Astuti N. P. W, Manuaba I. B. P, Jawi I. M, Putra A. A. B, Wiradana P. A, Widhiantara I. G, Permatasari A. A. A. P, Ansori A. N. M, Kharisma V. D. Phytoconstituents Analysis and Anti-Diabetic Potential of Sembung Leaf Extract (Blumea balsamifera L. DC.) through Inhibition of NF-KB p65, GLP-1, and DPP-4 Proteins with In-Silico Approaches. Biomed Pharmacol J 2024;17(2).

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Manuscript received on :08-03-2024
Manuscript accepted on :22-04-2024
Published online on: 04-06-2024

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Plagiarism Check: Yes
Reviewed by: Dr. Akhtar Ali
Second Review by: Dr. Maysaa Kadhim Al-Malkey
Final Approval by: Dr. Hans-Joachim Freisleben

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Ni Putu Widya Astuti^1,2* , Ida Bagus Putra Manuaba³, I Made Jawi⁴, Anak Agung Bawa Putra³, Putu Angga Wiradana⁵, I Gede Widhiantara⁵, Anak Agung Ayu Putri Permatasari⁵, Arif Nur Muhammad Ansori⁶, and Viol Dhea Kharisma⁷

¹Doctoral Study Program, Faculty of Medicine, Universitas Udayana, Denpasar City, Bali Province (80232), Indonesia

²Study Program of Public Health, Faculty of Health, Science, and Technology, Universitas Dhyana Pura, Jalan Raya Padangluwih, Dalung, North Kuta, Badung Regency, Bali Province (80351), Indonesia

³Department of Chemistry, Faculty of Mathematical and Natural Sciences, Universitas Udayana, Badung Regency, Bali Province (80361), Indonesia

⁴Department of Pharmacology, Faculty of Medicine, Universitas Udayana, Jalan P.B. Sudirman, Dangin Puri Klod, Denpasar City, Bali Province (80232), Indonesia

⁵Research Group of Biological Health, Study Program of Biology, Faculty of Health, Science, and Technology, Universitas Dhyana Pura, Jalan Raya Padangluwih, Dalung, North Kuta, Badung Regency, Bali Province (80351), Indonesia

⁶Postgraduate School, Universitas Airlangga, Kampus B, Jalan Airlangga, Surabaya, East Java (60286), Indonesia

⁷Study Program of Biology, Faculty of Science and Technology, Universitas Airlangga, Kampus C, Jln. Dr. Ir. H. Soekarno, Mulyorejo, Surabaya, East Java (60115), Indonesia

Corresponding Author E-mail: widyaastuti@undhirabali.ac.id

Abstract

Traditional herbal remedies have an important role in human health. Empirically, Blumea balsamifera is often used as a traditional beverage to alleviate fever symptoms, lower cholesterol levels, and maintain body immunity. The purpose of this study was to discover the phytoconstituent profile that contributes to the anti-diabetic properties of B. balsamifera leaf extract (BBLE) using in silico approaches.LCMS/MS was used to identify the constituent profile of BBLE, and the ability of these compounds against diabetes-related proteins was analyzed computationally.Three proteins related to diabetes are NF-KB p65, GLP-1, and DPP-4. A total of 18 compounds were successfully identified through LCMS/MS, including 4 compounds known to be flavonoid derivatives and can be used as markers of BBLE. Pheophorbide A and 1,1-Cyclopentanediacetic acid were reported for the first time to inhibit the NF-KB p65, GLP-1, and DPP-4 proteins in docking simulation studies. Based on these findings, it can be confirmed that the bioactive compounds in BBLE show strong inhibitory potential against anti-diabetic proteins.

Keywords

Antidiabetic activity; Blumea balsamifera; In Silico; LCMS/MS; Phytoconstituent

Introduction

Diabetes mellitus is a widespread metabolic condition in which glucose levels in the blood rise due to beta cells’ inability of producing enough insulin (Type-1) or ineffective release of insulin (Type-2). Both are caused by insulin resistance, production issues, or both, and alter the way proteins, lipids, and carbohydrates are synthesized¹. Diabetes raises the risk of amputation, renal failure, stroke, cardiovascular disease, and lifelong blindness.Diabetes is influenced by genetic and environmental variables, and the incidence rate varies among ethnic groups and specific communities². According to estimates, diabetes affected about 463 million people globally in 2019³.  

Interestingly, urban areas have a higher incidence rate of 10.8% compared to rural areas at 7.2%. Similarly, high-income nations have a greater frequency of 10.4% than nations with low incomes at 4% ³. Despite the fact that extensive research has been conducted to control diabetes and a number of oral medications, gene therapies, and stem cell therapies have been successfully implemented, the development of new diabetes medications through the search for chemical compounds with specific bioactive features that help manage glucose homeostasis and improve insulin sensitivity is necessary⁴.

