Positive modulation of cognition and mood in the healthy elderly volunteer following the administration of Centella asiatica Jintanaporn Wattanathorn a,∗ , Lugkana Mator b , Supaporn Muchimapura a , Terdthai Tongun a , Orapin Pasuriwong a , Nawanant Piyawatkul c , Kwanchanok Yimtae d , Bungorn Sripanidkulchai e , Jintana Singkhoraard c a Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand b Department of Physiology and Biomedical Science Program, Graduate School, Khon Kaen University, Khon Kaen 40002, Thailand c Department of Psychiatry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand d Department of Otolaryngology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand e Center for Research and Development of Herbal Health Product, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand Received 21 February 2007; received in revised form 21 November 2007; accepted 28 November 2007 Available online 4 December 2007 Abstract Aims of this study: Centellaasiaticahasareputationtorestoredeclinecognitive function in traditional medicine and in animal model. However, little evidence regarding the efficacy of Centella asiatica from systematized trials is available. Therefore, the present randomized, placebo-controlled, double-blind study investigated the effect of Centella asiatica on cognitive function of healthy elderly volunteer. Materials and methods: Twenty-eight healthy elderly participants received the plant extract at various doses ranging 250, 500 and 750 mg once daily for 2 months. Cognitive performance was assessed using the computerized test battery and event-related potential whereas mood was assessed using Bond–Lader visual analogue scales prior to the trial and after single, 1 and 2 months after treatment. Results: The results showed that the high dose of the plant extract enhanced working memory and increased N100 component amplitude of event-related potential. Improvements of self-rated mood were also found following the Centella asiatica treatment. Conclusion: Therefore, the present findings suggest the potential of Centella asiatica to attenuate the age-related decline in cognitive function and mood disorder in the healthy elderly. However, the precise mechanism(s) underlying these effects still require further investigation. © 2008 Elsevier Ireland Ltd. All rights reserved. Keywords: Centella asiatica; Cognitive function; Event-related potential; Mood; Elderly 1. Introduction During aging, a gradual deficit is present with regard to cog- nitive functions, which normally does not severely disturb the daily life activities related to the physical, mental, or social functioning in elderly adults. The age-related cognitive decline has been characterized as a diminution of attentional processes, episodic and working memory, and processing and psychomo- tor speed (Nolan and Blass, 1992; Salthouse, 1994; Earles and Salthouse, 1995; Grady and Craik, 2000; Christensen, 2001). However, this condition can eventually progress to clinically ∗ Corresponding author. Tel.: +66 43 348394. E-mail address: jintanapornw@yahoo.com (J. Wattanathorn). recognizable dementia (Chen et al., 2005; Amieva et al., 2005). Previous studies have demonstrated that age-related impair- ment in working memory is accompanied by the alteration of the event-related potential in respond to the auditory oddball tasks, a most frequent parameters examined in psychophys- iological studies of cognitive aging (Bashore et al., 1989). It was found that the latencies of P300 and N100 compo- nents increased (Oken and Kaye, 1992; Kugler et al., 1993; Anderer et al., 1996) whereas the amplitudes of these two components were decreased (Coyle et al., 1991; Nordin et al., 1999). Recently, numerous studies have suggested that the age- related cognitive decline can be prevented (Solfrizzi et al., 1999). Therefore, a new approach aimed at controlling the decrease in cognitive function was focused. Numerous previous studies had 0378-8741/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.11.038 demonstrated that fruits, vegetables and medicinal plants could prevent the occurrence of the neurochemical and behavioral changes that occurred in aging (Veerendra Kumar and Gupta, 2002; Joseph et al., 2003; Goyarzu et al., 2004). Centella asiatica (L) urban, a creeping plant in a family of Apiaceae, is found almost all over the world including in China, India and Thailand. This plant is claimed to be bene- fit for managing the central nervous system disorders, skin and gastrointestinal disorders (Subathra et al., 2005). In addition, Centella asiatica is also claimed to possess memory enhancing effect (Veerendra Kumar and Gupta, 2002), anti-inflammatory (Suguna et al., 1996), wound healing (Suguna et al., 1996), anti- cancer (Babu et al., 1995) and antioxidant properties (Zainol et al., 2003). Recently, Centella asiatica products are widely available in market as cognitive booster. However, supported document with valid biological data is still very limited. There- fore, the current study is carried out to determine the effect of Centella asiatica on the cognitive function and mood in the elderly. 