The palm oil-based chlorophyll removal and the evaluation of antiaging properties on Centella asiatica ethanolic extract

Centella asiatica is an essential medicinal herb in numerous traditions. The high chlorophyll concentration of C. asiatica makes dosage form formulation challenging. On the other hand, palm oil is feasible to eliminate the chlorophyll from plant-based products. This research aim to analysis the phytochemical profile and in vitro anti-aging effects on chlorophyll removal of C. asiatica extract. The extract was soxhletated in 70% ethanol. The extract was partitioned with 50% ethanol and palm oil to create a dechlorophyllation extract. Asiaticoside was analyzed using TLC-densitometry. The cytoprotective effect of H2O2-induced oxidative stress in Vero cells was assessed using MTT assays. Flow cytometer analysis was used for quantifying the intracellular ROS. The senescence-associated-β-galactosidase assay was used to identify senescent cells. The efficiency of chlorophyll removal by palm oil was 90.94±0.62%. The dechlorophyllation extract (DE) showed a concentration of 1.58±0.02% for asiaticoside, but it was not found in the original extract (OE). DE significantly reduced H2O2-induced cytotoxicity in Vero cells compared to OE. Both DE and OE reduced intracellular ROS and senescent cells. Palm oil-based chlorophyll removal improved the phytochemical content and anti-aging effects of C. asiatica in ethanolic extract.


Introduction
Centella asiatica (L.) Urb., also known as gotu kola, pegagan, Indian pennywort, and madukaparni, is a valuable medicinal herb.It has antioxidant, anti-inflammatory, anti-cancer, cardioprotective, and wound-healing properties.Years of tradition highlighted its medical uses.It is particularly famous in Ayurveda and other traditional systems as a "brain tonic" [1].As an antioxidant and anti-inflammatory, C. asiatica protects mitochondria from oxidative stress.Asiaticoside, asiatic acid, madecassoside, and madecassic acid are active triterpenoid compounds in the leaves [2].

2
C. asiatica treats vitiligo, hair loss, wound healing, and inflammation.Although it can treat skin conditions topically, its efficacy is constrained by a low transdermal absorption rate [3].Additionally, C. asiatica is a source of chlorophyll, a natural green pigment used in dietary supplements, cosmetics, and pharmaceuticals.The natural color chlorophyll is fat-soluble and widespread [4].Organic solvents, including ethanol, acetone, ether, and chloroform, dissolve chlorophyll, which is waterinsoluble.The chlorophyll molecule comprises an ester-bound porphyrin ring and a long hydrocarbon chain called phytol.Brown or green colors result from strong porphyrin rings.Despite its many biological functions, chlorophyll can photo-oxidize, compromise stability, and generate undesirable odors in medical formulations.Therefore, lowering chlorophyll levels is necessary to improve the acceptability and stability of pharmaceutical preparations, particularly in topical dosage forms [5].Jumpatong et al. (2006) late research showed the ethanolic extract of C. asiatica contained a high level of chlorophyll; hence the dechlorophyllation procedure is carried out by electrocoagulation.However, the electrocoagulation technique is less effective due to the long duration and complex equipment requirements [5].Phaisan et al. (2020) utilized an oil-based method to extract chlorophyll from Chromolaena odorata (L.) R.M. King & H. Rob.Palm oil was more effective than hexane at removing chlorophyll and recovering essential phytochemicals.This approach applies to developing nature-based pharmaceutical ingredients and phytochemical research [6].
Research related to the use of dechlorophyllization using palm oil on C. asiatica extract is still very limited.However, C. asiatica extract is extensively used in a variety of medicinal, nutritional, and cosmetic products.Therefore, the present study applied partitioning with palm oil to dechlorophyllize C. asiatica extract in a simple and environmentally friendly approach.Dechlorophyllation with palm oil will be evaluated for its phytochemical and anti-aging effects in Vero cello lines through cytoprotective, intracellular ROS, and senescent cells.Vero is an ideal cell modeling for testing the molecular safety and toxicity of drugs, poisons, and other compounds [7].

Material and Methods
Herbs from C. asiatica were collected and identified at the Medicinal Plants and Traditional Medicines Research and Development Center (MPTMRDC) in Tawangmangu, Central Java, Indonesia with specimen number KM.04.02/02/1025/2020.Culture and maintenance of Vero cells were conducted in Dulbecco's Modified Eagle Medium (DMEM (Gibco, New York)) supplemented with 10% fetal bovine serum (FBS (Gibco, New York)), 10% penicillin-streptomycin (Gibco, New York), and 2% fungizone (Gibco, New York).The cells were incubated at 37°C with 5% CO2 in an incubator.