Antioxidants have an important function in the treatment of disorders like diabetes mellitus ⁵. This is because the formation of free radicals in cells due to oxidative stress exposure has been linked to the occurrence of diabetes, especially Type-2⁶. Natural compounds have attracted considerable attention over the past few decades for their biological and pharmacological properties as sources of antioxidants⁷. Natural compounds are widely known to provide anti-inflammatory, anti-cancer, anti-bacterial, anti-fungal, and anti-viral activities⁸. Since they have been scientifically proven to exhibit hypoglycemic activity, these active compounds are of great interest for exploration in the development of new diabetes medications.

Indonesia is a center of mega-biodiversity for medicinal plants in the Asia region, and their traditional use is employed by many ethnic community societies across various regions in Indonesia to support their health needs⁹. The Sembung plant (Blumea balsamifera) is one of the plants used in traditional medicine practices as an anti-hypercholesterol¹⁰, anti-bacterial¹¹, anti-cancer¹², anti-neuroinflammation¹³, gastroprotectant¹⁴, and as a source of natural antioxidants¹⁵. Empirically, in the Bali Province, B. balsamifera can be processed into a health drink known as “Loloh,” which is considered capable of maintaining immune function. Previous reports revealed that the extract of B. balsamifera (BBLE) contains a profile of secondary metabolites such as flavonoids, saponins, phenols, tannins, and steroids¹⁶. Apart from Indonesia, several regions around the world also utilize plants from the Blumea species as traditional medicine. For example, in China, dried preparations of Blumea riparia have been standardized and used to treat irregular menstruation, postpartum hemorrhage, infertility, and vulva wounds. These plants are also commercialized because their phenolic compounds, flavonoids, acetylenes, and sesquiterpenes are considered to play a crucial role in health^17,18.

However, so far, reports on the metabolite profile of BBLE aimed at anti-diabetic purposes are very limited. Phytochemical screening of Blumea spp. is crucial, given their wide abundance in the wild, and the morphological similarity of the B. balsamifera species with other Blumea spp. such as B. megacephala and B. riparia¹⁹. As a result, the aim of this research is to identify the secondary metabolites of BBLE using High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS)  investigations and discover its anti-diabetic mechanism by in silico analysis. This study is valuable for developing new biologically active molecules, especially for controlling diabetes through the inhibition of related proteins.

Materials and methods

Preparation of crude extract

Fresh Sembung (B. balsamifera) leaves were collected from a plantation in Luwus Village, Tabanan Regency, Bali Province. Types of B. balsamifera L. (DC.) leaf samples were determined at the Bali Botanical Gardens, National Research and Innovation Agency (BRIN) with Sample Registration Number B. 206/IPH.7/AP/VIII/2020. The same kind of extract had been used in our previous publication¹⁶. Sembung leaves were then cleaned of organic material or contaminants using running water several times. The samples were then air-dried at room temperature to reduce moisture content and subsequently dried for 24 hours at 50℃ using an oven, resulting in dried samples or simplicia for further processing. The simplicia were then ground using a blender and sieved through a 20 mesh screen to obtain a powdered sample.

A total of 250 grams of sembung leaf powder preparation was weighed and moistened using 70% ethanol solvent, then left for 4 hours in a glass container covered with sterile gauze and wrapped in plastic. The maceration process was carried out for 24 hours, and during this process, stirring was performed to evenly extract the metabolites. The macerate was then separated by filtration method using sterile flannel cloth. The filtration process was performed three times with the same type and amount of solvent.The collected macerate was evaporated using a vacuum rotary evaporator at 40℃ and 100 rpm until a viscous extract was produced.This viscous extract of sembung, referred to as BBLE in this study, was adjusted according to the standards of the Indonesian Herbal Pharmacopoeia of 2017, which includes a yield of not less than 10.6%, flavonoid/quercetin identity compound (+), moisture content of no more than 14%, and total ash of no more than 6.7%^10,20.

