Clinical trial NCT03929250
Conditions: Healthy Elderly
Sponsor: Oregon Health and Science University
Start date: 2019-07-05; Completion date: 2024-01-03
Enrollment: 8 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
This study will measure the oral bioavailability and pharmacokinetics of known bioactive compounds from a standardized Centella asiatica water extract (CAW) product (CAP) in cognitively healthy elders.

Detailed description:
PRIMARY OBJECTIVES:

1. To assess the bioavailability and rate of clearance of Centella asiatica derived compounds in the plasma and urine of cognitively healthy elders over 12 hours.
2. To determine the acute tolerability of a Centella asiatica product in cognitively healthy elders.

OUTLINE:

Participants will orally consume a single administration of a standardized Centella asiatica water extract product (CAP). Two doses (2g and 4g CAW) will be administered on separate occasions, at least two weeks apart. The levels of known bioactive compounds present in Centella asiatica will be measured in human plasma and urine over 12 hours after administration of each of the doses.

Arms: arm 1: Participants first receive a single dose of a product containing 2g CAW, then after a minimum washout period of 2 weeks, they receive a single dose of a product containing 4g CAW. arm 2: Participants first receive a single dose of a product containing 4g CAW, then after a minimum washout period of 2 weeks, they receive a single dose of a product containing 2g CAW.
Interventions: intervention 1: 2g Centella asiatica water extract product intervention 2: 4g Centella asiatica water extract product
Intervention descriptions: intervention 1: 2g Centella asiatica water extract product is a powder containing Centella asiatica water extract (CAW) and excipients to improve palatability, color matching and dispersability in water. It will be consumed on an empty stomach orally suspended in 10-12 ounces of water. intervention 2: 4g Centella asiatica water extract product is a powder containing Centella asiatica water extract (CAW) and excipients to improve palatability, color matching and dispersability in water. It will be consumed on an empty stomach orally suspended in 10-12 ounces of water.

Protocol documents:
Prot_SAP_000.pdf: 
Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 1 of 33 
 
 
Pharmacokinetics of Centella asiatica in the Elderly  
 
 
NCT03929250  
 
 
STUDY PROTOCOL INCLUDING STATISTICAL ANALYSIS PLAN 
 
 
Document Version Date: February 28, 2024  
 
 
 
 
 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 2 of 33 
 
Title: A pharmacokinetic study of Centella asiatica in the elderly 
 
 
A. OBJECTIVES  
A.1. Purpose 
The purpose of this pilot study is to measure the oral bioavailability and pharmacokinetics of 
known bioactive compounds from a standardized Centella asiatica water extract product (CAP) 
in cognitively healthy elders. Compound levels will be measured in human plasma and urine 
over 12 hours after acute oral administration of two doses of the botanical extract product. The 
dose giving maximum plasma levels (Cmax) closest to those observed in our mouse studies, as 
well as the rate of clearance (t ½) of the known compounds, will be identified. These data will be 
used to inform decisions on the dosage and dosing frequency for future phase 1 and phase 2 
clinical trials.  
 
A.2. Primary goal 
To determine the oral bioavailability and pharmacokinetics of known compounds from two single 
administrations of a Centella asiatica product in non-demented elders.  
 
A.3. Endpoints  
• 
The primary endpoints for this study are the maximum concentration (Cmax), the area under 
the curve (AUC), and the time of maximum concentration (tmax) of known compounds from 
Centella asiatica (triterpenes and caffeoylquinic acids and their metabolites) in human 
plasma.  
• 
A secondary endpoint is the half-life (t1/2) of the known bioactive compounds and their 
metabolites to help determine dosage intervals.  
• 
Additional secondary endpoints include temporal changes in ferric reducing ability of plasma 
(FRAP) as an indicator of antioxidant potential over time, tolerability and detection of acute 
adverse events through monitoring by participant interviews, biometrics, vital signs and 
questionnaires.  
 
B. SPECIFIC AIMS 
Aim 1: To assess the bioavailability and rate of clearance of Centella asiatica derived 
compounds in cognitively healthy elders through a pharmacokinetic study.  
• 
Hypothesis: We hypothesize that the triterpene and caffeoylquinic acid components 
from Centella asiatica will be bioavailable, and a dose giving similar plasma levels to 
those associated with cognitive effects in mice can be achieved. 
 
Aim 2: To determine the acute tolerability of a Centella asiatica product in cognitively 
healthy elders.  
• 
Hypothesis: We hypothesize that acute usage of the Centella asiatica product will be 
well tolerated and produce no severe adverse events. 
 
 
C.BACKGROUND 
C.1. Alzheimer’s disease 
Alzheimer’s disease is a severe form of cognitive impairment and one of the most expensive 
and debilitating conditions known to modern medicine. The National Institute of Aging suggests 
that greater than five million Americans may have Alzheimer’s disease, making it the sixth 
leading cause of death in the United States.1 In 2016 alone, government health agencies 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 3 of 33 
 
(Medicare and Medicaid) spent an estimated $160 billion on Alzheimer’s disease with a 
projected 365% increase by 2050.2  
 
The pathogenesis of Alzheimer’s disease is highly complex. Current evidence suggests it is a 
combination of genetics, environment and lifestyle factors making the development of 
treatments very challenging. Its most striking pathological feature is the accumulation of -
amyloid (A) plaques within the brain.3 Current pharmaceutical investigation is aimed at 
preventing the accumulation of, or promoting the clearance of these neurotoxic plaques (anti-
amyloid immunotherapy)4,5; however, recent trials of such agents have failed to produce a 
significant clinical effect on Alzheimer’s disease.6,7 Drugs currently FDA-approved for the 
symptomatic treatment of Alzheimer’s disease are either cholinesterase inhibitors8 or act at the 
N-methyl-D-aspartate (NMDA) receptor.9,10 Unfortunately, these treatments do not influence 
disease progression, and their effectiveness is highly variable.  
 
Beyond cognitive impairment, Alzheimer’s disease also has significant comorbidities including 
insomnia, depression11 and anxiety.12 Multiple interventions are often needed to manage these 
symptoms affecting patient compliance and safety. This warrants further investigation into 
effective, inexpensive and well-tolerated treatments for Alzheimer’s disease and its 
comorbidities.  
 
Gender differences have been observed in the incidence, development and progression of 
Alzheimer’s disease, and in response to interventions targeting cognitive impairment. Women 
develop cognitive disability more rapidly compared to men.13,14 Previous studies of intranasal 
insulin for cognition showed functional abilities were better preserved in women compared to 
men.15 These differences highlight the importance of evaluating responses to any agent in both 
males and females. As part of this proposed project we will evaluate potential gender 
differences in bioavailability and pharmacokinetics. 
 
C.2. Centella asiatica 
Centella asiatica is a highly regarded botanical reputed in Eastern medicine to increase 
intelligence and memory.16 In Western countries, it is sold as the dietary supplement “gotu 
kola”17 for use in improving brain health and cognitive function. Preclinical studies have shown 
that Centella asiatica extracts have biological effects of relevance to memory, learning, aging, 
mood and potentially disease progression in Alzheimer’s disease.18 Water extracts of Centella 
asiatica have improved learning and memory in wild-type rats,19 wild-type mice,20,21 and rats 
subjected to central nervous system toxicity.22,23 In addition, similar extracts have been shown to 
modify the brain structure by increasing hippocampal neuron dendritic arborization in adult 
rats,24 neonatal rats,25 and neonatal mice,21 potentially contributing to the observed cognitive 
changes. With regards to mood and behavior, ethanol extracts given to mice subjected to 
chronic and acute stress26 and sleep deprivation27 demonstrated anxiolytic properties. Similar 
ethanol extracts revealed antidepressant properties in rats.28  
 
Figure 1. Centella asiatica water extract improves learning and memory in aged female Tg2576 mice without 
altering β-amyloid accumulation. Tg2576 mice (20 month old) were exposed to Centella asiatica water extract 
(200mg/kg/day) in their drinking water for 2 weeks before and during testing (total 5 weeks). Left – Morris water 
maze escape times. Right – Soluble and insoluble fractions of Aβ were extracted from cortical tissue and analyzed 
via ELISA (bars represent means +/- standard error).  


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 4 of 33 
 
With applications specific to Alzheimer’s disease, one study showed that long-term usage of a 
Centella asiatica extract (six months) reduced -amyloid plaque burden in a mouse model of 
Alzheimer’s disease29; however, studies performed at Oregon Health and Science University 
(OHSU) suggest that water extracts of Centella asiatica may to achieve its cognitive effects 
without a direct action on β-amyloid (Figure 1).30 Rather, Centella asiatica appears to affect 
downstream targets protecting neurons from β-amyloid induced neurotoxicity.20,31,32 This 
suggests that Centella asiatica may be able to limit disease progression even when -amyloid 
deposition has already taken place, thereby representing a novel treatment mechanism, 
potentially complementary to the medications in development. 
 
Human studies, although limited, support Centella asiatica’s ability to affect cognition and mood. 
Placebo-controlled trials showed that herbal extracts, or dried herb, improved cognitive function 
in healthy elderly33 and middle-aged34 volunteers. In elderly participants with mild cognitive 
impairment, investigators found improvements in cognitive test results (Mini Mental State 
Examination) following use of dried Centella asiatica for six months35; however, no control group 
was incorporated. Unfortunately, these studies used highly variable Centella preparations that 
were poorly characterized and were either performed in cognitively normal individuals thereby 
limiting clinical applicability, or were not placebo-controlled. This warrants a robust, randomized, 
double blind, placebo-controlled study in cognitively impaired humans to examine the effects of 
a chemically well-characterized Centella asiatica extract on cognition.  
 
