The proportion of people aged 65 and older in the United States (US) was estimated at 16.8% in 2020 and that number is predicted to be over 20%, or about 65 million people, by the year 2040 (Institute, 2020; Aging Ao, 2021). By 2030, older people are projected to outnumber children in the US for the first time in history (Vespa et al., 2020). Aging is associated with a variety of multi-system changes that significantly affect quality of life. Alterations in cognitive function and mood are two such changes that can have a significant impact on the lives of elderly people. A majority of elderly individuals experience some form of memory loss that affects their activities of daily life (Murman, 2015). These include episodic and source memory deficits (Cansino, 2009; Johnson et al., 2016; Park et al., 2017), decreased sensitivity to novelty (Fandakova et al., 2014) and impairment in executive function tasks such as attention, planning, inhibitory control and cognitive flexibility (Park et al., 2002; Buckner, 2004; Methqal et al., 2017). Changes in mood are also widespread in the aging population. It is estimated that 29% of people over the age of 55 experience some type of mental health concern, among which anxiety and depression are most common (CfDCaPaNAoCD, 2008). The proportion of older adults with depressive symptoms increases with age (Services UDoHaH, 1999) and the prevalence of anxiety disorders is estimated to be as high as 17% in the elderly population. Despite the prevalence of these age-related changes, therapeutic options remain limited, which has led many in the elderly population to seek out alternative therapies. It is estimated that about 60% of older individuals in the US have used at least one complementary and alternative medicine in the last year (Golden et al., 2023). In particular, botanical interventions are popular as they have been purported to promote resilience to age-related changes (Nascimento et al., 2016; Ward and Pasinetti, 2016).

Centella asiatica (CA) is an edible plant used in traditional Chinese and Ayurvedic medicine to improve brain function and boost memory (Kapoor, 1990; Shinomol and Muralidhara, 2011) and is marketed in the United States as the dietary supplement “gotu kola” (Newall et al., 1996). CA has many reported neurobehavioral effects, including anxiolytic, anti-depressant and cognitive enhancing properties. Anxiolytic and anti-depressant-like effects of the plant have been observed in healthy rodents as well as multiple models of stress including olfactory bulbectomy, chronic immobilization stress and sleep deprivation-induced stress (Wijeweera et al., 2006; Kalshetty et al., 2012; Selvi et al., 2012; Wanasuntronwong et al., 2012; Chanana and Kumar, 2016; Jagadeesan et al., 2019). Similar effects have been noted in a handful of human trials where CA treatment reduced anxiety in elderly adults with mild cognitive impairment (Tiwari et al., 2008) and decreased symptoms of anxiety and depression in middle-aged adults with generalized anxiety disorder (Jana et al., 2010). A substantial literature also supports the cognitive enhancing effects of CA. In human tests, CA extract or dried herb improved cognitive function of healthy middle-aged and elderly (Wattanathorn et al., 2008; Dev et al., 2009) individuals as well as elderly subjects with mild cognitive impairment (Tiwari et al., 2008). These effects have been recapitulated in rodent models as well, where CA was shown to improve learning and memory in healthy rats (Veerendra Kumar and Gupta, 2002) and in mouse models of neurotoxicity (Gupta et al., 2003; Veerendra Kumar and Gupta, 2003). Our own group has also shown that a low dose of the water extract of CA (CAW) in the drinking water can improve cognitive function in mouse models of aging and Alzheimer’s disease (Soumyanath et al., 2012; Gray et al., 2016; Gray et al., 2018a; Gray et al., 2018b).

This study builds on previous work to further investigate the effects of CAW on cognition, measures of anxiety and depressive-like behavior in healthy aged mice. For the first time the effects of increasing concentrations of CAW on cognitive performance in aged mice will be evaluated. Additionally, this is the first study investigating the effects of CAW on anxiety and depression.

The first part of the study evaluated the effects of 5 weeks of treatment with increasing concentrations of CAW integrated in the diet on learning, memory, executive function, measures of anxiety and depressive-like behavior in 3- and 18-month-old C57BL6 mice. The study then went on to compare the behavioral effects in 18-month-old C57BL/6J mice of the highest concentration of CAW administered in the diet versus in the drinking water.

