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Liposomal Vitamin C Clinical Trial: 4x Higher Absorption


An independent, open label, randomised, oral bioavailability comparison study of Zooki liposomal vitamin C VS. non-liposomal vitamin C in healthy adults under fasting conditions.

The clinical trial found 3.91 x more Vitamin C in the bloodstream over a 48h period when using Zooki Vitamin C compared to conventional Vitamin C.


liposomal vitamin c absorption


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Please note this is a draft report - we maintain the right to edit or change this report at any time until it has been submitted to academic journals for publication.

List of abbreviations

β-HCG: Beta Human Chorionic Gonadotropin

°F: degree Fahrenheit

°C: degree Celsius

AE: Adverse Events

ALP: Alkaline phosphatase

ALT: Alanine Amino Transferase

ANOVA: Analysis of Variance

AST: Aspartate Amino Transferase

AUC: Area Under the Curve

BP: Blood Pressure

Cmax: Peak plasma concentration

CRF: Case Report Form

ESR: Erythrocyte Sedimentation Rate

GCP: Good Clinical Practice

GGT: Gamma Glutamyl Transferase

GLP: Good Laboratory Practice

GMP: Good Manufacturing Practices

Hb: Haemoglobin

hr: Hour

ICD: Informed Consent Document

ICH: International Council for Harmonization

IEC: Independent Ethics Committee

IMP: Investigational Medicinal Products

Kel: Elimination rate constant

Kg: Kilogram

LC-MS/MS: Liquid Chromatography Mass Spectrometry/Mass Spectrometry

µg: Microgram

mg: Milligram

mL: Milli Litre

mm: Millimetre

PK: Pharmacokinetics

Pvt Ltd: Private Limited

RBC: Red Blood Cells

RPM: Revolutions per minute

RPR: Rapid Plasma Reagin

RR: Respiratory Rate

RS: Respiratory System

SAE: Serious Adverse Event

SAS: Statistical Analysis System

SGOT: Serum Glutamic Oxaloacetic Transaminase

SGPT: Serum Glutamic Pyruvic Transaminase

SOPs: Standard Operating Procedures

t½: Terminal elimination half-life

tmax: Time to reach peak plasma concentration

tlag: tlag is the time prior to the time corresponding to the first measurable (non-zero) concentration.

WBC: White Blood Cells


Vitamin C (ascorbic acid) occurs naturally in foods such as citrus fruit, tomatoes, potatoes, and leafy vegetables. Vitamin C is important for bones and connective tissues, muscles, and blood vessels. Vitamin C also helps the body absorb iron, which is needed for red blood cell production. Ascorbic acid is used to treat and prevent vitamin C deficiency.

Vitamin C is a water-soluble vitamin that is commonly found in food. It is also used as an additive, because of its reducing and antioxidant properties. The usage of ascorbic acid in food is associated with a variety of biological activities such as the protection of oxidizable molecules, the retardation of enzymatic browning, oxygen scavenging, the prevention of nitrosamine production, etc. Vitamin C is highly reactive, and it is susceptible to oxidation which leads to rapid exhaustion in a variety of food products. Vitamin C cannot be produced by humans; it must only be consumed from fruits and vegetables. The deficiency in vitamin C results in scurvy, which presents with haemorrhage, hyperkeratosis, and haematological abnormalities. In addition, poor dietary intake of vitamin C, alcoholism, elderly age, socioeconomic deprivation, mental illness, malabsorption disorders, kidney failure, haemodialysis, and peritoneal dialysis have been identified as risk factors for low vitamin C endogenous levels and developing clinical symptoms of scurvy.

The bioavailability and shelf life of bioactive components have both been improved by using encapsulation technologies. Liposomes are vesicles that are simple models of highly complex cell membranes, consisting of lipid bilayers surrounding an aqueous core. Due to their unique biocompatibility and biodegradability, liposomes have attracted significant interest in the delivery and protection of both hydrophilic and hydrophobic compounds, such as vitamins. Liposomal encapsulated vitamin C helps to overcome the degradation of vitamin C due to high reactivity. It also improves the absorption into intestine by the controlled release of vitamin C.

