WO2009103093A1

WO2009103093A1 - Use of aloe vera for increasing the bioavailability of poorly absorbable medicinal drugs

Info

Publication number: WO2009103093A1
Application number: PCT/ZA2009/000012
Authority: WO
Inventors: Josias Hendrik Hamman; Alvaro M. Viljoen
Original assignee: Tshwane University Of Technology
Priority date: 2008-02-15
Filing date: 2009-02-16
Publication date: 2009-08-20

Abstract

A method of using an aloe vera product for increasing bioavailability of a medicinal drug by co-administration of the product with the drug. Use of an aloe vera product in the manufacture of a medicament which medicament includes a poorly absorbable medicinal drug.

Description

USE OF ALOE VERA FOR INCREASING THE BIOAVAILABILITY OF POORLY ABSORBABLE MEDICINAL DRUGS

Field of the Invention

This invention relates to the use of aloe vera to increase the bioavailability of poorly absorbable medicinal drugs, when co-administered with such medicinal drug or pharmaceutical.

Background to the Invention

Bioavailability is used to describe the fraction of an administered dose of unchanged drug that reaches the systemic circulation. When a drug is administered intravenously, its bioavailability is 100 %. When a drug is administered via other routes, such as orally, its bioavailability decreases due to incomplete absorption of the drug. Bioavailability must therefore be considered when calculating dosages for non-intravenous routes of administration.

Some drugs are absorbed so poorly when administered orally that patients in need of such a drug have no option but to administer the drug by means of injection (e.g. intravenously, intramuscularly or subcutaneously). Examples of such drugs are insulin, gentamicin, and amikacin. Administration by injection may however lead to non-compliance with a required dosage regimen due to discomfort and inconvenience experienced by the patient.

Absorption of orally administered drugs takes place mainly in the small intestine where absorption takes place transcellularly (i.e. across the lipid bilayer membrane of the intestinal cells) or paracellularly (i.e. between cells via the tight junctions and through the intercellular spaces).

Poorly absorbable drugs are unable to be absorbed (or are only poorly absorbed) transcellularly due to factors such as their molecular weight, their hydrophilic nature, and strongly charged functional groups. Paracellular absorption is also limited by these factors as well as by the presence of tight junctions between cells and the relatively small surface area (about 0,01%) of the intestinal epithelium presented by the paracellular pathway.

The enhancement of transcellular and paracellular absorption has been investigated and various drug absorption enhancers have been identified. However, most of these enhancers do not increase bioavailability sufficiently and/or are toxic.

Accordingly, a need exists for providing a non-toxic absorption enhancer which is able to increase bioavailability of poorly absorbable drugs to such an extent that they can be administered orally rather than intravenously.

Summary of the Invention

According to the invention, there is provided a method of using an aloe vera product for increasing bioavailability of a medicinal drug by coadministration of the product with the drug.

The aloe vera product may be a whole leaf powder of an aloe vera plant and/or a gel extruded from the inner pulp of an aloe vera leaf. The powder and gel may be obtained from the International Aloe Science Council in Texas, USA.

The aloe vera product and drug may be co-administered orally.

The drug to be targeted for increasing of its bioavailability by means of absorption enhancement may be selected from the group including: insulin, calcitonin, vasopressin, leucine, acyclovir, chlorpromazine, cyclosporine, erythromycin, gentamicin, isosorbide dinitrate, pyridostigmine, labetalol, methyldopa, morphine, and propranolol. It is to be appreciated, that the drugs listed above are examples and do not place a limitation on the drugs that may be co-administered with the aloe vera product as envisaged by the invention.

The aloe vera product may be co-administered together with an additional absorption enhancer such as, for example, chitosan and/or its derivative N- trimethyl chitosan chloride (TMC). In addition or alternatively, an enzyme inhibitor for inhibiting degradation of the drug in the intestinal tract may be coadministered.

It is believed that the aloe vera product is able to open the tight junctions between adjacent intestinal epithelial cells to allow for enhanced drug transport across the intestinal epithelium.

The opening of the tight junctions by the aloe vera product is furthermore believed to be completely reversible once the aloe vera product is no longer in contact with the epithelial cells and that no damage to cell structures is caused by it.

The invention extends to the use of an aloe vera product in the manufacture of a medicament which includes a poorly absorbable medicinal drug so as to enhance the bioavailability of the drug. The medicament may be administered to a patient in need of the drug included therein.

The aloe vera product may be a whole leaf powder of an aloe vera plant and/or a gel extruded from the inner pulp of the of an aloe vera leaf. The powder and gel may be obtained from the International Aloe Science Council in Texas, USA.

The medicament may be administered orally and may be in a form selected from the group including: a capsule, a tablet, a solution, and a suspension.

The drug may be selected from the group including: insulin, calcitonin, . vasopressin, leucine, acyclovir, chlorpromazine, cyclosporine, erythromycin, gentamycin, isosorbide dinitrate, pyridostigmine, labetalol, methyldopa, morphine, propranolol, and terbutaline.

The medicament may include an additional absorption enhancer such as, for example, chitosan and/or its derivative N-trimethyl chitosan chloride (TMC). The medicament may alternatively or additionally include an enzyme inhibitor for inhibiting degradation of the drug in the intestinal tract.

A medicament including an aloe vera product, which medicament includes a poorly absorbable medicinal drug.

The aloe vera product may be a whole leaf powder of an aloe vera plant.

The aloe vera product may be a gel extruded from the inner pulp of an aloe vera leaf.

The powder and gel may be obtained from the International Aloe Science Council in Texas, USA.

The medicament may in use be administered orally and is in a form selected from the group including: a capsule, a tablet, a solution, and a suspension.

The drug may be selected from the group including: insulin, calcitonin, vasopressin, leucine, acyclovir, chlorpromazine, cyclosporine, erythromycin, gentamycin, isosorbide dinitrate, pyridostigmine, labetalol, methyldopa, morphine, propranolol, and terbutaline.

The medicament may includes an additional absorption enhancer, for example chitosan and/or its derivative N-trimethyl chitosan chloride (TMC).

The medicament may include an enzyme inhibitor for inhibiting degradation of the drug in the intestinal tract. Detailed Description of the Invention

A study was conducted to determine the potential of Aloe vera gel and whole leaf components as intestinal absorption enhancing agents. This was done by conducting different in vitro transport studies across Caco-2 intestinal epithelial cell monolayers as well as the effects of the Aloe vera components on the transepithelial electrical resistance (TEER) of Caco-2 cell monolayers.

MATERIALS

Aloe vera gel and whole leaf powder were obtained from the International Aloe Science Council (IASC051309, Texas, USA), N-trimethyl chitosan (TMC) was synthesized from chitosan (91% deacetylated chitosan, Warren Chem Specialities, South Africa), insulin (from bovine pancreas, Sigma-Aldrich, Germany), luteolin and rutin (Sigma-Aldrich, Germany)

Caco-2 cells (originally obtained from the American Tissue Culture Society, USA), Dulbeco's Modified Eagle's Medium (DMEM) (Bio Whittaker, Walkersville, MD, USA), 10% fetal bovine serum (Delta Byproducts, Johannesburg, South Africa), 1% non-essential amino acids (Bio Whittaker, Walkersville, MD, USA), 1 % pen/strep fungizone mixture (10 000 U penicillin/ml, 10 000 μg streptomycin/ml and 25 μg fungizone/ml, Bio Whittaker, Walkersville, MD, USA), Hank's Balanced Salt Solution (HBSS) (Bio Whittaker, Walkersville, MD, USA), trypsin-versene solution (Bio

Whittaker, Walkersville, MD, USA), HEPES [n-(2-hydroxyethyl) piperazine-N- (2-ethanesulfonic acid)] (Bio Whittaker, Walkersville, Maryland, USA)

DESIGN OF THE STUDY

The study was a quantitative research study with a true experimental design where the dependant variables (i.e. TEER and drug transport) were manipulated to measure the effect (i.e. absorption enhancement potential of

Aloe vera components), but all other conditions were kept constant. Control groups were included to indicate that the measured effect was indeed caused by the manipulation and not by chance interferences or external factors. The experiments were done in triplicate and the averages as well as standard deviations were calculated to indicate repeatability. Analytical methods were validated to ensure they meet proper standards of accuracy and reliability by means of parameters such as linearity, precision, specificity, selectivity, sensitivity and system suitability.

THE STUDY

In the first part of the study, the effect of two Aloe vera components (i.e. gel and whole leaf extract) on the TEER of Caco-2 cell monolayers was measured in five different concentrations and at two pH values. Reversibility of the effect of the plant components on the tight junctions of the epithelial cells was investigated by continual measurement of the TEER after removal of the components from the cell monolayers. This evaluation of the effect on the TEER by the plant components was done to determine their potential to open tight junctions as a mechanism of action for drug absorption enhancement.

Transepithelial electrical resistance (TEER) is calculated from the measured potential difference between the apical and the basolateral sides of a cell monolayer and is inversely related to the ion flow across the monolayer (Powell, 1987:1267). The movement of ions between the apical and basolateral sides of epithelial cells through the intercellular spaces depends on the integrity of the tight junctions and a decrease in the TEER value is therefore associated with the opening of the tight junctions. TEER studies have previously been used to predict the paracellular transport of hydrophilic compounds across epithelial cell monolayers (Borchard et a/., 1996:131 and Schipper et a/., 1997:923).

The effect of Aloe vera gel and whole leaf extract in different concentrations on the in vitro transport of three different model-compounds across Caco-2 cell monolayers followed in the second part of the study. The apparent permeability coefficients were calculated for the model compounds in the presence and absence of the plant components. The absorption enhancement ratios were calculated from the transport results and utilised to indicate the absorption enhancement ability of the Aloe plant components in different concentrations and pH values. TEER STUDY

Aloe vera gel and whole leaf extract powder and TMC (degree of quaternisation of 61.7%) were prepared in single compound solutions or in combination solutions in serum-free DMEM to give a series of solutions with concentrations as listed in Table 4.1. All the TEER experiments were carried out in acidic (pH 5.8) and neutral (pH 7.4) environments, which were obtained by means of adjusting the solutions with addition of 0.1 M HCL or 0.1 M NaOH. Test solutions were freshly prepared on the day that the TEER experiments were performed. Table 4.1 : Concentrations of test solutions used in the TEER study

Concentration % (w/v) Test solutions

0.10 0.25 0.50 1.00 2.50 5.00

Aloe vera gel

TMC

Aloe vera whole leaf extract -/

Aloe vera gel + 0.25 % TMC •/

Whole leaf + 0.25 % TMC S •/

The percentage reduction in TEER (% of initial value) was calculated and these TEER values were plotted as a function of time to determine the effect of the Aloe vera gel and whole leaf solutions on the TEER of the Caco-2 cell monolayers.

TRANSPORT STUDIES

The effect of Aloe vera gel and whole leaf extract on Caco-2 cell monolayer permeability was investigated by monitoring the transport of three different model compounds in presence of these Aloe vera components. All experiments were performed in the apical to basolateral direction at two different pH conditions (i.e pH 5.8 and pH 7.4, respectively).

Insulin is a macromolecular protein compound with limited absorption after oral administration due to enzymatic degradation and poor membrane permeability. One approach that has been investigated to increase its intestinal absorption is the modulation of tight junctions to allow paracellular transport across the mucosal epithelium (Tirumalasetty et a/., 2005:246).

Aloe vera gel and whole leaf extract powder was dissolved in Hank's Balanced Salt Solution (HBSS) containing 170 ug/ml insulin, which was first dissolved in a small volume of 0.1 N HCI and then made up to the required quantity with HBSS. The pH of the solutions was adjusted to 5.8 or 7.4 with addition of 0.1 M HCL and/or 0.1 M NaOH. The concentrations of the Aloe vera components used in the insulin transport studies are shown in table 4.2.

Table 4.2: Concentrations of Aloe vera solutions used in the insulin transport study

Concentration w/v)

Test solution

0.10 0.50 1.00 2.50 5.00

Aloe vera gel

Aloe vera whole leaf extract

Aloe vera gel and whole leaf extract powder was dissolved in Hanks balanced salt solution (HBSS) containing 60 μg/ml luteolin, which was dissolved in ethanol then made up to the required quantity with HBSS. The final ethanol concentration was 50 mg/ml (Laitinen et al., 2004:1906). The concentrations of the Aloe vera gel and whole leaf components used in the luteolin transport studies are shown in table 4.3.

