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In vitro transport of the steroidal glycoside p57 from hoodia gordonii across excised porcine intestinal and buccal tissue.



Buccal transport

Hoodia gordonii

Steroidal glycoside P57

Intestinal transport

In vitro



Hoodia gordonii was traditionally used by the Khoisan people as a thirst and appetite suppressant and is currently commercially available as a popular weight-loss supplement. The perceived active ingredient isolated from this plant is a steroidal glycoside named P57. This study aimed at investigating the in vitro transport of P57 across excised porcine intestinal and buccal mucosa in a Sweetana-Grass diffusion apparatus. For both the intestinal and buccal experiments, the transport of pure P57 was compared to that obtained from a crude plant extract. Bi-directional transport experiments were conducted across the intestinal tissue in two different media namely Krebs-Ringer bicarbonate buffer and simulated intestinal fluid. Apical-to-basolateral transport experiments were conducted across the buccal tissue in two different media namely Krebs-Ringer bicarbonate buffer and artificial saliva. Apparent permeability coefficient (Papp)and flux values were calculated and analysed by means of a one-way repeated analysis of variance (ANOVA) to determine if differences were significant (p less than 0.05). The transport of pure P57 across intestinal tissue was significantly higher in the secretory direction than in the absorptive direction indicating efflux by membrane transporters. Much higher intestinal transport was obtained for P57 in both directions when applied in the form of a crude extract, possibly due to inhibition of efflux as indicated by lower secretory transport compared to absorptive transport. For the buccal tissue, no transport was obtained for the pure P57, while relatively high transport was obtained when applied in the form of a crude extract. Furthermore, the intestinal transport of P57 was significantly decreased when the crude extract was prepared in simulated intestinal fluid compared to when it was prepared in buffer. On the other hand, buccal transport was higher in artificial saliva than in buffer. It is therefore evident that the transport of P57 across mucosal tissues is significantly affected on exposure to conditions simulating the in vivo situation.

[c] 2011 Elsevier GmbH. All rights reserved.


Hoodia gordonii (Masson) Sweet ex Decne is a spiny succulent plant that was traditionally used by the Khoisan people of South Africa and Namibia to suppress hunger and thirst while on long hunting trips or in times of famine. The fresh stems were peeled to remove the thorns and the flesh was chewed and/or retained in the buccal cavity before swallowing (Van Wyk and Gericke 2000; Lee and Balick 2007; Glasl 2009). Not surprisingly, research and commercial interest was ignited by the purported anti-obesity activity of this plant. Obesity has become one of the most common health concerns of modern times. In 2005 it was estimated that 1.6 billion adults were overweight globally with at least 400 million classified as obese. Obesity is associated with and can lead to many disease conditions including type-2 diabetes, cardiovascular disease, hypertension, sleep apnoea and cerebrovascular accidents amongst many others (WHO 2006). The multibillion dollar anti-obesity industry expands almost on a daily basis and H. gordonii supplements are extremely popular for purposes of weight-loss. After the isolation of the steroidal glycoside P57 (Fig. 1) from H. gordonii by Van Heerden et al. (2002), and the publication of scientific data on its anti-obesity effect (Van Heerden et al. 2007) more than 40 other glycosides have been isolated from H. gordonii (Abrahamse et al. 2007; Dall'Acqua and Innocenti 2007; Pawar et al. 2007a, b; Shukla et al. 2009). Despite the isolation of these structural analogues, pharmacological data is only available for P57 and it is therefore still regarded as the only active ingredient in H. gordonii. Research on aspects that should be investigated prior to commercialisation of products claiming biological activity such as the bioavailability of the active compound has been lacking, though recently some of the biopharmaceutical aspects have been the focus of in vitro and in vivo investigations in animals. A mouse model was used to determine the bioavailability, pharmacokinetic profile and tissue distribution of pure P57 (Madgula et al. 2010a). In addition, the in vitro metabolic stability of P57 and interactions with drug metabolising enzymes (Madgula et al. 2008), the pharmacokinetic profile of hoodigogenin A, the aglycone of P57, and the stability of P57 in simulated gastrointestinal fluids (Madgula etal. 2010b) has been studied. The intestinal transport of pure P57 using the Caco-2 cell model has been determined (Madgula et al. 2010b), but the current study is the first to compare the intestinal transport of pure P57 to that obtained from a crude extract across porcine intestinal mucosa. Permeability data obtained using porcine intestines correlate well to that of human intestines (Nejdfors et al. 2000). In addition, no transport studies across buccal tissue have been conducted for P57, which may contribute to the bioavailability of P57 specifically when the traditional use of the plant is considered. Porcine buccal mucosa is similar to human mucosa in term of structure, morphology, composition and thickness and is the most commonly used tissue model to assess buccal permeation of compounds (Nicolazzo and Finnin 2008). It was therefore the aim of this study to expand the current biopharmaceutical knowledge of P57 by investigating the in vitro transport of P57 across porcine intestinal and buccal tissues in an ex vivo model and to compare the transport obtained for pure P57 with the transport of P57 when applied in the form of a crude plant extract.