Identification of the phytochemical from BBLE

The chemical profile of BBLE was identified and quantified using the pure compound as a reference by High Performance Liquid Chromatography (HPLC) (LC: ACQUITY UPLC® H-Class System, Waters, USA) and mass spectrometry (MS) (Xevo G2-S QTof, Waters, USA). Briefly, an AC18 column measures 1.8 μm 2.1 × 100 mm at 50°C (column) and 25°C (ambient temperature). HPLC analysis used a mobile phase of water + 5 mM ammonium formate and acetonitrile + 0.05% formic acid, with a flow rate of 0.2 mL/min running for 23 minutes (mobile phase) and an injection volume of 5 μL. Mass spectrometer (MS) investigations were carried out using electrospray ionization in positive mode with a mass range of 50–1200 m/z and source and dissolution temperatures of 100 and 350°C. The conical gas and dissolution flows were 0 L/h and 793 L/h, respectively, with collision energies ranging from 4 to 60 eV. MassLynx software version 4.1 was used for data collection, analysis, and instrument control^21–23.

In silico analysis

Molecular docking analysis

The phytochemical profile, consisting of 18 compounds detected by BBLE extract analysis with HPLC/MS, was used in molecular docking studies. The PubChem database was utilized to obtain compound names, PubChem IDs, molecular weights, and structures of the obtained compounds²⁴. ADMET profiling was carried out for assessing the pharmacokinetic characteristics of the compounds utilizing online pkCSM and Swiss ADME ^25–27.The 3D structures of each compound were downloaded in .sdf format, and the respective proteins were obtained from the RCSB PDB website. NF-kB p65, Glucagon-like peptide (GLP-1), and Dipeptidyl peptidase-4 (DPP-IV) were identified as potential target receptors.Analysis was performed using PyRx 0.8 software with specific coordinates corresponding to the active site of each protein. The strength of the bond between the ligand and protein was measured based on binding energy and RMSD from the molecular docking results. The smaller/negative the binding energy value, the more stable the bond between the ligand and protein.

Ligand-Protein Interaction Analysis

The types of chemical bond interactions formed in the ligand-protein complex were further analyzed using Discovery Studio software. This analysis aims to identify the position of the active site and the amino acids that bond with the compounds through hydrogen bonding.

Druglikeness and Toxicity Analysis

The druglikeness analysis of compounds in sembung leaves was conducted using the websitehttp://www.swissadme.ch/index.php by copying the Simplified Molecular Input Line Entry System (SMILES) of each compound. Subsequently, the toxicity (LD50) of the compounds was assessed using the websitehttps://pubchem.ncbi.nlm.nih.gov/compound/.

Bioactivity Analysis with PASS Online Server

The bioactivity analysis of compounds contained in sembung leaves was performed by copying the Simplified Molecular Input Line Entry System (SMILES) notation of each compound onto the website https://www.way2drug.com/PassOnline/predict.php. The Pa (probability of activity) and Pi (probability of inactivity) values were determined for each ligand. Finally, only activities involved in diabetes were considered²⁸.

Results and Discussion

Chemical profiling

The phytochemical profiling results of BBLE using HPLC/MS revealed the presence of 18 chemical compounds at various retention times (RT) (Table 1). The compound 2,3-Dihydroxypropyl (9Z,12Z,15Z) – 9,12,15-octadecatrienoate (C₂₁H₃₆O₄) with an RT of 11.61 had the highest peak area percentage of 20.89%. Similarly, the compound 2-Hexyl-3,5-dipentylpyridine (C₂₁H₃₇N) with an RT of 11.80 had a peak area percentage of 20.89%. The BBLE chromatogram in this study is shown in Figure 1 below.

Figure 1: Chromatogram of peaks from B. balsamifera leaf extract (BBLE) analyzed using LCMS/MS.   Click here to view Figure

The chemical 2,3-Dihydroxypropyl, also called Octadecatrienoic Acid, is an a link in the production of arachidonic acid and an important component of essential oils in plants ²⁹. Reports indicate that conjugated Octadecatrienoic Acid can cause a decrease in the viability of LNCaP and PV-3 (human prostate cancer cells) cells, depending on the concentration, but is not toxic to normal human prostate epithelial cells RWPE-1, which are normal epithelial cells³⁰. 

Table 1: Phytoconstituent content of B. balsamifera leaf extract (BBLE) characterized by LCMS/MS.

The compound 2-Hexyl-3,5-dipentylpyridine was also previously found in mango peel waste extract analyzed using LC-MS with a retention time (RT) of 19.266 minutes and a molecular weight of 303.29137 g/mol³¹. This compound is capable of inhibiting the ACE2 receptor, which is a membrane protein on alveolar cells acting as an entry point for viruses into the human body^32–34. The compound 2-Hexyl-3,5-dipentylpyridine was successfully identified in the extract of the tuber of Merremia mammosa with bioactivities as an antiviral, antioxidant, anti-inflammatory, and anti-tuberculosis agent³⁵.