Table 4.3: Concentrations of Aloe vera gel and whole leaf extract solutions used in the luteolin transport study

Concentration (% w/v)

Test solution —

0.50 1.00 2.50 5.00

Aloe vera gel •/

Aloe vera whole leaf extract S Rutin (3,3',4',5,7-pentahydrohyflavone-3-rhamnoglucosicie) is a flavonoid found in many typical nutrimental plants. It is an important dietary constituent of food and plant-based beverages. Like many other flavonoid derivatives, which all display numerous biological and pharmacological activities, rutin exhibits antioxidant, anti-inflammatory, anti-carcinogenic, anti-thrombic, cytoprotective and vasoprotective activities (Kuntic eif a/., 2007:718).

Aloe vera gel and whole leaf powder was dissolved in Hanks Balanced Salt Solution (HBSS) containing 50 μg/ml rutin (Sigma-Aldrich, Gemany), which was dissolved in ethanol then made up to the required quantity with HBSS. The final ethanol concentration was 50 mg/ml. The concentrations of the Aloe vera components used in the rutin transport studies are shown in table 4.4.

Table 4.4: Concentrations of Aloe vera gel and whole leaf extract solutions used in the rutin transport study

Concentration (% w/v) Test solution

0.50 1.00 2.50 5.00

Aloe vera gel V V V V

Aloe vera whole leaf extract V V V V

RESULTS

EFFECT OF ALOE VERA GEL AND WHOLE LEAF EXTRACT ON THE TRANSEPITHELIAL ELECTRICAL RESISTANCE OF CACO-2 CELL MONOLAYERS

Aloe vera gel and whole leaf extract solutions prepared in concentrations of 0.1 , 0.5, 1.0, 2.5 and 5.0% (w/v) were used to investigate their effects on TEER of confluent Caco-2 cell monolayers, respectively. The Aloe vera gel and whole leaf extract solutions were prepared in serum-free DMEM and the pH was adjusted to 5.8 with 0.1 M HCI and to 7.4 with 0.1 M NaOH. After addition of the test solutions to the apical side of the cells, the TEER was measured every 20 min until removal of the solution after which the measurements continued.

Effect of Aloe vera gel on TEER at pH 5.8 and 7.4

The TEER values of Caco-2 cell monolayers measured at different time points after incubation with Aloe vera gel at pH 5.8 are shown in Table 5-1 , while those at pH 7.4 are shown in Table 5-2.

Aloe vera gel was able to significantly (p < 0.05) reduce the TEER of the Caco-2 cell monolayers in a concentration dependent way. The effect on the TEER was also pH dependent, but to a lower extent compared to concentration. After removal of the test gel solutions from the apical side of the cell monolayers, the TEER recovered relatively fast over the first 20 min and eventually returned to its original value. A 100% recovery of the cell monolayer integrity was already obtained at 120 min after removal of the gel solutions from the cell surface, which may be an indication that no damage occurred to the cells or their tight junctions. Aloe vera gel therefore opens the tight junctions of intestinal epithelia in a reversible manner and thereby demonstrates the potential to act as a safe paracellular absorption enhancer.

Table 5.1 : The effect of Aloe vera gel on the TEER of Caco-2 cell monolayers at pH 5.8

Time TEER (% of initial value)

(min) Control 0.1 % w/v 0.5 % w/v 1.0 % w/v 2.5% w/v 5.0 % w/v

102.86 ± 100.58 ± 100.39 ± 99.93 ±

-60 100.40 ± 100.21 + 1.38 0.21 5.02 8.12 6.53 7.90

105.54 ± 99.34 ± 100.57 ± 100.14 ±

-40 101.44 ± 98.58 + 5.61 1.23 1.15 9.11 0.21 6.61

98.48 ± 99.17 ±

-20 99.00 + 96.43 ± 98.94 ± 100.67 ± 5.02 2.89 6.70 6.60 5.36 4.99

0 100.00 + 100.00 ± 100.00 ± 100.00 ± 100.00 + 100.00 + 0.00 0.00 0.00 0.00 0.00 0.00

99.62 ± 89.00 ±

20 82.52 + 75.80 ± 69.01 ± 57.73 ± 4.21 4.33 2.73 5.21 3.20 4.75

98.87 ± 84.90 + 78.25 +

40 71.96 ± 67.63 ± 54.27 ± 4.23 5.56 5.92 2.99 1.56 1.68

97.24 +

60 81.98 ± 72.23 ± 63.80 ± 59.44 ± 46.31 ± 6.13 7.80 4.03 4.88 0.74 3.65

96.10 ±

80 81.97 ± 71.46 + 66.09 ± 55.16 ± 49.54 ± 4.76 7.45 3.63 1.96 3.60 3.64 100 98.29 ± 87.07 ± 73.86 ± 66.73 ± 56.45 ± 54.10 ± 4.09 5.31 4.84 4.71 5.17 2.19

80.89 ± 57.34 ±

120 99.89 ± 87.23 ± 75.26 ± 62.86 + 1.54 2.27^* 1.85^* 3.14^* 4.93^* 3.60^*

140 102.04 ± 104.09 ± 95.03 ± 92.71 + 92.89 ± 93.85 ± 6.18 4.50 8.29 4.21 2.30 3.55

160 101.66 + 103.41 ± 105.10 ± 99.36 ± 98.09 ± 97.72 ± 3.48 4.65 5.20 2.93 4.79 2.11

180 99.72 ± 97.89 + 103.40 ± 99.23 ± 99.39 ± 98.98 ± 5.05 3.39 1.83 4.49 7.35 4.32

200 101.24 ± 98.19 ± 98.92 ± 99.36 ± 98.27 ± 101.91 ± 1.13 1.32 7.98 4.26 5.95 4.14

220 100.67 + 100.64 + 98.06 + 102.19 + 102.06 + 100.06 ± 3.03 4.03 4.23 1.56 7.54 4.28

240 100.06 ± 100.57 + 98.91 ± 100.10 + 102.10 1 99.03 ± 3.28 6.28 4.03 8.35 1.69 7.55

*Statistically significantly different from control at time 120 min (p < 0.05).

Table 5.2: The effect of Aloe vera gel on the TEER of Caco-2 cell monolayers at pH 7.4

Time TEER (% of initial value)

(min) Control 0.1 % w/v 0.5 % w/v 1.0 % w/v 2.5% w/v 5.0 % w/v

97.24 ± 99.29 ± 99.02 + 99.57 ±

-60 101.80 ± 97.06 ± 3.65 2.11 4.69 0.81 1.03 4.26

101.57 ± 99.90 ± 101.48 ± 101.92 + 102.23 ± 98.10 +

-40 2.65 0.86 4.65 3.03 2.03 1.40

97.81 ± 95.30 ± 98.39 ± 100.33 ± 100.08 ± 97.25 ±

-20 2.14 1.69 4.86 1.29 1.63 2.10

100.00 ± 100.00 ± 100.00 ± 100.00 + 100.00 ± 100.00 ±

0 0.00 0.00 0.00 0.00 0.00 0.00

96.58 ± 90.91 ±

20 84.75 ± 92.63 ± 91.38 ± 82.61 + 2.48 1.14 2.69 2.52 1.08 1.29

99.11 + 90.50 + 85.97 ± 68.63 ± 59.26 ± 52.78 +

40 8.52 2.38 1.73 2.50 2.60 0.86

97.23 ± 86.47 ± 82.97 ± 65.00 + 56.01 ± 48.51 ±

60 4.25 1.50 1.11 2.49 3.09 3.43

98.62 ± 87.63 + 84.00 ± 68.53 ± 62.19 ± 53.74 +

80 5.35 2.97 3.57 2.80 3.69 3.52

102.39 ± 88.57 ± 83.43 ± 69.80 ± 61.871 53.97 ±

100 5.13 3.29 2.61 2.87 3.09 1.38

99.72 + 91.06 + 84.29 ± 73.73 ± 70.70 + 58.53 +

120 2.66 7.33 4.28^* 2.17^* 1.74^* 3.33^*

103.81 ± 104.55 ± 106.12 + 96.22 + 93.43 + 88.77 ±

140 1.01 4.10 6.70 7.82 1.80 5.35

100.66 ± 103.87 ± 107.24 ± 93.66 ± 90.46 + 91.76 +

160 5.20 4.38 3.29 10.09 5.82 5.67 98.13 + 98.33 ± 99.33 ± 96.51 ± 94.99 ± 93.75 +

180 2.11 3.01 2.13 5.51 5.26 5.42

97.81 ± 98.63 ± 103.64 + 97.53 ± 103.60 ± 98.24 ±

200 5.45 1.20 6.35 4.13 4.26 1.13

101.82 ± 101.09 + 101.59 ± 100.24 ± 102.61 ± 101.71 +

220 2.17 3.86 4.31 4.19 5.13 3.82

99.57 + 101.02 ± 100.00 ± 98.30 ± 103.04 ± 98.59 ±

240 3.82 6.11 6.27 6.53 1.66 2.79

*Statistically significantly different from control at time 120 min (p < 0.05).

Effect of Aloe vera whole leaf extract on TEER at pH 5.8 and 7.4

The TEER values of Caco-2 cell monolayers at different time points after incubation with Aloe vera whole leaf extract at pH 5.8 are shown in Table 5-3 and those at pH 7.4 in Table 5-4.

The Aloe vera whole leaf extract solutions were able to significantly (p < 0.05) reduce the TEER of the Caco-2 cell monolayers from a concentration of 0.5 % w/v and higher and this effect was concentration dependent. Furthermore, the effect on the TEER was pH dependent, albeit to a lesser extent than that for concentration. The TEER reduction was higher at a lower pH value probably indicating that protonation of phytoconstituents in the whole leaf extract influenced their interaction with the Caco-2 cell monolayers. After removal of the whole leaf extract solutions from the apical side of the cell monolayers, the TEER recovered relatively fast and a 100% recovery of the cell monolayer integrity was obtained at 20 - 40 min after removal of the whole leaf extract solutions from the cell monolayer surface, which may be an indication that no damage occurred to the tight junctions or cells. Aloe vera whole leaf extract showed the ability to open the tight junctions of intestinal epithelia in a reversible way and thereby demonstrated the potential to act as a paracellular absorption enhancer.

However, the Aloe vera gel reduced the TEER to a higher extent (i.e. a concentration of 5% w/v reduced the TEER to 46.31 ± 3.65 % of the initial value at pH 5.8 and 48.51 ± 3.43 % at pH 7.4) compared to the Aloe vera whole leaf extract (i.e. a concentration of 5% w/v reduced the TEER to 56.83 ± 1.44 % of the initial value at pH 5.8 and 65.43 ± 2.62 % at pH 7.4). This may be explained in terms of the different composition of phytoconsituents in the two Aloe vera products.

