MMP-9/NGAL Modulation by Piper Crocatum in Early Diabetic Nephropathy (MMP-9/NGAL)

Modulation of MMP-9/NGAL Ratio in Diabetic Patients With Early Renal Dysfunction by Piper Crocatum Functional Foods

The goal of this clinical trial is to determine whether functional foods made with Piper crocatum (red betel leaf) can reduce kidney damage markers in people with diabetes who have early kidney dysfunction. Researchers aim to find out if these foods can help decrease inflammation and oxidative stress, which are known to worsen kidney problems in diabetes.

Participants are divided into two groups: one group will consume cookies containing Piper crocatum extract, while the other group will consume similar cookies without the extract (placebo). They will eat these cookies twice a day for 12 weeks.

This study will measure changes in two main kidney damage markers-MMP-9 and NGAL-before and after the intervention to see if Piper crocatum helps lower these markers and supports kidney health.

Study Overview

• Status: Completed
• Conditions: Diabetic Nephropathy; Early Renal Dysfunction in Diabetes; Inflammation and Oxidative Stress in Diabetic Patients
• Intervention / Treatment: Dietary supplement: Piper crocatum Extract Cookie; Dietary supplement: Placebo Comparator

Detailed Description

This clinical trial investigates the potential of Piper crocatum-based functional foods to modulate renal biomarkers in diabetic patients with early renal dysfunction. Diabetic nephropathy, a common and severe complication of diabetes, is largely driven by chronic inflammation and oxidative stress. These conditions exacerbate renal damage and increase the risk of end-stage renal disease (ESRD). Current therapeutic approaches often fail to address these underlying inflammatory and oxidative pathways adequately, creating a need for complementary or alternative interventions.

Study Design and Intervention This study is a double-blind, randomized, placebo-controlled trial conducted in Indonesia. A total of 387 diabetic participants with early signs of renal dysfunction were recruited, and participants were randomly assigned to one of two groups: an intervention group and a placebo group. The intervention group receives cookies containing standardized extracts of Piper crocatum, a plant known for its high antioxidant and anti-inflammatory content, particularly flavonoids and glycoside polyphenols. The placebo group receives cookies identical in taste and appearance but without the active Piper crocatum ingredients.

Participants in both groups are instructed to consume two cookies per day for 12 weeks. The active cookies are carefully formulated to maintain bioactive integrity during production, ensuring that the concentration of Piper crocatum's key compounds is consistent and effective. The formulation includes 10% of an ethyl acetate fraction of Piper crocatum extract, chosen for its high antioxidant activity.

Objectives and Hypotheses The primary objective of this trial is to assess whether the consumption of Piper crocatum cookies reduces levels of matrix metalloproteinase-9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL) in diabetic patients with early renal dysfunction. MMP-9, an enzyme involved in extracellular matrix (ECM) remodeling, is a known marker for renal inflammation and fibrosis in diabetic nephropathy. NGAL, a marker of tubular injury, provides an indicator of oxidative stress and renal health. The secondary objective is to examine whether the MMP-9/NGAL ratio improves with Piper crocatum intervention, indicating a potential protective effect on both glomerular and tubular health.

Mechanism of Action and Prior Evidence The therapeutic action of Piper crocatum is hypothesized to involve the modulation of oxidative stress and inflammation through several key pathways. Flavonoids and glycoside polyphenols in Piper crocatum have demonstrated strong antioxidative properties, capable of scavenging free radicals and stabilizing cellular structures. These compounds also appear to influence regulatory proteins, including p53, SOD1, and E-cadherin, which play roles in cellular repair, apoptosis, and oxidative defense.

Recent studies have shown that Piper crocatum extracts can downregulate MMP-9 expression, thus preventing excessive ECM degradation and promoting tissue stability. Additionally, by reducing NGAL levels, Piper crocatum may protect against tubular injury, potentially lowering the risk of nephropathy progression. The current study expands on these findings by exploring Piper crocatum's effects specifically within a diabetic population with early renal dysfunction.