Study parameters

Primary parameters


Maximum measured plasma concentration.


The area under the plasma concentration versus time curve from time zero to last measurable concentration as calculated by linear trapezoidal method.


The area under the plasma concentration versus time curve from time zero to infinity. Where AUC0-∞ = AUC0-t + Ct/ Kel, Ct is the last measurable concentration and Kel is the terminal elimination rate constant.

Secondary parameters


Time of the maximum measured plasma concentration.

The elimination or terminal half-life will be calculated as 0.693/ Kel.


Elimination rate constant will be calculated using the formula by two different time points after elimination of the drug.

Elimination rate constant

Elimination rate constant

Investigational plan

Overall study design and plan 


This was an open label, randomized, single dose, two-treatment, two sequence, two-period, two-way crossover, oral bioavailability study in healthy, adult, human subjects under fasting conditions.


The randomisation schedule was generated using SAS® software, Version 9.4 of SAS Institute Inc., USA by a Bio-statistician. The order of receiving the test and reference products for each subject during each period of the study was determined according to a randomisation schedule. The randomisation schedule was made available to the Principal Investigator, Clinical Investigator, Duty Physician, Medical Officer, Pharmacist, Sponsor Monitor, IEC and/or the Regulatory authority only. The bio-analytical personnel did not have access to the randomisation schedule during the entire course of analysis.

Figure 1: Participant’s Flow Diagram

Selection of study population

Potential subjects were screened by study investigators at the selected study site. Subject eligibility was determined on basis of following inclusion-exclusion criteria.

Inclusion Criteria

Volunteers meeting all of the following criteria will be considered for enrolment in the study:

  • Normal, healthy, adult, male and female human subjects of age between 21-65 years with Body Mass Index (BMI) ranges between 18.50 kg/m2 to 24.99 kg/m2 (according to the formula of BMI = weight (kg) / [height (m)]2).
  • Plasma ascorbic acid (vitamin C) at screening <75μmol/1.
  • Subject agrees to avoid vitamin C containing medications and dietary supplements from screening until last visit.
  • Subject agrees to avoid high activity physical exercise 72  hours prior to last visit
  • Subjects who have no evidence of underlying disease during screening and check-in and whose screening is performed within 29 days of check in.
  • Subjects whose screening laboratory values are within normal limits or considered by the physician or principal/clinical investigator to be of no clinical significance.
  • Healthy as documented by the medical history, physical examination (including but may not be limited to an evaluation of the cardiovascular, gastrointestinal, respiratory, musculoskeletal and central nervous systems) and vital sign assessments.
  • Generally healthy as documented by 12-lead electrocardiogram (ECG), X-Ray and clinical laboratory assessments.
  • Non-smokers or ex-smokers. Ex-smokers are defined as someone who has completely stopped smoking for at least the past 3 months.
  • Willing to consume ova-lacto vegetarian diet.
  • Willing to comply to all requirements of this study protocol as well as instructed by the study personnel
  • Female subjects within normal limits or clinically non-significant laboratory evaluation results for FSH & LH. 
  • Generally healthy as documented by gynaecological examination and breast examination (for female subjects during Period-I check-in only).
  • Female subjects of childbearing potential;
  • Practicing an acceptable non-hormonal contraceptive method of birth control after consulting with principal investigator; and/or
  • Surgically sterile (bilateral tubal ligation)

Exclusion Criteria

Subjects who met any of the following criteria were excluded from the study.

