Potensi Adenostemma lavenia, A. madurense, dan A. platyphyllum sebagai Antimikroba: Pendekatan Metabolomik, In silico dan In vitro, serta Optimasi Ekstraksi

Abstract

Adenostemma (legetan warak) secara tradisional digunakan sebagai ramuan obat untuk mengobati penyakit yang disebabkan oleh bakteri seperti radang paru-paru, pneumonia, demam, hepatitis, inflamasi, luka pada kulit, dan gangguan kesehatan mulut. Senyawa diterpenoid kaurena hasil isolasi A. lavenia mampu menginduksi ekspresi protein antioksidan Heme Oxygenase (HO-1) melalui faktor transkripsi Nuclear Factor-E2-Related Factor 2 (NRF2) pada sel melanoma mencit yang diusulkan sebagai salah satu target seluler bagi penyembuhan pasien Corona Virus Disease 2019 (COVID-19). Namun, belum ada penelitian mengenai potensi antimikroba dari Adenostemma. Maka, fokus penelitian ini adalah mengkaji potensi antimikroba khususnya antivirus dan antibakteri terhadap tiga spesies Adenostemma yang paling sering diteliti, yaitu A. lavenia, A. madurense, dan A. platyphyllum melalui pendekatan metabolomik, in silico, dan in vitro, serta optimasi ekstraksi. Penelitian ini dibagi dalam lima tahap, yaitu (1) literature review mengenai genus Adenostemma, (2) evaluasi karakter morfologi, fenolik, dan flavonoid pada tiga spesies Adenostemma (3) uji in silico antivirus COVID 19, (4) pemprofilan metabolit tiga spesies Adenostemma dengan pendekatan metabolomik dan uji in silico antibakteri, (5) optimasi ekstraksi untuk mendapatkan ekstrak teraktif antibakteri secara in vitro.

Kajian literatur mengenai genus Adenostemma menunjukkan bahwa A. lavenia, A. madurense, dan A. platyphyllum merupakan spesies Adenostemma yang paling sering diteliti dan dilaporkan. Beberapa laporan mengenai kandungan senyawa kimia dan bioaktivitasnya telah dipublikasi seperti antioksidan, anti-inflamasi, antimelanogenesis, dan antitumor, tetapi bioaktivitas lain seperti antimikroba belum pernah dilaporkan. A. lavenia terdapat di beberapa provinsi di Indonesia, tetapi belum dibudidayakan secara luas. A. madurense dan A. platyphyllum memiliki morfologi yang mirip dengan A. lavenia, dilaporkan memiliki senyawa aktif berpotensi antibakteri. Hasil evaluasi karakter morfologi mengungkapkan bahwa terdapat perbedaan ciri morfologi pada ketiga spesies Adenostemma. Panjang dan lebar daun, tinggi tanaman dan jarak antar nodus dapat digunakan sebagai kriteria seleksi penting untuk menentukan jenis Adenostemma tinggi kandungan fenolik dan flavonoid. Senyawa fenolik dan flavonoid merupakan metabolit sekunder yang memiliki potensi antimikroba. A. lavenia mengandung total fenolik dan total flavonoid tertinggi. Hasil penelitian ini diharapkan dapat menjadi informasi yang bermanfaat untuk memperoleh sediaan biofarmaka Adenostemma yang tinggi kandungan fenolik dan flavonoidnya, untuk dimanfaatkan sebagai pangan fungsional dan obat herbal.

Pada kajian potensi senyawa-senyawa diterpenoid kaurena sebagai antivirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) melalui pendekatan in silico menunjukkan bahwa senyawa diterpena kaurena glikosida dan adenostemmoside B memiliki aktivitas penghambatan terbaik terhadap protein SARS CoV-2 dan lebih tinggi dibandingkan obat antivirus seperti remdesivir dan favipiravir. Simulasi dinamika molekul menunjukkan hampir semua kompleks ligan-protein stabil. Analisis ADMET menunjukkan semua diterpenoid kaurena diserap oleh usus manusia dengan baik, nonkarsinogenik, dan tidak menyebabkan mutasi pada DNA. Hasil studi ini dapat menjadi informasi awal untuk penelitian lebih lanjut dalam pengembangan obat antivirus baru.