Alterations in cognition and mood are highly prevalent in the aging population and negatively affect quality of life. Approximately two out of three Americans experience at least some form of cognitive impairment by age 70 (Hale et al., 2020). Changes in mood are similarly common in the aging population, with a prevalence of depressive symptoms and anxiety of nearly 1 in 5 in elderly people (Services UDoHaH, 1999; CfDCaPaNAoCD, 2008). All of these conditions also often severely curtail daily life activities in older people further underscoring the need for therapies that can increase resilience to these age-related challenges.

The cognitive, antidepressant and anxiolytic effects of C. asiatica have been demonstrated many times in both rodent and human studies (Veerendra Kumar and Gupta, 2003; Wijeweera et al., 2006; Tiwari et al., 2008; Wattanathorn et al., 2008; Dev et al., 2009; Jana et al., 2010; Kalshetty et al., 2012; Selvi et al., 2012; Wanasuntronwong et al., 2012; Chanana and Kumar, 2016). Our group has previously shown that the water extract of C. asiatica (CAW) administered in the drinking water at 2 mg/mL (calculated to deliver 200 mg/kg/d CAW) improves cognitive function in mouse models of aging and neurodegenerative disease (Soumyanath et al., 2012; Gray et al., 2016; Gray et al., 2018a; Gray et al., 2018b; Matthews et al., 2019). This study builds on that previous research to explore the ability of CAW to reverse age-related changes in anxiety and depression. The effects of multiple doses of CAW on cognitive and mood endpoints and assessed sex differences in response to the extract was also evaluated.

Impaired performance in aged mice (compared to young mice) was seen in ODRL, NORT and OF, although for OF the age effect was only observed in female mice. The impaired performance seen in these tasks is in line with previous reports that also demonstrated diminished ODRL, NORT and OF performance in aged mice relative to young ones (Patel and Larson, 2009; Shoji and Miyakawa, 2019; Yang et al., 2019). There was not, however, any observed age-related deficits in LD-Box or FST or for male mice in the OF. This is in contrast to previous reports in the literature where poorer performance was observed in LD-BOX, FST and OF (Stilling et al., 2014; Lim et al., 2018; Shoji and Miyakawa, 2019) and could be related to differences in the ages tested or it might reflect subtle differences in testing protocol and metrics recorded. For instance, in one study using LD-BOX, reduced distance traveled in the dark was seen in 23-month-old mice but not 17-month-old mice as compared to 3-month-old animals (Shoji and Miyakawa, 2019). In the same study time immobile in the FST test was increased in 24-month-old mice relative to 3-month- old animals but no differences were seen between the 3- and 18-month-old groups (Shoji and Miyakawa, 2019). This suggests that perhaps a larger difference in age is required to observe differences in performance in the FST and LD-BOX.

When CAW was administered in the diet, dose-dependent attenuation of age-related deficits in learning and cognitive flexibility were observed. Old mice receiving the highest concentration of CAW in the diet (1,000 mg/kg/d) showed improved performance in the ODRL test compared to what was seen in the young, 3-month-old mice. The effect of CAW in the diet on recognition memory was less robust. Significant improvements were seen in old male mice treated with 500 mg/kg/d in the 2 h NORT test and in old female mice treated with the same concentration in the 24 h test but no significant deficits were detected between old controls and young controls. Surprisingly, there was also a significant impairment in NORT performance in the old male mice treated with 200 mg/kg/d in the 24 h test. The lack of a consistent effect of CAW in the diet on NORT performance is in contrast to the clear benefit observed in the ODRL. It is possible this discrepancy could be explained by a differential effect of CAW in distinct brain regions. The ODRL is mediated by the medial prefrontal cortex (McAlonan and Brown, 2003; Dalley et al., 2004), while NORT performance relies on inputs from perirhinal cortex, hippocampus, medial prefrontal cortex and medial dorsal thalamus (Warburton and Brown, 2015). While further studies are needed to confirm that the levels of CAW constituent compounds are consistent across various brain regions, regional differences seem unlikely given our previous report of similar effects of CAW on antioxidant gene expression in the cortex, hippocampus and cerebellum of aged mice (Gray et al., 2016). It is more probable that the different effects seen in the ODRL and NORT reflect differences in the sensitivity and variability of each test. Another consideration is that anxiety towards exploring novel objects (neophobia) might have contributed to these divergent findings as well. There was a much greater amount of variability observed between animals in NORT performance which likely prevented the detection of more significant and consistent findings.