C.3. Phytochemistry 
Botanical agents are complex mixtures that show considerable variability due to growth 
conditions, geography, the parts utilized and preparation method.36,37 Limited regulation of the 
production of these agents makes the efficacy of commercial products unpredictable. To this 
end, it is imperative that pharmacokinetic and clinical studies of botanicals use botanically 
authenticated, chemically well-characterized, stable products that are standardized to a specific 
content of active ingredients.  
 
Chemical analyses of Centella asiatica performed by our group and others have identified 
unique substances known as triterpenoid saponins (asiatic acid, madecassic acid, asiaticoside 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 5 of 33 
 
and madecassoside),38,39 which are believed to be associated with Centella asiatica’s 
neuroprotective and neurotropic effects.40-43 Previous pharmacokinetic studies of Centella 
products have focused primarily on these compounds (most specifically asiatic acid) in plasma 
using purified extracts or synthesized compounds.44-46 Our group has found, using high 
performance liquid chromatography coupled to mass spectral detection (HPLC-MS), additional 
active phenolic components known as caffeoylquinic acids in water extracts of Centella.31 These 
acids have been shown to protect against excitotoxic and hypoxic damage47 and β-amyloid 
toxicity in primary neurons.31,48 To date, there are no pharmacokinetic studies of caffeoylquinic 
acids and triterpenes from water extracts of Centella asiatica in humans. This project aims to 
investigate the bioavailability of all of the aforementioned compounds from a water extract of 
Centella asiatica in human plasma and urine. 
 
C.4. Safety 
Centella asiatica is an edible plant and the Botanical Safety Handbook49 classifies it as a Class 
1 herb that can safely be consumed when used appropriately. The widespread use of Centella 
as a dietary supplement and the available human studies support its safety.17,33-35 The 
recommended dose of this herb, which is consumed in India and Sri Lanka as a healthy addition 
to the diet, is between 0.5 and 1.5 g dried leaf daily.16,17 In multiple human studies using the 
fresh or dried whole herb, or in many clinical studies using extracts of Centella asiatica, no 
adverse effects were reported.35,50-53 There have also been several clinical studies in which 
multi-component products containing Centella asiatica herb or extracts were found to be well 
tolerated.54-60 One study in which elderly participants consumed dried herb (1000 mg per day for 
6 months) found no change in their liver enzymes (Serum glutamic oxaloacetic transaminase 
(SGOT) or serum glutamic-pyruvic transaminase (SPGT)).35 In another study, administration of 
a concentrated extract at 750mg or 1000 mg per day daily for six weeks caused no change in 
liver enzymes Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) levels.61 
Two clinical studies on polyherbal preparations containing Centella asiatica (albeit in small 
quantities) also reported no changes in safety laboratory results.54,57 
 
TTFCA (a purified triterpene mixture), was well-tolerated at all of the following doses in 
microcirculation studies: 30 or 60mg daily for 7 days62; 30 or 60 mg administered twice daily for 
6 months63; and 60mg twice daily for 12 months.64 Two studies on femoral and carotid plaque 
formation respectively65,66 both used TTFCA at 180mg daily for 12 months. Again, TTFCA was 
well tolerated at this highest reported dose.     
 
C.4.1. Reports of adverse effects in humans: It is noteworthy that despite its presence in dietary 
supplements in the United States, there are no reports of adverse events associated with 
Centella asiatica found in the FDA CFSAN Adverse Event Reporting System (CAERS accessed 
8/08/2017). A recent review67 of hepatotoxicity linked to herbs or dietary supplements lists two 
reports associated with Centella asiatica. In the first report, hepatotoxicity was seen in three 
women following ingestion of Centella asiatica products.68 The publication does not give any 
details of the products (dose, whole herb or extract, etc), nor does it mention whether the 
products contained other ingredients in addition to Centella asiatica. Email correspondence 
between Dr. Amala Soumyanath (Principal Investigator on the present study) and the author 
(Dr. Oliver Jorge) established that the products contained “extracts” of Centella asiatica, and 
these were not the Centella asiatica mixed triterpene products similar to CAST known as 
TTFCA or TECA. The second report69 was of a 15-year-old girl who experienced acute hepatitis 
after taking a product containing “several ingredients, one of them Gotu kola (Centella asiatica) 
20mg for 6 weeks”. The patient had also been on lymecycline for 8 weeks. While these reports 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 6 of 33 
 
are concerning, the lack of detailed information on the products used in the first report,68 and the 
presence of additional ingredients, low Centella asiatica dose and concomitant use of 
lymecycline in the second report69 make it unclear what role (if any) Centella asiatica played in 
the hepatotoxicity seen in these individuals.    
 
In a recent clinical trial conducted at OHSU (unpublished), one female participant receiving 
CAST (a product similar to TTFCA) at 240mg per day withdrew due to abnormal liver enzyme 
levels, which returned to normal on stopping CAST. However, CAST was well tolerated by other 
participants who completed the 52-week study (dose escalated from 120mg and180 mg during 
weeks 1-8, to 240mg CAST daily for weeks 9-52). In this study, 29 of 43 randomized subjects 
(67%) experienced at least one adverse event (AE). The proportion of patients who experienced 
at least one AE in the Placebo (56%) and in the CAST (80%) groups was not significantly 
different (p-value = 0.1). AEs included transient abnormal liver and kidney function or 
gastrointestinal symptoms, which resolved on their own. Abnormal electrocardiograms (ECGs) 
were noted in some subjects and were linked to either pre-existing conditions, or returned to 
normal on subsequent tests. All AEs were graded as minor.  
 
Two studies where Centella asiatica herb or extract was taken for six months35,70 noted 
decreases in systolic and/or diastolic blood pressure, and one also reported improved sleep 
patterns among participants.70 Due to their relatively mild nature, these effects were mentioned 
as potentially beneficial rather than noted as adverse effects of the intervention. In a study 
involving 48 subjects, one case each of constipation, abdominal bloating and itchiness were 
reported following 750mg or 1000mg daily of a hydroethanolic extract of Centella asiatica.61 
Topical administration of CA extracts and triterpenes is reported to cause contact dermatitis in 
some individuals.71-73 
 
C.4.2. Non-clinical pharmacology/toxicology 
Rats administered dried Centella asiatica herb:74 Oral administration of dried Centella asiatica 
aerial parts to rats at 250, 500, and 1000 mg/kg body weight over 30 days did not produce any 
clinical signs of toxicity, morbidity or mortality or show any behavioral effects.  However, 
significant increases in serum levels of liver enzymes (ALT and AST) and kidney markers (BUN 
and creatinine) were observed, mostly in a dose dependent manner, suggesting effects on both 
liver and kidney. No gross pathological lesions were seen in any organs after necropsy; 
however, histopathology revealed tissue alterations on microscopical examination in liver, 
kidney, and spleen particularly at the highest dose. A decrease in viable cell count of the liver 
was seen particularly with the highest dose. Spleen weight was increased by Centella asiatica 
treatment; however, the statistical comparisons given in this paper are not clear.  
 
Rats administered Centella asiatica extract “INDCA”:75 For acute toxicity studies, INDCA* was 
administered orally to male and female rats at a single oral dose of 2000mg/kg body weight. No 
deaths, weight loss or treatment-related gross pathological changes occurred in the 14 days 
following treatment. This suggested that the LD50 of INDCA was >2000mg/kg body weight. For 
subchronic studies, INDCA was given orally at 250, 500 or 1000 mg/kg body weight for 90 days. 
No deaths were observed, nor were any clinical signs of toxicity observed over this period. 
While some significant differences to vehicle control were seen in hematological and 
biochemical parameters, these were not dose-dependent and the authors conclude that these 
were not indicative of an adverse effect. Mild focal lymphocytic infiltration and focal necrosis 
were seen in both liver and kidneys of INDCA treated animals. In this study, INDCA did not 
exhibit mutagenic activity in an Ames test either before or after metabolic activation. *INDCA 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 7 of 33 
 
was prepared by initial isopropyl alcohol extraction of dried Centella asiatica followed by 
concentration of the compounds of interest by solvent:solvent partition to yield a product 
containing 45.75% of the triterpene asiaticoside.76 
 
Mice administered Centella asiatica extract:77 The title and some sections of this paper state 
“acetone leaf extract” or “acetone extract”, while the method describes the Centella asiatica 
extract as being made with 50% ethanol. The extract is stated as 5.75% of the original dried 
plant material. For the acute study, mice were administered 100, 500, 1000, 2000, or 
4000mg/kg body weight of extract by gastric intubation. No deaths occurred over a 24 hour 
observation period after administration of the extracts, showing that the LD50 is > 4000 mg/kg. 
For subacute toxicity, animals were treated with 500, 1000, 2000 or 4000 mg/kg/day of extract 
for 15 days. No significant adverse effects were observed in these animals, nor were any gross 
pathological changes to organs seen at necropsy. The liver showed a significant decrease in 
weight in animals treated with 2000 or 4000 mg/kg/day. Hematological parameters were 
unaltered, and liver enzymes (ALT and AST) were not significantly different from control at any 
dose. Creatinine showed a significant decrease from control at the 1000 mg/kg/day dose only. 
The authors conclude that Centella asiatica is “destitute of toxic effects”.  
 
Effect on reproductive system in rats:78 An extract of Centella asiatica (solvent could not be 
verified as the original paper was not available) was administered to male rats at 100, 200 or 
300 mg/kg body weight for 42 days. All treatment groups showed some degeneration of 
spermatogenic cells, reduction of sperm count, and reduced testosterone levels compared to 
control rats.  
 