Volunteers with history or significant presence of the following will be excluded from participation/enrolment in the study:

  1. Evidence of allergy or known hypersensitivity to vitamin C or other related medicines.
  2. Subjects with hepatic encephalopathy, cholestasis, myasthenia, pre-existing liver disease, alcohol abuse, existing tinnitus, renal or liver impairment and pre-existing gallbladder disease.
  3. Any major illness in the last three months or any significant ongoing chronic medical illness.
  4. Any disease or condition which might compromise the haemopoietic, gastrointestinal, renal, hepatic, cardiovascular, musculoskeletal, respiratory, central nervous system, diabetes, psychosis or any other body system.
  5. History of alcohol addiction or abuse.
  6. Malabsorption syndrome that affects vitamin C metabolism.
  7. Heart failure, angina pectoris, ventricular arrhythmias or atrial fibrillation with >100/min ventricular rate.
  8. Gastrointestinal bleeding in past three months.
  9. Uncontrolled diabetes mellitus.
  10. Active psychiatric disorder, intention for suicidal, disorders with Unconsciousness.
  11. Psychopathic disorder, lack of cooperation.
  12. Chronic obstructive lung disease or active smoking (more than 2 cigarettes in the past 6 months)
  13. Taking more than 100 mg vitamin C daily within 2 weeks to screening.
  14. Consumption of caffeine and /or xanthine containing products (i.e. coffee, tea, chocolate, and caffeine-containing sodas, colas, etc.), tobacco containing products for at least 24 hours prior to check-in and throughout the entire study
  15. Consumption of grapefruit and its juice and poppy containing foods for at least 72 hours prior to check-in and throughout the study.
  16. Subjects who taken any prescription medications, over the counter medicinal products, herbal medications within 14 days prior to study check in and throughout the study.
  17. History of dehydration from diarrhoea, vomiting or any other reason within a period of 24 hours prior to study check-in of each period.
  18. An unusual or abnormal diet within 48.00 hours prior to study check-in of each period, for whatever reason e.g. because of fasting due to religious reasons.
  19. Subject who had participated in any other study within the 90 days of check-in.
  20. History of difficulty in swallowing.
  21. Positive results for drugs of abuse (Marijuana-THC, amphetamine-AMP, barbiturates-BAR, cocaine-COC, benzodiazepines-BZD and morphine-MOR) in urine prior to check-in of this study period.
  22. Positive results for alcohol breath test prior to check-in of any study period
  23. Any blood donation / excess blood loss within 90 days of check-in.
  24. Systolic blood pressure less than 90 mmHg or more than 140 mmHg and diastolic blood pressure less than 60 mmHg or more than 90 mmHg.
  25. Ingestion of any hormonal agent at any time within 14 days prior to start of study check-in.
  26. Female subjects demonstrating a positive pregnancy screen.
  27. Female subjects whose menstruation cycle coincides with the study periods.
  28. Female subjects who are currently lactating.
  29. Females likely to become pregnant during conducting of the study
  30. Use of hormone replacement therapy for a period of 6 months prior to dosing.
  31. Use of any oral contraceptives including oestrogen and progestin combined pills and progestin only pills within 28 days prior to period I dosing)

 Withdrawal Criteria 

Following criteria were set to withdraw the subjects from the study. 

  1. Subject who is willingly to withdraw from the study at any point in time. 
  2. In case of development of any systemic condition that in the opinion of the investigator, rendered the subject ineligible for further participation in the study. 
  3. Based on any other condition or circumstance as per the discretion of the investigator.


Study products

Test Product (T): Zooki Liposomal Vitamin C (1000 mg) liquid 

Reference Product (R): Non-liposomal Vitamin C (1000mg) liquid 

Storage and Dosage form

Storage conditions: Store in a cool, dry place away from direct sunlight.

Dosage and regimen: Oral Administration – 1 sachet (15ml) per day.

Subject Assignment to Treatment Groups


In order to preserve the blinding, test and reference packaging were matched for size, shape, colour, texture as well as labelling. The Participant IDs were arranged in a chronological order as per the randomisation chart. The blinding codes were secured in tamper-evident sealed envelopes with limited access at the site. Each envelope carried a slip identifying the participant ID and the treatment allocation (test or reference product).  The Master Randomisation Chart was sealed in an envelope and maintained in the Trial Master File (TMF) and the secured soft copy (electronic record) of the same was stored under respective project folder.

