	141.115364	141.11508	2.0	44,001,566.81
	Pyridines	Trigonelline	0.968	C₇H₇NO₂	137.047679	137.04743	1.8	22,244,006.33
	Pyrrolidines	DL-Stachydrine	0.962	C₇H₁₃NO₂	143.094629	143.09435	1.9	20,384,634.55
	Quinolines	6-Methylquinoline	5.271	C₁₀H₉N	143.073499	143.07324	1.8	18,254,920.68
	Purines	Caffeine	5.005	C₈H₁₀N₄O₂	194.080376	194.08025	0.6	11,090,480.45
	Piperidines	4-Piperidone	1.692	C₅H₉NO	99.068414	99.06857	-1.6	8,899,957.00
	β-Phenyl-ethylamines	N-Acetyltyramine	4.837	C₁₀H₁₃NO₂	179.094629	179.09451	0.7	2,498,298.21
Phenolics	Coumarins	7-Hydroxy-coumarine	3.210	C₉H₆O₃	162.031695	162.03150	1.2	13,406,442.09
	Flavonols	Kaempferol-3-O-β-glucopyranosyl-7-O-α-rhamno-pyranoside	7.561	C₂₇H₃₀O₁₅	594.158475	594.15819	0.5	12,234,273.36
		Rutin	7.224	C₂₇H₃₀O₁₆	610.153390	610.15300	0.6	8,121,030.26
		Kaempferol	7.561	C₁₅H₁₀O₆	286.047740	286.04714	2.1	2,370,680.02
		Quercetin	7.222	C₁₅H₁₀O₇	302.042654	302.04198	2.2	1,158,392.56
	Phenolic acids	Chlorogenic acid	5.101	C₁₆H₁₈O₉	354.095085	354.09471	1.1	14,892,726.50
		Caffeic acid	5.463	C₉H₈O₄	180.042260	180.04206	1.1	6,034,960.35
		Ferulic acid	7.321	C₁₀H₁₀O₄	194.057910	194.05781	0.5	2,923,936.08
		4-Coumaric acid	6.797	C₉H₈O₃	164.047345	164.04713	1.3	2,897,255.73
Nucleobases and derivatives		Adenine	1.365	C₅H₅N₅	135.054495	135.05422	2.0	12,630,152.91
		Adenosine	1.465	C₁₀H₁₃N₅O₄	267.096755	267.09624	1.9	11,394,283.55
		Uracil	1.435	C₄H₄N₂O₂	112.027278	112.02729	-0.1	4,989,613.51
		Guanine	1.467	C₅H₅N₅O	151.049410	151.04931	0.7	4,548,778.84
		Cytidine	1.376	C₉H₁₃N₃O₅	243.085522	243.08505	1.9	1,809,831.46
Fatty acids and derivatives		9S,13R-12-Oxophytodienoic acid	10.893	C₁₈H₂₈O₃	292.203845	292.20339	1.6	4,175,846.27
		α-Eleostearic acid	17.378	C₁₈H₃₀O₂	278.22458	278.22417	1.5	3,660,741.08
		Oleamide	22.325	C₁₈H₃₅NO	281.271864	281.27123	2.3	3,350,576.89
Organic acids		Indole-3-acrylic acid	3.901	C₁₁H₉NO₂	187.063329	187.06308	1.3	26,130,838.47
Organic acids		(3R,5R)-1,3,5-Trihydroxy-4-{[(2E)-3-(4-hydroxy-3-methoxyphenyl)-2-propenoyl]oxy} cyclohexane-carboxylic acid	6.311	C₁₇H₂₀O₉	368.110735	368.11031	1.2	1,420,786.28
Steroids		Withanolide A	12.441	C₂₈H₃₈O₆	470.266840	470.26628	1.2	1,709,017.92
Miscellaneous		(+/-)-C75	5.017	C₁₄H₂₂O₄	254.151810	254.15148	1.3	3,548,537.83
		Pyridoxine	1.454	C₈H₁₁NO₃	169.073894	169.07359	1.8	3,176,125.50
		4-Indole-carbaldehyde	7.935	C₉H₇NO	145.052764	145.05263	0.9	2,835,961.24
		Heptanophenone	14.411	C₁₃H₁₈O	190.135765	190.13556	1.1	2,124,974.85
		Shogaol	13.617	C₁₇H₂₄O₃	276.172545	276.17208	1.7	1,868,740.87
		3-Hydroxy-3,5,5-trimethyl-4-(3-oxo-1-buten-1-ylidene) cyclohexyl β-D-glucopyranoside	5.595	C₁₉H₃₀O₈	386.194070	386.19359	1.2	1,434,043.83
		Sedanolide	10.952	C₁₂H₁₈O₂	194.130679	194.13040	1.4	1,264,413.83