Other studies: A manufacturer’s brochure on Centella asiatica selected triterpenes (CAST; 
Indena SpA) refers to two studies, which report that asiaticoside was not toxic at up to 1g/kg on 
oral administration to rabbits, whereas toxicity was seen at 50-50mg/kg given intramuscularly.  
Rabbits given a standardized extract of Centella asiatica were reported not to show any 
teratogenic effects. These data could not be verified. We have ordered the two original 
publications and hope to include more details in our formal FDA IND application that is currently 
being composed. Tests in guinea-pigs show that the three triterpenes in CAST are very weak 
sensitizers.79 The author concludes that the risk of developing contact sensitivity to the plant or 
its constituents is low. An increased incidence of skin papillomas compared to controls was 
observed in hairless mice painted for about 20 months with asiaticoside (0.1%) dissolved in 
benzene compared to benzene painted controls.80 The authors conclude that asiaticoside is a 
weak tumor promoter in this model, its effects only appearing with repeated applications over a 
long period. A number of Internet sites erroneously quote a report of an increase in blood 
glucose in animals administered Centella asiatica citing a paper by Ramaswamy et al, 1970.81 
Careful reading of the original paper revealed no mention of this effect. Rats administered an 
ethanol extract of Centella asiatica (300-330mg/kg per day for 18 days) or water extract of 
Centella asiatica (200 mg/kg per day for 5 weeks) in our preclinical studies20,30,82 showed no 
obvious adverse effects. These rodent doses are comparable, by interspecies scaling83,84 to the 
proposed 2g and 4g per day human doses in this study.   
 
C.4.3. Influence of CA on drug metabolizing enzymes: In vitro studies suggest that Centella 
asiatica may contain compounds that are weak inhibitors of Phase I drug metabolizing CYP450 
isoenzymes. These need to be evaluated in vivo. In studies on Phase I metabolic reactions, 
Centella asiatica methanolic extract and asiaticoside were reported to weakly inhibit 
recombinant human Phase I metabolizing isoenzymes CYP3A4, CYP2D6, CYP2C9 and 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 8 of 33 
 
CYP1A2 compared to standard inhibitors of these isoforms.85 In another study, Centella asiatica 
was sequentially extracted with hexane, dichloromethane, ethanol and water. When tested 
against human CYP isoforms expressed by E. Coli, the ethanol and dichloromethane extracts 
showed a higher inhibitory effect on CYP2C19, CYP2C9, CYP2D6 and CYP3A4 compared to 
the water or hexane extracts. Madecassic and asiatic acid were stronger inhibitors compared to 
asiaticoside.86,87 A standardized Centella asiatica extract (ECa223) containing mostly the 
triterpenes madecassoside (43%) and asiaticoside (39%) inhibited purified CYP3A4, CYP2C19 
and CYP2B6, but did not affect CYP1A2, CYP2C9, CYP2D6 and CYP2E1.88 Extracts of 
Centella asiatica were inhibitory to CYP 1A2 and CYP2C989 CYP3A4 and CYP 2D690 in human 
liver microsomes. When ECa223 was administered to rats at 10, 100, and 1000 mg/kg/d for 90 
days, there was no significant effect on the Phase II metabolizing enzymes UDPGT, SULT, 
GST, and NQOR.91 The effect of a water extract of Centella asiatica on these Phase I and 
Phase II enzymes is yet to be determined.  
C.4.4. Conclusions: Based on the extensive use of Centella asiatica herb globally as a food, tea, 
traditional medicine or dietary supplement, and the lack of serious adverse effects in clinical 
studies of TTFCA, CAST or concentrated extracts of Centella asiatica, we expect that the 
product to be used in our trial (CAP) will be well-tolerated and safe. In particular, since hot water 
extracts of Centella asiatica are consumed regularly in teas,92-94 it appears that harmful 
components are not extracted by hot water. However, no human safety data is available on 
concentrated water extracts at the doses (2g and 4g) proposed in this study. There is some 
concern about possible hepatotoxicity, but a study in which 1g per day of concentrated 
hydroalcoholic extract of Centella asiatica was administered for six weeks, did cause any 
change in liver enzymes.61 The total triterpenes (approximately 4% w/w) delivered by our 
proposed CAP doses is expected to be 80, 160 and 320 mg/day. The lower doses are 
comparable to the TTFCA and CAST studies described earlier where these doses were well 
tolerated. We are also encouraged by the fact that no obvious adverse effects have been 
observed in our preclinical studies where mice were administered Centella asiatica water extract 
at 200mg/kg/day20,30 or 500 mg/kg/day (unpublished) for five weeks. These murine doses are 
comparable, by interspecies scaling83,84 to the proposed 2g and 4g per day human doses in this 
study. This study aims to provide preliminary safety data on this water extract product. 
 
D. SIGNIFICANCE 
Alzheimer’s disease is a severe form of memory loss, initially manifesting as mild cognitive 
impairment and followed by a decline in cognitive function. There is an acknowledged need95 to 
develop novel disease modifying agents to prevent progressive cognitive decline as patients 
and health providers are currently without viable pharmaceutical options for this debilitating 
disease. Concurrently, an interest in alternative therapies has increased substantially over the 
past two decades, especially among the elderly.96-98 Centella asiatica may offer an effective way 
to bridge this gap, providing a botanical-based therapy to reduce cognitive impairment in 
Alzheimer’s disease.  
 
Centella asiatica shows remarkable cognitive enhancing and neuroprotective properties in 
extensive preclinical research performed by our group and by others (Section C.2.). The small 
number of human clinical studies favors the use of Centella asiatica as a dietary supplement to 
improve cognitive function and brain health; however, limited regulation of these agents makes 
efficacy of commercial products unpredictable. Our ultimate goal is to develop an FDA-


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 9 of 33 
 
approved, standardized, botanical extract of Centella asiatica for evidence-based treatment of 
mild cognitive impairment and Alzheimer’s disease.  
 
This is the first human pharmacokinetic study of a standardized crude water extract of Centella 
asiatica. Previous pharmacokinetic studies have used refined combinations of Centella’s unique 
triterpenes.44-46 Use of such extracts eliminates possible chemical interactions and bioactive 
synergistic compounds that may result from crude extract preparation. Mouse studies have 
shown improvements in cognition in wild-type and Alzheimer’s disease models using crude 
water extracts with or without triterpenes20,30 suggestive of other neuroactive ingredients within 
Centella asiatica water extract. These appear to include caffeoylquinic acids and potentially 
other unidentified compounds. This study will preserve possible synergistic effects by using a 
crude water extract, but also standardize the product to the known bioactive constituents, which 
has not been done in previous human trials. In addition to studying the triterpenes, this study will 
investigate the pharmacokinetics and bioavailability of Centella derived caffeoylquinic acids and 
their metabolites, which has not been previously evaluated. The observed pharmacokinetic 
profile will inform dosage decisions and dosing frequency for future human clinical trials.  
 
E. PRELIMINARY DATA 
Centella asiatica water extract is comprised of 0.94-2.41% triterpenes and 0.01-0.46% 
caffeoylquinic acids.31 The abundant triterpenes asiaticoside and madecassoside are known to 
metabolize to the neuroactive metabolites asiatic acid and madecassic acid in vivo.99 Similarly, 
the caffeoylquinic acids metabolize to caffeic acid, ferulic acid, isoferulic acid, and their 
dihydroderivatives.100,101 Working with OHSUs’ Bioanalytical Shared Resource/Pharmacokinetic 
Core (BSR/PKC) we have developed a sensitive methodology using HPLC-MS/MS to separate 
and analyze the triterpenes, caffeoylquinic acids, and their further metabolites within a human 
plasma matrix. Linear calibration curves have been obtained for each of these analytes (25–
2000 ng/mL for asiatic acid, 5-2000 ng/mL for madecassic acid, and 5-100 ng/mL for several 
caffeoylquinic acids and their metabolites).  
 
Previous pharmacokinetic literature in rodents and dogs has reported maximum concentrations 
of the polar caffeoylquinic acid compounds within one hour of ingestion101,102 and the less polar 
triterpenes at 4-5 hours.103 We have performed a pharmacokinetic study in wild-type mice on a 
low phytochemical diet to identify the Cmax and time to reach Cmax (tmax) of Centella’s known 
bioactive compounds using the standardized water extract. We were able to detect the bioactive 
caffeoylquinic acids and their metabolites in plasma following oral administration of the extract 
using reversed phase HPLC-MS/MS (Figure 2). Maximum plasma concentrations (Cmax) of 
CQAs and metabolites (15 – 2350 ng/ml) primarily occurred within 45 minutes of oral 
administration (Figure 2) and there were no observable adverse events after extract 
administration. These results demonstrate Centella asiatica water extract is orally bioavailable 
and well-tolerated in mice warranting further evaluation in humans. They also confirm the 
proposed metabolism of caffeoylquinic acids from Centella asiatica water extract into their 
respective metabolites. To assess for changes in the bioavailability of the standardized 
manufactured product to be used for this study (CAP), we will perform another mouse 
bioavailability study. 
Table 1. Pharmacokinetic parameters of caffeoylquinic acids and metabolites in mouse plasma after single oral 
administration of Centella asiatica water extract. 
Analyte 
Cmax (ng/mL) 
tmax (min) 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 10 of 33 
 
-40
10
60
110
160
0
100
200
mean ng/mL + SEM
Time (minutes)
Dicaffeoylquinic Acids
-5
0
5
10
15
20
0
100
200
mean ng/mL + SEM
Time (minutes)
Ferulic Acid
  
 
  
 
 
 
 