Biomarker Analysis Biomarker measurements for MMP-9 and NGAL will be conducted at baseline (Day 0) and after the 12-week intervention (Day 84). Blood samples will be collected following an 8-hour fasting period, and serum levels of MMP-9 and NGAL will be quantified using validated ELISA kits. These biomarker measurements provide a direct assessment of the intervention's impact on key pathways of renal injury and repair.

Statistical Methods The analysis will use both paired and independent statistical tests to compare biomarker levels between and within groups. The primary analysis will involve comparing the mean change in MMP-9, NGAL, and the MMP-9/NGAL ratio between the intervention and placebo groups. Effect sizes will be calculated to determine the magnitude of change associated with the Piper crocatum intervention. All statistical analyses will use a significance threshold of p < 0.05.

Expected Outcomes and Clinical Implications The anticipated outcome is a significant reduction in MMP-9, NGAL, and the MMP-9/NGAL ratio in the intervention group compared to the placebo group, indicating a potential protective effect on renal health. If successful, this study could establish Piper crocatum-based functional foods as a novel adjunct therapy for diabetic nephropathy, offering a dietary approach to mitigate the progression of renal dysfunction in diabetic populations.

By providing insights into a natural, bioactive intervention, this study may open new avenues for integrating functional foods into diabetic care to improve outcomes and enhance quality of life for patients at risk of advanced renal complications.

• Study Type: Interventional
• Enrollment (Actual): 387
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Indonesia
  • South Sulawesi
    • Makassar, South Sulawesi, Indonesia, 90245
      • Public Health Center in Makassar, Indonesia

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

• Adults aged over 20 years.
• Diagnosed with Diabetes Mellitus (DM).
• Serum urea levels above 43 mg/dL or serum creatinine levels above 0.90 mg/dL.

Exclusion Criteria:

• Pregnant or breastfeeding individuals.
• Patients with other medical conditions that require intensive care.
• Patients currently undergoing therapy for hypoalbuminemia.
• Known allergies to any ingredients in the study cookies.
• Other chronic conditions that might interfere with the study protocol (e.g., severe cardiovascular disease).

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Quadruple

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Piper crocatum Cookie Group; Participants in this group will receive bioactive-rich cookies containing Piper crocatum extract, known for its antioxidant and anti-inflammatory properties. They will consume two cookies daily for 12 weeks, each formulated with a standardized 10% concentration of the ethyl acetate fraction of Piper crocatum extract. The intervention aims to modulate renal biomarkers associated with diabetic nephropathy	Dietary supplement: Piper crocatum Extract Cookie; Participants in the intervention group will receive cookies containing a standardized 10% ethyl acetate extract of Piper crocatum, known for its antioxidant and anti-inflammatory properties. These cookies will be consumed twice daily for 12 weeks. The intervention aims to assess the effects of Piper crocatum on renal biomarkers, such as MMP-9 and NGAL, in diabetic patients with early renal dysfunction
Placebo Comparator: Placebo Cookie Group; Participants in this group will receive placebo cookies that are identical in appearance, taste, and texture to the Piper crocatum cookies but do not contain the active Piper crocatum extract. They will consume two cookies daily for 12 weeks. This group serves as a comparison to evaluate the effects of the Piper crocatum extract in the intervention group	Dietary supplement: Placebo Comparator; Participants in the placebo group will receive cookies that are identical in appearance, taste, and texture to the active Piper crocatum cookies but do not contain the Piper crocatum extract. These cookies will be consumed twice daily for 12 weeks, serving as a control to assess the effects of the active intervention on renal biomarkers associated with diabetic nephropathy.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Change in MMP-9 and NGAL Levels in Diabetic Patients with Early Renal Dysfunction	This measure evaluates the change in serum levels of matrix metalloproteinase-9 (MMP-9) and neutrophil gelatinase-associated lipocalin (NGAL) in diabetic patients with early renal dysfunction after 12 weeks of intervention. MMP-9 is associated with renal inflammation and extracellular matrix remodeling, while NGAL is a marker of tubular injury and oxidative stress. Blood samples will be collected at baseline and at the end of the 12-week period, and serum levels of MMP-9 and NGAL will be quantified using validated ELISA kits. This assessment aims to determine the potential effects of the Piper crocatum intervention on renal health by observing significant changes in these biomarkers	Baseline and 12 weeks