Extraction and palm oil-based chlorophyll removal
Herbs were rinsed, drained, oven-dried at 50°C, and ground into a powder.Soxhletation with 70% ethanol yielded the original ethanolic (OE) dried extract.Ten grams of extracts were diluted in 85 mL of 50% ethanol and 120 mL of palm oil and centrifuged at 5,000 rpm for 10 min in room temperature.The clear brown supernatant at the bottom was pipetted, filtered, and dried to generate dechlorophyllation extract (DE).A spectrophotometry method was used to evaluate the efficiency of chlorophyll removal.The extract was dissolved in 75% (v/v) ethanol at 20 mg/mL.A microplate spectrophotometer (Multiskan Sky, Thermo Scientific, Singapore) measured extract absorbance and transmittance.The chlorophyll removal efficiency was calculated using Equation 1 [6].

Quantification of asiaticoside compound
Ten mg of extract was combined with 10 mL of ethanol, sonicated for 15 min, and left overnight in a laboratory bottle.Up to 2 mL of the settling sample is collected and evaporated at 50 °C.Dissolved the dried material in 4 mL methanol and sonicated for 15 min.The chloroform: methanol: water (65:25:4) mixture was used to elute the asiaticoside standard and sample from a TLC plate.Elution results were handled with 1.5 mL anhydrous acetic acid and Liebermann-Burchard (Sigma Aldrich, Germany) spot reagent and incubated at 110 °C for 10 min.The plate was observed using a Camag TLC Scanner 3 (CAMAG (Switzerland)) at 510 nm [8].
The extract was preserved in dimethyl sulfoxide ((DMSO) Merck, Germany) solvent before being diluted with culture media in various concentrations and incubated for 24 h.At the end of incubation, the culture media was withdrawn, and 100 µL of MTT reagent was added per well, followed by a 3hour incubation.The reaction was terminated by adding 10% SDS in 0.01N HCl left at room temperature and protected from light overnight.Solution absorbance was measured at 595 nm with an ELISA reader (BioTek, USA) [9,10].Cell was obtained from MPTMRDC Laboratory in Tawangmangu.

Intracellular ROS analysis using 2',7'-dichlorofluorescein diacetate (DCFDA) staining
DMEM culture media cultivated 5x10 4 Vero cells/wells on 24 well plates in a 5% CO2 incubator at 37°C for 24 h.Cells were separated using trypsin-EDTA for 3-5 min, then inactivated with 500 µL of FBS 10% in phosphate buffer saline (PBS).Cells were collected in microtubes treated with 25 µM DCFDA solution in 5% CO2 at 37°C for 30 min.Three h of extract treatment followed by one hour of 500 mM H2O2 ROS induction.ROS analysis with a BD Accuri C6 (BD Biosciences, Germany) flow cytometer set to 458 nm/535 nm.The concentration of intracellular ROS was calculated using the average fluorescence intensity [11].

Senescence-Associated-Galactosidase (SA-β-GAL) assay
Vero cells were cultivated in 24 well plates at 37°C in 5% CO2 for 24 h.The cells were stimulated with six mM H2O2 for 2 h and treated with the extract for 24 h at 37 o C in a CO2 incubator.After 4% paraformaldehyde (PFA) fixation at room temperature for 10 min, the cells were rinsed twice with 1x PBS.Treatment involved administering 1 mL SA-β-GAL (BioVision, USA) dye solution and incubating at 37°C in a carbon dioxide-free incubator.An inverted microscope (Olympus, Japan) viewed cells at 24, 48, and 72 h.Senescence cells are blue-green.ImageJ software (USA) showed data as a fraction of senescence [11].

Data analysis
One-way ANOVA and t-test using Excel365 software (version Microsoft Office Home and Student 2019) were used to examine each different treatment group.The data are the mean SD of three independent experiments.Significant statistical differences were determined using p<0.05.