2. Materials and methods 2.1. Participants Four male, and twenty-four female healthy elderly volunteers (mean age 65.05 ± 3.56 years) were recruited to participate in this study. The Ethical Committee of Faculty of Medicine, Khon Kaen University, have approved the study, and all subjects gave written informed consent. Prior to participation, each volun- teer signed an informed consent form and completed a medical health questionnaire. All subjects were also undergone exten- sive medical evaluation in order to ascertain subject suitability for entering the double-blind phase of trial. Additionally, they were free of any herbal or prescribed medication that interfered on the function of nervous system. Habitual smokers consuming more than 10 cigarettes/day were excluded from this study. All participants should abstain from caffeine containing products throughout each study day and alcohol for a minimum of 12 h prior to the test session. They were divided into four groups: placebo, CA250, CA500 and CA750 mg/day. Each group con- tained 1 male and no one dropout from the project. Moreover, no significant difference about mean age, education and body mass index among groups were observed. 2.2. Centella asiatica preparation A standardized extract of Centella asiatica was prepared by the Center for Research and Development of Herbal Health Product, Faculty of Pharmaceutical Sciences, Khon Kaen Uni- versity. All Centella asiatica used in this study was obtained from Tambon Sila, Khon Kaen Province. The plant was authenti- cated and kept as voucher specimen at Faculty of Pharmaceutical Sciences, Khon Kaen University. Standardization and confor- mity of the extract is assured by strict in-process controls during manufacture and complete analytical control of the resulting dry extract. A-day capsule contained a specialized aerial part extract containing total phenolic content equivalent to tannic acid = 29.9 mg/g. In addition, the extract also contained asiati- coside and asiatic acid were presented at concentration of 1.09 and 48.89 mg/g of crude extract, respectively. 2.3. Procedures and treatments This study was a pilot study conducted as 8-week, double- blind, placebo-controlled, randomized trial. A random list of numbers was generated by computer. After being randomly assigned to various treatment groups. Each participant received one capsule of placebo or Centella asiatica extract at various doses ranging 250, 500 and 750 mg once daily. Placebo and Centella asiatica capsules had the same colour, texture, size and smell. All participants were assessed baseline data about cognitive function, mood and quality of life and then they were assessed cognitive performance and mood after the single administration, 1 and 2 months of treatment whereas the quality of life was assessed after 2 months of treatment. The code for study allocation was only broken when the last participant completed the entire follow up. Staff involved in the collection of the study’s endpoints was instructed to follow a rigorous protocol and not to discuss any issues related to the use of medication. The review of compliance with medication and side effects was performed independently by the investigators, who were also blinded to group allocation. Subjects were asked to call the study center if they experienced any medical problems during the 60-day study period. At the end of the study, they were also asked about adverse events. Laboratory tests were drawn at baseline and follow-up visits and compared to see whether any changes suggested adverse events. 2.4. Event-related potential measurement Event-related potential (N100 and P300 amplitude and laten- cies) of all subjects were measured using an “oddball paradigm” (Kennedy et al., 2003). N100 and P300 amplitude and laten- cies were elicited with a standard auditory oddball paradigm. Frequent and target tones were presented binaurally through headphones. Participants were instructed to listen for and count infrequent target tones (650 Hz, 60 dB, 200 ms), which occurred randomly amongst 82–90 frequent non-target tones (1 kHz, 60 dB, 200 ms). Inter-stimulus intervals varied randomly between 1250 and 3000 ms. The latency range in which the N100 and P300 maximum amplitudes and latencies were determined was between 65–135 and 280–375 ms, respectively. However, any peaks outside this range were measured manually, with preliminary visual checking of all peaks prior to measurement. 