Formulasi Nanoemulsi Daun Alpukat dan Tanaman Kumis Kucing Sebagai Antioksidan Secara In Vitro

Abstract

Stres oksidatif merupakan kondisi patologis yang timbul akibat ketidakseimbangan antara produksi spesies oksigen reaktif (ROS) dan kapasitas sistem antioksidan endogen tubuh, sehingga dapat memicu berbagai penyakit kronis, termasuk gangguan ginjal. Kondisi tersebut dapat dicegah melalui pemanfaatan senyawa antioksidan alami, seperti flavonoid dan fenolik, yang terkandung dalam daun alpukat (Persea americana) dan kumis kucing (Orthosiphon aristatus). Namun, formulasi ekstrak konvensional dari kedua tanaman tersebut masih memiliki keterbatasan dalam hal kelarutan, bioavailabilitas, dan stabilitas senyawa aktifnya. Penelitian ini bertujuan untuk memformulasikan nanoemulsi kombinasi ekstrak daun alpukat dan kumis kucing menggunakan Self-Nanoemulsifying Drug Delivery System (SNEDDS), serta mengevaluasi kandungan fenolik total, flavonoid total, kapasitas antioksidan, karakteristik fisik, dan stabilitas fisiknya secara in vitro. Formulasi nanoemulsi diawali dengan uji kelarutan untuk menentukan pelarut, surfaktan, dan kosurfaktan terbaik, diperoleh propilen glikol sebagai kosolven serta kombinasi Tween 80 dan Span 80 sebagai Smix. Nanoemulsi diformulasikan pada rasio ekstrak daun alpukat dan kumis kucing (DA:KK) 1:4 dan 4:1, dengan ekstrak non-nanoemulsi dengan rasio yang sama sebagai kontrol. Kandungan fenolik total, flavonoid total, dan kapasitas antioksidan berturut-turut ditentukan menggunakan metode Folin-Ciocalteu, kolorimetri AlCl3, dan DPPH. Karakterisasi fisik nanoemulsi meliputi Z-average, indeks polidispersitas, dan zeta potensial menggunakan Particle Size Analyzer, sedangkan stabilitas fisik dievaluasi melalui uji heat-cool cycle dan freeze-thaw cycle masing-masing tiga siklus. Hasil penelitian menunjukkan bahwa nanoemulsi rasio 1:4 (DA:KK) menghasilkan kandungan fenolik total tertinggi sebesar 138,40 ± 2,72 mg GAE/g, kandungan flavonoid total sebesar 160,69 ± 0,22 mg QE/g, dan kapasitas antioksidan sebesar 53,27 ± 0,24 mg TE/g, yang secara signifikan lebih tinggi dibandingkan rasio 4:1 maupun ekstrak non-nanoemulsi (p < 0,05). Sebaliknya, nanoemulsi rasio 4:1 menunjukkan kandungan fenolik total lebih rendah dibandingkan ekstrak tanpa nanoemulsi pada rasio yang sama, diduga disebabkan oleh proses enkapsulasi SNEDDS terhadap keterbacaan analitik senyawa fenolik terhadap reagen Folin-Ciocalteu. Karakteristik fisik menunjukkan nanoemulsi rasio 1:4 memiliki Z-average sebesar 273,6 nm, PDI sebesar 0,56, dan zeta potensial sebesar -12,28 mV, sedangkan rasio 4:1 memiliki Z-average sebesar 251,8 nm, PDI sebesar 0,52, dan zeta potensial sebesar -2,96 mV. Kedua formulasi nanoemulsi tetap stabil secara fisik selama uji heat-cool dan freeze-thaw cycle tanpa disertai pemisahan fase, meskipun nilai PDI yang tinggi (>0,5) dan zeta potensial yang rendah pada kedua rasio mengindikasikan stabilitas termodinamika yang belum optimal dan memerlukan optimasi lebih lanjut.

Oxidative stress is a pathological condition resulting from an imbalance between the production of reactive oxygen species (ROS) and the body’s endogenous antioxidant capacity, which can trigger various chronic diseases, including kidney disorders. This condition can be prevented by using natural antioxidant compounds, such as flavonoids and phenolics, found in avocado (Persea americana) leaves and cat’s whiskers (Orthosiphon aristatus). However, conventional extract formulations from these two plants still have limitations in solubility, bioavailability, and stability of their active compounds. This study aims to formulate a combined nanoemulsion of avocado leaf and cat’s whiskers extracts using the Self-Nanoemulsifying Drug Delivery System (SNEDDS), and to evaluate the total phenolic content, total flavonoid content, antioxidant capacity, physical characteristics, and physical stability in vitro. The nanoemulsion formulation began with solubility tests to determine the optimal solvent, surfactant, and cosurfactant; propylene glycol was selected as the cosolvent, and a combination of Tween 80 and Span 80 was chosen as the Smix. The nanoemulsions were formulated at avocado leaf extract to cat’s whiskers extract (AL:CW) ratios of 1:4 and 4:1, with non-nanoemulsion extracts at the same ratios serving as controls. Total phenolic content, total flavonoid content, and antioxidant capacity were determined using the Folin-Ciocalteu method, AlCl3 colorimetry, and the DPPH assay, respectively. Physical characterization of the nanoemulsions included Z-average, polydispersity index, and zeta potential using a Particle Size Analyzer. In contrast, physical stability was evaluated through heat-cool and freeze-thaw cycle tests, each consisting of three cycles. The results of the study showed that the 1:4 nanoemulsion (AL:CW) produced the highest total phenolic content of 138.40 ± 2.72 mg GAE/g, a total flavonoid content of 160.69 ± 0.22 mg QE/g, and an antioxidant capacity of 53.27 ± 0.24 mg TE/g, which were significantly higher than those of the 4:1 ratio and the non-nanoemulsion extract (p < 0.05). In contrast, the 4:1 nanoemulsion exhibited a lower total phenolic content than the non-nanoemulsified extract at the same ratio, presumably because the SNEDDS encapsulation process reduced the analytical detectability of phenolic compounds by the Folin-Ciocalteu reagent. Physical characteristics showed that the 1:4 nanoemulsion had a Z-average of 273.6 nm, a PDI of 0.56, and a zeta potential of -12.28 mV, while the 4:1 nanoemulsion had a Z-average of 251.8 nm, a PDI of 0.52, and a zeta potential of -2.96 mV. Both nanoemulsion formulations remained physically stable during heat-cool and freeze-thaw cycle tests without phase separation, although the high PDI values (>0.5) and low zeta potentials for both ratios indicate that thermodynamic stability is not yet optimal and requires further optimization.