Blood pressure

As presented in Figure 1, compared to the sham group, the injection of L-NAME starting on G9 significantly (p<0.05) increased systolic and diastolic blood pressures on G12–G19 in the preeclampsia group. Compared to the preeclampsia group, Physalis angulata leaf extract treatment at 1500 and 2500 mg/kg BW significantly (p<0.05) reduced systolic and diastolic blood pressures on G15. Furthermore, extract treatment at 500, 1500, and 2500 mg/kg BW significantly (p<0.05) lowered systolic and diastolic blood pressures on G19, approaching the sham level (p>0.8).

Figure 1. Blood pressure.

The panels show the mean±SD of (A) systolic and (B) diastolic blood pressures on G7, G12, G15, and G19 for each group (n=6). **p<0.01 vs. Sham; ***p<0.001 vs. Sham; ^#p<0.05 vs. PE; ^##p<0.01 vs. PE; ^###p<0.001 vs. PE; ^####p<0.0001 vs. PE; D1–D3, preeclampsia rats treated with Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW, respectively; G, gestational day; PE, preeclampsia.

Total urine protein levels

The injection of L-NAME starting on G9 significantly (p<0.0001) elevated total urine protein levels on G12–G18 in the preeclampsia group compared to the sham group. Physalis angulata leaf extract administration at 500, 1500, and 2500 mg/kg BW significantly (p<0.05) reduced total urine protein levels on G18 compared to the preeclampsia group (Figure 2).

Figure 2. Total urine protein levels.

The 24-h total urine protein levels on G12 and G18 for each group (n=6) are presented as mean±SD. ****p<0.0001 vs. Sham; ^#p<0.05 vs. PE; ^##p<0.01 vs. PE; ^####p<0.0001 vs. PE; D1–D3, preeclampsia rats treated with Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW, respectively; G, gestational day; PE, preeclampsia.

Serum sFlt-1 levels

As presented in Figure 3, the injection of L-NAME significantly (p<0.0001) elevated serum sFlt-1 levels compared to the sham group. Physalis angulata leaf extract administration at 500, 1500, and 2500 mg/kg BW significantly (p<0.01) reduced serum sFlt-1 levels compared to the preeclampsia group.

Figure 3. Serum sFlt-1 levels.

The serum sFlt-1 levels on G19 for each group (n=6) are presented as mean±SD. **p<0.01; ***p<0.001; ****p<0.0001; D1–D3, preeclampsia rats treated with Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW, respectively; PE, preeclampsia; sFlt-1, soluble fms-like tyrosine kinase.

Serum and placental MDA levels

The injection of L-NAME significantly (p<0.01) raised serum and placental MDA levels compared to the sham group, as seen in Figure 4A and B. Physalis angulata leaf extract treatment at 1500 and 2500 mg/kg BW dose-dependently reduced (p<0.01) serum and placental MDA levels compared to the preeclampsia group.

Figure 4. Oxidative stress markers.

The serum and placental MDA levels and SOD activities on G19 for each group (n=6) are presented as mean±SD. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; D1–D3, preeclampsia rats treated with Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW, respectively; G, gestational day; PE, preeclampsia; MDA, malondialdehyde; SOD, superoxide dismutase.

Serum and placental SOD activities

L-NAME injection significantly (p<0.0001) lowered serum and placental SOD activities compared to the sham group. Compared to the preeclampsia group, treatment of Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW significantly (p<0.01) increased serum and placental SOD activities (Figure 4C and D).