2.5. Computerized assessment battery test The computerized assessment battery test used in this study has been modified from the CDR computerized assessment bat- tery test used in hundreds of European and North American drug trials and sensitive to acute cognitive improvements as well as impairments with a wide variety of substances (Moss et al., 1998; Wesnes, 2001). Presentation was performed using note- book computers with high-resolution VGA colour monitor and, with the exception of written word recall tests, all responses were recorded via two-button (yes/no) response box. The entire selection of tasks took approximately 20 min. Tests were administered in the following order: Word presentation: Fifteen words, matched for frequency and concreteness, were presented in sequence on the monitor for the participant to remember. The stimulus duration was 1 s, as was the inter-stimulus interval. Picture presentation: A series of 20 photographic images was presented on the monitor at the rate of 1 every 3 s, with a stimulus duration of 1 s, for the participant to remember. Simple reaction time: The participant was instructed to press the ‘yes’ response button as quickly as possible every time the word ‘yes’ was presented on the monitor. Fifty stimuli were presented with an inter-stimulus interval that varied randomly between 1 and 3.5 s. Reaction times were recorded in millisec- onds. Digit vigilance task: A target digit was randomly selected and constantly displayed to the right of the monitor screen. A series of digits was presented in the centre of the screen at the rate of 80 min −1 and the participant was required to press the ‘yes’ button as quickly as possible every time the digit in the series matched the target digit. The task lasted 1 min and there were 15 stimulus–target matches. Task measures were accuracy (%), reaction time (milliseconds), and number of false alarms. Choice reaction time : Either the word ‘no’ or the word ‘yes’ was presented on the monitor and the participant was required to press the corresponding button as quickly as possible. There were 50 trials, of which the stimulus word was chosen randomly with equal probability, with a randomly varying inter-stimulus interval of between 1 and 3.5 s. Reaction times (millisecond) and accuracy (%) were recorded. Spatial working memory: A pictorial representation of a house was presented on the screen with four of its nine windows lit. The participant was instructed to memorize the position of the illuminated windows. In 36 subsequent presentations of the house, one of the windows was illuminated and the participant decided whether or not this matched one of the lighted windows in the original presentation. The participant made their response by pressing the ‘yes’ or ‘no’ response button as quickly as pos- sible. Mean reaction times were measured in milliseconds and the accuracy of responses to both original and novel (distractor) stimuli were recorded as percentages that were used to derive a ‘percentage greater than chance performance’ score. Numeric working memory: Five digits were presented sequentially for the participant to hold in memory. This was followed by a series of 30 probe digits for each of which the participant decided whether or not it had been in the original series and pressed the ‘yes’ or ‘no’ response button as appropri- ate, as quickly as possible. This was repeated two further times with different stimuli and probe digits. Mean reaction times were measured in milliseconds and the accuracy of responses to both original and novel (distractor) stimuli were recorded as percent- ages that were used to derive a ‘percentage greater than chance performance ’ score. Delayed word recognition: The original words plus 15 dis- tractor words were presented one at a time in a randomized order. For each word, the participant indicated whether or not he or she recognized it as being included in the original list of words by pressing the ‘yes’ or ‘no’ button as appropriate and as quickly as possible. Mean reaction times were measured in milliseconds and the accuracy of responses to both original and novel (dis- tractor) stimuli were recorded as percentages that were used to derive a ‘percentage greater than chance performance’ score. Delayed picture recognition: The original pictures plus 20 distractor pictures were presented one at a time in a randomized order. For each picture, participants indicated whether or not it was recognized as being from the original series by pressing the ‘yes’ or ‘no’ button as appropriate and as quickly as possible. Mean reaction times were measured in milliseconds and the accuracy of responses to both original and novel (distractor) stimuli were recorded as percentages that were used to derive a ‘percentage greater than chance performance’ score. 2.6. Subjective mood measurement The 16 visual analogue scales of Bond–Lader were combined as recommended by the authors to form three mood factors: ‘alert’, ‘calm’ and ‘content’. 2.7. Statistical analysis All data are expressed as mean ± S.E.M. Comparisons between placebo and various doses of Centella asiatica at differ- ent time points were made using analysis of variance (ANOVA). Statistical significance was set at p-value < 0.05. 