Table 5.3: The effect of Aloe vera whole leaf extract on the TEER of Caco-2 cell monolayers at pH 5.8

Time TEER (% of initial value)

(min) Control 0.1 % w/v 0.5 % w/v 1.0 % w/v 2.5% w/v 5.0 % w/v

102.86 ± 102.86 ± 97.40 + 101.34 + 102.49 ± 103.62 +

-60 1.38 3.74 4.01 4.20 1.91 2.93

105.54 + 105.44 + 100.62 ± 107.67 ± 106.41 ± 106.09 +

-40 5.61 2.85 3.64 3.60 1.12 0.20

98.48 ± 99.08 ± 94.26 + 96.1 ± 96.11 + 97.66 ±

-20 5.02 2.69 5.80 2.08 3.65 2.16

100.00 ± 100.00 + 100.00 ± 100.00 + 100.00 ± 100.00

0 0.00 0.00 0.00 0.00 0.00 +0.00

99.62 ± 88.24 ± 85.66 + 91.80 ± 87.60 + 79.51 +

20 4.21 5.10 4.21 5.40 5.69 1.68

98.87 ± 87.88 + 83.72 ± 78.18 ± 75.60 ± 67.36 ±

40 4.23 4.88 5.60 3.41 0.85 2.79

97.24 ± 81.76 + 79.86 ± 62.89 ± 61.25 ± 56.83 ±

60 6.13 5.15 5.74 2.04 0.62 1.44

96.10 ± 84.52 ± 82.77 ± 71.28 ± 68.79 ± 58.54 ±

80 4.76 2.73 6.07 4.07 3.18 2.91

98.29 + 85.24 + 82.91 ± 71.71 + 71.64 + 62.82 ±

100 4.09 5.27 5.13 2.38 0.76 2.21

99.89 + 91.85 ± 89.06 ± 74.90 ± 76.85 ± 71.72 ±

120 1.54 6.10 5.63^* 3.28^* 0.72^* 0.55^* 102.98 ± 104.96 ± 99.68 ± 93.75 ±

140 102.04 ± 100.35 ± 6.18 3.69 7.25 4.02 2.66 4.96

101.66 ± 103.04 ± 103.32 ± 105.18 ± 101.28 ± 103.26 +

160 3.48 2.81 6.83 3.05 9.27 7.45

99.72 ± 100.43 ± 102.48 ± 100.48 ± 103.64 ± 101.01 +

180 5.05 1.61 9.95 4.91 7.34 7.88 24 ± 99.46 ± 102.21 + 102.25 + 102.31 ± 99.97 ±

200 101. 1.13 3.45 9.74 4.71 9.46 4.47

220 100.67 ± 100.27 ± 97.40 ± 103.42 + 102.60 ± 102.09 + 3.03 4.31 3.68 5.08 3.00 2.92

100.06 ± 101.66 ± 102.67 ± 101.85 ± 98.89 ± 99.85 ±

240 3.28 7.95 2.53 1.28 6.33 5.22

*Statistically significantly different from control at time 120 min (p < 0.05).

Table 5.4: The effect of Aloe vera whole leaf extract on the TEER of Caco-2 cell monolayers at pH 7.4

Time TEER (% of initial value) (min) Control 0.1 % w/v 0.5 % w/v 1.0 % w/v 2.5% w/v 5.0 % w/v

97.24 ± 99.17 + 103.57 ± 101.21 ± 97.00 ± 99.70 ±

-60 3.65 1.03 1.98 3.93 2.06 4.94

101.57 ± 104.70 ± 103.02 + 101.33 ± 100.47 + 101.76 ±

-40 2.65 4.71 1.53 2.65 2.71 2.44

97.81 ± 98.58 ± 98.87 ±

-20 97.28 ± 102.93 ± 98.36 ± 2.14 2.64 2.58 2.31 7.02 2.44

100.00 ± 100.00 + 100.00 ± 100.00 ± 100.00 + 100.00

0 0.00 0.00 0.00 0.00 0.00 ±0.00

96.58 ± 101.53 ± 86.38 + 81.58 ±

20 94.16 + 92.32 ± 2.48 5.44 3.39 3.09 1.72 1.69

99.11 ± 102.01 + 87.67 ± 88.71 ± 83.01 + 76.71 ±

40 8.52 4.14 4.38 3.16 2.96 5.61

97.23 ± 100.69 ± 82.37 ± 80.48 ± 76.78 ± 66.24 ±

60 4.25 5.86 1.93 4.86 2.53 2.43

98.62 ± 67.20 +

80 92.85 ± 80.04 ± 76.51 ± 74.65 + 5.35 4.16 6.00 0.97 1.54 1.57

102.39 + 92.14 ± 80.35 ± 75.53 ± 71.28 ± 65.43 ±

100 5.13 4.46 9.52 2.37 1.12 2.72

99.72 ± 98.14 + 82.66 ± 78.93 ± 71.75 ± 65.43 ±

120 2.66 8.67 12.24^* 1.00^* 2.13^* 2.62^*

103.81 ± 98.29 ± 103.81 ± 101.88 ± 101.57 ± 103.38 ±

140 1.01 8.07 6.67 3.41 4.52 4.77

100.66 ± 95.99 ± 98.35 ± 102.08 ± 97.67 + 98.42 ±

160 5.20 2.13 5.82 1.55 2.72 5,73

98.13 + 96.68 ± 98.57 ± 97.47 ± 100.91 ± 97.51 ±

180 2.11 1.58 12.24 0.51 2.84 3.46

97.81 ± 101.68 ± 99.89 ± 99.22 ± 101.97 ± 100.43 ±

200 5.45 3.25 4.99 2.48 7.24 2.45

101.82 + 104.14 ± 98.96 + 100.34 ± 100.58 + 102.31 ±

220 2.17 1.76 5.38 2.63 1.96 7.37

99.57 ± 104.00 ± 100.36 + 98.90 ± 100.35 98.34 ±

240 3.82 2.98 8.48 3.11 ±1.21 3.21

*Statistically significantly different from control at time 120 min (p < 0.05).

Effect of Aloe vera αel combined with TMC on TEER at pH 5.8 and 7.4

The TEER values of Caco-2 cell monolayers measured at different time points after incubation with Λ/-trimethyl chitosan chlorite (TMC) alone as well as in combination with Aloe vera gel at pH 5.8 are shown in Table 5-5, while those at pH 7.4 are shown in Table 5-6.

From the results it is clear that a combination of Aloe vera gel (5 % w/v) and TMC (0.25 % w/v) reduced the TEER of the Caco-2 cell monolayers to a larger extent (e.g. a reduction in TEER to 39.36 ± 2.90 % of the initial value at pH 5.8) as compared to TMC alone (at a corresponding TMC concentration of 0.25 % w/v the TEER reduction was to 59.92 ± 0.97 % of the initial value at pH 5.8) or to the Aloe vera gel alone (at a concentration of 5 % w/v the TEER reduction was to 46.31 ± 3.65 % of the initial value at pH 5.8) at both pH values. This indicates a possible synergistic effect between Aloe vera gel and TMC in terms of opening the tight junctions between epithelial cells and in addition, the reversibility improved for the combinations compared to TMC alone. This synergism may be employed to lower the concentrations needed of these compounds for effective absorption enhancement that is also potentially safer as indicated by a higher extent of reversibility. It was previously shown that the reduction in TEER obtained with TMC did not recover fully after removal, which was explained by the difficulty to remove all of the mucoadhesive TMC from the cell surface (Kotze, et al., 1997:1199).

The test solutions containing TMC were slightly more effective in reducing TEER at a pH of 5.8 as compared to the effect on TEER at pH 7.4, which can be explained by protonation of the un-substituted amino groups in an acidic environment with an influence on its interaction with the epithelial cells and thereby an increase in the effect on the TEER.

Table 5.5: The effect of Aloe vera gel in combination with TMC on the TEER of Caco-2 cell monolayers at pH 5.8

TEER (% of initial value)

Tim

0.1 % 0.5 % 1.0 % 2.5% 5.0 % e 0.25

Contr (mi 0.1% 0.5% 2.5% gel + gel + gei + gel + gel + ol TIWC % TMC 0.25%T TMC TMC 0.25%T 0.25%T 0.25%T 0.25%T n) MC MC MC MC MC

102.8 102. 102.

99.8 96.6

-60 6 ± 77 105.72 ± 96.77 ± 96.87 ± 96.40 ± 99.63 ± 64 ± 7 ± 5 ± 1.38 ±4.4 2.77 5.24 1.88 1.75 1.68 2.54 2.72 3.54 6

105.5 97.4 96.8 99.3 98.4 104.80 ±

-40 4 ± 93.84 ± 102.11 ± 98.72 ± 95.97 ± 2 ± 1 ± 7 ± 9 ± 5.61 4.68 5.77 0.84 1.46 1.49 3.70 2.42 1.75 2.98

97.6 100.

98.48 99.6 96.6

-20 7 ± O ± 10 99.85 ± 99.49 ± 102.79 ± 99.62 ± 98.83 ± ± 5.02 O ± ±3.3 1.02 0.89 1.80 1.02 0.65 3.75 3.16 0.48 0

100.

100.0 100. 100. 100. 00

0 O ± 00 ± 00 00 100.00 ± 100.00 ± 100.00 ± 100.00 ± 100.00 0.00 ±0.0 0.00 ±0.0 ±0.0 0.00 0.00 0.00 0.00 ±0.00 0 0 0

75.9 69.5

99.62 66.0 57.3

20 81.69 ± 73.87 ± 71.48 ± 65.60 ± 56.75 ± 3 ± 2 ± ± 4.21 O ± 4 ± 4.87 4.05 3.77 1.78 2.32 1.24 3.97 2.74 2.57

40 98.87 69.1 69.5 63.0 54.1 77.49 ± 70.17 ± 67.71 ± 61.36 ± 51.91 ± ±4.23 3± 1 ± 3± 6± 4.57 3.56 6.60 3.15 2.03 2.12 2.34 2.01 1.92

70.6 69.3 63.5 51.3

97.24 77.22 ± 69.92 ± 63.21 ± 56.48 ± 49.99 ±

60 8± 7± 3± 6± ±6.13 3.90 4.66 5.57 1.77 38 2.86 1.40 4.11 0.14 2.

71.0 66.5 58.9 48.3

96.10 70.07 ± 65.75 ± 61.59 ± 53.26 ± 46.67 ±

80 6± 8± 4± 2± ±4.76 4.39 2.91 3.11 3.77 1.02 1.30 1.82 2.13 1.22

69.2 59.9 58.8 48.0

98.29 67.15 ± 60.93 ± 59.57 ± 53.14 ± 43.31 ±

100 2± 3± ±4.09 5± 7± 7 1.68 3.77 2.54 2.74 3.22 1.51 0.97 2.8 2.14

68.7 62.4 60.7 46.9

99.89 62.85 ± 60.12 ± 58.38 ± 49.82 ± 39.36 ±

120 1 ± 4± 9± 6± ±1.54 91 1.78 0.81 3.58 2.28 0.55 2. 0.76 3.67* 2.90*

102.0 85.0 80.4 78.0 75.0 102.41

140 4± 6± 2± 9± .54 ± 3± 102.27 ± 101.26 ± 106.59 ± 106 4 4.11 6.16 2.38 1.45 3.71 6.00 ±6.20 6.18 5.90 0.7

101.6 86.5 82.4 81.0 75.4

103.62 ± 102.11 ± 100.91 ± 99.62 ± 101.32

160 6± 1 ± 2± 8± 6± 3.61 4.96 1.14 6.02 ±1.40 3.48 3.40 2.81 2.79 4.22

84.0 84.9 80.9 77.6

99.72 4± 107.74 ± 99.70 ± 101.34 ± 104.37 ± 100.85

180 4± 2± 8± ±5.05 5.81 4.36 ±4.70 4.11 4.07 1.76 4.04 1.42 1.57

85.4

101.2 84.9 9 80.7 74.7

106.46 ± 101.73 ± 103.08 ± 102.56 ± 103.44

200 4± 4± 0± 5± ±6.3 7.21 3.01 2.87 1.60 ±3.01 1.13 3.12 3.15 5.64 1

100.6 84.9 82.4 82.7 77.8

99.78 ± 103.45 ± 102.15 ± 100.00 ± 107.49

220 7± 6± 2± 2± 2± 4.62 5.46 2.04 1.54 ±4.70 3.03 3.12 2.39 1.51 4.21

84.6

100.0 82.7 76.5 8 81.0

97.69 ± 99.00 ± 95.53 95.90 ± 101.11

240 6± 3± 1 ± ±2.5 6± 2.96 2.45 2.49 ±4.95 3.28 4.50 1.98 3.03 ±0.72 7

*Statistically significantly different from 0.25% TMC at 120 min (p < 0.05).

Table 5.6: The effect of Aloe vera gel in combination with TMC on the TEER of Caco-2 cell monolayers at pH 7.4

TEER (% of initial value) e 0.25 0.1 % 0.5 % 1.0% 2.5% 5.0 % (mi Contr 0.1% 0.5% 2.5% % gel gel gel gel gel n) ol TMC TMC TMC 0.25%T 0.25%T 0.25%T 0.25%T 0.25%T TWlC MC MC MC MC MC

102. 101. 100. 105. 40 30 64

-60 97.24 94 101.16 ± 99.76 ± 99.24 ± 100.92 ± 104.23 ±3.65 ±2.8 ±1.4 ±2.0 ±4.2 0.77 1.42 2.53 4.59 ±1.01 5 0 9 2

100. 101. 102.