Serum NO levels

As presented in Figure 5A, the injection of L-NAME significantly (p<0.001) decreased serum NO level compared to the sham group. Physalis angulata leaf extract administration at 500, 1500, and 2500 mg/kg BW significantly (p<0.01) increased serum NO levels compared to the preeclampsia group.

Figure 5. NO levels and eNOS expression levels.

(A) The serum NO levels, (B) tail artery and (C) placental eNOS expression levels on G19 for each group (n=6) are presented as mean±SD. (D) The representative micrographs of the tail artery and (E) placental eNOS expression from five independent experiments in which the same results were obtained are shown. (A) The serum NO level was determined by colorimetry. (B) Tail artery eNOS expression was evaluated in tunica intima by measuring the percentage of eNOS-positive area relative to total area. (C) Placental eNOS expression was evaluated in the basal zone by measuring the optical density (OD, arbitrary unit). (D) The nucleus of an eNOS-positive endothelial cell is brown, marked with a red arrow. Endothelial cell detachment is marked with a black arrow. Magnification: 200×. (E) The cytoplasm of an eNOS-positive cell is brown, marked with a red arrow. Magnification: 200×. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; D1–D3, preeclampsia rats treated with Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW, respectively; G, gestational day; PE, preeclampsia; NO, nitric oxide; eNOS, endothelial nitric oxide synthase.

Tail artery eNOS expression

Tail artery endothelial nitric oxide synthase (eNOS) expression was evaluated in tunica intima by measuring the percentage of eNOS-positive area relative to total area. Figure 5B and D show that the injection of L-NAME significantly (p<0.0001) reduced the tail artery eNOS expression compared to the sham group. Physalis angulata leaf extract administration at 1500 and 2500 mg/kg BW dose-dependently elevated (p<0.05) the tail artery eNOS expression compared to the preeclampsia group. There was endothelial cell detachment in the tail artery of the preeclampsia group, and it was repaired by the administration of the extract (Figure 5D).

Placental eNOS expression

Placental eNOS expression was evaluated in the basal zone by measuring the optical density (arbitrary unit). Figure 5C and E show that the injection of L-NAME significantly (p<0.0001) decreased placental eNOS expression compared to the sham group. Physalis angulata leaf extract administration at 500, 1500, and 2500 mg/kg BW dose-dependently increased (p<0.5) placental eNOS expression compared to the preeclampsia group.

Placental HIF-1α expression

Placental HIF-1α expression was evaluated in the basal zone by counting the brown-stained nuclei of trophoblast giant cells. As shown in Figure 6, the injection of L-NAME significantly (p<0.001) raised placental HIF-1α expression compared to the sham group. Physalis angulata leaf extract administration at 500, 1500, and 2500 mg/kg BW significantly (p<0.001) lowered placental HIF-1α expression compared to the preeclampsia group.

Figure 6. Placental HIF-1α expression levels.

(A) Placental HIF-1α expression was evaluated in the basal zone by counting the brown-stained nuclei of trophoblast giant cells. The HIF-1α-positive trophoblast giant cells number on G19 for each group (n=6) are presented as mean±SD. (B) The representative micrographs of placental HIF-1α expression (brown-stained nuclei, red arrows) from five independent experiments in which the same results were obtained are shown. Magnification: 200×. ***p<0.001; ****p<0.0001; D1–D3, preeclampsia rats treated with Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW, respectively; G, gestational day; PE, preeclampsia; HIF-1α, hypoxia-inducible factor-1α.

Circulating EPCs

The injection of L-NAME significantly (p<0.05) increased the percentage of CACs (CD34⁺/CD309⁺/CD45⁺) compared to the sham group. Although not statistically significant (p>0.05), the injection of L-NAME also increased the percentage of circulating ECFCs (CD34⁺/CD309⁺/CD45^-) compared to the sham group. Physalis angulata leaf extract administration at 500, 1500, and 2500 mg/kg BW tended to reduce the percentages of CACs and ECFCs compared to the preeclampsia group (Figure 7).

Figure 7. Circulating endothelial progenitor cells.