3. Results 3.1. Characteristics of subjects The baseline data about characteristic of subjects in all groups were shown in Table 1. No significant difference of all parame- ters among various groups was observed. Table 1 Characteristics of subjects Baseline data Placebo Centella asiatica 250 Centella asiatica 500 Centella asiatica 750 Age 65.88 ± 5.11 67.25 ± 1.39 62.00 ± 4.34 64.75 ± 2.71, p = 0.0511 Education year 11.50 ± 6.21 12.25 ± 5.50 10.38 ± 5.80 14.14 ± 4.91, p = 0.5393 Full scale IQ 102.50 ± 10.61 99.17 ± 16.68 91.40 ± 17.80 99.33 ± 11.91, p = 0.7605 Blood sugar 93.29 ± 9.32 93.25 ± 6.61 89.75 ± 5.95 96.75 ± 11.13, p = 0.4500 Blood pressure 120.13 ± 10.26/77.75 ± 5.52 121.50 ± 10.89/78.13 ± 7.28 123.50 ± 7.76/80.38 ± 2.77 124.75 ± 6.76, p = 0.7465/81.25 ± 4.30, p = 0.4794 Table 2 Mean amplitudes and latencies (S.D.) of event-related potential elicited by oddball paradigm for each group at Fz electrode Wave Pre-dose baseline Post-dose Single dose 1 month 2 month P300 latency Placebo 352.25 ± 27.74 339.63 ± 31.52 342.13 ± 25.67 330.63 ± 27.07 CA250 333.38 ± 31.18 337.75 ± 25.39 353.13 ± 36.99 348.50 ± 31.27 CA500 345.00 ± 20.65 336.00 ± 34.90 343.50 ± 28.16 332.88 ± 29.94 CA750 365.00 ± 31.48 337.38 ± 37.89 343.50 ± 35.27 369.88 ± 42.97 F(3,28) = 1.7785, p = 0.1741 F(3,28) = 0.0167, p = 0.9970 F(3,28) = 0.2035, p = 0.8931 F(3,28) = 2.4673, p = 0.0828 P300 amplitude Placebo 574.38 ± 410.53 557.75 ± 538.67 893.75 ± 871.72 1401.00 ± 1325.39 CA250 1595.00 ± 1918.49 1119.38 ± 960.73 801.00 ± 456.01 816.50 ± 527.87 CA500 890.13 ± 1178.20 1187.25 ± 1181.66 715.50 ± 543.00 1456.50 ± 1367.99 CA750 754.25 ± 686.50 863.00 ± 923.14 914.38 ± 823.24 844.63 ± 523.17 F(3,28) = 1.1192, p = 0.3580 F(3,28) = 0.7552, p = 0.5287 F(3,28) = 0.1381, 0.9364 F(3,28) = 0.1381, p = 0.9364 N100 latency Placebo 109.75 ± 29.40 100.69 ± 27.74 93.75 ± 22.63 106.63 ± 2359 CA250 103.56 ± 16.27 108.25 ± 22.71 107.25 ± 17.77 107.56 ± 15.67 CA500 100.56 ± 10.13 98.31 ± 8.09 100.25 ± 16.86 95.38 ± 12.20 CA750 94.56 ± 13.36 102.25 ± 12.26 102.38 ± 13.85 106.75 ± 12.74 F(3,28) = 0.5893, p = 0.6271 F(3,28) = 0.4079, p = 0.7485 F(3,28) = 0.7694, p = 0.5209 F(3,28) = 0.9727, p = 0.4195 N100 amplitude Placebo 3241.25 ± 2752.75 3915.38 ± 3059.96 3621.88 ± 1122.45 3655.88 ± 1903.34 CA250 5408.75 ± 3108.07 4297.50 ± 2017.30 4836.25 ± 1961.17 5005.00 ± 1639.17 CA500 4437.50 ± 2414.69 4515.00 ± 2114.88 3265.00 ± 2340.02 4028.13 ± 1944.52 CA750 4895.39 ± 2422.85 4218.75 ± 1219.84 4151.88 ± 1464.45 6691.25 ± 1542.46** F(3,28) = 0.9687, p = 0.4213 F(3,28) = 0.1684, p = 0.9168 F(3,28) = 1.1748, p = 0.3370 F(3,28) = 4.7179, p = 0.0087 N100 amplitude **p-value < 0.01 compared with placebo (F(3,28) = 4.7179, p < 0.01). N100 latency of CA750 compared with placebo (F(3,28) = 0.9727, p = 0.4195). 3.2. Event-related potential components (ERP) The effects of Centella asiatica on ERP components were shown in Table 2. The pre-dose baseline data of latency and amplitude of both N100 [F(3,28) = 0.9068, p = 0.4503; F(3,28) = 1.0372, p = 0.3913] and P300 [F(3,28) = 1.7785, p = 0.1741; F(3,28) = 1.1192, p = 0.3580] showed no significant difference. After 2 months of treatment with Centella asiatica at dose of 750 mg/day, the subject’s N100 amplitude significantly increased [F(3,28) = 4.7179, p = 0.0087] whereas no signifi- cant change in N100 latency was observed [F(3,28) = 0.9727, p = 0.4198]. In addition, there were no significant changes in either the amplitude or latency of P300 [F(3,28) = 0.9180, p = 0.4449; F(3,28) = 2.4673, p = 0.0828]. 3.3. Cognitive measures Prior to the determination of Centella asiatica on cognitive function, baseline data and mean pre-dose raw baseline scores for all four conditions (placebo, 250, 500 and 750 mg Centella asiatica) for each individual task scores were subjected to a one-way ANOVA. There were no significant differences on any measure. Mean pre-dose baseline raw scores and change from base- line scores for each condition at each post-dose time point on the individual task outcome measures are represented in Table 3. The results showed that the Centella asiatica extract appeared to decrease the reaction time while increased the %accuracy of working memory, which indicated that the plant extract exerted its influence on both speed and quality of working mem- ory. However, the significant changes in reaction times were observed only in choice, spatial, numeric and picture reaction times while the significant changes of %accuracy were observed only in spatial, numeric, word and picture recognition due to the high variation of other parameters and limited number of our subjects. 3.4. Bond–Lader mood scales Mean raw scores on the ‘alert’, ‘content’ and ‘calm’ factors obtained from the Bond–Lader visual analogue scales for each condition across each session are displayed in Table 4. 3.4.1. Alert factor It was found that the participants’ subjective ratings score about alert factor of Centella asiatica treated group at dose of 750 mg/day showed a significant increase, in compari- son to placebo rating, after 1 and 2 months of treatment [F(3,28) = 4.0828, p = 0.0016; F(3,28) = 3.7149, p = 0.0229]. 