In addition to those three compounds, there are phytochemical compounds identified in BBLE that belong to the flavonoid group, such as 3′,4′,5-Trihydroxy-6,7,8-trimethoxyflavone (TTF); 3′,5-Dihydroxy-3,4′,6,7-tetramethoxyflavone (TMF); 5-Hydroxy-3′,4′,6,7,8-pentamethoxyflavone (PMF); and 3,3′,4′,7-Tetramethylquercetin (TMQ). Compounds from the flavonoid group have been reported as standardization markers for BBLE phytochemistry and are known for their role as sources of natural antioxidants with various important bioactivities^10,36. Similar research revealed that the compound 3,5,4′-trihydroxy-6,7,3′-trimethoxyflavone (TTF) isolated from Achillea fragrantissima extract could prevent cell damage due to oxidative stress and inhibit protein phosphorylation that signals cells, including the mitogen-activated protein kinase (MAPK) family³⁷. Compounds from this flavonoid group were also found in Loranthus acutifolius extract with antityrosinase bioactivity³⁸.

The compound 3′,5-Dihydroxy-3,4′,6,7-tetramethoxyflavone (TMF), also known as Casticin, is a bioactive compound that can be found in various parts of plants. Casticin’s therapeutic properties include anti-tumor, anti-inflammation, neuroprotective activity, and natural analgesic³⁹. Casticin isolated from Larrea tridentata has shown antibacterial activity against Mycobacterium tuberculosis , including sensitive and multidrug-resistant strains⁴⁰. The compound 5′ – Hydroxy -6, 7,8,3′,4′-pentamethoxyflavone (PMF), isolated from the mandarin orange Citrus reticulata, has anti-inflammatory properties and modulates immune function. Recent studies also report the effect of this compound in preventing psoriasis, a chronic and benign proliferative skin disease, through the regulation of several gene expressions related to immunity and inflammation⁴¹.

The compound 3,3′,4′,7-Tetramethylquercetin (TMQ) is one of the parent compounds expected to be found in the phenolic hydroxyl groups within quercetin. The bioactivity demonstrated by TMQ includes acting as an anti-prostate cancer agent through the activation of apoptosis in PC-3 cells⁴². On the other hand, this quercetin derivative is capable of multi-drug resistance as well as human breast cancer cells (MCF-7) by inhibiting the activity of TrxR, which activates cell death through apoptosis⁴³. TMQ has also been reported in extracts from Cissus quadrangularis extracted with various solvents⁴⁴.

Ligand Compounds

Phytochemical compounds acting as ligands must meet inclusion criteria that fulfill both pharmacological and pharmacodynamic criteria. Based on their similarity as drug candidate materials, each compound successfully identified using HPLC/MS is used as a ligand compound in this study (Table 2).

Table 2: Samples of ligand compounds from BBLE accessed from the PubChem database and SMILES notation

The results of the molecular docking analysis of each BBLE compound against the target protein NF KB-p65 (1LE5) show that the best binding energy was obtained by the compound Pheophorbide-A, with a binding energy value of -8.1 kcal/mol and an RMSD of 0 Å. Pheophorbide A is a breakdown product of chlorophyll A, reported to be used as a photosensitizer and utilized in photodynamic therapy to reduce tumor growth^45,46. The inhibition of Pheophorbide A detected in BBLE against the target protein NF KB-p65 (1LE5) is the first report of its kind (Table 3).

Table 3: Molecular Docking analysis of the compounds contained in BBLE against NF protein KB-p65 (1LE5).

In several cell-based and animal experimental systems, it has been proven that NF-KB activation has a role in the early pathobiology of diabetes⁴⁷. NF-KB is activated by increased oxidative stress, which in diabetes patients is caused by high glucose levels and advanced glycation end products⁴⁸. Inhibition of this protein can offer new opportunities in the treatment process of diabetes played by BBLE through the compound Pheophorbide-A. The 2D interaction and 3D visualization of the Pheophorbide A compound against the NF KB-p65 protein (ILE5) are shown in Figure 2 below. 

Figure 2: 2D Interaction and 3D Visualization of the Pheophorbide A Compound with the NF KB-p65 protein (1LE5)   Click here to view Figure

The Glucagon-like peptide-1 (GLP-1) protein is one of the important incretin hormones for preventing postprandial hyperglycemia⁴⁹. The results of the molecular docking analysis of the compounds contained in BBLE against the GLP1 protein show that the best binding energy was obtained by the compound Cyclopentanediacetic acid with a binding energy value of -7.2 kcal/mol and an RMSD of 0 Å (Table 4).

 Table 4: Molecular Docking analysis of the compounds contained in BBLE against GLP 1 (5NIQ) protein.