Result and Discussion
Pharmaceutical dose formulations need less chlorophyll to be stable [12].The high chlorophyll content of C. asiatica could contribute to the progressive degradation of the ultimate product.The 70% ethanol soxhletation method yielded 8.5% sticky mass, dark green color, and herbal odor.The palm oil eliminated chlorophyll from the ethanolic extract, producing a light brown, slightly damp material with a 75.2% yield.The extracted substance was known as dechlorophyllation extract (DE).Table 1 shows that the approach removed chlorophyll 90.94% efficiently.Figure 1 illustrates a TLC analysis followed by Liebermann-Burchard application in visible and UV light at 366 nm.In its TLC profile, OE has indistinct spots and a lower hRf than asiaticoside.DE demonstrated sharper and better TLC compound separation than OE.The asiaticoside level also could not be found in OE, although DE yielded 1.58±0.02%.These results showed that palm oil-based chlorophyll removal eliminated impurities from the extract.Our results are comparable to Ruksiriwanich et al., 2020, who used supercritical CO2 pre-treatment and 85% ethanol maceration to produce 1.179% asiaticoside [12].
Exposure to H2O2 is a standard method for inducing cellular oxidative damage [14].Through specialized transporters, H 2 O 2 diffuses across cell membranes and becomes highly reactive hydroxyl radicals.Oxidative stress results from free radical production and antioxidant defense imbalance [15,16].
The biological sciences are uncovering the physiological and pathophysiological functions of reactive oxygen species (ROS).Obstructive lung, renal, neurological, cancer, sarcopenia, and frailty are clinical pathologic aging-related diseases connected to oxidative stress [14].Natural antioxidants like enzymes and small compounds interact with individual ROS [17].Antioxidants have a therapeutic effect on aging by scavenging free radicals and, as a result, reducing damage to cell molecules and adjacent tissues [18].
The intracellular ROS on H2O2-induced oxidative stress in Vero cells was measured using DCFDA small-molecule fluorescent probes.Induction with 500 mM H2O2 enhanced intracellular ROS fourfold.Table 2 shows considerable ROS reduction from OE and DE 100 and 500 µg/mL, comparable to ascorbic acid 100 µg/mL.At all concentrations, OE inhibited intracellular ROS production more effectively than DE.At 100 µg/mL DE, ROS production can be prevented at the same level as ascorbic acid.High levels of chlorophyll in OE are thought to be involved in a higher ROS inhibitory effect.Chlorophyll may produce less ROS and directly trap free radicals [19].The planar shape of chlorophyll traps mutagens and reduces cell-damaging substances, which has therapeutic effects [20].
ROS are linked to growth factor-stimulated and inflammatory signaling cascades.They are also essential regulators of cellular processes such as differentiation, proliferation, apoptosis, and senescence.Senescence, also known as biological aging, is a physiological condition that leads to mortality through aging and the deterioration of mature organs.Cell cycle arrest and apoptosis resistance characterize cellular senescence.Although inactive, senescent cell formation has metabolic activity and alterations at the cellular and molecular levels, which could impact the microenvironment.Cellular senescence is a hallmark of aging and contributes to chronic degenerative disorders [21,22].Zhu et al.Identifying specific markers to identify senescent cells in living tissues represented the initial challenge.Notably, many senescent cells have lysosomes that are damaged.Increased lysosomal SA-βgal levels in senescent cells may indicate lysosomal malfunction [23].The increase in the number and activity of lysosomes, likely due to the accumulation of damaged macromolecules in senescent cells, causes an increase in GLB-1 mRNA and protein levels.SA-β-gal activity rises with GLB-1 gene expression [22].SA-β-gal activity was one of the first biomarkers discovered [1].De Mera-Rodríguez et al. (2021) suggest that SA-β-gal activity is a common biomarker for cellular senescence staining in cultivated cells and tissue sections [22].
The ability of the extracts to prevent cell senescence was next tested in this study utilizing the SAgal assay, and two h of exposure to 6 mM H2O2 induced an increase in SA-β-gal activity in Vero cells.Figure 3 shows that OE, DE, and ascorbic acid treatments reduced senescent cells by 3.3%, 3.1%, and 0.3% vs. untreated control cells (6.6%).Dechlorophyllation gently reduced senescent cell growth overextract.In H2O2-induced Vero cells, OE and DE extracts function as cytoprotectants and decrease intracellular ROS and cell senescence, suggesting anti-aging properties.Cell senescence and mitochondrial malfunction are linked to aging.Cellular senescence is both a cause and a consequence of mitochondrial failure, which is essential in numerous feedback loops that generate and sustain the senescent phenotype.Operationally, it is characterized as a lower mitochondrial membrane potential and respiratory capacity per mitochondrion, often accompanied by increased oxygen free radicals like ROS [24].

Conclusion
Palm oil-based chlorophyll removal from C. asiatica ethanolic extract improves the phytochemical profile, asiaticoside detection, cytoprotective action, and eradication of cell senescence in Vero cells.Compared to the original extract, chlorophyll removal lessens the suppression of the intracellular ROS action, but it still exhibits a similar effect to ascorbic acid.Further research is needed on the extract's medicinal use and anti-aging effects.

Figure 1 .
Figure 1.Asiaticoside TLC profile of original and palm oil-based chlorophyll removal extract.Spots S1-S5 were asiaticoside standard, D1 was DE, and E1 was OE (2018)  found that senescent cells exhibit expanded and flattened phenotypes, giant vacuoles, occasional multi-nuclear phenomena, and positive β-galactosidase staining.

Figure 2 .
Figure 2. The morphology of Vero cells treated with original and dechlorophyllation extract 1000 µg/mL compared to untreated cells (A).The cells were observed under an inverted microscope, 400x magnification.The white arrows showed morphological alterations that harmed cells.The graph showed the relationship of viability vs. extract concentrations for 24 h, (B) without H2O2 induction, (C) with H2O2 induction.

Table 1 .
The palm oil-based chlorophyll removal efficiency of C. asiatica extract

Table 2 .
Intracellular ROS level of Vero cells treated by original and dechlorophyllation extract based on flow cytometry DCFDA staining