3.4.2. Content factor The present data showed that the rating score about content factor obtained from Bond–Lader visual analogue scale showed no significant difference between placebo and various groups of Centella asiatica treatment. 3.4.3. Calm factor The effect of Centella asiatica extract on clam factor was also determined. The results showed that Centella asiatica at all dosage range used in this study significantly increased the calm factor after 1 and 2 months of treatment [F(3,28) = 3.3943, p = 0.0316; F (3,28) = 4.2124, p = 0.0141]. Table 3 Mean pre-dose baseline raw scores and change from baseline scores for each condition at each post-dose time point on the individual task outcome measures Measure Pre-dose baseline score Post-dose 1 h 1 month 2 month Simple reaction time (ms) Placebo 626.09 ± 99.94 620.87 ± 88.74 671.21 ± 141.30 693.47 ± 180.39 CA250 640.46 ± 223.62 637.74 ± 233.15 621.38 ± 249.05 573.87 ± 151.92 CA500 636.61 ± 152.38 592.38 ± 134.25 584.19 ± 139.50 580.01 ± 133.32 CA750 615.47 ± 154.01 641.05 ± 227.45 616.98 ± 142.59 586.17 ± 156.73 F(3,28) = 0.0378, p = 0.9900 F(3,28) = 0.1200, p = 0.9476 F(3,28) = 0.3386, p = 0.7975 F(3,28) = 1.0594, p = 0.3820 Digit vigilance accuracy (%) Placebo 40.95 ± 25.94 60.00 ± 26.10 49.05 ± 31.43 64.45 ± 23.35 CA250 58.89 ± 22.08 70.00 ± 19.20 56.67 ± 37.71 67.52 ± 31.61 CA500 50.47 ± 26.35 64.76 ± 34.58 68.57 ± 24.26 66.67 ± 29.81 CA750 54.58 ± 26.42 54.17 ± 30.95 71.43 ± 26.31 68.31 ± 32.61 F(3,24) = 0.6100, p = 0.6150 F(3,24) = 0.3848, p = 0.7649 F (3,25) = 0.8173, p = 0.4965 F(3,25) = 0.0207, p = 0.9958 Digit vigilance reaction time (ms) Placebo 691.21 ± 37.71 684.53 ± 12.28 590.90 ± 190.51 676.01 ± 22.59 CA250 642.43 ± 46.81 678.70 ± 109.13 671.47 ± 75.84 651.12 ± 57.73 CA500 640.38 ± 16.18 762.54 ± 123.85 662.76 ± 45.27 672.85 ± 30.08 CA750 654.24 ± 53.19 654.44 ± 50.38 635.99 ± 38.53 658.75 ± 47.85 F(3,24) = 0.7618, p = 0.5266 F(3,24) = 0.3650, 0.7789 F(3,25) = 0.8499, p = 0.4798 F(3,25) = 0.5326, p = 0.6641 Choice reaction time (ms) Placebo 931.22 ± 171.64 883.01 ± 155.60 1012.41 ± 161.47 1031.06 ± 219.37 CA250 967.15 ± 320.46 957.08 ± 338.17 874.25 ± 218.00 834.25 ± 208.19* CA500 922.34 ± 119.26 911.10 ± 185.14 885.27 ± 213.84 798.65 ± 100.20* CA750 842.24 ± 124.81 860.17 ± 109.42 854.42 ± 119.19 838.74 ± 106.04* F(3,28) = 0.5486, p = 0.6532 F(3,28) = 0.2936, p = 0.8296 F(3,28) = 1.2318, p = 0.3167 F(3,28) = 3.1369, p = 0.0411 Spatial memory (%accuracy) Placebo 81.94 ± 14.47 78.45 ± 21.10 78.47 ± 17.36 80.21 ± 17.09 CA250 77.78 ± 16.80 86.81 ± 15.55 88.54 ± 11.35 82.29 ± 20.19 CA500 83.68 ± 24.10 91.67 ± 13.28 84.38 ± 15.99 86.78 ± 10.55 CA750 84.79 ± 24.04 84.37 ± 21.92 90.28 ± 12.69 92.36 ± 16.18 F(3,28) = 0.1839, p = 0.9064 F(3,28) = 0.7194, p = 0.5488 F(3,28) = 1.0419, p = 0.3893 F(3,28) = 0.8645, p = 0.4710 Spatial memory reaction time (ms) Placebo 2385.29 ± 761.48 2020.62 ± 441.03 1866.21 ± 357.60 1822.57 ± 594.97 CA250 2116.33 ± 580.06 1954.26 ± 556.26 1906.65 ± 595.45 1714.48 ± 424.70 CA500 2088.62 ± 847.33 1604.90 ± 339.26 1690.60 ± 404.47 1429.49 ± 220.02 CA750 1590.78 ± 220.39 1506.79 ± 218.17* 1486.29 ± 286.23 1290.17 ± 219.74* F(3,28) = 2.0860, p = 0.1247 F(3,28) = 3.0928, p = 0.0430 F(3,28) = 1.6188, p = 0.2073 F(3,28) = 3.0860, p = 0.0433 Numeric working memory (%accuracy) Placebo 85.42 ± 11.50 88.33 ± 11.13 88.33 ± 13.92 83.75 ± 15.37 CA250 98.33 ± 3.56* 97.50 ± 3.45* 97.09 ± 4.52 96.67 ± 3.09* CA500 92.50 ± 10.04 95.00 ± 9.09 90.00 ± 19.19 95.00 ± 7.56* CA750 96.25 ± 5.17* 97.08 ± 3.75* 97.92 ± 1.72 97.50 ± 3.45* F(3,28) = 3.7734, p = 0.0216 F(3,28) = 2.4765, p = 0.0820 F(3,28) = 1.2977, p = 0.2947 F(3,28) = 4.1683, p = 0.0147 Numeric working memory reaction time (ms) Placebo 1527.65 ± 154.88 1325.31 ± 160.15 1286.51 ± 203.46 1366.07 ± 237.31 CA250 1429.32 ± 707.37 1329.09 ± 467.90 1205.05 ± 382.51 1209.00 ± 271.95 CA500 1531.55 ± 406.20 1268.84 ± 252.98 1251.14 ± 213.54 1170.19 ± 219.44 CA750 1195.95 ± 165.16 1211.97 ± 196.32 1075.45 ± 173.86 984.49 ± 217.75* F(3,28) = 1.1061, p = 0.3632 F(3,28) = 0.2814, p = 0.8384 F(3,28) = 1.0344, p = 0.3925 F(3,28) = 3.4838, p = 0.0288 Word recognition (%accuracy) Placebo 75.42 ± 13.21 76.25 ± 7.65 79.58 ± 14.41 81.24 ± 9.73 CA250 85.00 ± 11.13 79.58 ± 10.15 76.67 ± 14.47 83.33 ± 9.59 CA500 88.33 ± 10.69 82.08 ± 7.95 88.34 ± 5.91 90.83 ± 6.36* CA750 86.67 ± 7.97 83.75 ± 5.18 90.00 ± 6.42 90.42 ± 4.15* F(3,28) = 2.2514, p = 0.1043 F(3,28) = 1.3539, p = 0.2771 F(3,28) = 2.7586, p = 0.0609 F(3,28) = 3.1333, p = 0.0412 Table 3 (Continued ) Measure Pre-dose baseline score Post-dose 1 h 1 month 2 month Word recognition reaction time (ms) Placebo 2164.17 ± 441.54 1815.69 ± 369.90 1652.98 ± 403.65 1647.23 ± 324.29 CA250 2011.64 ± 619.17 1745.24 ± 546.15 1767.28 ± 389.70 1626.07 ± 627.49 CA500 1953.14 ± 705.49 1582.76 ± 334.46 1459.81 ± 309.07 1439.16 ± 278.40 CA750 1658.19 ± 436.07 1555.17 ± 490.51 1450.09 ± 250.96 1400.85 ± 316.46 F(3,28) = 1.1355, p = 0.3517 F(3,28) = 