Collaborators and Investigators

• Sponsor: Hasanuddin University

Publications and helpful links

General Publications

• Serra R, Grande R, Butrico L, Buffone G, Calio FG, Squillace A, Rizzo BA, Massara M, Spinelli F, Ferrarese AG, de Caridi G, Gallelli L, de Franciscis S. Effects of a new nutraceutical substance on clinical and molecular parameters in patients with chronic venous ulceration. Int Wound J. 2016 Feb;13(1):88-96. doi: 10.1111/iwj.12240. Epub 2014 Feb 25.
• Rojas-Quintero J, Wang X, Tipper J, Burkett PR, Zuniga J, Ashtekar AR, Polverino F, Rout A, Yambayev I, Hernandez C, Jimenez L, Ramirez G, Harrod KS, Owen CA. Matrix metalloproteinase-9 deficiency protects mice from severe influenza A viral infection. JCI Insight. 2018 Dec 20;3(24):e99022. doi: 10.1172/jci.insight.99022.
• Rodriguez-Sanchez E, Navarro-Garcia JA, Aceves-Ripoll J, Abarca-Zabalia J, Susmozas-Sanchez A, Bada-Bosch T, Hernandez E, Merida-Herrero E, Andres A, Praga M, Fernandez-Ruiz M, Aguado JM, Segura J, Ruilope LM, Ruiz-Hurtado G. Variations in Circulating Active MMP-9 Levels During Renal Replacement Therapy. Biomolecules. 2020 Mar 26;10(4):505. doi: 10.3390/biom10040505.
• Latronico T, Petraglia T, Sileo C, Bilancia D, Rossano R, Liuzzi GM. Inhibition of MMP-2 and MMP-9 by Dietary Antioxidants in THP-1 Macrophages and Sera from Patients with Breast Cancer. Molecules. 2024 Apr 10;29(8):1718. doi: 10.3390/molecules29081718.
• Kumar M, Dev S, Khalid MU, Siddenthi SM, Noman M, John C, Akubuiro C, Haider A, Rani R, Kashif M, Varrassi G, Khatri M, Kumar S, Mohamad T. The Bidirectional Link Between Diabetes and Kidney Disease: Mechanisms and Management. Cureus. 2023 Sep 20;15(9):e45615. doi: 10.7759/cureus.45615. eCollection 2023 Sep.
• Jones JI, Nguyen TT, Peng Z, Chang M. Targeting MMP-9 in Diabetic Foot Ulcers. Pharmaceuticals (Basel). 2019 May 22;12(2):79. doi: 10.3390/ph12020079.
• Setyawati A, Wahyuningsih MSH, Nugrahaningsih DAA, Effendy C, Fneish F, Fortwengel G. Piper crocatum Ruiz & Pav. ameliorates wound healing through p53, E-cadherin and SOD1 pathways on wounded hyperglycemia fibroblasts. Saudi J Biol Sci. 2021 Dec;28(12):7257-7268. doi: 10.1016/j.sjbs.2021.08.039. Epub 2021 Aug 19.
• Setyawati A, Saleh A, Tahir T, Yusuf S, Syahrul S, Aminuddin A, Raihan M, Jafar N, Hamzah H, Arfian N. Matrix Metalloproteinase-9 Testing of Golden Rice Cookies With Piper Crocatum Active Extract for Preventing Foot Ulcers in Patients With Diabetes: Protocol for a Randomized Controlled Trial. JMIR Res Protoc. 2024 Feb 29;13:e49940. doi: 10.2196/49940.
• Papadopoulou-Marketou N, Whiss PA, Eriksson AC, Hyllienmark L, Papassotiriou I, Wahlberg J. Plasma levels of tissue inhibitor of metalloproteinase-1 in patients with type 1 diabetes mellitus associate with early diabetic neuropathy and nephropathy. Diab Vasc Dis Res. 2021 Mar-Apr;18(2):14791641211002470. doi: 10.1177/14791641211002470.
• Pan SC, Li CY, Kuo CY, Kuo YZ, Fang WY, Huang YH, Hsieh TC, Kao HY, Kuo Y, Kang YR, Tsai WC, Tsai ST, Wu LW. The p53-S100A2 Positive Feedback Loop Negatively Regulates Epithelialization in Cutaneous Wound Healing. Sci Rep. 2018 Apr 3;8(1):5458. doi: 10.1038/s41598-018-23697-5.
• Nevo A, Armaly Z, Abd El Kadir A, Douvdevani A, Tovbin D. Elevated Neutrophil Gelatinase Lipocalin Levels Are Associated With Increased Oxidative Stress in Hemodialysis Patients. J Clin Med Res. 2018 Jun;10(6):461-465. doi: 10.14740/jocmr3360w. Epub 2018 Apr 13.
• Danquah M, Owiredu WKBA, Jnr BAE, Serwaa D, Odame Anto E, Peprah MO, Obirikorang C, Fondjo LA. Diagnostic value of neutrophil gelatinase-associated lipocalin (NGAL) as an early biomarker for detection of renal failure in hypertensives: a case-control study in a regional hospital in Ghana. BMC Nephrol. 2023 Apr 26;24(1):114. doi: 10.1186/s12882-023-03120-6.
• Bae MJ, Karadeniz F, Oh JH, Yu GH, Jang MS, Nam KH, Seo Y, Kong CS. MMP-Inhibitory Effects of Flavonoid Glycosides from Edible Medicinal Halophyte Limonium tetragonum. Evid Based Complement Alternat Med. 2017;2017:6750274. doi: 10.1155/2017/6750274. Epub 2017 Sep 20.
• Alsawaf S, Alnuaimi F, Afzal S, Thomas RM, Chelakkot AL, Ramadan WS, Hodeify R, Matar R, Merheb M, Siddiqui SS, Vazhappilly CG. Plant Flavonoids on Oxidative Stress-Mediated Kidney Inflammation. Biology (Basel). 2022 Nov 26;11(12):1717. doi: 10.3390/biology11121717.
• Kunadu AP, Aboagye EF, Colecraft EK, Otoo GE, Adjei MYB, Acquaah E, Afrifa-Anane E, Amissah JGN. Low Consumption of Indigenous Fresh Dairy Products in Ghana Attributed to Poor Hygienic Quality. J Food Prot. 2019 Feb;82(2):276-286. doi: 10.4315/0362-028X.JFP-18-146.