 
F. RESEARCH DESIGN AND METHODS 
F.1. Study design 
This is a phase 1 pilot clinical study using a blinded randomized crossover design. 
Randomization will use an arm equivalence design to promote equal numbers of participants for 
each order schema. We will dispense a single administration of a Centella asiatica water extract 
-20
0
20
40
60
80
100
120
140
0
50
100
150
200
250
mean ng/mL + SEM
Time (minutes)
Monocaffeoylquinic Acids
0
10
20
30
40
50
60
0
50
100
150
200
250
mean ng/mL + SEM
Time (minutes)
Caffeic Acid
Monocaffeoylquinic acids 
84.3 
15 
Dicaffeoylquinic acids 
37.8 
60 
Caffeic acid 
39.6 
15 
Ferulic acid 
15.2 
7.5 
Isoferulic acid 
2.5 
7.5* 
Dihydrocaffeic acid 
13.1 
240* 
Dihydroferulic acid 
Not detected 
Not detected 
Dihydroisoferulic acid 
124 
30* 
4-methyl catechol 
4.9 
120* 
4-ethyl catechol 
0.94 
90* 
3-(3-hydroxyphenyl)propionic acid  
84.5 
7.5* 
* Inconsistent time curve 
 
Figure 2. Pharmacokinetic profile of monocaffeoylquinic acids, dicaffeoylquinic acids and metabolites caffeic acid 
and ferulic acid in wild-type female mice. Six-month-old female mice (n=39) were administered Centella asiatica water 
extract (200 mg/kg) via oral gavage. Four mice were euthanized per time point and blood collected via cardiac puncture for 
analysis with HPLC-MS/MS. 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 11 of 33 
 
product to the research participants at two doses (2g and 4g) on two separate occasions two to 
four weeks apart (Figure 3). Participants will be required to adhere to a “low phytochemical diet” 
known as a “low plant diet” designed by the Oregon Clinical and Translational Research 
Institute’s (OCTRI) Bionutrition Department for 48 hours immediately preceding each study visit 
and for the duration of each study visit (Section I.4.). We will provide participants a handout at 
screening outlining the diet with meal examples. Serial blood samples (2 teaspoons; 10 
mL/sample) will be obtained through a peripheral intravenous catheter, once prior to Centella 
asiatica consumption and then over a 12-hour post-administration period (15, 30, 45, 60, 90, 
120, 180, 240, 360, 480, 600, and 720 min). Participants will also perform a 12-hour urine 
collection at each study visit to assess for excretion of Centella asiatica metabolites. Adverse 
events and tolerability will be assessed with questionnaires in-person at each study visit, by 
phone 24 hours after each study visit, and one week following study completion (Figure 3).  
F.2. Study Population 
F.2.1. Number of participants 
Eight non-demented elderly volunteers (n=4 each female and male) 65-85 years of age will be 
screened and enrolled in the study. We estimate that we will need to screen approximately 
twelve potential participants to meet the eight volunteers needed to complete the study. We 
expect a 10% drop out rate. We are still powered to see changes from baseline and differences 
between doses with a 25% drop out rate resulting in 6 subjects.  
 
F.2.2. Inclusion and exclusion criteria 
Inclusion criteria: 
1. Age 65-85, male and female 
2. Sufficient English language skills to complete all tests 
3. Sufficient vision and hearing to complete all tests 
4. No known allergies to Centella asiatica,  (excipients redacted) or any of their 
derivatives 
5. Willingness to discontinue all botanical dietary supplements for one week prior to and 
during each study visit  
6. Willingness to comply with a 48-hour low plant diet for each study visit  
7. Absence of significant depression symptoms (Geriatric Depression Scale-15 score of 
<12) 
8. Body Mass Index (BMI) greater than 17 and less than 35 at screening 
9. Non-demented, defined as Clinical Dementia Rating (CDR) score of zero and Mini 
Mental State Examination (MMSE) score >28 
10. General health status that will not interfere with the ability to complete the study  
 
Exclusion criteria: 
1. Current smoking, alcohol or substance abuse according to DSM-V criteria 
2. Women who are pregnant, planning to become pregnant or breastfeeding 
3. Men who are actively trying to conceive a child or planning to within three months of 
study completion 
4. Severe aversion to venipuncture 
5. Abnormal laboratory evaluation indicating asymptomatic and untreated urinary tract 
infection 
6. Cancer within the last five years, with the exception of localized prostate cancer 
(Gleason Grade <3) and non-metastatic skin cancers 
7. Comorbid conditions such as diabetes mellitus, kidney failure, liver failure, hepatitis, 
blood disorders, clinical symptomatic orthostatic hypotension, and unstable or 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 12 of 33 
 
significantly symptomatic cardiovascular disease 
8. Significant disease of the central nervous system such as brain tumor, seizure disorder, 
subdural hematoma, cranial arteritis, or clinically significant stroke 
9. Major depression, schizophrenia, or other major psychiatric disorder defined by DSM-V 
criteria  
10. Medications: sedatives (except those used occasionally for sleep), central nervous 
system active medications that have not been stable for two months (including beta 
blockers, cimetidine, SSRIs, SNRIs), anticoagulants (i.e. Warfarin), investigational drugs 
used within five half-lives of baseline visit, systemic corticosteroids, neuroleptics, anti-
Parkinsonian agents, narcotic analgesics, nicotine (tobacco, patches, gum, lozenges, 
etc.), Cannabis sativa (herb or edibles) 
11. Diseases associated with dementia such as Alzheimer’s disease, vascular dementia, 
normal pressure hydrocephalus or Parkinson’s disease with a CDR score >0.5 and 
MMSE score <28  
12. Current drug or alcohol use or dependence that, in the opinion of the site investigator, 
would interfere with adherence to study requirements.  
13.  


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 13 of 33 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Telephone Screening 
Eligibility Criteria 
Phone Adverse Events Check 
Phone Adverse Events Check  
Phone Adverse Events Check 
Study End 
Screening Measures 
Cognitive assessment*, biometrics∧, 
physical exam, lab measures+ 
48h Low Plant Diet 
Dose 1 Measures 
12h serial plasma collection, 12h 
urine collection, biometrics**, 
adverse events questionnaire, EKG 
Dose 2 Measures 
12h serial plasma collection, 12h 
urine collection, biometrics∧, 
adverse events questionnaire, EKG 
Tolerability Measures 
Adverse event questionnaire 
Phone Adverse Events Check 
48h Low Plant Diet 
Tolerability Measures 
Adverse event questionnaire 
Screening Visit 
•
Consent 
•
Eligibility 
Visit 1: Dose A 
•
Bioavailability assessment 
•
Tolerability assessment 
Visit 1: Dose B 
•
Bioavailability assessment 
•
Tolerability assessment 
Visit 2: Dose B 
•
Bioavailability assessment 
•
Tolerability assessment 
Visit 2: Dose A 
•
Bioavailability assessment 
•
Tolerability assessment 
24 hours 
1 week 
24 hours 
 
Randomization 
48h Low Plant Diet 
 
 
2-4 weeks  
Figure 3. Study design. *Cognitive assessments: Clinical Dementia Rating (CDR), Mini Mental State Examination (MMSE), 
Geriatric Depression Scale (GDS); ∧Biometrics: weight, height, body mass index (BMI), age, blood pressure, pulse rate, 
temperature; +Lab measures: complete blood count (CBC), comprehensive metabolic panel (CMP), urinalysis. 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 14 of 33 
 
 
F.2.2.1. Concomitant interventions 
 
F.2.2.1.1. Allowed interventions: diphenhydramine, temporary use of sedatives for sleep (such as 
Ambien), probiotic supplements, non-plant based laxatives such as magnesium citrate 
or milk of magnesia, gastroesophageal reflux disorder medications such as antacids 
(non-flavored), central nervous system active medications that have been stable for 
greater than two months, analgesics 
 
F.2.2.1.2. Required interventions: none   
 
F.2.2.1.3. Prohibited interventions: sedatives (except those used occasionally for sleep), central 
nervous system active medications that have not been stable for two months (including 
beta blockers, cimetidine, SSRIs, SNRIs), anticoagulants (i.e. Warfarin), investigational 
drugs used within five half-lives of baseline visit, systemic corticosteroids, neuroleptics, 
anti-Parkinsonian agents, narcotic analgesics, nicotine (tobacco, patches, gum, 
lozenges, etc.), Cannabis sativa (herb or edibles), any over the counter or prescribed 
products containing plant extracts such as herbal supplements, plant based over the 
counter vitamin supplements, plant based over the counter protein powder 
supplements 
 
F.2.3. Vulnerable populations 
Based upon the age range and cognitive status needed in our study population, children and 
decisionally impaired adults will be excluded. We will exclude women who are pregnant, 
planning to become pregnant or breast-feeding. We will also exclude prisoners. We will not 
exclude any ethnic group. 
 
F.3. Setting 
All participants will be enrolled at Oregon Health and Science University (OHSU) and the OHSU 
Institutional Review Board (IRB) will oversee the study. There will be no additional sites for this 
study. All screening and study visits will be conducted at the Oregon Clinical and Translational 
Research Institute’s Clinical and Translational Research Center (CTRC). Screening visits will 
occur in the equipped outpatient rooms and the study visits will occur in the infusion suite of the 
CTRC. All plasma and urine storage will occur in a -70°C freezer in Dr. Quinn’s laboratory in the 
Biomedical Research Building and processing will occur in Dr. Soumyanath’s laboratory in 
Richard Jones Hall at OHSU. All sample analysis will occur at the Bioanalytical Shared 
Resource/Pharmacokinetic Core (BSR/PK) in Richard Jones Hall at OHSU and/or the mass 
spectrometry service at Oregon State University.  
 