CAW given in the diet did not appear to affect metrics of anxiety in aged mice. However, there was a statistically significant decrease in time spent in the center of the open field in young female mice at all concentrations of CAW tested, suggesting elevated levels of anxiety. This effect in young mice and the lack of effect in aged mice was surprising given the purported anxiolytic effects of CAW. To the best of our knowledge, there have not been prior reports of anxiogenic effects of C. asiatica in young mice. CAW administered in the diet also had no effect on depressive-like behavior as quantified in the FST.

The limited behavioral effects of CAW in the diet were unexpected. Although our group had not previously investigated the effects of CAW on measures of anxiety or depressive like behavior, based on our many published studies on the cognitive effects of CAW a more robust effect at least on those endpoints was expected. Previous experiments have shown improvements in cognitive function in aged mice following CAW administered in the drinking water at a concentration equivalent to the 200 mg/kg/d dose of CAW used in this study (Gray et al., 2016; Gray et al., 2018a) and in a mouse model of Alzheimer’s disease increasing concentrations of CAW in the drinking water (equivalent to the 500 mg/kg/d and 1,000 mg/kg/d) resulted in even greater improvements in cognitive function (Matthews et al., 2019). Interestingly, NORT performance was also improved in both healthy aged mice treated with 200 mg/kg/d equivalent in the drinking water (Gray et al., 2018a) and with all three concentrations administered in the drinking water in the Alzheimer’s disease mouse model (Matthews et al., 2019) whereas that was not seen in the current study where CAW was administered in the diet. This led us to hypothesize that mode of administration could affect behavioral response. Therefore the next evaluation looked at the effects of the equivalent dose (1000 mg/kg/d) of CAW administered in the drinking water to old mice on the same battery of tests of cognitive function, anxiety and depression.

When old mice were treated with 1,000 mg/kg/d CAW equivalent in the drinking water, there was a significant improvement in both the ODRL and the NORT, and the improvement in ODRL elicited by CAW in the drinking water was even greater than what was seen with CAW (1,000 mg/kg/d) in the diet. There were also significant reductions in anxiety in the open field test but this affect again varied by sex, with aged female mice exhibiting significantly less time in the center than young female mice and aged female mice treated with CAW in the drinking water showing a robust increase in this metric. The apparent sex-specificity of this response is interesting. There are many reports in the literature, in both humans and rodents, of females exhibiting increased anxiety-related behavior relative to males (Bahrami and Yousefi, 2011; McLean et al., 2011; Marchette et al., 2018; Bicakci et al., 2022). It is possible therefore that the increased anxiety evident in the aged female mice made it easier to detect treatment effects than in male mice. Future studies using more sensitive assays of anxiety behavior could help clarify this issue.

The age-related increase in time immobile in the open field was attenuated with CAW in the drinking water in both sexes however, this effect is not necessarily related to changes in anxiety exclusively and might instead reflect a change in overall activity with CAW treatment. Intriguingly, CAW in the drinking water significantly increased time in the dark for aged mice in the LD-BOX test suggesting a potential anxiogenic effect. These apparent contradictory effects of CAW in the drinking water on anxiety-like behavior are puzzling. Previous reports of anxiolytic effects of various extracts of the plant have demonstrated improvements in open field and elevated plus maze (Wijeweera et al., 2006; Chanana and Kumar, 2016; Jagadeesan et al., 2019). LD-BOX was used in a study of a standardized formulation of triterpene compounds (ECa233) from C. asiatica and a reduction in anxiety was observed (Wanasuntronwong et al., 2012). However, the concentration of individual triterpenes in ECa233 was much higher (30%–50%) than in CAW (<5%) and the mice tested had been exposed to chronic immobilization stress; both factors may account for the differences in the effect of the CA extracts. Importantly, none of these reports evaluated anxiety in aged animals. In our study age-related increases in anxiety were only detected in the open field and not the LD-BOX. It would be interesting in future studies to employ the elevated plus maze and determine if deficits are evident in aged mice and if CAW in the drinking water attenuates or exacerbates them.