0.6446, p = 0.5928 F(3,28) = 1.6157, p = 0.2080 F(3,28) = 0.7551, p = 0.5287 Picture recognition (%accuracy) Placebo 84.38 ± 10.84 86.88 ± 7.99 93.75 ± 5.18 89.38 ± 5.63 CA250 86.88 ± 7.99 91.25 ± 9.54 88.75 ± 9.54 89.38 ± 11.16 CA500 88.75 ± 9.16 85.00 ± 11.34 88.75 ± 6.94 90.00 ± 3.78 CA750 87.50 ± 5.98 83.13 ± 20.17 92.50 ± 9.64 96.88 ± 3.72* F(3,28) = 0.3600, p = 0.7823 F(3,28) = 0.5614, p = 0.6449 F (3,28) = 0.8207, p = 0.4934 F(3,28) = 2.3220, p = 0.0967 Picture recognition reaction time (ms) Placebo 2081.19 ± 787.08 1654.23 ± 245.86 1938.81 ± 240.94 1586.45 ± 470.62 CA250 2072.40 ± 480.36 1765.95 ± 409.49 1633.44 ± 282.12*** 1677.62 ± 426.63 CA500 2140.18 ± 958.35 1504.79 ± 321.24 1473.51 ± 209.53*** 1364.52 ± 160.51 CA750 1619.25 ± 285.32 1512.22 ± 290.68 1448.87 ± 198.84*** 1390.04 ± 177.10 F(3,28) = 1.0065, p = 0.4045 F(3,28) = 1.2032, 0.3267 F(3,28) = 7.3583, p = 0.0009 F(3,28) = 1.6066, p = 0.2101 (*) (**) p-value < 0.05 and 0.01 when compared with placebo group, respectively. 4. Discussion The present study investigated the effect of Centella asiatica extract on the cognitive performance and mood in the elderly. The results from this study suggest that the ingestion of single dose of Centella asiatica can modulate only the reaction time of spatial memory and %accuracy of numeric working memory in the healthy elderly. Recently, it was found that the numeric working memory process involved the function of lateral prefrontal cortex (PFC) (D’Esposito et al., 1999; Smith and Jonides, 1999) whereas the spatial memory was reported to involve hippocampus (Moser and Moser, 1998). Previous studies demonstrated that the numeric working memory could be modulated by dopamine, norepinephrine and other main neurotransmitters (Goldman- Rakic et al., 2000; Arnsten and Robbins, 2002) and during spatial memory task, both acetylcholine and serotonin in hip- pocampus were simultaneously activated (Stancampiano et al., 1999). Therefore, the acute effect of Centella asiatica on the numeric memory and spatial memory might partly occur via the modulation of dopamine and norepinephrine in prefrontal cor- tex together with the modulation of acetylcholine and serotonin in hippocampus. However, the possible underlying mecha- nisms for acute effect of Centella asiatica on the numeric Table 4 Mean raw scores on the ‘alert’, ‘content’ and ‘calm’ factors obtained from the Bond–Lader visual analogue scales for each condition across each session Mood Pre-dose baseline score Post-dose 1 month 2 month Alertness Placebo 67.88 ± 7.92 62.00 ± 8.55 62.50 ± 7.96 CA250 64.88 ± 11.78 68.00 ± 7.13 71.88 ± 6.83 CA500 66.13 ± 6.62 69.75 ± 7.96 68.25 ± 10.50 CA750 71.88 ± 5.11 74.63 ± 5.07** 75.88 ± 7.59* F(3,28) = 1.0976, p = 0.3665 F(3,28) = 4.0825, p = 0.0159 F(3,28) = 3.7149, p = 0.0229 Contentedness Placebo 40.63 ± 6.16 37.75 ± 7.50 38.25 ± 5.85 CA250 39.00 ± 6.72 40.00 ± 3.89 41.88 ± 3.80 CA500 41.25 ± 3.06 40.88 ± 2.75 40.13 ± 4.79 CA750 42.38 ± 3.54 42.63 ± 3.07 42.38 ± 3.50 F(3,28) = 0.6046, p = 0.6175 F(3,28) = 1.4889, p = 0.2390 F(3,28) = 1.3368, p = 0.2824 Calmness Placebo 15.00 ± 2.00 14.25 ± 2.19 13.38 ± 2.07 CA250 15.38 ± 3.25 16.00 ± 2.00 15.88 ± 2.10* CA500 15.38 ± 1.19 16.13 ± 0.99 15.63 ± 2.56* CA750 16.25 ± 2.12 16.75 ± 1.04* 17.00 ± 1.51* F(3,28) = 0.4398, p = 0.7263 F(3,28) = 3.3943, p = 0.0316 F(3,28) = 4.2124, 0.0141 *p < 0.05 compared with placebo, **p < 0.01 compared with placebo. memory and spatial memory still required further investiga- tion. The repetitive administration of Centella asiatica further to 2 months showed the significant increase in %accuracy of both numeric working memory and word recognition. In addition, Centella asiatica also showed significant increase in reaction time of both numeric working memory and spatial memory. Thus, the present findings suggest that Centella asiatica can improve both speed and accuracy of working memory. Since the results showed the dissociation of temporal profiles in %accu- racy of numeric working memory and spatial memory reaction time, thus, it was reasonable to suggest that the relationship between the Centella asiatica treatment and working memory following repetitive administration of this substance might not be a simple relationship. Interestingly, Centella asiatica also increased the amplitude of N100 component after 2 months of treatment with high dose of Centella asiatica whereas no significant changes in N100 latency and P300 latency and amplitude were observed. Previous studies have demonstrated that N100 component is interpreted as unitary “sensory gain effect” (Wijers et al., 1996; Hillyard et al., 1998) and related to attention (Wood et al., 2006) especially the enhanced processing of the attended location including the spatial properties of the attend stimulus (Mangun et al., 1993). Recently, it has been reported that this activity involves the function of temporal lobe (Sabri et al., 2004). Previous study demonstrated that hippocampus, an important area in the