F.3.1. Data management 
We will use the Oregon Clinical and Translational Research Institute’s installment of REDCap to 
manage the data and prevent data corruption or loss. All analyzed data will be stored in a 
password-protected folder on OHSU’s X-drive. All paper case report forms, consent forms and 
questionnaires will be scanned into the participant’s REDCap entry. Only people directly 
involved with the study will be granted access to the REDCap database and X-drive with access 
specific to their role in the project as determined by the Principal Investigator. 
 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 15 of 33 
 
G. RECRUITMENT 
Participants will be enrolled on a rolling basis until the desired number of participants is 
achieved. We will recruit from the Oregon Clinical and Translational Research Institute’s 
volunteer repository, with over 40 qualifying participants identified with preliminary searches, the 
National Institute of Neurological Disorders and Stroke (NINDS) NeuroNext database, and the 
Oregon Alzheimer Disease Center’s participant database. Potential participants available 
through the above databases will be mailed a study advertisement describing the study and 
requesting they call if they are interested in participation. The study will be advertised using 
fliers in OHSU clinics and local retirement communities/elder care facilities and an online 
posting in the March Wellness “March Stride” e-mail newsletter. In addition, it will be advertised 
using the NIH Clinical Trials website, local newspapers and radio announcements. It will be 
emphasized that participation in the study is completely voluntary. Copies of all advertisements 
have been submitted with this application for IRB approval .  
 
G.1. Incentives 
Participants will be given the option of complimentary transportation by taxi to and from their 
study visits (up to $50 per visit) or a parking permit for each study visit. They will not be provided 
with transportation for screening visits. To compensate them for their time and effort, 
participants will receive $80 for completion of the study via the ClinCard. Prorated amounts of 
$40 per study visit will be implemented for participants who do not complete the study and 
provided using the above card at the end of the first study visit. While not a direct incentive, 
participants will be provided with three complete meals and snacks during each study visit that 
comply with a low plant diet (section I.4.).  
 
H. INTERVENTION 
H.1. Centella asiatica intervention (Figure 4) (information on the preparation method is 
confidential/proprietary): Centella asiatica dried herb (aerial parts) has been purchased through 
Oregon’s Wild Harvest (OWH; Redmond, OR) and authenticated by organoleptic analysis, thin 
layer chromatography (TLC), and high-performance liquid chromatography-tandem mass 
spectrometry (HPLC-MS/MS). Heavy metal testing of the raw herb and a pilot hot water 
extraction at OHSU was performed to ensure low levels of all heavy metals and compliance with 
the Food and Drug Administration (FDA) and American Public Health Association’s (APHA) 
limits. Pharmachem Laboratories (PCL: Kearny, NJ), a Good Manufacturing Practice (GMP) 
certified facility, performed a large-scale hot water extraction of Centella asiatica using a 
standardized protocol. The water extract was spray dried onto (matrix, redacted)  at PCL and 
then shipped to OWH for blending with a food-grade sweetening and coloring agent before 
packaging into individual dose sachets. To yield different doses (equivalent to 2g and 4g of 
water extract) the matrix, coloring agent and flavoring agent (excipients) was blended in 
appropriate ratios so that each dose contains the same amount of the excipients but different 
amounts of the water extract. The final product is to be referred to as Centella asiatica product 
(CAP) and is to be reconstituted in eight to twelve ounces of room temperature water and 
consumed by mouth once daily. Samples of the raw herb, extract and product have been 
retained for fingerprinting using high-performance liquid chromatography-mass spectrometry 
(HPLC-MS) and voucher samples deposited in Dr. Soumyanath’s (PI) laboratory and the 
Oregon State University Herbarium.  
 
OWH will send the sachets of intervention to the OHSU Research Pharmacy for storage at 4°C 
and dispensing following the Pharmacy’s applicable policies and procedures. Dr. Soumyanath, 
members of the study team, and the mass spectrometry service at Oregon State University will 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 16 of 33 
 
perform quality control and stability assessments throughout the study. Investigational New 
Drug (IND) approval has been obtained from the FDA.The drug will be used in compliance with 
the requirements of HRP-815.  
 
Each sachet will be dispensed to a member of the study team just prior to the participant’s study 
visit and recorded by the Pharmacy staff including the date, participants ID number and dosage 
for accountability. Participants will receive two single administration doses of the Centella 
asiatica product equivalent to 2g and 4g of Centella asiatica water extract. The order in which 
they receive these doses will be random to prevent a tolerance effect. The doses selected are 
based on allometric scaling104 from previous murine studies, which showed that 1.4 g/day of 
Centella asiatica water extract for a 70 kg human was equivalent to the 200 mg/kg/day 
estimated intake in mice. Participants will consume CAP in the presence of a member of the 
research team following baseline measurements, in order to ensure adherence to the study 
protocol. Following study completion, the remaining intervention and placebo sachets will be 
provided to Dr. Soumyanath for destruction. 
 
H.2. Administration 
The study intervention will be administered in an outpatient setting. The research pharmacy will 
dispense one dose of CAP to a member of the study team, who will reconstitute the CAP 
powder in 8-12 ounces of warm water and provide it to the participant for oral consumption. One 
dose will be administered at each study visit in a random order. 
 
H.3. Modification to study intervention 
There will be no modifications to the study intervention during the study unless there are 
adverse events are noted and recommendations are made by the study’s Independent 
Monitoring Committee (IMC).  
 
 
 
 
 
Figure 4:  Redacted 
 
H.4. Criteria for intervention discontinuation and stopping guidelines 
Participants will be advised during the informed consent process that they have the right to 
permanently discontinue the Centella asiatica product and/or withdraw from the study at any 
time without negative repercussions. Participants will also be counseled during the consent 
process that they may be withdrawn from the study at the discretion of the Principal Investigator 
or designees. A participant may discontinue and/or withdraw and/or be withdrawn from the 
study for the following reasons: 
 
Administrative: 
1) Withdrawal of participant consent 
2) Request of site investigator or designees 
3) Request of primary care physician 
4) Non-compliance 
5) Protocol deviation 
6) Premature termination of the study 
 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 17 of 33 
 
Adverse event (AE)/experience: 
1) Worsening of pre-existing disease 
2) Intercurrent illness 
3) Death 
4) Major/clinically significant alteration in laboratory values or biometric assessments after 
beginning product 
5) Other AE 
6) Other reasons concerning the participant’s health or well-being 
 
The study will be terminated if two participants terminate due to intervention-related adverse 
events. Severe adverse events will be reviewed immediately by the Principal Investigator, 
clinical investigators and the Independent Monitoring Committee (IMC) to determine if severity 
and relatedness warrant termination of the study (Section N). 
 
I. SCREENING AND STUDY ACTIVITIES 
I.1. Phone screening 
Potential participants will be screened over the phone by a member of the study team, primarily 
the study coordinator, prior to coming in for the in-person screening visit. A standardized 
telephone script will be used and has been submitted for IRB approval. The telephone 
screening will take approximately 10-15 minutes. Potential participants that meet eligibility 
criteria (Section F.2.2) over the phone will be scheduled for a screening visit.  
 
I.2. Consent process 
To ensure full comprehension of all aspects of their participation in the study, all potential 
participants will be mailed a copy of the IRB approved consent and HIPAA form prior to their in-
person screening visit. They will be instructed to review the form ahead of time but not to sign it 
until the visit. All in-person screening visits and consenting will occur in a CTRC outpatient 
room. At the in-person screening visit, a member of the study team will review the 
consent/HIPAA form and then will ask the participant to describe key points in the form (e.g. 
purpose of study, dietary requirements, length of study) before signing. After the participant 
signs the consent, the participant will continue with screening assessments and baseline 
measurements. A physical copy of their consent form will be provided to each participant and 
kept in the affiliated study folders to be made available as necessary to an Independent 
Monitoring Committee or the OHSU IRB. To ensure ongoing consent, participants will be called 
three days prior to their second study visit to ask if they wish to continue their participation in the 
study. At this time, participants will also be reminded of their upcoming study visit, to stop 
necessary supplements, and begin the necessary dietary changes (Section I.4.). Appropriate 
language will be in included in the consent form to avoid coercion. We will underscore that they 
can decline to participate at any point in the study, even if they have signed the consent form, 
and it will not impact their participation in future research trials. 
 
I.3. Screening visit 
The participant’s health status will be evaluated by a study clinician using a biometric 
assessment (blood pressure, height, weight and body mass index), vital signs (pulse rate and 
temperature), a screening exam, a mood assessment (Geriatric Depression Scale-15) and 
cognitive assessments (Clinical Dementia Rating and Mini Mental State Examination). A non-
fasting plasma and urinary laboratory evaluation (complete blood count, comprehensive 
metabolic panel and urinalysis) will be performed and submitted to the OHSU clinical core 
laboratory to identify asymptomatic illnesses that could impact study results (Table 2). A 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 18 of 33 
 
member of the study staff with venipuncture training or CTRC nursing personnel will collect 
approximately two teaspoons (10 mL) of venous blood by peripheral venipuncture and the 
participant will collect half a cup of urine (20 mL) via a clean-catch method for analysis. 
Participants will be provided with a handout describing the “low plant diet” (Section I.4.) that they 
are to follow prior to each study visit, should they qualify for the study. The visit is expected to 
take 45-60 minutes.  
 
A study clinician will review screening and laboratory results prior to participant enrollment. 
Participants will be contacted by phone and told whether they qualify for the study or not. 
Participants meeting all final eligibility criteria will be invited to participate in the intervention 
phase of the study and will be scheduled for Study Visit 1. Any participants with abnormal health 
screening or laboratory evaluation will be directed to their primary care physician for treatment 
and excluded from the study. In the event that a participant does not qualify for the study, their 
data will be destroyed at the end of the study.  
 
I.4. Study visits 1 and 2  
Visit timing: Visit 1 will occur a minimum of 72 hours following the screening visit to ensure 
adequate time for the low plant diet (Section I.4.), the screening laboratory results and for 
scheduling of appropriate facilities. Visit 2 will occur approximately 2-4 weeks following Visit 1 to 
allow for complete wash out of the study intervention (Figure 3).  
 