(A) Percentages of CAC and (B) ECFC on G19 for each group (n=6) are presented as mean±SD. *p<0.05; D1–D3, preeclampsia rats treated with Physalis angulata leaf extract at 500, 1500, and 2500 mg/kg BW, respectively; G, gestational day; PE, preeclampsia; CAC, circulating angiogenic cells; ECFC, endothelial colony-forming cells.

Discussion

Consistent with Shu et al. (2018), our results indicated that subcutaneous injection of L-NAME starting at the ninth gestational day of pregnancy significantly elevated blood pressure and urinary protein excretion, the two symptoms typical in preeclampsia.²⁵ L-NAME is a nonselective NOS inhibitor, thereby decreasing NO production.¹⁸ In line with Motta-Mejia et al. (2017), rats injected with L-NAME had a reduced placental eNOS expression, thus diminishing NO production.²⁶ Preeclampsia rats’ placenta also had a higher MDA level, and a lower SOD activity indicated oxidative stress. Among the mechanisms involved, reduced NO bioavailability and increased oxidative stress play a critical role in placental dysfunction in preeclampsia. Increased reactive oxygen species (ROS) production inhibits the expression and function of eNOS. Scavenging of NO by ROS reduces NO bioavailability and forms the metabolite peroxynitrite (ONOO⁻). Both interfere with critical vascular signaling pathways, impairing vascular function and growth associated with poor placentation.¹⁴^,²⁷^,²⁸ This condition leads to placental hypoperfusion and hypoxic microenvironment, marked with increased placental HIF-1α expression.²⁹^,³⁰

Ischemia-reperfusion injury to the placenta causes the release of soluble mediators into the maternal circulation. Soluble fms-like tyrosine kinase (sFlt)-1, a soluble form of the vascular endothelial growth factor receptor (VEGFR)-1 generated by alternative splicing of VEGFR-1 mRNA, is one of the mediators.³¹ Overproduction of the antiangiogenic factor sFlt-1 may antagonize the vasodilatory properties of VEGF. The sFlt-1 binds free VEGF and inhibits activation of VEGFR-2 and eNOS so that NO synthesis decreases.³²^,³³ Furthermore, the NO bioavailability decreases due to rapid scavenging by ROS.²⁸ In the present study, preeclampsia rats had an increased serum sFlt-1 level, reduced tail artery eNOS expression, and lower serum NO level, indicating a systemic maternal endothelial dysfunction. Preeclampsia rats also had a higher serum MDA level, and lower serum SOD activity showed oxidative stress. These results align with previous studies.³⁴^–³⁶

The balance of stimulatory and inhibitory substances in peripheral blood may also be important in controlling the number and function of circulating EPCs, which leads to endothelial homeostasis. Its disruption may play a role in the pathophysiology of preeclampsia via endothelial dysfunction.¹⁰ There are two forms of EPCs: circulating angiogenic cells (CACs, CD34⁺/CD309⁺/CD45⁺) and endothelial colony-forming cells (ECFs, CD34⁺/CD309⁺/CD45^-).¹¹ Circulating angiogenic cells release paracrine factors that promote ECFCs’ migration, proliferation, and differentiation into mature endothelial cells at vessel formation or injuries.³⁷ There were higher percentages of CACs and ECFCs in preeclampsia rats in the present study. This condition might happen because preeclampsia causes maternal endothelial damage, which drives EPCs to mobilize from the bone marrow. However, the cells could not fulfill their normal function due to oxidative stress. A lower eNOS expression in tunica intima of the tail artery in preeclampsia rats indicated this condition. These results align with Parsanezhad et al. (2015), who used CD34, CD133, CD309, and CD45 as markers for EPCs.¹¹ In contrast, Matsubara et al. (2006) reported no difference in the number of circulating EPCs (CD34⁺/CD309⁺/CD133⁺), although the proliferation was significantly increased compared to the cells from women without preeclampsia.¹⁰ Although these studies are not easily comparable because of the different methods used, it is suggested that the EPC function is more important than the quantity.