Helpful Links

• This study examines the effect of Piper crocatum-enriched Golden Rice cookies on diabetic foot ulcer prevention by modulating MMP-9 levels. The cookies are designed to reduce inflammation and support diabetic foot health through dietary intervention

Study record dates

Study Major Dates

• Study Start (Actual): June 15, 2024
• Primary Completion (Actual): September 5, 2024
• Study Completion (Actual): September 16, 2024

Study Registration Dates

• First Submitted: November 4, 2024
• First Submitted That Met QC Criteria: November 4, 2024
• First Posted (Actual): November 6, 2024

Study Record Updates

• Last Update Posted (Actual): November 6, 2024
• Last Update Submitted That Met QC Criteria: November 4, 2024
• Last Verified: November 1, 2024

More Information

Terms related to this study

• Keywords: Antioxidants; Diabetes Complications; Anti-inflammatory; Piper crocatum; Functional Foods; Early Renal Dysfunction; Renal Biomarkers
• Additional Relevant MeSH Terms: Urogenital Diseases; Endocrine System Diseases; Pathologic Processes; Male Urogenital Diseases; Urologic Diseases; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Diabetes Mellitus; Diabetes Complications; Inflammation; Renal Insufficiency; Kidney Diseases; Diabetic Nephropathies
• Other Study ID Numbers: PROT-2024-UH; 1220/UN4.14.1/TP.01.02/2024 (Other Grant/Funding Number: Research and Community Service (LPPM) Universitas Hasanuddin)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO
• IPD Plan Description: Individual participant data (IPD) will not be routinely shared to protect participant privacy and confidentiality. However, data may be made available to qualified researchers upon request, subject to approval by the study's principal investigator and in compliance with relevant privacy regulations and institutional policies.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No