Low phytochemical “plant” diet: Due to the nature of the bioactive compounds from Centella 
asiatica, participants will be required to adhere to a “low plant diet” designed by the Oregon 
Clinical and Translational Research Institute Bionutrition Department at OHSU. They will comply 
with this diet for 48 hours prior to each study visit, for which they will be given a handout at the 
screening visit. The diet is essentially limiting intake of fruits, vegetables, coffee, whole grains, 
chocolate and tea, which may contain compounds similar to those found in Centella asiatica. 
Participants will be provided with food conforming to this diet for the duration of each study visit 
including three full meals and snacks as needed. To assess for success of the diet, a small 
amount of the blood obtained at the screening visit will be analyzed for the presence of the 
bioactive compounds while the participant is following their normal diet and then compared to 
baseline levels at each study visit. To monitor for dietary compliance, a 48-hour diet diary will be 
provided to each participant to be filled out each day and brought to each study visit. The diary 
will also include areas for documenting supplement and medication usage. This has been 
submitted with this protocol for approval.  
 
Sample collection: Participants will be asked to fast for 10-12 hours beginning at 9:00 PM the 
day prior to each study visit, excluding water, in an attempt to standardize gastrointestinal transit 
time and minimize delayed absorption of the study intervention due to the presence of food. A 
study clinician or CTRC nursing staff member will place a peripheral intravenous catheter in the 
participant’s arm or hand to allow for serial plasma specimen collection. Prior to administration 
of the Centella asiatica product, a baseline plasma sample of one tablespoon (15 mL) will be 
collected through the catheter using a syringe and transferred immediately into a heparinized 
Vacutainer tube. The participant will be asked to consume by mouth one of the two doses of 
Centella asiatica product dissolved in 8-12 ounces of warm water, on an empty stomach in the 
presence of study personnel to ensure compliance. Participants will be asked not to consume 
food for two hours following consumption of the study intervention to allow for transition into the 
small intestine and absorption. If signs of hypoglycemia present, participants will be allowed to 
consume a high glycemic food and monitored for improvement. Serial blood samples (two 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 19 of 33 
 
teaspoons (10 mL)/sample) will be obtained through the peripheral intravenous catheter using a 
syringe over a 12-hour post-administration period (15, 30, 45, 60, 90, 120, 180, 240, 360, 480, 
600, and 720 min) (Table 2). Participants will collect their urine for the duration of the study visit 
(12-hours) to assess for excretion of Centella asiatica metabolites. 
 
Tolerability and safety: We will record weight, height, body mass index, age, blood pressure, 
temperature and pulse rate at each study visit to monitor for adverse events and asymptomatic 
illnesses that may impact study results (Figure 3, Table 2). Participants will have an 
electrocardiogram (EKG) at the beginning of each study visit and six hours after intervention 
administration. Participants will be interviewed and administered a standardized multi-system 
adverse events questionnaire at the beginning and end of each study visit. This questionnaire 
has been adapted from the National University of Natural Medicine approved questionnaire and 
has been included in this IRB submission for approval. 
 
I.5. Visit 1 and 2 tolerability phone assessment 
Participants will be telephoned the day following each study visit to assess for delayed adverse 
events and/or complications from the intravenous catheter placement using interviews and a 
standardized multi-system adverse events questionnaire (Figure 3, Table 2). Participants will be 
instructed to contact a study clinician if they experience any reactions following the phone 
assessment, and prior to their second study visit or one-week exit interview.  
 
I.6. Exit interview tolerability assessment 
Participants will be telephoned one week following the completion of their second study visit, or 
first study visit if they elect to drop out of the study, to assess for delayed adverse events and/or 
complications from the intravenous catheter placement using interviews and a standardized 
multi-system adverse events questionnaire (Figure 3, Table 2). 
 
Table 2. Schedule of study visits and assessments. 
 
Screening 
 Visit 1 
Visit 1  
+ 1 
day 
Visit 2 
Visit 2   
+ 1 
day 
Visit 2    
+ 7 days 
Event 
 
Study 
visit 
Phone 
visit 
Study 
visit 
Phone 
visit 
Phone 
visit 
Informed consent 
 
 
 
 
 
 
Vitals✚ 
 
 
 
 
 
 
Health screening, biometric assessment 
 
 
 
 
 
 
Electrocardiogram (EKG) 
 
 
 
 
 
 
Blood draw (CBC, CMP*) and urinalysis 
 
 
 
 
 
 
Low plant diet (starting 48h prior to visit) 
 
 
 
 
 
 
Single dose of intervention  
 
 
 
 
 
 
Blood collected at intervals (0-12hr) for 
bioavailability and antioxidant 
assessment  
^ 
 
 
 
 
 
12-hour urine collection, baseline 
urinalysis 
 
 
 
 
 
 
Adverse events (AE) questionnaire 
 
 
 
 
 
 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 20 of 33 
 
  Vitals include blood pressure, temperature and pulse rate 
* CBC = Complete Blood Count with differential; CMP = Comprehensive Metabolic Panel 
^ Single time point 
 
I.7. Subject participation duration 
Including the screening visit, a two to four-week interval between doses, and an exit phone 
interview for adverse event monitoring, it will take a participant six to eight weeks to complete 
the study.   
 
 
 
 
 
J. STUDY DURATION 
Recruitment and data collection is expected to begin upon approval from the IRB and FDA and 
continue for seven months. We estimate a total of four months for final data analysis. Total 
estimated study duration is eleven months (Table 3). 
 
Table 3. Study timeline.  
                                                     Month 
Activity 
1-3 
4-6 
7-9 
10-12 
Finalize product manufacture/quality 
control 
  
  
  
  
  
  
  
  
  
  
  
  
Recruitment/enrollment 
  
  
  
  
  
  
  
  
  
  
  
  
Data collection 
  
  
  
  
  
  
  
  
  
  
  
  
Data analysis/summary 
  
  
  
  
  
  
  
  
  
  
  
  
Reports, manuscripts 
 
 
 
 
 
 
 
 
 
 
 
 
 
K. DATA AND SPECIMENS 
The principal investigator will oversee the management of the participants, intervention, and 
analysis, which will be carried out by the clinical investigators, research statistician, and 
research assistant. Data entry, quality control and preparation, and participant management will 
be ongoing throughout the study. 
 
K.1. Data collection methods  
The procedures for obtaining research material will include a telephone screening form, a 
medical history form, cognitive screening questionnaires (Mini Mental State Examination and 
Clinical Dementia Rating), a mood screening questionnaire (Geriatric-Depression Scale-15), a 
standardized adverse event questionnaire, a diet diary, electrocardiogram, urine collection and 
blood collection. Data will be collected directly from study participants specifically for research 
purposes. All blood collection will be performed at the CTRC. All data used for this project will 
be obtained only after receiving participant informed consent.  
 
K.2. Handling of data and specimens 
Upon enrollment, each participant will obtain a unique identification number. This number will be 
associated with all specimens and data collected from that participant. Following the collection 
of whole blood from the participant, a trained member of the study team will transfer the blood to 
the specified Vacutainer tubes and process the blood by centrifugation in the CTRC’s laboratory 
to separate the plasma for analysis. The isolated plasma will be transferred into labeled tubes 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 21 of 33 
 
and placed on ice until transported by a member of the study team via a sealed Styrofoam 
cooler labeled with biohazard to The Oregon Alzheimer Disease Center Biomarker Core Lab 
(Biomedical Research Building, OHSU). The plasma and a 50mL aliquot of the urine specimens 
will be stored in labeled boxes in Dr. Quinn’s -70°C freezer for up to five years. All written data 
will be stored in a locked cabinet in Dr. Soumyanath’s office (Richard Jones Hall, OHSU) for up 
to five years. All computerized data will be stored in OHSU’s installment of REDCAP and in a 
password protected folder on the OHSU X-drive for up to five years.  
 
K.3. Detection of Centella asiatica derived compounds in plasma and urine 
Biological levels of Centella asiatica triterpenes, caffeoylquinic acids, and their metabolites will 
be assessed in both plasma and urine using high-performance liquid chromatography coupled 
to tandem mass spectrometry (HPLC-MS/MS). We will also conduct additional targeted and 
untargeted analysis to fingerprint the Centella asiatica derived compounds found in plasma and 
urine samples.  
 
Plasma calibration curves will be made using pooled baseline human plasma from the study 
participants (one teaspoon (5 mL)/participant) separated by gender. The blank plasma will be 
spiked with increasing doses of commercially available reference triterpenes, caffeoylquinic 
acids and known metabolites, as well as appropriate internal standards (see below). If the blank 
plasma is found to contain traces of the analytes, we will calculate the true concentration by 
back extrapolation using the method of standard addition. A pooled baseline urine sample taken 
from participants (separated by gender) will be used as a “blank” urine sample for spiking with 
standards. 
 
Triterpenes; Plasma (200 µl) and urine samples (3000 µl) will be mixed with phosphoric acid 
and internal standard (chrysin) before incubation with E. coli glucuronidase and S. aerogene 
sulfatase to release the analytes of interest from glucuronide and sulfate conjugates. Incubated 
samples are passed through a Supelco SupelcleanTM C8 column, washed with dilute aqueous 
acetic acid to remove salts, and eluted with ammoniacal methanol. The eluant is vacuum dried, 
reconstituted in 200 µl of 1:1 methanol: ammonium acetate and filtered through a 0.2 µM 
spinfilter to remove residual particles before analysis. HPLC-MS/MS will be performed on an 
Applied Biosystems Q-Trap 4000 LC-MS instrument. The analytical method to be used is a 
modification of that described by Nair et al.(Nair, Menon et al. 2012) Chromatographic 
separation will be achieved using a Poroshell 120 EC18 column and methanol:ammonium 
acetate gradient. Triterpenes will be detected as their ammonium adducts with positive ion 
mode electrospray ionization using the following MS/MS transitions: AA (506/453) and MA 
(522/451), and chrysin will be detected as the molecular ion (255/255).  
 