101.5 98.5 49 00 92 101.16 ± 100.14 ± 100.91 ± 99.90 ± 102.25

-40 7± ±1.5 4± ±5.8 2.65 2.60 ±3.0 1.40 2.42 3.14 3.44 ±1.38 1 8 5

100. 100. 100.

98.5

97.81 25 98 99.87 ± 100.52 ± 100.13 ± 99.75 ± 101.01

-20 2± 66 ±2.14 ±0.2 ±4.0 ±1.8 1.46 1.23 1.81 1.55 ±0.87 2 9 2.45 4

100. 100. 100. 100.

100.0 00 00 00 00

0 00 100.00 ± 100.00 ± 100.00 ± 100.00 ± 100. O± ±0.0 ±0.0 ±0.0 ±0.0 0.00 0.00 0.00 0.00 ±0.00 0.00 0 0 0 0

90.7 89.5 81.6 74.4 85.18 ± 82.53 ± 73.63 ± 67.61 ±

20 96.58 9± O± 6± 8± 89.99 ± ±2.48 0.48 0.43 2.54 0.63 3.14 2.57 3.22 1.36 6.45 81.0 75.4 64.9 52.2

99.11 76.50 ± 70.97 ± 62.15 ± 55.69 ± 49.91 ±

40 1 ± 4± 5± 0± ±8.52 5.04 6.47 4.08 4.23 3.33 5.24 3.06 3.85 1.12

71.6 67.0 62.0 50.0

97.23 58.93 ± 54.67 ± 48.41 ±

60 1 ± 8±

±4.25 7± 68.68 ± 70.44 ± 5± 0.87 4.65 1.54 3.49 3.87 2.75 1.96 1.76 2.95

73.1 66.1 62.2 51.5

98.62 68.16 ± 66.14 ± 60.85 ± 49.95 ± 46.92 ±

80 6± 7± 0± 4± ±5.35 0.83 2.16 3.30 1.53 2.51 2.46 3.07 2.17 2.85

102.3 74.6 67.1 64.0 50.9

70.73 ± 69.13 ± 60.87 ± 49.69 ± 46.66 ±

100 9± 4± 8± 5± 3± 2.33 1.21 5.30 2.19 1.79 5.13 1.79 3.48 0.42 3.62

74.7 66.9 62.6 52.0

99.72 72.65 ± 67.64 ± 63.96 ± 50.69 ± 48.68 ±

120 8± 2± 6± 9± ±2.66 2.28 0.72 4.66 0.19 1.23 1.09 2.97 1.21* 3.6*

103.8 81.8 83.3 77.4 71.4

91.53 ± 89.10 ± 90.89 ± 88.42 ± 87.56 ±

140 1± 5± 0± 4± 3± 1.77 1.56 2.16 2.39 2.37 1.01 0.86 4.11 2.44 1.45

100.6 81.9 80.9 75.9 69.5

99.11 ± 95.60 ± 97.04 ± 96.20 ± 93.03 ±

160 6± 0± 8± 5± 6± 1.35 1.57 2.62 2.25 2.46 5.20 6.36 3.74 3.58 2.04

84.9 81.6 76.8 73.2

98.13 98.08 ± 93.85 ± 95.25 ± 93.63 ± 93.20 ±

180 2± 4± 1 ± 9±

±2.11 1.54 2.04 3.08 1.47 1.90 5.45 1.86 3.04 1.15

84.0 82.4 77.0 74.4

97.81 97.30 ± 100.15 ± 97.82 ± 100.13 ± 99.43 ±

200 3± 3± 4± 0± ±5.45 1.16 2.32 2.11 2.55 3.16 4.43 0.69 1.25 0.23

101.8 85.2 84.0 81.5 75.4

96.01 ± 101.04 ± 99.49 ± 99.76 ± 102.25

220 2± 8± 9± 6± 1 ± 2.53 3.81 0.88 1.91 ±1.74 2.17 3.62 2.04 3.88 0.58

84.7 82.3 80.4 72.2

99.57 96.67 ± 98.53 ± 98.86 ± 98.22 ± 98.53 ±

240 5± 9± 3± 9± ±3.82 4.06 2.94 2.69 1.89 1.45 0.70 2.07 2.59 0.31

*Statistically significantly different from 0.25% TMC at time 120 min (p < 0.05). A comparison of the reduction in TEER (% of initial value) obtained for TMC alone (0.25 % w/v) with that of the solutions containing a combination of TMC at a concentration of 0.25 % w/v and Aloe vera gel at different concentrations ranging from 0.1 to 5 % w/v is shown in Figure 5-1.

□ 0 25% TMC (control) ■ 0 1% gel, 025% TMC DO 5% gel 025 TMC □ 1 0% gel, 025% TMC ■ 25% gel, 025% TMC ■ 50% gel 025% TMC

Figure 5-1: Reduction in TEER (% of initial value) of Aloe vera gel in combination with TMC on Caco-2 cell monolayers at 120 minutes. *Statistically significantly different from control.

Effect of Aloe vera whole leaf extract combined with TMC on TEER at pH 5.8 and 7.4

The TEER values of Caco-2 cell monolayers measured at different time points after incubation with TMC alone as well as in combination with Aloe vera whole leaf extract at pH 5.8 are shown in Table 5-7, while those at pH 7.4 are shown in Table 5-8.

Similar to the results obtained for the combination of Aloe vera gel and TMC, the combination of TMC with Aloe vera whole leaf extract also led to a reduction in the TEER of the Caco-2 cell monolayers to a larger extent (e.g. a reduction in TEER to 52.53 ± 1.51 % of the initial value at pH 5.8) as compared to TMC alone (at a corresponding TMC concentration of 0.25 % w/v the TEER reduction was to 59.92 ± 0.97 % of the initial value at pH 5.8) or to the Aloe vera whole leaf extract alone (at a concentration of 5 % w/v the TEER reduction was to 56.83 ± 1.44 % of the initial value at pH 5.8) at both pH values. This, as stated before, indicates a possible synergistic effect between Aloe vera whole leaf extract and TMC in terms of opening the tight junctions between epithelial cells. As in the case with the combination study of Aloe vera gel and TMC, combination of Aloe vera whole leaf extract with TMC also improved the reversibility of the cell monolayers compared to TMC alone. This indicates therefore not only an increase in absorption enhancement effect but also a reduction in the inability of the TEER of the cell monolayers to return to the original value after incubation with TMC.

Similarly to the results obtained for the TMC combination with Aloe vera gel solutions, the test solutions containing TMC and Aloe vera whole leaf extract were slightly more effective in reducing TEER at a pH of 5.8 as compared to the effect on TEER at pH 7.4. This was previously explained by protonation of the un-substituted amino groups in an acidic environment and thereby increasing the degree of quaternisation of the TMC with an influence on its interaction with the epithelial cells and thereby an increase in the effect on the TEER.

Table 5.7: The effect of Aloe vera whole leaf extract combined with TMC on the TEER of Caco-2 cell monolayers at pH 5.8

TEER (% of initial value) e 0.1 % 0.5 % 1.0% 2.5% 5.0 %

0.25 (mi Contr 0.1% 0.5% 2.5% WL WL WL WL WL

% TMC TW n) ol lC TWIC 0.25%T 0.25%T TWlC 0.25%T 0.25%T 0.25%T MC MC MC MC MC

102. 102.

102.8 99.8 96.6 64 77 103.75 ± 101.41 ± 97.53 ± 94.59 ± 98.95 ±

-60 6± 7± 5± ±2.5 ±4.4 4.07 1.56 1.59 2.14 1.83 1.38 2.72 3.54 4 6

105.5 97.4 96.8 99.3 98.4

101.92 ± 98.52 ± 100.13 ± 98.58 ± 99.61 ±

-40 4± 2± 1 ± 7± 9± 4.43 5.37 0.60 0.59 2.22 5.61 3.70 2.42 1.75 2.98

100.

97.6 99.6 96.6

98.48 10 102.56 ± 96.83 ± 102.09 ± 98.84 ± 100.27 ±

-20 7± O± O± ±5.02 ±3.3 3.09 2.90 0.19 1.77 1.51 3.75 3.16 0.48 0

100. 100. 100. 100.

100.0 00 00 00 00 100.00 ± 100.00 ± 100.00 ± 100.00 ± 100.00

0 O± ±0.0 ±0.0 ±0.0 ±0.0 0.00 0.00 0.00 0.00 ±0.0 0.00 0 0 0 0

75.9 69.5 66.0 57.3

99.62 81.62± 77.18 ± 78.69 ± 73.29 ± 68.95 ±

20 3± 2± O± 4± ±4.21 0.88 1.48 3.03 1.28 5.36 1.24 3.97 2.74 2.57

69.1 69.5 63.0 54.1

98.87 69.92 ± 67.31 ± 66.27 ± 66.20 ± 63.25 ±

40 3± 1 ± 3± 6± ±4.23 3.38 0.96 4.03 1.24 3.15 2.12 2.34 2.01 1.92

70.6 69.3 63.5 51.3

97.24 71.38 ± 67.22 ± 62.58 ± 61.30 ± 57.42 ±

60 8± 7± 3± 6± ±6.13 3.46 3.57 1.80 3.16 2.21 0.14 2.38 2.86 1.40

71.0 66.5 58.9 48.3

96.10 70.43 ± 66.27 ± 62.33 ± 57.80 ± 55.44 ±

80 6± 8± 4± 2± ±4.76 1.07 3.00 2.88 1.54 1.31 1.30 1.82 2.13 1.22

69.2 59.9 58.8 48.0

98.29 62.94 ± 61.29 ± 59.05 ± 54.72 ± 53.58 ±

100 5± 2± 3± 7± ±4.09 2.99 1.88 2.80 2.74 0.98 1.51 0.97 2.87 2.14

68.7 62.4 60.7 46.9

99.89 64.52 ± 60.63 ± 59.32 ± 54.97 ± 52.53 ±

120 1 ± 4± 9± 6± ±1.54 3.76 1.07 3.17 0.55 2.91 1.78 0.76 2.16* 1.51^*

102.0 85.0 80.4 78.0 75.0

100.16 ± 105.02 ± 102.75 ± 101.93 ± 101.20

140 4± 6± 2± 9± 3± 3.21 2.01 1.72 0.38 ±1.06 6.18 5.90 0.74 4.11 6.16

101.6 86.5 82.4 81.0 75.4

102.17 ± 104.39 ± 101.57 ± 98.84 ± 102.79

160 6± 1 ± 2± 8± 6± 4.43 3.47 0.76 0.77 ±1.44 3.48 3.40 2.81 2.79 4.22

84.0 84.9 80.9 77.6

99.72 102.17 ± 100.93 ± 102.11 ± 102.19 ± 99.48 ±

180 4± 4± 2± 8± ±5.05 3.47 2.72 1.51 0.46 2.76 4.11 4.07 1.76 4.04

101.2 85.4 84.9 80.7 74.7

99.53 ± 101.56 ± 102.37 ± 103.10 ± 101.20

200 4± 9± 4± O± 5± 2.78 1.80 1.45 2.34 ±1.74 1.13 6.31 3.12 3.15 5.64

100.6 84.9 82.4 82.7 77.8

96.38 ± 103.25 ± 101.45 ± 99.62 ± 99.90 ±

220 7± 6± 2± 2± 2± 5.93 3.89 0.84 3.17 8.15 3.03 3.12 2.39 1.51 4.21

100.0 82.7 84.6 81.0 76.5

100.02 ± 96.96 ± 98.42 ± 100.51 ± 101.06

240 6± 3± 8± 6± 1 ± 9.01 2.74 1.72 1.57 ±1.28 3.28 4.50 2.57 1.98 3.03

*Statistically significantly different from 0.25% TMC at time 120 min (p < 0.05).

Table 5.8: The effect of Aloe vera whole leaf combined with TMC on the TEER of Caco-2 cell monolayers at pH 7.4 TEER (% of initial value) e 0.1 % 0.5 % 1.0% 2.5% 5.0 %

0.25 (mi Contr 0.1% 0.5% 2.5% WL WL WL WL WL

% n) ol TNIC TWIC TNIC TNIC 0.25%T 0.25%T 0.25%T 0.25%T 0.25%T MC MC NIC MC MC

102. 101. 100. 105.

97.24 40 30 64 94 107.31 ± 99.51 ± 101.66 ± 100.99 ± 101.60

-60 ±3.65 ±2.8 ±1.4 ±2.0 ±4.2 2.38 2.81 1.97 1.57 ±0.79 5 0 9 2

100. 101. 102.