inner temporal lobe, played a crucial role on working memory processing both in human and in primate. Moreover, the quality of memory also depended on many factors particularly efficiency of storage and retrieval processes which in turn influ- enced by attention process (Cohen and Squire, 1980;Tulvingand Schacter, 1990). In addition, it had been reported that attention could modify the function of hippocampus (Kentros et al.,2004). Therefore, the increase in N100 component amplitude in this study was corresponding to the results which demonstrated the improve working memory induced by Centella asiatica. Based on these pieces of evidence, we did suggest that the effect of Centella asiatica to improve working memory might be associ- ated with the improve attention manifested as the increase N1 amplitude. It is very striking that Centella asiatica improves not only the cognitive performance but also the mood. The high dose of Centella asiatica could increase calmness and alertness after 1 and 2 months of treatment. In addition, the significant increase in calmness was also observed after Centella asiatica treatment at medium and high doses for 2 months. Our present data demon- strated the improvement of both alertness and calmness together with the increase attention, therefore we suggested that the plant extract might possibly improve positive emotion such as alert- ness and calmness, which in turn improve attention and finally improve working memory. However, further study about the pre- cise relationship among various parameters mentioned above and possible underlying mechanism are still essential. In conclusion, this study is the first study to demonstrate the scientific document with many valid biological markers to support the positive modulation effect of Centella asiatica on the cognitive function and mood in the healthy elderly. However, the precise mechanism(s) underlying these effects still require further investigation. Acknowledgements This study was supported by Faculty of Medicine, Center for Research and Development of Herbal Health Product and Graduate School, Khon Kaen University. Moreover, gratitude was also extends to the Cognitive Drug Research Company who first developed the CDR battery test. References Amieva, H., Jacqmin-Gadda, H., Orgogozo, J.M., Le Carret, N., Helmer, C., Letenneur, L., Barberger-Gateau, P., Fabrigoule, C., Dartigues, J.F., 2005. The 9-year cognitive decline before dementia of the Alzheimer type: a prospective population-based study. Brain 128, 1093–1101. Anderer, P.P., Semlitsch, H.V., Saletu, B., 1996. Multichannel auditory event- related brain potentials: effect of normal aging on the scalp distribution of N1, P2, N2 and P300 latencies and amplitude. Electroencephalography and Clinical Neurophysiology 99, 458–472. Arnsten, A.F.T., Robbins, T.W., 2002. Neurochemical modulation of prefrontal function in humans and animals. In: Stuss, D.T., Knight, R.T. (Eds.), Prin- ciples of Frontal Lobe Function. Oxford University Press, New York. Babu, T.D., Kuttan, G., Padikkala, J., 1995.Cytotoxic and anti-tumourproperties of certain taxa of Umbelliferae with special reference to Centella asiatica (L.) Urban. Journal of Ethnopharmacology 48, 53–57. Bashore, T.R., Osman, A., Hefley, E.F., 1989. Mental slowing in elderly per- sons: a cognitive psychophysiological analysis. Psychology and Aging 4, 235–244. Chen, Y., Han, T., Rui, Y., Yin, M., Qin, L., Zheng, H., 2005. Effects of total triterpenes of Centella asiatica on the corticosterone levels in serum and contents of monoamine in depression rat brain. Journal of Chinese Medicinal 28, 492–496. Christensen, H., 2001. What cognitive changes can be expected with normal ageing? The Australia and New Zealand Journal of Psychiatry 35, 768– 775. Cohen, N.J., Squire, L.R., 1980. Preserved learning and retention of pattern- analyzing skill in amnesia: dissociation of knowing how and knowing that. Science 210, 207–210. Coyle, S., Gordon, E., Howson, A., Meares, R., 1991. The effect of age on auditory event-related potential. Experimental Aging Research 17, 103–111. D’Esposito, M., Postle, B.R., Ballard, D., Lease, J., 1999. Maintenance versus manipulation of informationheld in working memory: an event-related fMRI study. Brain Cognition 41, 66–86. Earles, J.L., Salthouse, T.A., 1995. Interrelations of age, health, and speed. The Journals of Gerontology and Psychological Social Sciences 50, 33–41. Goldman-Rakic, P.S., Muly III, E.C., Williams, G.V., 2000. D1 receptors in prefrontal cells and circuits. Brain Research Reviews 31, 295–301. Goyarzu, P., Malin, D.H., Lau, F.C., Taglialatela, G., Moon, W.D., Jennings, R., Moy, E., Moy, D., Lippold, S., Shukitt-Hale, B., Joseph, J.A., 2004. Blue- berry supplemented diet: effects on object recognition memory and nuclear factor-kappa B levels in aged rats. Nutritional Neuroscience 7, 75–83. Grady, C.L., Craik, F.I.M., 2000. Changes in memory processing with age. Current Opinion in Neurobiology 10, 224–231. Hillyard, S.A., Vogel, E.K., Luck, S.J., 1998. Sensory gain control (ampli- fication) as a mechanism of selective attention: electrophysiological and neuroimaging evidence. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 353, 1471–2970. Joseph, J.A., Arendash, G., Gordon, M., Diamond, D., Shukitt-Hale, B., Mor- gan, D., 2003. Blueberry supplementation enhances signaling and prevents behavioral deficits in an Alzheimer disease model. Nutritional Neuroscience 6, 153–162. Kennedy, D.O., Scholey, A.B., Drewery, L., Marsh, V.R., Moore, B., Ashton, H., 2003. Electroencephalograph effects of single doses of Ginkgo biloba and Panax ginseng in healthy young volunteers. Pharmacology, Biochemistry, and Behavior 75, 701–709. Kentros, C.G., Agnihotri, N.T., Streater, S., Hawkins, R.D., Kandel, E.R., 2004. Increased attention to spatial context increases both place field stability and spatial memory. Neuron 42, 283–295. Kugler, C.F.A., Taghavy, A., Platt, D., 1993. The event-related P300 potential analysis of cognitive human brain aging: a review. Gerontology 39, 280– 303. Mangun, G.R., Hillyard, S.A., Luck, S.J., 1993. Electrocortical substrates of visual selective attention. Attention and Performance, 219–243. Moser, M.B., Moser, E.I., 1998. Distributed encoding and retrieval of spa- tial memory in the hippocampus. The Journal of Neuroscience 18, 7535– 7542. Moss, M.C., Scholey, A.B., Wesnes, K.A., 1998. Oxygen administration selectively enhances cognitive performance in healthy young adults: a placebo-controlled double-blind crossover study. Psychopharmacology 138, 27–33. Nolan, K.A., Blass, J.P., 1992. Preventing cognitive decline. Clinics in Geriatric Medicine 8, 19–34. Nordin, S., Quinonez, C., Morgan, C.D., Geisler, M.W., Plich, J., Musphy, C., 1999. Olfactory event-related potentials in young and elderly adults: eval- uation of tracking task versus eye open/closed recording. Chemical Senses 24, 459–464. Oken, B.S., Kaye, J.A., 1992. Electrophysiologic function in healthy extremely old. Neurology 42, 519–526. Sabri, M., Liebenthal, E., Waldron, E.J., Medler, D.A., Binder, J.R., 2004. Increased attention to spatial context increases both place field stability and spatial memory. Neuron 42, 283–295. Salthouse, T.A., 1994. Aging associations: influence of speed on adult age differ- ences in associative learning. Journal of Experimental Psychology.Learning, Memory, and Cognition 20, 1486–1503. Smith, E.E., Jonides, J., 1999. Storage and executive processes in the frontal lobes. Science 283, 16571–1661. Solfrizzi, V., Panza, F., Torres, F., Mastroianni, F., Del Paridi, A., Venezia, A., Capurso, A., 1999. High monounsaturated fatty acids intake protects against age-related cognitive decline. Neurology 52, 1563–1569. Stancampiano, R., Cocco, S., Cugusi, C., Sarais, L., Fadda, F., 1999. Serotonin and acetylcholine release response in the rat hippocampus during a spatial memory task. Neuroscience 89, 1135–1143. Subathra, M., Samuel, S., Marimuthu, S., Muthuswamy, A.D., Chinnakkannu, P., 2005. Emerging role of Centella asiatica in improving age-related neu- rological antioxidant status. Experimental Gerontology 40, 707–715. Suguna, L., Sivakumar, P., Chandrakasan, G., 1996. Effects of Centella asiatica extract on dermal wound healing in rats. Indian Journal of Experimental Biology 34, 1208–1211. Tulving, E., Schacter, D.L., 1990. Priming and human memory systems. Science 247, 301–306. Veerendra Kumar, M.H., Gupta, Y.K., 2002. Effect of different extracts of Cen- tella asiatica on cognition and markers of oxidative stress in rats. Journal of Ethnopharmacology 79, 253–260. Wesnes, K.A., 2001. The use of cognitive tests to facilitate drug and dose selection in phase I and to optimize drug dosing in phase IV. International Congress Series 1220, 35–50. Wijers, A.A., Mulder, G., Gunter, T., Smid, H.G.O.M., 1996. Brain potential analysis of selective attention. In: Neumann, O., Sanders, F. (Eds.), Hand- book of Perception and Action. Academic Press Ltd., London. Wood, S.M., Potts, G.F., Hall, J.F., Ulanday, J.B., Netsiri, C., 2006. Event- related potentials to auditory and visual selective attention in schizophrenia. International Journal of Psychophysiology 60, 67–75. Zainol, M.K., Abd-Hamid, A., Yusof, S., Muse, R., 2003. Antioxidant activity and total phenolic compounds of leaf, root and petiole of four accessions of Centella asiatica (L) Urban. Food Chemistry 81, 575–581. . Positive modulation of cognition and mood in the healthy elderly volunteer following the administration of Centella asiatica Jintanaporn Wattanathorn a,∗ , Lugkana. memorize the position of the illuminated windows. In 36 subsequent presentations of the house, one of the windows was illuminated and the participant decided whether or not this matched one of the. determine the effect of Centella asiatica on the cognitive function and mood in the elderly. 2. Materials and methods 2.1. Participants Four male, and twenty-four female healthy elderly volunteers (mean