In our study, CAW also had no effect on depressive-like behavior quantified in the FST, regardless of whether it was administered in the diet or in the drinking water. These results are not consistent with the existing literature showing anti-depressant effects of C. asiatica extracts on performance in the FST in a mouse model of olfactory bulbectomy and in healthy young rats (Kalshetty et al., 2012; Selvi et al., 2012). Again, however, these studies were not directly investigating the effects in aged rodents and used other types of CA extracts. It is possible the FST is not sensitive enough to detect age-related changes in depressive-like behavior. If that is the case, then it is unsurprising that a treatment effect would also not be seen in the FST. Future studies could employ other tests of depressive behavior such as the tail suspension, sucrose preference test or conditioned place preference (Scheggi et al., 2018), or add an additional stressor to potentially magnify age-related differences, such as restraint stress, cage tilt or noise exposure (Atrooz et al., 2021).

One consistent result observed in the study was that effects of CAW were more often observed in aged animals than young ones. This is in line with our previous findings in younger animals where cognitive improvements were not observed at either 2 or 6 months of age (Gray et al., 2016; Zweig et al., 2021a). One reason that could account for this is that mice at those ages do not display any cognitive deficits. If a ceiling effect of maximal performance is being observed in untreated young mice, then it is not surprising that no improvement could be detected with CAW treatment. Although our group has not previously evaluated the effects of CAW on anxiety and depression in young animals it is possible that the same phenomenon is occurring in those tests as well.

Another of the most consistent findings of the present study was that CAW effects were more pronounced in the animals that received the extract in the drinking water than those that received it in the diet. While individual consumption was not assessed in this study due to the group housing set up, food and water consumption was monitored by cage and no differences were observed between treatment groups (data not shown). This suggests that palatability was not an issue in the CAW treated animals. A possible explanation for the differences in behavioral effect resulting from water or diet administration could be differing bioavailability of the constituent compounds of CAW depending on the mode of administration. In fact, a recent paper published by our group demonstrated exactly that showing that 5xFAD mice treated with CAW in the same manner as in this study (10 mg/mL in the water or the calculated equivalent in the diet for 5 weeks) had elevated plasma concentrations of constituent compounds when CAW was delivered in the drinking water compared to in the diet (Speers et al., 2024). Studies are underway to quantify constituent compounds from CAW in the plasma of treated animals to confirm whether this could explain the behavioral differences observed in this study as well.

Centella asiatica has a number of chemical components (Alcazar et al., 2020) of which triterpenes and caffeoylquinic acids (CQAs), appear to be the most relevant for its neurological effects (Gray et al., 2017). There is evidence in the literature for the ability of these compounds to penetrate the blood-brain barrier. The triterpenes asiatic acid and madecassic acid (found in C. asiatica) were detected in mouse brain following their administration as pure compounds (Yin et al., 2012) and their corresponding glycosides asiaticoside and madecassoside were observed in rat brain following administration of a C. asiatica extract or the pure compounds (Anukunwithaya et al., 2016; Hengjumrut et al., 2018). Mono and di-CQAs (Su et al., 2014), and their potential metabolites caffeic and ferulic acid have been detected in rat brain after administration of the pure compounds or botanical sources (Su et al., 2014; Gasperotti et al., 2015; Zhang et al., 2015). The regional distribution of these compounds within the brain has not been reported. Levels of constituent compounds from CAW have not been measured in the tissue of treated animals before but previous studies have reported changes in antioxidant gene expression and mitochondrial activity in the brains of mice treated with CAW (Gray et al., 2016; Gray et al., 2018b; Zweig et al., 2021b) which might suggest compound penetration although it is also possible that those are secondary effects induced by peripheral changes. Work is ongoing in to determine whether compounds from the CAW extract administered orally do in fact cross the blood brain barrier and whether differences may exist in their abundance depending on route of administration.

In summary, these findings suggest a beneficial effect of CAW on age-related changes in cognition and measures of anxiety, but not on depressive-like behavior. The magnitude of these effects was greater when CAW was administered in the drinking water as compared to when it was given in the diet. Future studies are needed to elucidate the mechanism by which CAW elicits its effects on cognition and measures of anxiety as well as to determine if active compounds from the extract reach greater systemic levels when given in the drinking water than in the diet and if these compound levels vary across different brain regions.