101.5 98.5 49 00 92 102.87 ± 99.25 ± 98.50 ± 99.50 ± 102.60

-40 7± 4± 2.65 ±1.5 ±5.8 2.60 ±3.0 0.48 1.63 1.37 0.56 ±2.10 1 8 5

100. 100. 100.

98.5

97.81 25 98 106.01 ± 99.63 ± 98.66 ± 100.65 ± 99.89 ±

-20 2± 66 ±2.14 ±0.2 ±4.0 8 3.20 0.64 2.63 2.45 1.67 2.45 ±1. 2 9 4

100. 100. 100. 100.

100.0 00 00 OO 00 100.00 ± 100.00 ± 100.00 ± 100.00 ± 100.00

0 O± ±0.0 0.00 0.00 0.00 ±0.0 ±0.0 0.00 0.00 0.00 ±0.00 0 0 ± 0

90.7 89.5 81.6

96.58 74.4 87.38 ±

20 9± O± 80.11 ± 73.68 ± 70.67 ± 67.39 ± 6± 8± ±2.48 0.61 0.63 2.97 1.98 3.58 5.03 0.48 2.54 6.45

81.0 75.4 64.9

99.11 52.2 83.60 ± 74.39 ±

40 66.66 ± 64.24 ± 58.94 ± 1 ± 4± 5± O± ±8.52 2.32 1.40 1.77 1.10 2.88 5.24 3.06 3.85 1.12

71.6 67.0 62.0 50.0

97.23 75.66 ± 66.57 ± 5± 62.64 ± 61.37 ± 55.34 ±

60 8± 7±

±4.25 1± 2.11 3.21 1.46 4.27 0.90 2.75 1.96 1.76 2.95

73.1 66.1 62.2 51.5

98.62 71.73 ± 64.44 ± 4± 63.34 ± 61.62 ± 56.21 ±

80 6± 7± O± ±5.35 1.62 2.10 3.12 3.13 1.30 2.46 3.07 2.17 2.85

102.3 74.6 67.1 64.0 50.9 71.44± 66.18 ± 64.92 ± 59.71 ±

100 4± 8± 5± 3± 53.98 ± 9± 3.99 3.87 3.42 2.79 2.25 5.13 1.79 3.48 0.42 3.62

74.7 66.9 62.6 52.0

120 99.72 75.61 ± 67.04 ± 63.77 ± 61.82 ± 54.61 ± 8± 2± 6± 9± ±2.66 0.19 3.40 0.22 2.84 3.06 1.23 1.09 0.93^* 2.97

103.8 81.8 83.3 77.4 71.4 94.26 ± 88.32 ± 88.89 ± 87.51 ± 81.88 ±

140 1 ± 5± O± 4± 3± 1.01 0.86 5.02 1.45 3.65 4.76 3.60 4.11 2.44 1.45

100.6 81.9 80.9 75.9 69.5

160 94.25 ± 98.17 ± 94.95 ± 94.49 ± 97.34 ± 6± O± 8± 5± 6± 36 4.66 4.88 5.09 1.52 3.67 5.20 6. 3.74 3.58 2.04

84.9 81.6 76.8 73.2

180 98.13 99.36 ± 97.99 ± 96.88 + 100.18 ± 99.42 ± ±2.11 2± 4± 1± 9± 1.11 2.75 5.04 4.21 2.47 5.45 1.86 3.04 1.15

84.0 82.4 77.0

97.81 74.4

200 98.18 ± 99.27 ± 97.09 ± 99.29 ± 99.06 ± 3± 3± 4± O± ±5.45 4.43 1.46 1.70 5.04 3.45 2.87 0.69 1.25 0.23

101.8 85.2 84.0 81.5 75.4

220 2± 8± 98.88 ± 100.14 ± 97.31 ± 101.02 ± 98.70 ± 9± 6± 1± 4.43 2.82 3.42 1.57 4.56 2.17 3.62 2.04 3.88 0.58

84.7 82.3 80.4 72.2 96.13 ± 97.65 ± 96.64 ± 100.11 ± 99.56 ±

240 99.57 5± 9± 3± ±3.82 9± 3.70 0.70 2.07 2.59 2.04 2.01 2.62 3.72 0.31

A comparison of the reduction in TEER (% of initial value) obtained for TMC alone (0.25 % w/v) with that of the solutions containing a combination of TMC at a concentration of 0.25 % w/v and Aloe vera whole leaf extract at different concentrations ranging from 0.1 to 5 % w/v is shown in Figure 5-2.

pH5 8 pH74

DO 25% TMC (control) ■ 0 1 % whole leaf, 025% TMC □ 0 5% whole leaf, 025 TMC

D 1 0% whole leaf, 025% TMC ■ 2 5% whole leaf, 025% TMC ■ 5 0% whole leaf, 0 25% TMC

Figure 5-2: Reduction in TEER (% of initial value) of Aloe vera whole leaf extract in combination with TMC on Caco-2 cell monolayers at 120 minutes. *Statistically significantly different from control.

EFFECT QF ALOE VERA GEL AND WHOLE LEAF EXTRACT ON INSULIN PERMEABILITY ACROSS CACO-2 CELL MONOLAYERS

Effect of Aloe vera gel on insulin transport at pH 5.8 and pH 7.4

The cumulative transport values of insulin across Caco-2 cell monolayers when administered with Aloe vera gel solutions in different concentrations at pH 5.8 are shown in Table 5.14 and those obtained at pH 7.4 are depicted in Table 5.15. The calculated apparent permeability coefficient values (P_apP) and transport enhancement ratios (R) for insulin transport across the cell monolayers in the presence of different concentrations of Aloe vera gel are listed in Table 5.16.

As expected and predicted by the TEER results, Aloe vera gel was able to significantly (p < 0.05) enhance the transport of insulin across Caco-2 cell monolayers in concentrations higher than 0.5 % w/v. The influence of pH on the transport enhancement effect was more pronounced compared to that observed in the TEER experiment, with a higher effect on insulin transport in the slight acidic environment (pH 5.8). Since the transcellular pathway is excluded for insulin transport across intestinal epithelia (and therefore it is usually limited to the paracellular pathway) and because of the TEER reduction by Aloe vera gel, the transport enhancement of insulin by Aloe vera gel most probably occurred via the transient opening of the tight junctions to allow for increased paracellular transport. This should, however, be investigated further by making use of fluorescent labelled high molecular compounds (e.g. FITC- dextran) and confocal laser scanning microscopy to elucidate the pathway of trans-epithelial movement when administered with Aloe vera gel to epithelial cell monolayers.

The calculated transport enhancement ratios (R) show that the Aloe vera gel increased insulin transport 1.92-fold at a concentration of 0.5% w/v, 2.35-fold at 1.0% w/v, 2.35-fold at 2.5% w/v and 2.54-fold at 5.0% w/v in an environment with a pH of 5.8 compared to the control. At a pH of 7.4, the increase in insulin transport was 1.86-fold at a concentration of 0.5% w/v, 2.02-fold at 1.0% w/v, 2.31 -fold at 2.5% w/v and 2.48-fold at 5.0% w/v compared to the control. These values represent statistically significant differences for insulin transport between the Aloe vera gel test solutions (i.e. concentrations higher than 0.5% w/v) and the control group.

Table 5.16: Apparent permeability coefficient values (P_apP) and transport enhancement ratios (R) for insulin transport in the presence of Aloe vera gel

Aloe vera gel concentration Papp X iO ⁶ (cm/s) R P_app x 10-⁶ (cm/s) R (% w/v) at pH 5.8 at pH 7.4

Control 1.94 ± 0.43 1.00 1.66 ± 0.22 1.00

0.1 1.84 ± 0.38 0.95 2.74 ± 0.34* 1.65

0.5 3.73 ± 0.13* 1.92 3.09 ± 0.37* 1.86

1.0 4.56 ± 0.30* 2.35 3.36 ± 0.20* 2.02

2.5 4.55 ± 0.29* 2.35 3.84 ± 0.12* 2.31

5.0 4.93 ± 0.38* 2.54 4.11 ± 0.32* 2.48 Each value represents the mean ± S. D. of 3 experiments; *Statistically significantly different from control (p < 0.05).

Table 5.14: The effect of Aloe vera gel on the transport of insulin across Caco- 2 cell monolayers at pH 5.8

Cumulative insulin transport (% of initial dose)

Time 2.5 %

0.1% w/v 5.0 %

0.5% w/v 1.0% w/v

(minutes) w/v

Control Aloe Aloe w/v

Aloe Aloe Aloe vera gel vera gel vera gel vera gel vera gel

9.76 + 10.49 ± 11.12 ± 15.39 ± 17.17 +

30 9.08 + 1.17 3.11 1.15 1.72 1.44 0.92

11.30 ± 12.50 ± 15.44 + 16.96 ± 20.67 ± 23.87 ±

60 1.16 2.81 2.09 2.36 0.80 4.64

11.44 ± 12.53 + 19.28 ± 21.56 ± 23.50 + 26.09 ±

90 1.23 2.16 0.34 1.58 1.37 2.55

10.87 + ± 19.72 ± 24.84 ± 25.79 ± 31.80 ±

120 13.36 3.05 1.55 1.18 1.24 1.15 1.69

14.78 ± 15.25 ± 24.64 ± 30.34 + 31.25 + 37.27 ±

180 3.80 0.78 0.98 1.06 1.09 2.00

16.66 + 16.30 + 28.84 ± 33.29 + 36.18 + 37.64 +

240 2.41 0.97 0.93 2.13 2.85 0.99

Table 5.15: The effect of Aloe vera gel on the transport of insulin across Caco- 2 cell monolayers at pH 7.4

Cumulative insulin transport (% of initial dose)

Time 2.5 % 5.0 %

0.1% w/v 0.5% w/v 1.0% w/v

(minutes) w/v

Control Aloe Aloe Aloe w/v

Aloe Aloe vera gel vera gel vera gel vera gel vera gel

9.67 + 9.99 ± 14.20 + 16.11 ± 17.96 ±

30 7.18 ± 2.55 2.05 0.63 1.36 0.23 0.80

11.00 + 12.85 + 12.95 + 17.13 ± 18.33 ± 20.82 ±

60 1.22 3.74 0.90 2.90 0.37 1.56

12.14 + 16.36 + 17.07 ± 19.58 + 21.35 ± 23.05 ±

90 0.68 1.10 2.19 3.08 3.10 3.78

12.31 + 17.35 ± 18.15 ± 23.17 + 25.32 ± 28.08 ±

120 0.78 1.40 1.14 2.55 3.28 3.73

12.72 ± 20.06 + 21.15 ± 24.86 + 28.23 ± 30.44 ±

180 0.91 0.13 3.21 2.54 2.10 4.16 14.33 ± 21.40 + 24.08 + 27.53 ± 31.18 ± 33.90 +

240 1.27 C I.87 1.17 1.13 C 1.41 0.39

Effect of Aloe \ /era whole leaf extract on insulin transport at pH 5.8 and pH 7.4

The cumulative transport values of insulin across Caco-2 cell monolayers when administered with Aloe vera whole leaf extract solutions in different concentrations at pH 5.8 are shown in Table 5.17 and those obtained at pH 7.4 are depicted in Table 5.18. The calculated apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for insulin transport across the cell monolayers in the presence of different concentrations of Aloe vera whole leaf extract are listed in Table 5.19. The transport enhancement effect obtained with Aloe vera whole leaf extract on insulin was expected as it was able to decrease the TEER of the Caco-2 cell monolayers, which indicates opening of the tight junctions and thereby allow for increased paracellular transport across the epithelial cell layer. As in the case with the Aloe vera gel transport studies, the influence of pH on the transport enhancement effect of Aloe vera whole leaf extract was higher compared to that observed in the TEER experiment, with a higher effect on insulin transport in the slight acidic environment (pH 5.8). From the calculated transport enhancement ratios (R) it was shown that the Aloe vera whole leaf extract increased insulin transport 1.82-fold at a concentration of 0.1% w/v, 2.02-fold at 0.5% w/v, 2.03-fold at 1.0% w/v, 2.19-fold at 2.5% w/v and 3.05-fold at 5.0% w/v in an environment with a pH of 5.8 compared to the control. At a pH of 7.4, the increase in insulin transport was 1.21 -fold at a concentration of 0.1% w/v, 1.75-fold at 0.5% w/v, 2.07-fold at 1.0% w/v, 2.13-fold at 2.5% w/v and 2.16-fold at 5.0% w/v compared to the control. These values represent statistically significant differences for insulin transport between the Aloe vera whole leaf extract solution groups (i.e. concentrations higher than 0.5% w/v) and the control group. Table 5.17: The effect of Aloe vera whole leaf extract on the transport of insulin across Caco-2 cell monolayers at pH 5.8