Endothelial progenitor cells play a role in reproductive organ neovascularization for implantation and pregnancy maintenance. A decrease in the quantity and function of EPCs may lead to inadequate endothelial cell regeneration in compromised uteroplacental circulation.³⁸^–⁴⁰ As in the peripheral blood, the endothelial injuries in uteroplacental circulation may induce EPCs mobilization. However, the cells were dysfunctional due to oxidative stress, thereby not regenerating injured endothelial cells or promoting neovascularization. Thus, the placental eNOS expression was lower, and the placental HIF-1α expression was higher in preeclampsia rats.

Physalis angulata leaf ethanol extract contains active compounds. They are trigonelline, stachydrine (alkaloid), chlorogenic acid (polyphenol), quercetin, kaempferol (flavonoid), and withanolide (steroidal lactone). Many studies have revealed that these compounds have antioxidant activity.⁴¹^–⁴⁷ Quercetin improves the angiogenic function of EPCs, i.e., migration, proliferation, differentiation, and survival, by increasing the SIRT1/AMPK/Akt/eNOS,⁴⁸ ERK/eNOS,⁴⁹ and PI3K/Akt/eNOS signaling pathways.⁴⁹^,⁵⁰ Quercetin has also been shown to prevent the reduction of eNOS phosphorylation, intracellular NO, and cGMP production in EPCs.⁴⁸ In vitro study by Li et al. (2016) has revealed that withaferin A, a withanolide, dose-dependently increases VEGF secretion in endothelial cells, thereby increasing the proliferation and migration of endothelial cells.⁵¹ Many studies showed that stachydrine,⁵² chlorogenic acids,⁴⁶^,⁵³^,⁵⁴ quercetin,⁵⁵^–⁵⁷ and kaempferol ⁵⁸ improve endothelial cell function by increasing the eNOS/NO signaling pathway. Trigonelline has also increased the Ca²⁺-dependent eNOS/NO signaling pathway.⁵⁹ In this study, the administration of Physalis angulata leaf ethanol extract in preeclampsia rats was proved to reduce oxidative stress and maintain NO bioavailability. It restored EPC function in improving the damaged endothelial cells. Thus, vascular signaling pathways and vascular function and growth are restored, placentation is improved, marked with decreased placental HIF-1α expression and reduced production of the antiangiogenic factor sFlt-1. This condition leads to improved preeclampsia symptoms, i.e., reduced blood pressure and proteinuria.

Furthermore, various compounds in Physalis angulata leaf ethanol extract can help decrease blood pressure. Quercetin and kaempferol inhibit cGMP hydrolysis by the Ca²⁺-dependent phosphodiesterase.⁶⁰^,⁶¹ Kaempferol has been shown to reduce myosin light-chain kinase activity,⁶² inducing myosin light-chain dephosphorylation and triggering vasorelaxation.⁶³ Besides, chlorogenic acid and quercetin inhibit angiotensin-converting enzyme (ACE) in endothelial cells.⁶⁴ Chlorogenic acid⁶⁵ and quercetin ⁶⁶^,⁶⁷ have also been proven to improve endothelial-dependent vasodilation and reduce blood pressure in hypertensive subjects. The presumption mechanism of action of active compounds in Physalis angulata leaf extract in ameliorating the EPC and endothelial cell function is presented in Figure 8.

Figure 8. The predicted mechanism of action of active compounds in Physalis angulata leaf extract in ameliorating the EPC and EC function.

ADMA, asymmetric dimethylarginine; ADP, adenosine diphosphate; AMPK, adenosine monophosphate-activated protein kinase; CaM, calmodulin; cAMP, cyclic adenosine monophosphate; CGA, chlorogenic acid; cGMP, cyclic guanosine monophosphate; EC, endothelial cell; EDHF, endothelium-derived hyperpolarizing factor; eNOS, endothelial nitric oxide synthase; EPC, endothelial progenitor cell; GTP, guanosine-5'-triphosphate; inositol 1,4,5-trisphosphate; KAE, kaempferol; L-Arg, L-arginin; L-Cit: L-citrulline; MLC, myosin light-chain; MLCK, MLC kinase; MLCP, MLC phosphatase; NO, nitric oxide; PDE5, phosphodiesterase type 5; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PKG, protein kinase G; PLCγ, phospholipase C; QUE, quercetin; ROS, reactive oxygen species; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; sGC, soluble guanylate cyclase; SIRT1, sirtuin1; TRI, trigonelline; VEGF, vascular endothelial growth factor; VEGFR2, VEGF receptor-2; VSMC, vascular smooth muscle cell; WIT, withanolide.