Caffeoylquinic acids and metabolites: Plasma (200 µl) and urine samples (3000 µl) will be mixed 
with ascorbic acid and isotopic internal standards (13C9-caffeic acid, 13C3-ferulic acid, and d3-
isoferulic acid) before incubation with E. coli glucuronidase and S. aerogene sulfatase to release 
the analytes of interest from glucuronide and sulfate conjugates. Incubated samples will be 
applied to a Phree® column along with acidified acetonitrile (1 mL) and passed through by 
centrifugation, precipitating proteins and removing lipids. The eluant will be vacuum dried, 
reconstituted in 200 µl of 66% aqueous acetonitrile with 0.66% formic acid and filtered through a 
0.2µM spinfilter to remove residual particles. LC-MS/MS will be performed on an Applied 
Biosystems 5500 QTRAP HPLC-MS instrument. Chromatographic separation will be achieved 
using a C8 reversed-phase column and acidified acetonitrile:water gradient. Caffeoylquinic 
acids (CQAs), their metabolites and internal standards will be detected using negative ion mode 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 22 of 33 
 
electrospray ionization and the following MS/MS transitions: mono-CQAs (353/191); di-CQAs 
(515/353; 515/191); caffeic acid (179/135); ferulic acid and isoferulic acid (193/134); 
dihydrocaffeic acid (181/109); 4-methylcatechol (123/108); 4-ethylcatechol (137/122); 13C9-
caffeic acid (188/143); 13C3-ferulic acid (196/136); d3-isoferulic acid (196/134). 
 
Additional targeted and untargeted analysis of Centella asiatica derived compounds: Frozen 
aliquots of de-identified plasma and urine samples will be shipped on dry ice to Dr. Claudia 
Maier, Director of the Mass Spectrometry Center at Oregon State University according to OHSU 
biological materials shipping procedures. Targeted or untargeted high-performance liquid 
chromatography coupled to high resolution tandem mass spectrometry (HPLC-MS) analysis will 
be performed to fingerprint the Centella asiatica derived compounds present in the samples. 
The analytical method applied will be based on a method published in Alcazar Magana et al as 
described below.105 
 
Samples will undergo chromatographic separation using an Intersil Phenyl-3 column with an 
ejection volume of 10µL. Triplicate analysis will be performed. Gradient elution will be performed 
using a mobile phase consisting of solvent A (water containing 0.1% v/v formic acid) and solvent 
B (methanol containing 0.1% v/v formic acid), with a flow rate at 0.4mL/min. HPLC-MS analysis 
will be performed using a Shimadzu Nexera UHPLC system connected to an AB SCIEX 
TripleTOF® 5600 mass spectrometer equipped with a Turbo V ionization source operated in the 
electrospray ionization (ESI) mode will be used. Data-dependent acquisitions (DDAs) will be 
conducted to obtain precursor and fragment ion information using the negative (ESI-) and 
positive ionization (ESI+) modes. Level 1 annotations we will be based on accurate mass, 
fragment ion spectral similarity, and retention time co-elution based on authentic commercially 
available standards. Putative Level 2 annotations will be based on exact mass, isotopic pattern, 
and MS/MS spectral data.  
 
K.4. Analysis of total antioxidant compounds 
Total antioxidant compounds in plasma will be measured at each time point using the 
OxiSelect™ kit (Cellbiolabs Inc.) and following the manufacturer’s instructions. The assay, 
performed in 96-well plates, is a quantitative assay for measuring antioxidant potential within 
plasma samples. Following the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+) by 
antioxidants in the sample, the kit colorimetric probe develops a blue color that is read at 540-
600 nm. The antioxidant potential of samples is determined based on an iron standard curve 
and results are calculated at Fe2+ equivalents (µM) or FRAP value.  
 
K.5. Sharing of results with participants 
All screening laboratory results, performed by the CLIA certified OHSU Core Laboratory, will be 
provided to the participant. If a screening result is found abnormal (an incidental finding), the 
participant will be instructed to share their result with their primary care provider. 
Electrocardiograms (EKGs) will be performed at each study visit. If the EKGs are found to be 
abnormal, the participant will be instructed to share their result with their primary care provider. 
As there will be no additional laboratory assessments with clinical implications beyond the 
screening visit, no results from the study will be shared with the research participant or their 
provider(s).  
 
K.6. Data and specimen banking 
All data obtained from this study will be used for research purposes only and will comply with 
HIPAA regulations. No genetic research will be performed on these specimens. The specimens 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 23 of 33 
 
and data collected relate specifically to this research project; however, plasma samples may be 
stored at -70°C in Dr. Quinn’s laboratory or shipped to collaborators at Oregon State University 
for future analysis of bioactive compounds of Centella asiatica.  No specimens will be sent to or 
used in a repository. All specimens will be labeled with each participant’s unique numeric ID 
code but will not contain any identifiable participant information, and will be maintained at 
OHSU. 
 
L. DATA ANALYSIS 
L.1. Specimen analysis 
Each analyte of interest from Centella asiatica will be identified by its retention time and 
characteristic mass spectral fragmentation pattern using selected reaction monitoring (MS/MS). 
The peak area ratio to the appropriate internal standard will be identified and evaluated against 
the standard calibration curve of each analyte. Each analyte’s concentration will be used to 
generate a concentration-time curve for pharmacokinetic profiling. Pharmacokinetic profiling will 
use non-compartmental analysis to calculate common pharmacokinetic parameters such as 
peak concentration (Cmax), the time at peak concentration (tmax) and the total 12-hour 
bioavailability represented by the total area under the curve (AUC0-12). These will be compared 
to our mouse data in order to assess appropriateness of dosing level. Other calculable non-
compartmental analysis metrics will also be determined, such as the elimination half-life (t1/2) 
calculated from the profile tails and the mean residency time of the compounds in plasma. 
Descriptive assessment will be done at each of the doses to give basic pharmacokinetic 
summarization of the various Centella asiatica compounds and total antioxidant compounds 
measured by ferric reducing ability of plasma. Dose dependence of the study outcomes will be 
determined by evaluation of the within-subject differences in study outcomes between the 
different doses. For all continuous variables, dose effects will be evaluated both naively using 
paired t-tests and with ordinary least-squares regression with correction for commonly cited 
confounders including but not limited to baseline age and gender. Assumptions of normality will 
be confirmed using outcome density distributions alongside utility from formal tests, such as the 
Anderson-Shapiro. In instances of normality violation, statistical transformation such as natural 
logs and square roots will be considered, as will non-parametric equivalent tests such as the 
paired Wilcoxon rank-sum test. Standard diagnostics will be very conservative given the limited 
number of available participants but the effect of removing clearly obvious outliers will be 
evaluated. Although all Centella asiatica compounds of interest and pharmacokinetic 
parameters are defined a priori, multiple comparison correction to p-values will be applied as 
necessary, with final two-tail significance defined at p<0.05. 
 
L.2. Sample size 
Power analysis and sample size estimation for the dose-dependent pharmacokinetics of 
Centella asiatica analytes were derived from the literature, specifically the plasma dose 
response analysis of the triterpene asiatic acid by Grimaldi et. al.45 after single acute 
administration of 30 mg and 60 mg of a Centella asiatica triterpene fraction. In a repeated 
measures cohort of 12 males, they found significant differences (p<0.05) in asiatic acid’s peak 
plasma concentration (Cmax) (30 mg: 0.70 μg/ml ± 0.38; 60 mg: 1.36 μg/ml ± 0.45; d=1.57) and 
24-hour total bioavailability (AUC0-24) (30 mg: 4.16 ± 3.81; 60 mg: 9.39 ± 3.53; d=1.42) between 
the two doses. This paper was chosen as an analog for the current study since a proportionally 
equimolar dose response shift in the pharmacokinetic profile of asiatic acid can be expected of 
the proposed Centella asiatica product doses. Under that assumption, a significant within-group 
dose response at α=0.05 with at least 80% power would be found for Cmax with six participants 
and for AUC0-12 with seven participants.  Even if the inclusion of additional compounds in the 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 24 of 33 
 
water extract reduces the dose effect anticipated in the current study, a significant difference in 
the pharmacokinetic outcomes would still be seen with eight participants. We therefore, propose 
a cohort of four men and four women, which will enable dose responses to be seen within each 
gender individually, and allow for evaluation of an attenuated response by pooling gender or 
provide protection against study dropout.  
 
L.3. Data quality and management 
The Principal Investigator will oversee study progress including recruitment, retention, 
demographics, study participant status and error rates pertaining to inclusion/exclusion criteria 
and the study protocol. Tabulation of study data will have both physical and digital formats 
available. A member of the study team will record data from each study visit and phone visit on 
paper case report forms. Physical copies of the case report forms will be scanned into the 
participant’s REDCap encounter, kept in participant-specific binders and kept available for 
comparison against the final study database. Digital documentation will use the REDCap 
database system, which will maintain quality of integrity using prescribed logic rules inherent to 
the system. Data will be input immediately upon the participant’s visit and double-checked within 
48 hours for accuracy by a separate member of the study team or the Principal Investigator. 
This will include limited options for responses, protection against redundancy and double-data 
entry, and locking of the database for information entry upon study completion. This 
combination of steps will promote cohesiveness of study outcomes from study visit to data entry 
while maintaining the necessary protection of participant privacy. All plasma concentration 
calculations will be performed using a Microsoft Office Excel database by study personnel and 
double-checked within one week by a second member of the study team to ensure accuracy. 
The computer files will be protected with a password and only appropriate study personnel will 
have access to the database. 
 
