Supplementation of standardized extract of C. asiatica inhibited rotenone-induced-hepatotoxicity by inhibition of lipid peroxidation in vivo Altogether, extract from C. asiatica has a potential therapeutic utility on oxidative stress-related-disorders, including skin aging.

Naturally grown plant serves as source for secondary metabolites which can be used in cosmeceutical and pharmaceutical industries. However, the major limitations for production of compounds from wild natural plant are the dependence of geographical, seasonal and environmental factors.

In addition, further drawbacks for extraction and purification of secondary metabolites from intact plants include complex and time-consuming process; and low concentration of bioactive compounds To overcome these obstacles, in vitro callus culture has been proposed to be an alternative procedure to efficiently produce bioactive metabolites.

This in vitro method offers a manufacturing system which ensures the continuous supply of compounds with uniform quality and high yields. With this approach, cells of any plants, even rare or endangered species, can be easily manipulated and maintained to produce compounds of interest.

The active compounds can be produced independently of external factors. Moreover, plant cell culture uses aseptic technique so it will not be threatened by micro-organisms or pests The maintenance of aseptic conditions is essential for successful tissue culture procedures Taken together, plant cell culture represents an attractive platform for mass production of phytochemicals.

In this present study, we demonstrated the application of callus culture of C. asiatica to produce bioactive metabolites. The major goals of this study are 1 to evaluate the antioxidant capacities of callus extracts; 2 to investigate the beneficial activities of extracts against oxidative stress in human dermal fibroblasts.

Data from our study will provide an invaluable insight into potential application of C. asiatica callus extract in anti-aging cosmeceutical products. R , polyethylene glycol PEG; Cat no. D , sucrose Cat no. M , 2,2-diphenylpicrylhydrazyl DPPH; Cat no. A , p -anisaldehyde Cat no.

A , dimethyl sulfoxide DMSO; Cat no. DX , ethyl acetate Cat no. HPLC-grade acetonitrile Cat no. baker Phillipsburg, NJ, USA. All other chemicals in this study were analytical grade. Murashige and Skoog medium MS; Cat no.

M , 1-naphthaleneacetic acid Cat no. N , 6-benzylaminopurine Cat no. B and Agargellan TM Cat no. A were obtained from Phytotechnology Labs Lenexa, KS, USA. The explants were obtained from in vitro germinated C.

asiatica seedlings. asiatica is a commercially available plant and it is grown at the garden of Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand Latitude Experimental research on plants complies with relevant institutional, national, and international guidelines and legislation.

The seed of the plant used in the study was collected under the permission of the Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Thailand.

The plant was identical to a voucher specimen herbarium number deposited at Museum of Natural Medicines, Chulalongkorn University, Thailand. To remove dust particles, collected seeds were washed with detergent for 10 min. All subsequent steps were performed under a laminar flow cabinet.

The seeds were washed five times with sterile distilled water to remove all traces of sterilant. The sterilized seeds were then inoculated on Murashige and Skoog medium, supplemented with 0. Callus was obtained from in vitro germinated C. asiatica seedling using the protocol reported by Loc and An with slight modifications Briefly, about 1 cm long stolon explants of 15 days old seedling of C.

asiatica were cut and inoculated on MS medium pH 5. Callus were routinely sub-cultured onto the same medium every 30 days. The filtrate was then used for HPTLC and HPLC analysis. The compounds in extracts were separated by using HPTLC technique.

The plates were developed in the chamber saturation containing mobile phases. The development was done with a migration distance of 70 mm using an automatic development chamber ADC2 Camag; Muttenz, Switzerland.

For observation of triterpenoids, the plate was dipped with sulfuric reagent using Chromatogram Immersion Device III Camag; Muttenz, Switzerland and then heated at °C for 3 min on the TLC Plate Heater Camag; Muttenz,Switzerland.

The derivatized plates were visualized under white light using visualizer 2 Camag; Muttenz, Switzerland. The derivatized plates were visualized under UV light at nm using visualizer 2 Camag; Muttenz, Switzerland.

In order to estimate the antioxidant activity of tested samples, HPTLC-DPPH was performed as a screening assay. After development, the HPTLC plates were immersed with 0. The chromatogram was then examined under white light.