Penelitian selanjutnya, hasil analisis profil metabolit ketiga spesies Adenostemma dengan pendekatan metabolomik menggunakan LC-MS/MS menunjukkan 35 metabolit berhasil diidentifikasi secara putatif. Hampir semua senyawa tersebut pertama kali dilaporkan terkandung dalam Adenostemma. Analisis kemometrik berhasil mengelompokkan metabolit yang berbeda diantara ekstrak, yang menunjukkan variasi kimia yang signifikan di tiap spesies. Uji in silico antibakteri menunjukkan bahwa asam dikafeoilkuinat memiliki afinitas pengikatan terbaik terhadap Staphylococcus aureus UDP-GlcNAc 2-epimerase, pektolinarigenin terhadap Escherichia coli UDP-3O-(3- hidroksimiristoil)glukosamin-N-asiltransferase, dan eriodiktiol 7-O-soforosida terhadap Pseudomonas aeruginosa pyochelin sintase PchD. Simulasi dinamika molekuler menunjukkan bahwa semua kompleks ligan-protein stabil. Pektolinarigenin dan eriodiktiol 7-O-soforosida (flavonoid) diidentifikasi memiliki persentase luas puncak kromatogram tertinggi pada A. lavenia, sedangkan persentase luas puncak asam dikafeoilkuinat (fenolik) tertinggi di A. platyphyllum. Hasil analisis profil metabolit Adenostemma tidak menunjukkan adanya senyawa asam ent-6-11-dihidroksi-15-okso 16-kauren-19-oat β-D-glukopiranosil ester dan adenostemmosida B yang memiliki potensi antivirus SARS-CoV-2. Oleh karena itu, pada penelitian selanjutnya tidak dilakukan uji in vitro antivirus. Hasil analisis profil dan in silico menjadi dasar untuk optimasi ekstraksi guna memperoleh ekstrak teraktif antibakteri secara in vitro.

Pada tahap akhir, ekstraksi serbuk simplisia daun A. lavenia dan A. platyphyllum menggunakan metode Microwave Assisted Extraction (MAE) dan optimasi ekstraksi menggunakan metode permukaan respon (Response Surface Methodology, RSM) dengan desain eksperimen Box-Behnken Design. Didapatkan kondisi optimum ekstraksi A. lavenia adalah pada konsentrasi etanol 75%, rasio simplisia terhadap pelarut 1:15, dan waktu ekstraksi 3 menit. Kondisi optimum tersebut menghasilkan respon rendemen 15,89%, kandungan total fenolik 49,49 mg GAE/g ekstrak, kandungan total flavonoid 25,66 mg QE/g ekstrak, zona hambat terhadap S. aureus, E. coli, dan P. aeruginosa berturut-turut, yaitu 7,01; 1,45; dan 2,52 mm. Kondisi optimum ekstraksi A. platyphyllum adalah pada konsentrasi etanol 58%, rasio simplisia terhadap pelarut 1:7, dan waktu ekstraksi 3 menit. Kondisi optimum tersebut menghasilkan respon rendemen 15,29%, kandungan total fenolik 42,84 mg GAE/g ekstrak, kandungan total flavonoid 28,82 mg QE/g ekstrak, zona hambat terhadap S. aureus, E. coli, dan P. aeruginosa berturut-turut, yaitu 2,83; 1,62; dan 1,68 mm. Hasil verifikasi kondisi optimum yang direkomendasikan menghasilkan respon yang sesuai prediksi dan metode yang digunakan valid. Aktivitas antibakteri terbaik ditunjukkan oleh ekstrak A. lavenia terhadap S. aureus, yaitu aktivitas sedang hingga kuat.

Hasil uji in vitro antibakteri tersebut sesuai dengan hasil prediksi in silico. Dengan demikian, pendekatan metabolomik dan in silico mampu memprediksi bioaktivitas antibakteri. Penelitian lebih lanjut perlu dikembangkan, diantaranya pengujian in silico terhadap berbagai protein pada bakteri tersebut, dan melakukan isolasi senyawa target pada Adenostemma untuk lebih menegaskan bioaktivitasnya.

Adenostemma (known as legetan warak) is traditionally used as a herbal remedy for diseases caused by bacteria, such as lung inflammation, pneumonia, fever, hepatitis, inflammation, skin wounds, and oral health disorders. The isolated diterpenoid kaurene compounds from Adenostemma lavenia have demonstrated the induction of Heme Oxygenase (HO-1) antioxidant protein expression through the Nuclear Factor-E2- Related Factor 2 (NRF2) transcription factor in mouse melanoma cells. This suggests their potential as cellular targets for treating patients with Coronavirus Disease 2019 (COVID-19). However, there is limited research on the antimicrobial potential of Adenostemma. Therefore, this study explored the antimicrobial potential, especially antiviral and antibacterial properties, of three frequently studied Adenostemma species: A. lavenia, A. madurense, and A. platyphyllum. This exploration employs metabolomic, in silico, and in vitro approaches, along with extraction optimization. The research was divided into five phases: (1) literature review on the genus Adenostemma, (2) evaluation of morphological, phenolic, and flavonoid characteristics of the three Adenostemma species, (3) in silico analysis for COVID-19 antiviral activity, (4) metabolite profiling of the three Adenostemma species using metabolomic approaches and in silico antibacterial analysis, and (5) extraction optimization for obtaining the most active antibacterial extract in vitro.