Current medications and any medications taken in the 14 days prior to the start of the study was recorded as prior/concomitant medications with the corresponding indication and dose. Prescription and over-the-counter (OTC) medications, including non-steroidal anti-inflammatory drugs (NSAIDs) will be recorded. All medications taken on a regular basis was recorded prior to commencing the use of the study product. If concomitant medication was required during the study, participants were treated accordingly and a decision to continue or discontinue the participants will be made by the investigator, based on the pharmacology and pharmacokinetics of the study product and the concomitant medication. All prescription and over the counter medications and vaccinations were recorded and reported.

Treatment Compliance

Current medications and any medications taken in the 14 days prior to the start of the study was recorded as prior/concomitant medications with the corresponding indication and dose. Prescription and over-the-counter (OTC) medications, including non-steroidal anti-inflammatory drugs (NSAIDs) was recorded. All medications taken on a regular basis were recorded prior to commencing the use of the study product. No concomitant medication were required during the study, However, when required, participants were treated accordingly and a decision to continue or discontinue the participants were made by the investigator, based on the pharmacology and pharmacokinetics of the study product and the concomitant medication. All prescription and over the counter medications and vaccinations were recorded and reported.

Exposure and safety variables

Exposure Variables

Various pharmacokinetic parameters were set as primary and secondary exposure variables. 

Safety Variables

No specific safety variables were assessed in this study; however, study participants vitals were monitored throughout the study period.

Data quality assurance

All data generated during the course of the study and the technical reports, in addition to on-line monitoring of critical phases will be liable for inspection and quality audit for compliance to this Protocol and all the governing SOPs of the Quality Assurance departments of CRO.

Statistical methods

Determination of Sample Size

A sufficient number of participants were screened and 24 participants satisfying inclusion and exclusion criteria were enrolled.

Data Handling

All data generated during the conduct of the study were directly entered in the respective raw data sheets. All such forms and transcribed data forms were compiled by the study personnel designated for the study and was checked wherever applicable for its completeness. All data related to the project was in the custody of the designated personnel, until transferred to archives of CRO.

Statistical Methods for Study Outcome Analysis 

Demographic and Baseline Information

Mean and SD value were calculated

Analysis of Exposure Parameters

Statistical analyses were performed using SAS® system for windows version 9.4 or above (SAS® Institute Inc., USA). Mean and SD value were calculated and inter group statistical significance (p) was calculated. 

Analysis of Safety Parameters

Mean and SD value were calculated

Changes in the conduct of the study or planned analyses



Study subjects

Disposition of Subjects

In the current cross-over study, the same pool of subjects were randomised to 2 treatment groups in a sequential manner. The disposition of subjects is presented in Table 1.

Table 1: Disposition of Subjects
Subject Details Group 1 (Test) Group 2 (Reference)
Randomized 24 24
Total no. of subjects dropped out 00 00
No. of subjects discontinued 00 00
Lost to Follow-up 00 00
Withdrawal 00 00
No. of subjects analysed 24 24

No protocol deviation was recorded during the entire study duration and all subjects completed the study per planned investigation. Data derived from all subjects were considered for statistical evaluation.

Demographic and Other Baseline Characteristics

Demographics and other baseline characteristics remain same for both treatment groups (test and reference) as the same subjects were crossed-over to next treatment group (Table 2).

Table 2: Demographic Characteristics (for Group 1: Test and Group 2: Reference)
Parameters Mean (±SD)
Age (Years) 41.21 (13.28)
Height (cm) 167.04 (3.04)
Weight (Kg) 66.05 (3.57)
BMI (Kg/m2) 23.33 (1.83)

Exposure evaluation

Primary Exposure Variables

Cmax: This parameter is defined as the maximum measured plasma concentration (Table 3). Cmax was 5.79 (1.56) µg/mL and 1.60 (0.33) µg/mL for Group 1 and 2, respectively.

AUC0-t: This parameter is defined as the area under the plasma concentration versus time curve from time zero to last measurable concentration as calculated by linear trapezoidal method (Table 3). AUC0-48h was 189.50 µg.h/mL and 48.44 µg.h/mL for Group 1 and 2, respectively.