Generated bands with yellowish color on the purple backgrounds were considered as compounds with antioxidant activities.

The radical scavenging activities of tested extracts were evaluated by traditional DPPH assay Briefly, µL of DPPH solution 0. After incubation, the absorbance was recorded at nm using CLARIOStar microplate reader BMG Labtech; Ortenberg, Germany.

Seven standards, namely, asiaticoside, asiatic acid, madecassoside, madecassic acid, kaempferol, quercetin and rutin, along with the callus extract were used for the analysis. The analysis was performed using a Shimadzu HPLC LCA connected with a PDA detector Shimadzu; Kyoto, Japan.

The HPLC analysis was conducted according to the method of Buraphaka and Putalun The column temperature was controlled at 30 °C, and chromatograms were recorded at and nm. Human foreskin fibroblasts, BJ ATCC ® CRL , were obtained from American Type Culture Collection ATCC; Manassas, VA, USA.

The condition for cell culture was followed the standard protocol as described previously H 2 O 2 was used as an inducing agent for oxidative stress A solution of H 2 O 2 was freshly prepared in serum-free MEM prior each experiment. To examine the cytotoxicity of H 2 O 2 , the BJ cells were exposed to H 2 O 2 at µM for 1 h.

For a control group, the medium was replaced with a fresh serum-free MEM similar to the H 2 O 2 -treated cells.

The treatment protocol was slightly modified from our previous in vitro experiments on skin cells 21 , Control groups were incubated with an equivalent amount of DMSO 0. DMSO at 0. The cell viability was determined by using MTT assay After indicated treatments, exposure media were aspirated, and the cells were washed with phosphate buffer solution PBS; pH 7.

The absorbance of the formazan solution was then measured at nm using the CLARIOStar microplate reader. The BJ cells were seeded into multiple culture dishes 60 mm at a density of , cells per dish in 4 mL of the MEM.

Following the indicated treatments, treated cells were trypsinized and resuspended with PBS. Levels of intracellular ATP were immediately measured with CellTiter-Glo Luminescent Cell Viability Assay Promega, Madison, WI, USA; Cat no. G as previously described Briefly, a cell suspension 50, cells in µL of PBS was added into each well of opaque walled, well white microplate.

Subsequently, µL of the CellTiter-Glo reagent was homogenously mixed with the cell suspension at room temperature to induce cell lysis and start the luminescent reaction. After a min incubation, the luminescent signal was measured using the CLARIOStar microplate reader.

The generated luminescent signal is directly proportional to the amount of ATP in the sample. To estimate the intracellular level of ATP, standard curves for each experiment were produced with serial dilutions of ATP solutions 0— μM.

The amount of ATP in each sample was calculated from the corresponding standard curve and then further transformed to an intracellular concentration using the cell number. After treatments, total RNA was isolated from BJ cells by using GENEzol reagent Geneaid Biotech Ltd.

One microgram of extracted RNA was then reverse transcribed into cDNA by using RevertAid First Strand cDNA Synthesis Kit Thermo Fisher Scientific, Waltham, MA, USA; Cat no. The expression of interested genes were quantified by using ΔΔCq approach with normalization to house-keeping β-Actin.

The conditions for RT-qPCR were modified from previous study by Biagini and colleagues The PCR conditions, sequences of primer nucleotide Macrogen, Seoul, South Korea and analysis of RNA extraction are listed in Supplementary Table S1 , S2 , and S3 respectively.

All means and standard errors of the mean SEMs were calculated from three independent experiments. Statistical analysis was performed using GraphPad Prism version 9. To determine a suitable solvent for extraction process, callus culture and authentic plant of C.

Due to their abundance and pharmacological activities, asiaticoside, asiatic acid, madecassoside, and madecassic acid are considered to be major active components of C. asiatica 7 , To examine whether callus extract CE and authentic plant extract APE contains these triterpenoids, active compounds in extracts were separated with HPTLC approach.