Cumulative insulin transport (% of initial dose)

Time 0.1% w/v 0.5% w/v 1.0% w/v 2.5 % w/v 5.0 % w/v (minutes) whole whole

Control whole whole whole leaf leaf leaf leaf leaf extract extract extract extract extract

9.08 ± 9.76 ± 11.58 + 11.12 ± 15.39 + 20.91 +

30 1.17 3.11 0.85 1.72 1.44 1.12

11.30 ± 10.84 ± 15.53 + 16.96 + 20.67 + 29.06 ±

60

1.16 2.01 1.97 2.36 0.80 5.64

11.44 ± 8.56 + 19.28 + 23.53 + 23.50 + 31.76 ±

90 1.23 3.01 0.34 3.08 1.37 3.10

10.87 ± 8.72 + 18.81 ± 25.39 ± 25.79 ± 38.72 ±

120 3.05 3.05 1.17 1.78 1.15 2.06

14.78 ± 10.07 ± 24.56 ± 30.38 ± 31.25 + 45.37 ±

180 3.80 0.55 0.88 1.08 1.09 2.44

16.66 ± 12.92 ± 28.84 ± 33.29 + 36.18 ± 45.82

240 2.41 0.49 0.93 2.13 2.85 ±1.20

Table 5.18: The effect of Aloe vera whole leaf extract on the transport of insulin across Caco-2 cell monolayers at pH 7.4

Cumulative insulin transport (% of initial dose)

Time 0.1% w/v 0.5% w/v 1.0% w/v 2.5 % w/v 5.0 % w/v (minutes) whole whole whole whole whole

Control leaf leaf leaf leaf leaf extract extract extract extract extract

30 7.18 ± 7.68 ± 7.65 + 8.93 ± 15.17 + 19.00 ± 2.55 1.48 0.73 1.58 1.33 1.34

60 11.00 ± 11.17 ± 14.48 + 15.46 + 21.62 + 21.65 ± 1.22 0.35 0.44 0.41 1.19 2.61

90 12.14 ± 12.50 ± 16.16 ± 18.30 + 24.28 ± 23.83 ± 0.68 0.66 1.61 0.77 1.19 0.96

120 12.31 ± 13.51 ± 17.10 ± 20.16 + 26.85 ± 27.46 ± 0.78 0.60 1.24 0.78 1.96 3.57

180 12.72 ± 15.09 ± 20.58 ± 24.42 ± 27.48 ± 28.78 + 0.91 0.32 1.37 1.23 1.72 3.52

240 14.33 ± 16.30 + 22.08 ± 25.45 + 29.42 + 31.14 + 1.27 0.66 1.60 0.81 2.37 1.98 Table 5.19: Apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for insulin transport in the presence of Aloe vera whole leaf extract

Aloe vera whole leaf P_app x 1θ^"6 (cm/s) P_app x 1θ^"6 (cm/s)

R R concentration (% w/v) at pH 5.8 at pH 7.4

Control 1.91 ± 0.35 1.00 1.66 ± 0.22 1.00

0.1 3.48 ± 0.37* 1.82 2.01 + 0.15 1.21

0.5 3.86 ± 0.40* 2.02 2.91 ± 0.30* 1.75

1.0 3.87 ± 0.29* 2.03 3.44 ± 0.25* 2.07

2.5 4.18 ± 0.50* 2.19 3.53 ± 0.34* 2.13

5.0 5.83 ± 0.13* 3.05 3.59 ± 0.40* 2.16

Each value represents the mean±S.D. of 3 experiments; *Statistically

significantly different from control (p < 0.05).

Comparing the absorption enhancement effect of Aloe vera gel with whole leaf extract

In accordance with the TEER results, the Aloe vera gel was more effective in terms of insulin transport enhancement compared to Aloe vera whole leaf extract, however this difference was not statistically significant. This is in line with results previously obtained in an in vivo study where the absorption enhancing effects of Aloe vera gel on vitamins C and E was higher compared to that of Aloe vera whole leaf extract (Vinson et a/., 2005:760).

Since the whole leaf extract contains both the gel component and skin component of the aloe leaf, this phenomenon may be explained by a probable influence of the phytoconstituents in the skin part of the leaf on the interaction of the gel with the cell monolayer. These phytoconstituents may therefore inhibit the effect of the gel part on transport enhancement. However, further experimentation is needed to clarify the mechanism of this phenomenon. EFFECT OF ALOE VERA GEL AND WHOLE LEAF EXTRACT ON THE PERMEABILITY OF CACO-2 CELL MONOLAYERS FOR LUTEOLlN

Luteolin is a flavonoid found in food such as parsley, artichoke leaves, celery, peppers, olive oil, rosemary, lemons, peppermint, sage, thyme and many others. It has antioxidant, anti-inflammatory, anti-allergic, anticancer, and immune-modulating properties. Luteolin is also a potent hypoglycemic agent and improves insulin sensitivity.

A luteolin solution (i.e. the control group) and test solutions of luteolin containing 2.5% w/v Aloe vera gel or whole leaf extract at pH 5.8 and 7.4, respectively, were incubated under the same conditions as for the transport study. Table 5.23 shows the luteolin concentrations as percentage of initial concentration in the control and test solutions after 1 and 2 h of incubation at pH 5.8. Table 5.24 shows the results at pH 7.4.

Table 5.23: Luteolin (actual concentration and percentage of initial concentration) in the control and test solutions after 1 and 2 h of incubation at pH 5.8 cubated L««ooHn concentrations ,_μg/_ml, nt " time

(hours) _rnntrn, 2.5% w/v 2.5% w/v _rnntrn, 2.5% w/v 2.5% w/v Control _{ge| who|e |eaf} Control _{a|Qβ gβ| who|e} ,_eaf

O 71 .0 ± 5 .1 56.1 + 0.7 49 .8 + 3.1 100. 0 100. 0 100. 0

1 17 .1 ± 2 .0 34.1 ± 1 .1 34 .0 ± 1.0 24.1 ± 1 .7 60.8 ± 1 .0 68.2 ± 0 .8

2 7. 6 ± 0. 9 19.6 + 1 .2 24 .1 + 3.0 10.7 ± 1 .2 35.0 ± 1 .1 48.4 ± 2 .8

Each value represents the mean ± S. D. of 3 experiments.

Table 5.24: Luteolin (actual concentration and percentage of initial concentration) in the control and test solutions after 1 and 2 h of incubation at pH 7.4 Incubated ^Luteolin concentration (μg/ml) % of initial luteolin concentration

0 66. 7 ± 2. 8 54.3 ± 3 .2 62 .2 ± 1 .2 100.0 100.0 100. 0

1 68. 2 ± 1. 9 55.8 ± 2 .2 60 .7 ± 1 .7 102.3 ± 1 .7 102.9 ± 2 .1 97.5 ± 1.5

2 60. 8 ± 2. 1 53.4 ± 1 .1 57 .2 + 1 .2 91.2 ± 2. 0 98.3 ± 1. 0 92.0 + 1.1

Each value represents the mean ± S. D. of 3 experiments.

Luteolin is relatively unstable under the conditions for the transport studies at pH 5.8 and the concentration of luteolin in the control solution decreased severely. Degradation of luteolin was less pronounced at pH 7.4. Addition of Aloe vera gel or whole leaf extract protected the luteolin from degradation and the percentage of initial concentration was 3.3-fold higher compared to the control with the gel and 4.5-fold higher with the whole leaf extract at pH 5.8 compared to the control. In an environment with a pH of 7.4, the luteolin showed less degradation compared to the pH of 5.8 and in this case the Aloe vera gel increased the stability 1.08-fold and the whole leaf extract 1.01-fold.

Effect of Aloe vera gel on luteolin permeability across Caco-2 cell monolayers at pH 5.8 and 7.4

The cumulative transport values of luteolin across Caco-2 cell monolayers when administered with Aloe vera gel solutions in different concentrations at pH 5.8 are shown in Table 5.25 and those obtained at pH 7.4 are depicted in Table 5.26. The calculated apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for luteolin transport across the cell monolayers in the presence of different concentrations of Aloe vera gel are listed in Table 5.27.

At a pH of 5.8, Aloe vera gel enhanced the transport of luteolin in an apparent concentration dependent way, which is represented by a 3.13-fold (5.0% w/v); 1.86-fold (2.5% w/v); 1.30-fold (1.0% w/v) and 1.23-fold (0.5% w/v) increase compared to the control. This increase in luteolin transport was probably achieved by a combination of tight junction modulation (as indicated by the ability of Aloe vera gel to reduce TEER) and improvement of the chemical stability of luteolin. The extent of transport enhancement was less pronounced for luteolin compared to that of insulin. A possible explanation for this may be that luteolin is absorbed simultaneously by the transcellular and paracellular pathway, while insulin is only absorbed by the paracellular pathway and the latter is therefore influenced to a larger extent by opening of the tight junctions.

Luteolin alone (i.e. control group) showed relatively fast transport over the first 40 min at pH 7.4 and relatively low transport enhancement ratios (with R ranging between 0.84 and 1.62) were obtained for Aloe vera gel in this experiment. The effect of Aloe vera gel on luteolin transport was also not dependent on concentration in this transport experiment with a neutral environment, which is not consistent with the TEER results. The lower effect of the gel on the stability of luteolin may further contribute to a lower effect on the transport of this model compound. Other reasons for this low transport enhancement effect may include complex formation between the gel in higher concentrations (i.e. 2.5 % w/v and 5.0% w/v) and luteolin at a neutral pH environment, but this need to be investigated further before any conclusions can be made in this regard.

Table 5.27: Apparent permeability coefficient values (P_apP) and transport enhancement ratios (R) for luteolin transport in the presence of Aloe vera gel

Aloe vera gel concentration P_apP x 10'⁶ (cm/s) P_{aPp X} i0'⁶ (cm/s)

R R (% w/v) at pH 5.8 at pH 7.4

Control 0.91 ± 0.04 1.00 2.07 ± 0.01 1.00

0.5 1.12 + 0.02* 1.23 3.36 ± 0.04* 1.62

1.0 1.18 ± 0.11* 1.30 3.02 ± 0.22* 1.46

2.5 1.69 ± 0.05* 1.86 1.99 ± 0.24 0.96

5.0 2.85 + 0.23* 3.13 1.73 + 0.14* 0.84

Each value represents the mean±S.D. of 3 experiments; ^*Statistically significantly different from control (P < 0.05)

Table 5.25: The effect of Aloe vera gel on the transport of luteolin across Caco-2 cell monolayers at pH 5.8 Cumulative of luteolin transport (% of initial dose)

Time

0.5% w/v 1.0% w/v 2.5% w/v 5.0% w/v (minutes) Control Aloe vera Aloe vera Aloe vera Aloe vera gel gel gel gel

20 1.29 ± 0.07 1.90 ± 0.03 1.83 ± 0.04 3.05 ± 0.27 4.67 ± 0.09

40 2.03 + 0.06 2.87 ± 0.11 2.68 ± 0.20 3.84 ± 0.09 6.91 ± 0.40

60 2.42 ± 0.04 3.20 + 0.07 3.04 + 0.26 4.58 + 0.13 8.14 ± 0.42

90 2.90 ± 0.15 3.73 ± 0.04 3.89 ± 0.31 5.48 ± 0.08 9.50 ± 0.45

120 3.35 ± 0.04 4.28 + 0.06 4.36 + 0.32 6.51 ± 0.06 10.65 ± 0.69

Table 5.26: The effect of Aloe vera gel on the transport of luteolin across Caco-2 cell monolayers at pH 7.4

Cumulative of luteolin transport (% of initial dose)

Time

0.5% w/v 1.0% w/v 2.5 % w/v 5.0 % w/v (minutes) Control Aloe vera Aloe vera Aloe vera Aloe vera gel gel gel gel

20 5.21 ± 0.02 2.14 ± 0.03 3.00 ± 0.09 0.36 + 0.07 1.61 + 0.49

40 7.49 ± 0.03 3.00 ± 0.23 4.84 ± 0.64 2.07 ± 0.21 2.85 ± 0.11

60 7.67 ± 0.07 7.32 ± 0.18 7.12 ± 0.88 2.82 ± 0.73 3.38 ± 0.10

90 7.98 ± 0.16 9.60 ± 0.02 9.32 ± 0.68 4.34 ± 0.74 5.10 ± 0.71

120 8.61 ± 0.08 11.05 + 0.14 10.22 ± 0.65 6.71 ± 0.63 5.98 ± 0.64

Effect of Aloe vera whole leaf extract on luteolin permeability at pH 5.8 and pH IA

The cumulative transport values of luteolin (% of initial value) across Caco-2 cell monolayers when administered with Aloe vera whole leaf extract solutions in different concentrations at pH 5.8 are shown in Table 5.28 and those obtained at pH 7.4 are depicted in Table 5.29. The calculated apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for luteolin transport across the cell monolayers in the presence of different concentrations of Aloe vera whole leaf extract are listed in Table 5.30.