AUC0-∞: This parameter is defined as the area under the plasma concentration versus time curve from time zero to infinity. Where AUC0-∞ = AUC0-t + Ct/ Kel, Ct is the last measurable concentration and Kel is the terminal elimination rate constant (Table 3). AUC0-∞ was 419.42 µg.h/mL and 99.49 µg.h/mL for Group 1 and 2, respectively.

Table 3: Exposure Variables-Mean (±SD)
PK parameters Group 1 (Liposomal vitamin C) Group 2 (Non-liposomal vitamin C) Comparison Ratio
Cmax (µg/mL) 5.79 (1.56) 1.60 (0.33) 3.61
AUC0-t=AUC0-48h (µg.h/mL) 189.50 48.44 3.91
AUC0-∞ (µg.h/mL) 419.42 99.49 4.22

Secondary Exposure Variables

tmax: This parameter is defined as the time of the maximum measured plasma concentration. tmax was 2 hours for both Groups 1 and 2.

t½: This parameter is defined as the elimination or terminal half-life calculated as 0.693/ Kel. t1/2 was 52.17 hours and 45.89 hours for Group 1 and 2, respectively.

Kel: Elimination rate constant is calculated using the formula by two different time points after elimination of the drug.

Elimination rate constant

Elimination rate constant

Kel was 0.013 and 0.015 for Group 1 and 2, respectively.

Table 4: Secondary Exposure Variables
PK parameters Group 1 (Liposomal vitamin C) Group 2 (Non-liposomal vitamin C) Comparison Ratio
tmax (h) 2.00 2.00 -
t½ (h) 52.17 45.89 1.14
Kel 0.013 0.015 0.88

Figure 2: Plasma concentration vs Time Curves for Group 1 and Group 2

Relative bioavailability (F) in terms of percentage in comparison with Test product (Liposomal vitamin C) can be calculated as follows:

% F = (AUCTest/AUCReference)*100

So, % F = 391.20; which infers that liposomal vitamin C has a 391% relative bioavailability compared to non-liposomal Vitamin C.

Safety evaluation

All subjects were observed for occurrence of any adverse event throughout the study period. The following safety-related parameters were evaluated during current study.

Haematological Parameters

Table 5: Haematological Parameter [Mean ± SD] at Enrolment
Parameters Mean SD
ESR (<15) 5.71 3.38
Haemoglobin (13.5-18) 15.41 1.42
PCV (42-52) 46.13 3.89
RBC Count (4.7-6.0) 5.17 0.54
MCV (78-100) 89.61 6.59
MCH (27-32) 29.94 2.45
MCHC (32-36) 33.40 0.67
RDW-CV (11.5-16.0) 14.23 0.76
RDW-SD (39-46) 44.74 4.03
Total Leukocyte Count (TC) (4000-11000) 7925.00 1081.16
Neutrophils (40-75) 53.07 6.90
Lymphocytes (20-45) 34.43 6.44
Eosinophils (01-06) 4.23 3.73
Monocytes (01-10) 7.57 1.37
Basophils (0-02) 0.76 0.32
Absolute Neutrophil Count (1.5-6.6) 4.22 0.91
Absolute Lymphocyte count (1.5-3.5) 2.71 0.56
Absolute Eosinophil count (AEC) (0.04-0.44) 0.34 0.30
Absolute Monocyte count (<1.0) 0.60 0.11
Absolute Basophil count (<0.2) 0.06 0.03
Platelet count (150-450) 253.21 60.09
MPV (7.9-13.7) 8.64 1.05
PCT (0.18-0.28) 0.21 0.04
Glucose Random (RBS)(<200) 95.21 61.74
Blood Urea Nitrogen (BUN) (7.0-21) 13.55 4.67
Creatinine (0.9-1.3) 0.83 0.17
Cholesterol Total (200-239) 190.85 31.58
SGO/AST (5-400) 28.00 24.70
SGOT/ALT (5-40) 26.88 27.53
Alkaline Phosphatase (SAP) (53-128) 98.64 30.07
BILIRUBIN Total (0.1-1-2) 0.65 0.36
Bilirubin Direct (0.0-0.3) 0.23 0.18
Bilirubin Indirect (0.1-1.0) 0.45 0.26

Vital Signs

Table 6: Vital Signs [Mean ± SD]
Parameters Visit 1 Visit 2
Systolic BP (mm Hg) 123.92 (4.54) 126.38 (3.69)
Diastolic BP (mm Hg) 75.58 (4.74) 81.13 (21.75)
Pulse rate (beats per minute) 75.38 (2.36) 76.46 (2.28)
Body Temperature (°F) 96.86 (0.86) 97.06 (0.82)

No adverse events were observed throughout the study period.