All of four centelloids were observed in APE Lanes 5—8; Fig. To screen active antioxidants in the extracts, HPTLC plates were further derivatized with DPPH reagent. A stable free radical DPPH is a widely used substrate to evaluate free radical scavenging activities of tested compounds. On HPTLC plates, the compounds generating yellow zones against the purple background were considered as antioxidants.

The CE and APE exhibited strong free radical scavenging activities. Interestingly, all of tested triterpenoids did not show antioxidant activities Fig. The CE showed significant antioxidant activity in the absence of these standards. These HPTLC-DPPH data suggested that asiaticoside, asiaticoside, asiatic acid, madecassoside, and madecassic acid do not contribute to antioxidant properties of the extracts.

The antioxidant activities of Centella extracts are not due to asiaticoside, asiatic acid, madecassoside and madecassic acid.

A The HPTLC plate was derivatized with anisaldehyde sulphuric acid reagent and visualized with white light. B The HPTLC plate was dipped with DPPH reagent and visualized with white light. Kaempferol, quercetin, and rutin reported in C. asiatica 27 , 28 are flavonoid compounds with antioxidant activities.

It was suspected that these compounds may contribute to the antioxidant activity of the extracts. However, the chromatogram on the HPTLC plate demonstrated that these three flavonoids were not presented in any extracts Fig. The data from HPTLC-DPPH showed that CE and APE exhibited significant antioxidant activities, as evidenced by a great intensity of yellow spots Fig.

Moreover, Centella extracts exhibited potent radical scavenging properties without the presence of kaempferol, quercetin and rutin Fig. Taken together, these results strongly suggested that antioxidant activities of extracts are not due to these three flavonoids but possibly from other flavonoids.

The antioxidant activities of Centella extracts are not due to kaempferol, quercetin and rutin. B The HPTLC plate was immersed with DPPH reagent and visualized with white light. As demonstrated in Figs. As shown in Figs.

We exhibited that CE had approximately 2. These results suggest that extract from callus culture possesses a strong free radical scavenging ability, which is even greater than from native plant. HPLC was conducted to analyze and compare chemical compositions in Centella extracts Fig.

All the standards were detected in the APE, irrespective of the solvent used for extraction. These HPLC data are consistent with HPTLC results demonstrated in Figs.

Moreover, CE demonstrated different chemical profiles when compared to APE. Hence, the difference in antioxidant capacity between CE vs APE could possibly be due to variation in active components.

Centella extract from callus culture has unique chemical profiles. The retention time RT was shown in parenthesis. The compounds with antioxidant capacity have been proposed as promising candidate for anti-skin-aging agents According to cell-free based antioxidant assay, CE and APE were selected to further examine their beneficial activities on human dermal fibroblast BJ cells.

Firstly, we evaluated potential toxicity of CE and APE on dermal fibroblasts. Centella extracts prevent H 2 O 2 -induced cytotoxicity in dermal fibroblasts. C and D CE and APE inhibit oxidative damage from H 2 O 2 exposure.

The cell viability was evaluated with MTT assay immediately after the treatments. E and F CE inhibit depletion of intracellular ATP following H 2 O 2 exposure. The protocol for treatment was as A and C. To investigate the preventive effects of CE and APE against oxidative insults, BJ cells were pre-treated with CE or APE at non-toxic concentration for 24 h prior to H 2 O 2 treatment.

Pre-treatment with CE significantly inhibited H 2 O 2 -induced toxicity Fig. Interestingly, the protective effects of CE were comparable to APE pre-treatments Fig. These data suggested that CE and APE could exert their beneficial effects against H 2 O 2 on dermal fibroblasts via their antioxidant activities.

The imbalance of cellular bioenergetics due to oxidative stress is closely linked to aging processes of several tissues, including skin Exposure to H 2 O 2 caused an immediate depletion of steady-state levels of ATP in dermal fibroblasts Fig. Interestingly, pre-treatment with CE significantly inhibited reduction of intracellular ATP pool following H 2 O 2 treatment in dose-dependent fashion Fig.

The results from these bioenergetics studies were consistent with the protective effects of CE against oxidative damage from MTT assay, highlighting the contribution of antioxidant activity of CE on its protective effect against oxidative insults.