The novelty of this study is that the EPCs’ involvement in preeclampsia pathogenesis has just been widely studied in the past decade. The effect of antihypertensive drugs on improving the number and function of EPCs in preeclampsia patients was also recently published by Wang et al. in 2019.⁶⁸ In addition, studies of the benefits of herbs in preeclampsia are still focused on preventive models. Until now, no studies have proven the roles of herbs in improving EPCs in preeclampsia models. So, this is the first study.

The study has limitations that need to be considered while interpreting the results. Although prolonged NO suppression by L-NAME in pregnant rats leads to many clinical manifestations of preeclampsia, this is not a physiologically accurate representation of the pathogenesis of human preeclampsia. However, this approach emphasizes the significance of NO in pregnancy.⁶⁹ Furthermore, the development of animal models that mimic the pathogenesis of preeclampsia in humans still needs to be continued to discover treatments for preeclampsia.

Conclusions

In conclusion, we suggest that the ameliorative effects of Physalis angulata leaf ethanol extract on preeclampsia symptoms may be achieved by improving EPC and endothelial cell function due to reduced antiangiogenic factor and oxidative stress and also restored eNOS/NO signaling. As a result, the extract has the potential to be used as a treatment for preeclampsia. Furthermore, our data highlight the significance of EPCs as the target for biological therapy for preeclampsia.

Data availability

Underlying data

Figshare: Data for Journal F1000: Physalis angulata Leaf Extract Ameliorates L-NAME-induced Preeclampsia Symptoms in Rats through Improved Endothelial Progenitor Cells and Endothelial Cells due to Reduced Antiangiogenic Factor and Oxidative Stress. https://doi.org/10.6084/m9.figshare.20057093.¹⁹

This project contains the following underlying data:

• Data LC-HRMS 1 (Best Match).xlsx (mzCloud best match compounds identified by LC-HRMS)
• Data LC-HRMS 2 (Best Similarity Match).xlsx (mzCloud best similarity match compounds identified by LC-HRMS)
• Serial Data Systole-Diastole-Protein Urine.xlsx (Serial data of systole and diastole blood pressures on G7, G12, G15, G19, and also 24-h total urine protein levels on G12 and G18)
• All Data Gestational Day 19.xlsx (Raw data on s-Flt1, MDA, SOD, NO, eNOS, HIF-1α, CAC, and ECFC)
• Raw images_Placental eNOS expression.zip (Raw unedited microscope images of placental eNOS expression-200x magnification)
• Raw images_Placental HIF-1a expression.zip (Raw unedited microscope images of placental HIF-1a expression-200x magnification)
• Raw images_Tail artery eNOS expression.zip (Raw unedited microscope images of tail artery eNOS expression-200x magnification)

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Reporting guidelines

Figshare: ARRIVE checklist for ‘Physalis angulata leaf extract ameliorates L-NAME-induced preeclampsia symptoms in rats through improved endothelial progenitor cells and endothelial cells due to reduced antiangiogenic factor and oxidative stress’. https://doi.org/10.6084/m9.figshare.20057093.¹⁹

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

Appreciation goes to the Animal House at the Faculty of Dentistry; Laboratory of Pharmacology, Laboratory of Physiology, Laboratory of Biochemistry and Biomolecules, Laboratory of Anatomical Pathology, Laboratory of Clinical Pathology, and Central Laboratory of Biomedicine at the Faculty of Medicine; and also, Central Laboratory of Life Science and Bioscience Institute, Brawijaya University, for all laboratory facilities. Sincere appreciation goes to Wibi Riawan, Fitri Armania, Wahyudha Ngatiril Lady, Ferrida, Mochammad Abuhari, Budi Wicaksono, Lasmijan, Ami Maghfironi, Widiastuti, Choirunil Chotimah, and Midia Lestari Wahyu Handayani for the technical assistance.

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