M. PRIVACY, CONFIENTIALITY, AND DATA SECURITY 
All participant information, and even the fact that an individual is in the study, is considered 
confidential. Confidentiality will be assured in this study through several mechanisms. 
Individuals interested in participation in the study will call the study coordinator’s personal office 
telephone for a phone screening. The potential participant’s contact information (phone number, 
name, and address), telephone screening responses, and any other paper records will be 
documented in a secured binder that is kept in a secured and locked area when not in use and 
only accessible to study staff. During screening and study visits, the investigators will ensure 
physical privacy by conducting interviews and examinations in a closed room. After enrollment, 
each participant will be assigned an anonymous study ID number, which will be used on all case 
report forms, computerized data sheets and specimen containers. In the case of computerized 
information, access to the study data on computers will be password protected. Finally, 
participants will not be identified by name in any reports or publications, nor will data be 
presented in such a way that the identity of individual participants can be inferred. All staff are 
trained and annually re-certified regarding these procedures in Responsible Conduct of 
Research and HIPAA.   
 
N. PROVISIONS TO MONITOR THE DATA TO ENSURE THE SAFETY OF SUBJECTS 
Adverse event (AE) monitoring will occur in “real time” using a standardized Adverse Event 
Monitoring Form and reviewed weekly by the principal (Soumyanath) and clinical (Quinn) 
investigators. This form is a ninety-one point multi-system questionnaire that assesses all organ 
systems (eyes/ears/nose/throat, gastrointestinal, neurological/ musculoskeletal, 
psychological/general, cardiopulmonary, skin, genitourinary and whole body systems). It will be 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 25 of 33 
 
completed at the screening visit in order to collect data on any/all pre-existing symptoms and 
their severity, and will serve as baseline data to determine attribution to the study intervention. 
The Adverse Event Monitoring Form will be administered in person at the beginning and end of 
Visits 1 and 2, via telephone the day following Visit 1 and 2, and by telephone one week after 
Visit 2, to assess for acute and delayed adverse events. Electrocardiograms will be performed 
just prior to administration of Centella asiatica product and six hours after intervention to monitor 
for adverse events.  
 
Participants will be reminded and encouraged during clinic visits to contact a study clinician if a 
moderate or serious AE occurs. Participants will be given a telephone number they can call at 
any time to report potential adverse effects. All severe AEs will immediately be reported to the 
Independent Monitoring Committee (IMC) and the OHSU IRB by the study principal investigator. 
Severe AEs (SAEs) are defined as events that are life-threatening or result in hospitalization, 
permanent disability, cancer, death, or another significant outcome. 
 
N.1. Attribution to intervention 
At each administration, items identified on the Adverse Event Monitoring Form will be compared 
to baseline levels to determine if the symptom was pre-existing. If the symptom was pre-existing 
at the same or a previously greater severity, the symptom will not be considered attributable to 
the study intervention. If the symptom was pre-existing at lesser severity (or was not previously 
present), the symptom may be considered attributable to the study intervention. If the 
intervention is considered attributable, Dr. Quinn and Dr. Wright, in consultation with the 
Principal Investigator, will determine if an alternative clinical explanation exists for the adverse 
event. If an alternative explanation does not exist, the adverse event will be considered 
attributable to the study intervention. If an alternative explanation does exist, the adverse event 
will not be considered attributable to the study intervention. 
 
N.2. Independent Monitoring Committee (IMC) 
Data monitoring and safety oversight is to be carried out via an independent monitoring 
committee (IMC) composed of at least three members beyond the principal study team. A 
clinician with experience in natural supplement research will chair the IMC and the other two 
members will be a statistician and a second clinician. An IMC is planned over a Data Safety 
Monitoring Board (DSMB) based on the National Institute of Health (NIH) and National Center 
for Complementary and Integrative Health (NCCIH) listed criteria with respect to data safety and 
monitoring. This study is single-site and phase I trial in scope. In addition, Centella asiatica is 
expected to be largely safe based on pre-clinical work and its long-standing position and 
extensive usage in herbal medicine. However, the use of blinded randomization and an elderly 
study cohort predicates the use of a multi-person committee and not just an independent 
monitor.  
 
N.3. Plan for review and reporting 
The IMC will meet biannually to study un-blinded AE data coded by organ system, and on an 
ad-hoc basis as necessary, based on the cited reporting of severe AEs and continuing reviews 
to OHSU’s IRB. Should the protocol be amended as a result of data review, the OHSU IRB and 
monitoring body will be notified and the amendment approved prior to study amendment 
implementation, unless immediate implementation is required to protect the safety of the study 
participants. In such a case, the protocol amendment will be immediately implemented and the 
OHSU IRB and IMC will be notified directly after protocol amendment implementation. 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 26 of 33 
 
O. RISKS AND BENEFITS 
O.1. Risks to subjects 
Potential harm to participants will be minimized by excluding participants with the conditions 
detailed in section F.2.2. The risks to the subjects participating in this proposed research project 
are mainly those associated with venous access, potential toxicity of the test material, and time 
commitment. 
 
Venipuncture: The potential risks to participants are minimal and reversible. Likely risks include 
pain and bruising from the intravenous catheter or venipuncture needle insertion. Occasional 
risks include feeling lightheaded or faint during or immediately after a blood sample is collected. 
Unlikely risks include swelling, infection, transient hematomas, transient thrombophlebitis, a 
fractured catheter embolus, or an allergic reaction to the adhesives or sterilization materials 
used at the venous access point. To minimize risk, catheter insertions and blood draws will be 
conducted in a seated position and using aseptic technique by trained personnel. 
 
Centella asiatica product: Centella asiatica is an edible plant. The Botanical Safety Handbook49 
classifies Centella asiatica as a Class 1 herb, i.e. one that can safely be consumed when used 
appropriately. The widespread use of Centella as a dietary supplement and human studies 
performed with Centella asiatica (Appa Rao, Srinivasan et al. 1973, Tiwari, Singh et al. 2008, 
Dev, Mohamed et al. 2009, Wattanathorn, Mator et al. 2008, Roushan, Tiwari et al. 2013)12 
support its safety. At the doses given in this study, the risks are expected to be minimal and 
unlikely. The lowest dosage is based upon allometric scaling from mouse studies in which no 
toxicity was observed; however, the present product is a concentrated extract, and it is 
important to establish its safety, particularly in a vulnerable elderly population. We do not 
anticipate a great increase in adverse effects with the higher dosage, but are aware that they 
may occur. In particular, we anticipate potential transient gastrointestinal side effects such as 
nausea, gastric reflux/discomfort or diarrhea. Due to unknown interactions with pharmaceutical 
medications, we will exclude those on blood thinning medications (e.g. warfarin). See section 
C.4 above for further information.  
 
Treatment-emergent suicidal ideation and behavior have been identified as a concern for a 
number of drugs and drug classes. Because of these concerns, a prospective assessment for 
suicidal ideation and behavior will be monitored. Heisel et al. (2011)106 have identified a “suicide 
ideation” subscale in the Geriatric Depression Scale (GDS). We are using the GDS as part of 
study procedures, and will pay special attention to the identified subscale questions to assess 
potential risk of suicidal ideation or behaviors. 
Risks associated with a breach of confidentiality: There is a small risk that information about a 
study participant could be inadvertently disclosed to non-study personnel. Procedures to 
minimize this risk have been described in section M of this protocol. 
 
Other risks/discomforts associated with participation: The participants will experience a time 
burden by participation in the research study, as they will need to remain within the Clinical and 
Translational Research Center for 13 hours at each study visit (total 26 hours). Participants will 
be remunerated in a prorated fashion ($40 per visit) for their participation. They will also be 
asked to follow a low plant diet, which may present an economic burden. To mitigate the burden 
associated with bringing food that conforms to the diet or purchasing food during their study 
visits, participants will be provided with three meals and snacks that conform to the diet for each 
study visit at no expense. As this study is being conducted only at the OHSU Marquam Hill 
campus, there are travel and parking expenses associated with study participation. To address 


Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
Page 27 of 33 
 
this expense, participants will be provided with an option of a round trip taxi trip to their home 
(up to $50) or a validated parking pass for each study visit at no expense. 
 
O.2. Potential benefits to subjects 
As this is a pilot pharmacokinetic study with single administrations of the intervention, it is 
unlikely participants will experience any direct benefits. The data on dosing and tolerability will 
be extrapolated to trials of CAP in patients with mild cognitive decline or Alzheimer’s disease. 
 
 
P. IMPORTANCE OF KNOWLEDGE GAINED 
Alzheimer’s disease is a debilitating disease with very limited successful treatments. Due to a 
lack of effective interventions, physicians are looking to alternative therapies that can address 
comorbidities and cognitive changes. Centella asiatica water extract has shown promising 
changes in preclinical models warranting further robust investigation in humans. Because there 
are no previous studies of a well-characterized Centella asiatica product in Alzheimer’s disease, 
it is an important public health issue to scientifically evaluate this therapy to be able to perform 
further studies investigating its effect on cognition. This pharmacokinetic study of Centella 
asiatica will provide the bioavailability data for the selection of dosing and dosage intervals for 
future clinical trials. It will also provide preliminary acute safety and tolerability data on an extract 
of Centella asiatica that has not been previously investigated in humans.  
 
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Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
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Study Title: A pharmacokinetic study of Centella asiatica in the elderly 
Principal Investigator: Amala Soumyanath, PhD 
IRB#: 17697 
Date: Feb 28 2024 
Protocol v4.0 
 
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