At pH 5.8, Aloe vera whole leaf extract increased the transport of luteolin in a concentration dependent way that is represented by a 2.92-fold (5.0% aloe w/v); 2.80-fold (2.5% w/v); 2.56-fold (1.0% w/v) and 1.68-fold (0.5% w/v) compared to the control. As in the case with the Aloe vera gel, the transport enhancement effect on luteolin was lower as compared to that obtained in the insulin transport experiment. Furthermore, similar to the results obtained for the luteolin transport experiment in the presence of Aloe vera gel, luteolin alone (i.e. control group) showed relatively fast transport and to a large extent at pH 7.4, which probably contributed to the relatively low transport enhancement ratios (with R ranging between 0.71 and 1.11) that were obtained. As mentioned before, another reason is probably the role of transcellular transport of luteolin in conjunction to paracellular transport and therefore a smaller effect is achieved by modulation of tight junctions. The effect of Aloe vera whole leaf extract on luteolin transport was not dependent on concentration at pH 7.4, which is not consistent with the TEER results. The transport of luteolin was lower compared to the control for higher concentrations Aloe vera whole leaf extract, probably indicating complex formation with the model compound.

Table 5.28: The effect of Aloe vera whole leaf extract on the transport of luteolin across Caco-2 cell monolayers at pH 5.8

Cumulative luteolin transport (% of initial dose)

Time

0.5% w/v 1.0% w/v 2.5 % w/v 5.0 % w/v (minutes) Control whole whole whole whole leaf extract leaf extract leaf extract leaf extract

20 1.29 ± 0.07 2.20 ± 0.02 2.78 ± 0.01 1.60 ± 0.13 3.25 ± 0.65

40 2.03 ± 0.06 3.12 ± 0.04 3.99 ± 0.09 3.85 ± 0.03 5.39 + 0.50

60 2.42 ± 0.04 3.68 ± 0.09 4.99 + 0.09 5.37 ± 0.77 7.03 ± 0.88

90 2.90 ± 0.15 4.78 ± 0.02 6.90 + 0.12 7.18 ± 0.08 8.44 + 0.96

120 3.35 ± 0.04 5.67 ± 0.02 8.36 0.11 8.48 + 0.22 9.40 ± 0.61

Table 5.29: The effect of Aloe vera whole leaf extract on the transport of luteolin across Caco-2 cell monolayers at pH 7.4

Cumulative luteolin transport (% of initial dose)

Time

20 5.21 ± 0.02 2.88 ± 0.70 2.64 ± 0.13 0.33 + 0.09 1.23 ± 0.51

40 7.49 ± 0.03 4.66 ± 0.38 3.75 ± 0.11 2.17 + 0.96 2.28 0.64

60 7.67 ± 0.07 5.85 + 0.34 4.48 ± 0.03 2.75 ± 0.79 3.16 ± 0.44

90 7.98 + 0.16 7.15 ± 0.16 5.81 ± 0.58 3.65 ± 0.44 4.27 0.47

120 8.61 ± 0.08 8.21 ± 0.28 6.22 ± 0.62 4.86 ± 0.22 5.05 ± 0.85

Table 5.30: Apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for luteolin transport in the presence of Aloe vera whole leaf extract

Whole leaf concentration (% P_app x 10-⁶ {cm/s) ⁵ (cm/s)

R P_apP x 10^J R w/v) at pH 5.8 at pH 7.4

Control 0.91 ± 0.04 1.00 2.07 + 0.01 1.00

0.5 1.53 ± 0.01* 1.68 2.29 + 0.15 1.11

1.0 2.33 ± 0.04* 2.56 1.72 + 0.21* 0.83

2.5 2.55 ± 0.07* 2.80 1.47 ± 0.06* 0.71

5.0 2.66 ± 0.12* 2.92 1.48 ± 0.19* 0.71

Each value represents the mean±S.D. of 3 experiments; *Statistically significantly different from control (p < 0.05).

Conclusion

Both Aloe vera gel and whole leaf extract were able to increase the transport of luteolin significantly (i.e. in concentrations higher 0.5 % w/v) across Caco-2 cell monolayers at pH 5.8 in a concentration dependent manner. This effect was, however, lower compared to that on the transport of insulin and probably due to the transcellular transport of luteolin in conjunction with paracellular transport. In contrast to the results obtained at pH 5.8, the transport of luteolin at pH 7.4 was not concentration dependent and was only statistically significantly higher in a limited number of test solutions, while it was significantly lower in other test solutions compared to the control. This is probably due to the higher transport of luteolin at a neutral pH value as well as possible complex formation between the gel and the luteolin in higher concentrations of the Aloe vera gel.

EFFECT OF ALOE VERA GEL AND WHOLE LEAF EXTRACT ON THE PERMEABILITY OF CACO-2 CELL MONOLAYERS FOR RUTIN

The flavonoid, rutin, is a flavonol glycoside composed of quercetin and the disaccharide, rutinose. Rutin is extracted from the medicinal herb, Sophora Japonica, and is mainly used for high blood pressure treatment, vessel wall protection and against bleeding of internal organs. Rutin has shown to exhibit antioxidant, anti-inflammatory, anti-carcinogenic, antithrombotic, cytoprotective and vasoprotective activities.

Effect of Aloe vera gel on rutin permeability across Caco-2 cell monolayers at PH 5.8 and 7.4

The cumulative transport values of rutin across Caco-2 cell monolayers when administered with Aloe vera gel solutions in different concentrations at pH 5.8 are shown in Table 5.34 and those obtained at pH 7.4 are depicted in Table 5.35. The calculated apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for rutin transport across the cell monolayers in the presence of different concentrations of Aloe vera gel are listed in Table 5.36.

According to the transport enhancement ratios (R) for the rutin transport results, Aloe vera gel increased the transport of rutin in a concentration dependant order, which is represented by a 1.75-fold (5.0% w/v); 1.61-fold (2.5% w/v); 1.29-fold (1.0% w/v) and 0.94-fold (0.5% w/v) increase compared to the control at pH 5.8. However, this increase was much lower compared to the transport enhancement effect on insulin. The increase in rutin transport by Aloe vera gel at a pH of 7.4 was not concentration dependent and to a lower extent compared to pH 5.8, which is represented by a 1.26-fold (0.5% w/v); 1.15-fold (5.0% w/v); 1.15-fold (2.5% w/v); 1.12-fold (1.0% w/v) increase compared to the control.

Table 5.36: Apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for rutin transport in the presence of Aloe vera gel

Aloe vera gel concentration P_app x 10^"6 (crn/s) P_app x 1θ-⁶ (cm/s)

R R (% w/v) at pH 5.8 at pH 7.4

Control 6.47 ± 1.14 1.00 5.82 ± 0.22 1.00

0.5 6.05 ± 0.40 0.94 7.36 ± 0.37* 1.26

1.0 8.36 + 0.70* 1.29 6.50 ± 0.20 1.12

2.5 10.40 ± 0.53* 1.61 6.72 ± 0.12 1.15

5.0 11.34 + 1.70* 1.75 6.71 ± 0.32 1.15

Each value represents the mean±S.D. of 3 experiments; *Statistically significantly different from control (p < 0.05) A contributing factor for the lower effect of the Aloe vera gel on rutin transport across the intestinal cell monolayers is that this compound is probably transported by means of the transcellular and paracellular pathways. Furthermore, the transport of rutin in the control group was already relatively high, namely 23.74 + 4.09% at pH 5.8 and 20.22 ± 1.91% at pH 7.4 after 120 minutes of incubation.

Table 5.34: The effect of Aloe vera gel on the transport of rutin across Caco-2 cell monolayers at pH 5.8

Cumulative of rutin transport (% of initial dose)

Time

0.5% w/v 1.0% w/v 2.5 % wlv 5.0 % w/v (minutes) _Contro|

Aloe vera Aloe vera Aloe vera Aloe vera gel gel gel gel

20 7.: 27 ± 1.38 5. 11 ± 0. 73 6.44 : 0.91 8. 84 : t 0.47 11.21 + 1.05

40 11. .06 ± 1.37 8. 19 ± 0.22 11.30 + 1.32 16 .35 ± 1.09 18.46 ± 1.71

60 14.14 ± 2.24 10 .60 ± 0 .92 15.97 ± 2.62 24 .19 ± 1.18 27.16 + 2.29

90 17. .81 + 1.41 16.50 ± 1 .05 24.07 ± 2.65 29.23 ± 2.66 34.08 + 3.69

120 23. .74 + 4.09 21 .05 ± 1 .58 27.99 ± 2.23 35 .82 ± 1.16 38.82 ± 4.57

Table 5.35: The effect of Aloe vera gel on the transport of rutin across Caco-2 cell monolayers at pH 7.4

Cumulative of rutin transport (% of initial dose)

Time

. 0.5% w/v 1.0% w/v 2.5 % w/v 5.0 % w/v

{ in t s) Control Aloe vera Aloe vera Aloe vera Aloe vera gel gel gel gel

20 7.10 : i 1.55 7.75 ± 1.72 6.67 ± ( D.34 7.60 + 1.89 6.75 ± ^• 1. 15

40 11.90 ± 1.28 15.23 + 1.92 12.75 ± 2.23 11.39 ± 1.12 13.49 ± 2.98

60 16.28 ± 1.69 20.41 ± 1.59 16.97 ± 1.62 16.05 ± 2.21 18.66 ± 2 .58

90 19.00 ± 1.75 23.14 ± 1.85 20.54 ± 1.80 19.67 ± 1.59 21.04 ± 1 .11

120 20.22 ± 1.91 25.43 ± 1.36 22.21 ± 2.04 23.91 ± 0.82 22.97 + 1 .05 Effect of Aloe vera whole leaf extract on rutin permeability across Caco-2 cell monolayers at pH 5.8 and pH 7.4

The cumulative transport values of rutin across Caco-2 cell monolayers when administered with Aloe vera whole leaf extract solutions in different concentrations at pH 5.8 are shown in Table 5.37 and those obtained at pH 7.4 are depicted in Table 5.38. The calculated apparent permeability coefficient values (P_app) and transport enhancement ratios (R) for rutin transport across the cell monolayers in the presence of different concentrations of Aloe vera whole leaf extract are listed in Table 5.39.

According to the transport enhancement ratios (R) for the rutin transport results, Aloe vera whole leaf extract increased the transport of rutin in a concentration dependant order, which is represented by a 1.02-fold (5.0% w/v); 1.22-fold (2.5% w/v); 1.59-fold (1.0% w/v) and 1.92-fold (0.5% w/v) increase compared to the control at pH 5.8. As in the case with the Aloe vera gel, this increase was much lower compared to the transport enhancement effect on insulin. The increase in rutin transport by Aloe vera whole leaf at a pH of 7.4 was not concentration dependent and to a lower extent compared to pH 5.8, which is represented by a 1.15-fold (0.5% w/v); 1.22-fold (5.0% w/v); 1.16-fold (2.5% w/v); 1.00-fold (1.0% w/v) increase compared to the control.