The beneficial effects of CE and APE against oxidative damage as showed in Fig. To examine this hypothesis, the mRNA expression of key antioxidant enzymes, including catalase CAT , glutathione peroxidase 1 GPx1 , superoxide dismutase 1 SOD1 , and superoxide dismutase 2 SOD2 , were measured following h treatments.

Our RT-qPCR results demonstrated that CE and APE have different profiles on the induction of antioxidant machinery of fibroblasts; CE induced expression of CAT in dose-dependent fashion Fig. The expression of GPx1 did not alter with either CE or APE treatment Fig.

These RT-qPCR data suggested that an upregulation of cellular antioxidant enzymes is the principal factor for protective effects of CE and APE. Centella extracts elevate transcription of antioxidant enzymes. A — D CE promoted CAT expression, while APE enhanced SOD1 and SOD2 expression.

We also observed the effects of Centella extracts on expression of antioxidant enzymes after H 2 O 2 treatment. Exposure to H 2 O 2 leaded to dramatic decrease in GPx1 Fig. The increase of SOD2 expression is possibly due to adaptive mechanism of fibroblasts following H 2 O 2 treatment.

This phenomenon has been reported in several models of aged fibroblasts 31 , 32 , Again, CE and APE demonstrated distinctive effects on induction of antioxidant enzymes following H 2 O 2 exposure.

Pre-treatment with CE induced CAT Fig. Altogether, these results clearly exhibited that Centella extracts could prevent H 2 O 2 cytotoxicity by an enhanced capacity of fibroblasts to remove deleterious ROS. Centella extracts promote expression of antioxidant enzymes following H 2 O 2 treatment. A — D In response to H 2 O 2 treatment, CAT , GPX1 and SOD1 expressions were increased in CE-treated fibroblasts, while SOD2 transcription were elevated in APE-treated fibroblasts.

Matrix metalloprotease-9 MMP-9 is the gelatinase enzyme that is responsible for regulation of homeostasis of collagen. Formation of oxidative stress can lead to upregulation of MMP-9 in fibroblasts, which subsequently results in degradation of collagen.

The increased breakdown of collagen by MMP-9 appears to be the major factor for aging processes of skin tissue 34 , Here, we demonstrated that exposure to H 2 O 2 markedly upregulated the expression level of MMP-9 of dermal fibroblasts Fig.

Elevation of MMP-9 expression was significantly suppressed by CE and APE pre-treatment Fig. These data suggested that CE and APE may possess anti-aging activities via an inhibition of MMP-9 transcription. Centella extracts inhibit expression of MMP9 following H 2 O 2 treatment.

A Exposure to H 2 O 2 resulted in elevation of MMP9 expression, while CE and APE did not affect the transcription of MMP9. B CE and APE suppressed H 2 O 2 -induced MMP9 expression. asiatica is a medicinal plant with broad range of pharmacological effects e.

antioxidant 8 , 9 , 10 , 11 , anti-inflammation 36 , 37 , wound healing 38 , 39 , 40 , neuroprotective 8 , 9 and memory improvement 41 , Due to its high therapeutic potential, the demand for this plant in cosmeceutical and pharmaceutical industries has exceeded the supply from conventional cultivation.

Callus culture offers a manufacturing system which ensures the continuous supply of compounds with uniform quality and high yields asiatica due to i.

antioxidant activity of extracts; and ii. safety from reduced solvent residue. Compared to APE, the data from HPTLC Fig.

Interestingly, results from DPPH assay showed that the total antioxidant activity of CE is higher than APE Supplementary Fig. The greater antioxidant activity of CE is possibly due to the differences in active components in the extracts.

We demonstrated that the major triterpenoids of C. asiatica asiaticoside, asiaticoside, asiatic acid, madecassoside and madecassic acid are not responsible for antioxidant activities of CE Fig.

These findings are parallel to previous observation demonstrating that triterpene-free-extract of C. asiatica possesses greater in vitro radical scavenging properties as well as in vivo antioxidant activities than triterpene-enriched-extract.

Triterpene-free-fraction of C. asiatica has significantly stronger in free-radical scavenging activities than triterpene-enriched-extract as determined by several in vitro anti-radical assays, including DPPH, ABTS, NORAC, ORAC and NO scavenging assays.