This study is the first to report the inhibition of the GLP-1 protein by the active compound 1,1-Cyclopentanediacetic acid in an in silico manner. There are not many reports explaining the bioactivity of this compound against degenerative diseases like diabetes. However, similar compounds that are derivatives of diacetic acid have been reported to have bioactivity as anti-inflammatory agents. Interestingly, the compound regulated several pro-inflammatory cytokines in microglial BV-2 cells induced with lipopolysaccharide (LPS)⁵⁰. On a molecular level, the compound 1,1-Cyclopentanediacetic acid contained in BBLE, associated with anti-diabetic effects through the activation of GLP1 protein and its analogs, can be linked to the pancreatic GLP1R signaling that activates insulin production^51,52. Pre-clinical scale research is still needed to prove the activation of BBLE compounds on the expression of GLP-1 and GLP1R genes in the ileum of diabetic rats. The 2D interaction and 3D visualization of the compound 1,1-Cyclopentanediacetic acid binding with the GLP-1 protein are displayed in Figure 3 below.

Figure 3: 2D Interaction and 3D Visualization of Compound 1,1- Cyclopentanediacetic acid contained in BBLE with GLP 1 protein (5NIQ)   Click here to view Figure

The molecular docking analysis of the compound contained in BBLE against the DPP-IV protein (5YP1) shows the highest binding energy by the compound 1,1-Cyclopentanediacetic acid with a binding energy value of -10.5 kcal/mol and an RMSD of 0 Å (Table 5). DPPIV inhibitors have been proven to benefit various organs, including renal and cardiovascular health⁵³. DPP-IV inhibitors are widely contained in incretin-based oral hypoglycemic drugs intended for diabetes patients, which have been commercially available for nearly a decade. Several types of DPP-IV inhibitors, such as Litagliptin and Saxagliptin, are capable of controlling glycemia and reducing the risk of renal and cardiovascular complications in diabetic patients and have been approved by the US FDA ^54–56.

Table 5: Molecular docking analysis of the compound contained in BBLE with the DPP-IV protein (5YP1)

This study reveals that the compound 1,1-Cyclopentanediacetic acid from BBLE has good potential as a DPP-IV inhibitor in silico. The 2D interactions and 3D visualization of the compound 1,1-Cyclopentanediacetic acid with the DPP-IV protein are displayed in Figure 4. Several related studies have confirmed the role of herbal plant extracts and their constituents as DPP-IV inhibitors. Procyanidin compounds isolated from Vitis vinifera seed extracts were able to reduce DPP-IV levels and down-regulate its gene expression in the human intestinal cell model Caco-2.In vivo, procyanidin compounds also decrease the regulation of DPP-IV in the intestines of obese Wistar rats ⁵⁷. Similarly, the compound Emodin from the extract of Rheum palmatum L. shows inhibition against the DPP-IV enzyme in vitro and a dose-dependent decrease in DPP-IV levels in the plasma of Balb/c mice ⁵⁸. However, in vivo scale research is still needed to determine the effectiveness of the BBLE compound in reducing DPP-IV levels in diabetic rats, thereby clarifying the results from in silico studies.         

Figure 4: 2D Interaction and 3D Visualization of Compound 1,1-Cyclopentanediacetic acid with DPP IV protein (5YPI)   Click here to view Figure

Conclusion

In this study, LCMS/MS and molecular docking methods were used to analyze the phytoconstituent profile of BBLE with potential anti-diabetic properties. A total of 18 active compounds were identified in BBLE, including several that are flavonoid derivatives. Docking studies showed that Pheophorbide A could inhibit the NF KB-p65 protein (1LE5), and the compound 1,1-Cyclopentanediacetic acid inhibited the GLP 1 (5NIQ) and DPP-IV (5YP1) proteins. This study reveals the anti-diabetic effects of BBLE and further research is needed to determine the effectiveness of BBLE’s active compounds and to identify their molecular mechanisms of action on diabetic animal models.

Acknowledgement

The authors would like to thank the Institute for Research and Community Service (LPPM) at Universitas Dhyana Pura for supporting the implementation of this research. The authors also thank those who have helped carry out the research, such as students, workers at the Science and Health Laboratory, Universitas Dhyana Pura, and ASCAdemia who have provided proofreading services for this manuscript.

Conflict of Interest

The authors reported no declarations of interest.

Funding Source

This research was funded by the Institute for Research and Community Service (LPPM) Universitas Dhyana Pura through the Higher Education Excellence Research Scheme Research Funding Grant Program on 2023 with Contract Number: 011/UNDHIRA-LPPM/Lit./IX/2023.

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