The same factors that possibly played a role in the rutin transport studies for the Aloe vera gel test solutions may also contribute to the lower than expected transport enhancement of rutin by the Aloe vera whole leaf extract.

Table 5.37: The effect of Aloe vera whole leaf on the transport of rutin across Caco-2 cell monolayers at pH 5.8

Cumulative rutin transport (% of initial dose)

Time

0.5% w/v 1.0% w/v 2.5%w/v 5.0%w/v (minutes) Control whole leaf whole leaf whole leaf whole leaf extract extract extract extract

20 7.27: t 1.38 3.! 38: to. 94 6.! 98 ± 0.76 9.84: t: 2.14 11.31 + 1.14

40 11.06 ± 1.37 7.43: to. 64 11. .62 ± 1.11 18.50 ± 0.76 21.66 + 0.78

60 14.14 + 2.24 10. .59 ±0 .71 17 .11 ±1.68 24.66 ± 2.21 30.04 ± 2.67

90 17.81 ± 1.41 18. .03 ±1 .43 22 .25 ± 0.82 28.62 + 3.49 37.55 ± 4.06

120 23.74 ± 4.09 21 .73 ±1 .00 27 .35 ±1.30 36.64 + 1.80 42.26 + 3.72

Table 5.38: The effect of Aloe vera whole leaf extract on the transport of rutin across Caco-2 cell monolayers at pH 7.4

Cumulative of rutin transport (% of initial dose)

Time

0.5% w/v 1.0% w/v 2.5% w/v 5.0% w/v (minutes) Control whole leaf whole leaf whole leaf whole leaf extract extract extract extract

20 7. 10: t 1. 55 6. 63: 3. 16 8.: 32: t⁽ D.26 2. 20 ±0. 58 4.41 ± OJ 82

40 11 .90 ± 1 .28 13 .67 ± 0 .81 15. .75 ± 0.12 8. 73 ±1. 74 8.I 39 ± 0.75

60 16.28 ± 1 .69 15 .38 + 0 .64 18.44 ± 1.02 13 .87 ±2 .88 12. .34 + 1 .23

90 19 .00 ± 1 .75 20 .85 ± 1 .61 21. .73 ± 0.79 17 .94 ±1 .30 15. .16 ± 0 .23

120 20 .22 ± 1 .91 23 .16 ± 2 .21 25 .73 ± 1.01 21 .66 ±0 .81 20. .30 + .1 .37 Table 5.39: Effect of aloe whole leaf on the permeability of rutin (apparent permeability coefficients (P_app) and transport enhancement ratios (R))

Aloe vera gel concentration P_ap_p x 1θ^"6 (cm/s) P_app x 10 ^e (cm/s) (% w/v) R R at pH 5.8 at pH 7.4

Control 6.47 ± 1.14 1 .00 5.82 ± 0.22 1 .00

0.5 6.58 ± 0.22 1 .02 6.68 ± 0.72 1 .15

1.0 7.92 ± 0.19 1 .22 7.12 ± 0.17* 1 .22

2.5 10.28 ± 0.73 1 .59 6.76 ± 0.30 1 .16

5.0 12.44 ± 1.22* 1 .92 5.83 ± 0.28 1 .00

Each value represents the mean±S.D. of 3 experiments; *Statistically significantly different from control (p < 0.05). Conclusion

Both Aloe vera gel and whole leaf extract were able to increase the transport of rutin across Caco-2 cell monolayers at pH 5.8 in a concentration dependent manner. This effect was, however, lower compared to that on the transport of insulin probably because of the transcellular transport of rutin in conjunction with paracellular transport across intestinal epithelial cell monolayers. Furthermore, a relatively high rate and extent of rutin transport at pH 7.4 across epithelial cell monolayers was less influenced by the presence of Aloe vera gel or whole leaf extract. The effect on rutin transport decreased with an increase in Aloe vera gel and whole leaf extract, which probably indicate complex formation that hamper rutin transport at these concentrations of the >A/oe vera components.

CONCLUSION

Measurement of the TEER of Caco-2 cell monolayers revealed that Aloe vera gel and whole leaf extract are able to reduce the TEER significantly (although this is concentration dependent) compared to the control. This indicates their ability to open the tight junctions between adjacent epithelial cells and thereby allow better movement of ions through the intercellular spaces. It is therefore expected that these Aloe vera components will be able to enhance the paracellular absorption of poorly permeable drugs. Combining Aloe vera gel and whole leaf extract with TMC resulted in a synergistic effect on TEER reduction with improved reversibility of this effect compared to TMC alone.

Both the Aloe vera gel and Aloe vera whole leaf extract were able to reduce the TEER of the Caco-2 cell monolayers significantly at pH 5.8 and 7.4. This TEER reducing effect was dependent on the concentration of the Aloe products and to a lesser extent also dependent on the pH of the environment. Of high importance is the recovery of the TEER of the Caco-2 cell monolayer to reach the initial value after removal of the Aloe vera gel and whole leaf extract solutions. The results obtained from these TEER studies indicate the ability of Aloe vera gel and whole leaf extract to open tight junctions between adjacent epithelial cells in order to allow for paracellular movement of ions and possibly other compounds. The complete reversibility of this effect to reach the original resistance after removal of the gel and whole leaf extract indicates that no damage was caused to the cell layer and the integrity of the monolayer was preserved. It can be concluded from these results that Aloe vera gel and whole leaf extract are potentially safe and effective paracellular absorption enhancers across the intestinal epithelium.

/V-trimethyl chitosan chloride (TMC) is an absorption enhancer that causes a pronounced and immediate reduction in the TEER of Caco-2 cell monolayers, but only a slight recovery towards the initial value can be obtained after removal of the TMC from the cell surface. This may indicate potential damage to certain structures of the cells but was previously explained by the inability to remove this mucoadhesive polymer from the cell surface without causing physical damage. When TMC was combined with Aloe vera gel and whole leaf extract, respectively, a significant reduction in the TEER of Caco-2 cell monolayers was observed (i.e. in concentrations of 2.5 and 5.0% w/v for the Aloe products). Furthermore, the recovery of the TEER towards the initial value was markedly improved as compared to that obtained with TMC alone. This indicates a possible synergistic effect on the TEER reduction between TMC and the Aloe vera products as well as an improvement of the reversibility. As expected from the results of the TEER studies, both Aloe vera gel and whole leaf extract were able to significantly enhance the transport of the macromolecular model compound, insulin, across Caco-2 cell monolayers. Due to the physicochemical properties of the model compound and the fact that the Aloe vera products reduced the TEER significantly, it was concluded that this transport enhancement effect was most probably achieved by means of opening of the tight junctions to allow for increased paracellular movement of the insulin molecules. In accordance with the TEER results, the transport enhancement effect was higher at the lower pH value. This was explained by possible protonation of phytoconstituents that influences their interaction with the cell monolayer and thereby increase the effect on the tight junctions.

>A/oe vera gel and whole leaf extract were able to enhance the transport of luteolin significantly at pH 5.8 (at concentrations higher than 0.5% w/v), which was attributed to opening of the tight junctions as well as an increase in the stability of the luteolin. Only a slight increase was observed in the transport of luteolin at pH 7.4, which was not concentration dependent. In fact, a decrease of luteolin transport was observed at pH 7.4 for certain higher concentrations of the Aloe products, which can be explained by possible complex formation at these higher concentrations. It was also deduced from these results that luteolin is possibly also transported by the transcellular route and opening of the tight junctions by the Aloe products therefore had a less pronounced effect as compared to a model drug such as insulin. Similar results were obtained for rutin (as for luteolin), where transport enhancement was observed at pH 5.8 in a concentration dependent way but not at pH 7.4. Therefore, the same conclusions could be drawn from the results of the rutin transport studies as for the luteolin transport studies. It is to be appreciated, that the invention is not limited to any particular embodiment as hereinbefore generally described.

Claims

1. Use of an aloe vera product for increasing bioavailability of a medicinal drug by co-administration of the product with the drug.

2. Use as claimed in claim 1 , wherein the aloe vera product is a whole leaf powder of an aloe vera plant.

3. Use as claimed in claim 1 or claim 2, wherein the aloe vera product is a gel extruded from the inner pulp of an aloe vera leaf.

4. Use as claimed in any one of claims 1 to 3, wherein the aloe vera product and drug are co-administered orally.

5. Use as claimed in any one of the preceding claims, wherein the drug to be targeted for increasing of its bioavailability by means of absorption enhancement is selected from the group including: insulin, calcitonin, vasopressin, leucine, acyclovir, chlorpromazine, cyclosporine, erythromycin, gentamicin, isosorbide dinitrate, pyridostigmine, labetalol, methyldopa, morphine, and propranolol.

5. Use as claimed in any one of the preceding claims, wherein the aloe vera product is co-administered together with an additional absorption enhancer.

6. Use as claimed in claim 5, wherein the additional absorption enhancer is selected from: chitosan and/or its derivative N-trimethyl chitosan chloride (TMC).

7. Use as claimed in any one of the preceding claims, wherein the aloe vera product is co-administered together with an enzyme inhibitor for inhibiting degradation of the drug in the intestinal tract.

8. Use of an aloe vera product in the manufacture of a medicament which medicament includes a poorly absorbable medicinal drug so as to enhance the bioavailability of the drug.

9. Use as claimed in claim 8, wherein the aloe vera product is a whole leaf powder of an aloe vera plant.

10. Use as claimed in claim 8 or claim 9, wherein the aloe vera product is a gel extruded from the inner pulp of an aloe vera leaf.

11. Use as claimed in any one of claims 8 to 10, wherein the medicament is in use administered orally and is in a form selected from the group including: a capsule, a tablet, a solution, and a suspension.

12. Use as claimed in any one of claims 8 to 11 , wherein the drug is selected from the group including: insulin, calcitonin, vasopressin, leucine, acyclovir, chlorpromazine, cyclosporine, erythromycin, gentamycin, isosorbide dinitrate, pyridostigmine, labetalol, methyldopa, morphine, propranolol, and terbutaline.

13. Use as claimed in any one of claims 8 to 12, wherein the medicament includes an additional absorption enhancer.

14. Use as claimed in claim 13, wherein the additional absorption enhancer is selected from: chitosan and/or its derivative N-trimethyl chitosan chloride (TMC).

15. Use as claimed in any one of claims 8 to 14, wherein the medicament includes an enzyme inhibitor for inhibiting degradation of the drug in the intestinal tract.

16. A medicament including an aloe vera product, which medicament includes a poorly absorbable medicinal drug.

17. A medicament as claimed in claim 16, wherein the aloe vera product is a whole leaf powder of an aloe vera plant.

18. A medicament as claimed in claim 16 or claim 17, wherein the aloe vera product is a gel extruded from the inner pulp of an aloe vera leaf.

19. A medicament as claimed in any one of claims 16 to 18, wherein the medicament is in use administered orally and is in a form selected from the group including: a capsule, a tablet, a solution, and a suspension.

20. A medicament as claimed in any one of claims 16 to 19, wherein the drug is selected from the group including: insulin, calcitonin, vasopressin, leucine, acyclovir, chlorpromazine, cyclosporine, erythromycin, gentamycin, isosorbide dinitrate, pyridostigmine, labetalol, methyldopa, morphine, propranolol, and terbutaline.

21. A medicament as claimed in any one of claims 16 to 20, wherein the medicament includes an additional absorption enhancer.

22. A medicament as claimed in claim 21 , wherein the additional absorption enhancer is selected from: chitosan and/or its derivative N-trimethyl chitosan chloride (TMC).

23. A medicament as claimed in any one of claims 16 to 22, wherein the medicament includes an enzyme inhibitor for inhibiting degradation of the drug in the intestinal tract.

24. A process according to the invention for producing a medicament which medicament includes a poorly absorbable medicinal drug using an aloe vera product so as to enhance the bioavailability of the drug substantially as hereinbefore described or exemplified.

25. A process of producing a medicament which medicament includes a poorly absorbable medicinal drug using an aloe vera product so as to enhance the bioavailability of the drug including any new and inventive integer or combination of integers, substantially as herein described.

26. A medicament as claimed in any one of claims 16 to 23 whenever supplied with instructions for the use thereof.

27. A medicament as claimed in claim 26 when the instructions are in printed or written form.

28. A medicament as claimed in claim 27 supplied in a package or container having said instructions provided thereon or therein.