Moreover, pre-treatment with triterpene-free-extract of C. asiatica reduced levels of malonaldehyde, an oxidative stress marker, in brain tissues of scopolamine-treated-rat, while triterpene-enriched-fraction did not have these protective effects These preclinical findings support our results that the centelloids are not principal contributors for antioxidant activity of C.

Kaempferol, quercetin, and rutin have been reported to be major flavonoids with antioxidant properties of C. asiatica 27 , Hence, future studies are required to identify and characterize novel antioxidants presented in these extracts. The process of skin aging is closely associated with an induction of oxidative stress in dermal fibroblasts 1.

Hence, compound that can prevent oxidative damage in dermal fibroblasts could be a potential candidate to be used as anti-skin-aging in cosmeceutical product. We then further investigated biological activities of Centella extracts against oxidative stress on dermal fibroblasts.

Our data from MTT assay as well as bioenergetic study showed that CE significantly inhibited the cytotoxicity of H 2 O 2 on dermal fibroblasts Fig. The preventive effects of CE were comparable to APE.

Moreover, we found that the protective activities of these extracts could be due to an increase in capacity of fibroblasts to eliminate ROS. We used RT-qPCR approach to investigate the alterations of mRNA expression of key antioxidant enzymes in response to treatments.

Catalase is the major enzyme involved in the elimination of high fluxes of H 2 O 2 , while GPx1 is responsible for detoxification of low fluxes of H 2 O 2 RT-qPCR data revealed that CE and APE have different downstream targets on cellular antioxidant machineries. Treatment with CE promoted mRNA expression of H 2 O 2 -detoxifying enzyme, CAT.

In response to H 2 O 2 treatment, pre-exposure to CE induced CAT , GPx1 and SOD1 expression, whereas pre-treatment with APE upregulated SOD2 expression Fig. Upregulation of these antioxidant machineries due to Centella extracts could promote capacity of fibroblasts to eliminate harmful ROS, resulting in suppression of oxidative damage and prevention of cell death.

Further studies on protein levels and enzymatic activities of antioxidant machineries following treatments of CE and APE are necessary to better understand the protective mechanisms of these extracts on fibroblasts. In rat model of hepatic injury, administration of Centella extract prevented hepatotoxicity through an increased level of catalase, SOD and GPx in liver tissues In hamster model of hyperlipidemia, supplementation with ethanolic extract of C.

asiatica promote hepatic function by an enhanced expression of SOD and GPx in hepatic tissues In diabetic rat, aqueous extracts from C. asiatica ameliorate hippocampal dysfunction by an induction of catalase, SOD and GPx expression in hippocampus In stroke model, supplementation with ethanolic extract of C.

asiatica prevented brain injury through a restoration of glutathione level and augmentation of catalase, SOD and GPx activities in ischemic rat Nrf2 functions as a redox sensor for oxidative stress.

In the presence of oxidative insults, Nrf2 translocates into nucleus and binds to promoter regions of ARE, leading to transcriptional activation of a battery of cytoprotective and detoxification genes, including CAT , GPx and SOD 51 , 52 , 53 , The activation of this pathway by Centella extract is strongly accompanied by an improvement in neuronal health and cognitive function 8 , 55 , 56 , 57 , Recently, Park and colleagues demonstrated that pre-treatment with Centella extract can prevent the progression of age-related macular degeneration in vitro and in vivo.

They clearly showed that the cytoprotective activities against oxidative damage of Centella extract in cell culture and animal experiment is mainly due to the regulation of Nrf2 pathway Further pharmacological study is required to validate this hypothesis.

In addition to effects on cellular antioxidant enzymes, we also observed activities of Centella extracts on expression of MMP MMP-9 is zinc-containing-gelatinase which plays an important role in degradation of dermal extracellular matrix, especially collagen type IV. Environmental insults, e. UV irradiation, tobacco smoke, and pollutants, have been reported to induce expression of MMP-9 of skin cells through formation of oxidative stress Upregulation of MMP-9 leads to fragmentation of dermal collagen; thereby diminish skin elasticity and integrity; and eventually promote wrinkle and sagging formation of skin 34 , Hence, agents with MMP-9 inhibitory activities would be an attractive candidate to combat skin aging Our RT-qPCR data demonstrated that CE and APE significantly inhibited upregulation of MMP9 following H 2 O 2 exposure Fig.