Vitamin C is an essential micronutrient, necessary for a variety of metabolic processes.  Gastrointestinal absorption of low doses of vitamin C is efficient and occurs in the small intestine via a sodium-dependent active transport mechanism. The extent of absorption of vitamin C is 80-90% at the usual intakes from food of 30-180 mg/day (SCF, 1993), but because the transporter is saturable, absorption efficiency gradually decreases at higher intakes (Kallner et al, 1979 and 1985; Hornig and Moser, 1981; Blanchard et al, 1997). There is a non-linear relationship between daily intake of vitamin C and plasma concentrations, with a 5-fold increase in intake from 0.5 g/day to 2.5 g/day producing only a 20% increase in plasma levels (Levine et al, 1996 and 1999). 

This study was designed to evaluate whether encapsulating vitamin C in liposomes alters the uptake of vitamin C and elevates plasma concentrations of vitamin C over a 48h time period in comparison with non-liposomal vitamin C administration.

Twenty-four subjects were administered both liposomal and non-liposomal vitamin C formulations, with sufficient interim washout period. The vitamin C concentration in venous blood was measured from baseline (0 h), 0.5, 1, 2, 6, 12, 24 and 48 h. The maximum vitamin C concentration for liposomal vitamin C (5.79 ± 1.56 µg/mL) was significantly higher (p<0.05) than that for the non-liposomal vitamin C ( 1.60 ± 0.33 µg/mL). The liposomal vitamin C was found to have a relative bioavailability of 391.20 %. and a longer half-life (t1/2 was 52.17 hours and 45.89 hours for liposomal and non-liposomal vitamin C, respectively).

Overall, the results demonstrate that oral delivery of vitamin C encapsulated in Zooki liposomes promotes greater bioavailability than non-liposomal vitamin C.

Vitamin C is a water soluble vitamin that is an important anti-oxidant in the body. Insufficient intake results in the deficiency condition scurvy and publications by The Linus Pauling Institute suggest that daily intakes of 1 g or more of vitamin C can protect against the common cold (see Miller and Hayes, 1982). This has been followed by other claims of beneficial effects on a variety of conditions and as such there has been extensive human exposure to intakes up to 10 g/day (Miller and Hayes, 1982). However despite this extensive human exposure, there are only limited data that are appropriate for use in risk assessment. The available human data in the literature suggest that supplemental daily doses of vitamin C up to about 1000 mg in addition to normal dietary intakes are not associated with adverse gastrointestinal effects, but that acute gastrointestinal effects may occur at higher intakes (3000-4000 mg/day). While there is uncertainty whether high intakes of vitamin C increase renal excretion of oxalate, which could increase the risk of renal stones, an increased risk of kidney stones was not found in individuals with habitual intakes of 1500 mg/day. The absorption of vitamin C is saturated at high doses and therefore intakes above 1000 mg/day would be associated with negligible increased uptake and tissue levels, but an increased risk of adverse gastrointestinal effects.

The current study shows that encapsulating vitamin C in liposomes increases uptake and presumably increases tissue levels while reducing the risk of gastrointestinal effects. Indeed, no adverse effects were reported in this study even though the plasma concentrations are nearly 4 fold higher. 


In summary, the current study findings demonstrated that oral delivery of vitamin C encapsulated in liposomes (Zooki Liposomal Vitamin C Liquid; 1000mg) produced circulating concentrations of vitamin C greater than those elicited by oral delivery of non-liposomal vitamin C (1000mg).


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