A literature review on the genus Adenostemma revealed that A. lavenia, A. madurense, and A. platyphyllum are the most studied and reported species. Some reports on their chemical compounds and bioactivities, such as antioxidants, anti-inflammatory, antimelanogenesis, and antitumor properties, have been published. However, their antimicrobial bioactivity has not been reported. A. lavenia was found in several provinces in Indonesia but has not been extensively cultivated. A. madurense and A. platyphyllum, with morphological similarities to A. lavenia, were reported to contain potentially antibacterial active compounds. Morphological character evaluation exhibited differences among the three Adenostemma species. Leaf length and width, plant height, and internodal distance were important selection criteria for determining Adenostemma species with high phenolic and flavonoid content. Phenolic and flavonoid compounds are secondary metabolites with antimicrobial potential. A. lavenia contains the highest total phenolics and total flavonoids. The results of this research were expected to be valuable information for obtaining Adenostemma biopharmaceutical preparations with high phenolic and flavonoid content for use as a functional food and herbal medicine.

The investigation into the potential of diterpenoid kaurene compounds as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antivirals through in silico approaches showed that kaurene glycoside and adenostemmoside B had the best inhibitory activity against the SARS-CoV-2 protein, surpassing established antiviral drugs like remdesivir and favipiravir. Molecular dynamics simulations indicated the stability of almost all ligand-protein complexes. ADMET analysis showed that all kaurene diterpenoids are well-absorbed by the human intestine, non-carcinogenic, and do not induce DNA mutations. These findings served as preliminary information for further research in developing new antiviral drugs.

Subsequent research involved the analysis of metabolite profiles of the three Adenostemma species using LC-MS/MS metabolomics. Based on these results, 35 putative metabolites were identified, many of which have not been previously reported in Adenostemma species. Chemometric analysis revealed separate clusters for the three species, confirming the substantial chemical differences between their extracts. In silico antibacterial analysis showed that dicaffeoylquinic acid had the strongest binding for Staphylococcus aureus UDP-GlcNAc 2-epimerase (PDB ID:5ENZ). Eriodictyol 7-O sophoroside and pectolinarigenin exhibited the best docking scores for Pseudomonas aeruginosa pyochelin synthase PchD (PDB ID:7TYB) and Escherichia coli LpxD acyltransferase (PDB ID:6P86) respectively. Molecular dynamics simulations revealed that all ligand-protein complexes were stable. Pectolinarigenin and eriodictyol 7-O sophoroside (flavonoids) were identified with the highest peak area percentage in A. lavenia, while caffeoylquinic acid (phenolics) has the highest peak area percentage in A. platyphyllum. The profiling analysis did not show the presence of compounds such as ent-6-11-dihydroxy-15-oxo-16-kauren-19-oic acid β-D-glucopyranosyl ester and adenostemmoside B, which have potent SARS-CoV-2 antiviral properties. Therefore, in vitro antiviral analysis was not carried out in the subsequent research. The profiling and in silico analysis results served as a basis for extraction optimization to obtain the most active antibacterial extracts in vitro.

In the final phase, powdered leaves of A. lavenia and A. platyphyllum was extracted using microwave-assisted extraction (MAE), and extraction optimization was performed using response surface methodology (RSM) with Box-Behnken design experimental design. The optimal extraction conditions for A. lavenia were determined to be 75% ethanol concentration, a 1:15 ratio of simplicia to solvent, and a 3-minute extraction time. These conditions produced a yield of 15.89%, a total phenolic content of 49.49 mg GAE/g extract, a total flavonoid content of 25.66 mg QE/g extract, and inhibition zones against S. aureus, E. coli, and P. aeruginosa of 7.01, 1.45, and 2.52 mm, respectively. Meanwhile, the optimal extraction conditions for A. platyphyllum were determined to be 58% ethanol concentration, a 1:7 ratio of simplicia to solvent, and a 3-minute extraction time. These conditions produced a yield of 15.29%, a total phenolic content of 42.84 mg GAE/g extract, a total flavonoid content of 28.82 mg QE/g extract, and inhibition zones against S. aureus, E. coli, and P. aeruginosa of 2.83; 1.62; and 1.68 mm, respectively. The verification of the recommended optimum conditions showed that responses were consistent with predictions, validating the method used. The A. lavenia extract demonstrated the best antibacterial activity against S. aureus (moderate to strong activity).

The in vitro antibacterial results aligned with the in silico predictions, demonstrating the predictive capability of metabolomics and in silico approaches for antibacterial bioactivity. Further research is needed, including in silico analysis against various proteins in these bacteria and the isolation of target compounds in Adenostemma to confirm their bioactivity further.