The proposed mechanisms for antioxidant and anti-skin aging activities of CE and APE are summarized in Fig. Proposed mechanisms for antioxidant and anti-skin aging activities of Centella extract. Pre-treatment with Centella extracts prevent H 2 O 2 -induced cytotoxicity on dermal fibroblasts by upregulation of cellular antioxidant machineries.

Supplementation with CE upregulated CAT , GPx1 and SOD1 expression, whereas pre-treatment with APE induced SOD2 expression following H 2 O 2 treatment.

Moreover, pre-treatment with CE or APE suppressed H 2 O 2 -mediated-upregulation of MMP This figure was created with BioRender. In conclusion, our present study provides the potential application of callus culture platform to produce biomass and biochemical from C. Moreover, this study provides pharmacological evidence to support the use of Centella extracts as anti-skin-aging agents in cosmeceutical and pharmaceutical products.

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A , Cheng, Z. High-throughput relative DPPH radical scavenging capacity assay. Food Chem. Buraphaka, H. Stimulation of health-promoting triterpenoids accumulation in Centella asiatica L. Such antioxidant enzymes work much better than antioxidants small-molecules taken by mouth, but still they do not seem to have significant effects on lifespan.

Even more confusingly, mounting evidence shows that the substances antioxidants are supposed to neutralize, free radicals, can even have life extension effects.

How is this possible? These exercise-induced free radicals activate all kinds of repair and defense mechanisms in our cells, so that the cells can better protect themselves against the next time you exercise.

In the meantime, these revved-up defense and repair mechanisms also protect you against aging and aging-related diseases. Besides exercise, we know that foods like vegetables, fruits and green tea are healthy.

The classic, main explanation for this is that these foods contain antioxidants. But this is an oversimplification. A reason that healthy food is healthy is not because of its antioxidant activity, but because this food contains slightly toxic substances.

These substances upregulate detoxification and repair enzymes in the body, so that our body is better protected against damage. Healthy foods are also healthy due to reasons other than their oxidants. Healthy food contains substances that have epigenetic effects , that reduce inflammation , that are beneficial to the gut microbiome, that do not overstimulate aging pathways like mTOR or insulin receptors , that improve mitochondrial functioning.

And of course, healthy foods deliver vitamins and minerals our body needs to function properly. The whole notion that antioxidants can slow down aging is a huge oversimplification of the aging process.

We also age because of epigenetic dysregulation, protein accumulation, lysosomal dysfunction, telomere shortening, crosslinking, mitochondrial dysfunction in which most mitochondrial damage is not caused by oxidative damage, but by mutations in the mitochondrial DNA as a consequence of mitochondrial division , and so on.

Aging is much more complex than just free radicals damaging our cellular machinery. In conclusion, taking antioxidants is not a good way to extend your lifespan. Of course, when you are deficient in specific antioxidants, such as vitamin A, vitamin E or other vitamins, taking these antioxidants can be very useful.

To slow down aging and to extend human lifespan, we need to look beyond oxidants and their counterparts, antioxidants. We need to take in substances that act on various other aging mechanisms, like epigenetic dysregulation, protein accumulation and mitochondrial dysfunction.

For this, we created NOVOS Core. Our foundational formulation, NOVOS Core, targets all the root causes of aging to promote longevity, appearance, cognition, and energy. Slow down aging with these 12 highly-effective longevity ingredients in one daily dose, which you can mix with water to drink.

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Includes comprehensive guidance on how to improve your scores with lifestyle upgrades. This is the fifth and final installment of our series of articles that covered the world of longevity and explored the various pathways that intertwine lifespan and healthspan.

This article […]. Some people worry that pterostilbene increases LDL cholesterol and that this is a bad thing. A study done in found that people who took pterostilbene supplements had an increase […]. Both animal and human studies have painted a disappointing picture about the role of antioxidants in longevity.

Many studies have shown that antioxidants can even be dangerous.