Materials and methods

Plant material and chemicals

A crude plant extract was prepared with acetonitrile from a commercial H. gordonii product of which the quality (viz. P57 content) was determined by LC-MS analysis. Pure P57 was purchased from Chromadex Inc. (California, USA).

Tissue preparation

Fresh porcine buccal and intestinal tissue were obtained from a local abattoir (Pretoria, South Africa) immediately after slaughter and washed with and transported in cold Krebs-Ringer bicarbonate buffer (referred to henceforth as buffer). The serosal layer was removed from the intestinal mucosa with blunt dissection, whereafter the mucosal layer was cut along the mesenteric border and mounted (Legen et at 2005) between two halves of Sweetana-Grass diffusion chamber clamps with an exposed surface area of 1.13 cm2 (Grass and Sweetana 1988). Excess underlying tissue was removed from the buccal mucosa by dissection with a scalpel and mounted between two halves of Sweetana-Grass diffusion chamber clamps (Ceschel et al. 2002; Van Eyk and Van der Bijl 2004). The clamps were inserted into the diffusion apparatus with the mucosal side facing the apical chamber, and 5 ml of fresh warmed buffer (37[degrees]C) was added to both the apical and basolateral chambers. The tissues were continuously oxygenated with medical oxygen and an equilibration period of 30min was allowed before commencement of the transport studies.

Preparation of simulated intestinal fluid and artificial saliva

The artificial saliva consisted of 5.208 g [NaHCO.sub.3], 1.369g [K2HPO.sub.4]-3[H.sub.2]O, 0.877g NaCl, 0.447 g KC1, 0.441 g [CaCl.sub.2]-2[H.sub.2]O, 2.160g mucin and 200,000 U of hog pancreas a-amylase (Sigma Aldrich) in 11 distilled water. The resulting artificial saliva solution was adjusted to a pH of 7.0 [+ or -] 0.1 (Boland et al. 2004). Simulated intestinal fluid (SIF) was prepared by dissolving 6.8 g monobasic potassium phosphate (KH2PO4) in 250 ml water. After mixing 190 ml 0.2 M sodium hydroxide (NaOH), 400 ml water and 10.0 g pancreatin (Sigma Aldrich) were added. The pH was adjusted to 7.5 [+ or -] 0.1 with 0.2 M sodium hydroxide and distilled water was added to provide a volume of 1000 ml (USP 1990).

Transport studies

Bi-directional transport experiments were conducted across porcine intestinal tissue for pure P57 and for P57 in crude extract, while transport experiments were conducted across porcine buccal tissue in the apical-to-basolateral direction for pure P57 and for P57 in crude extract. In each case, 5% methanol was used to dissolve the pure P57 or the crude extract before it was made up to volume with the specific transport medium. Solutions made up to volume with buffer was adjusted to pH 7.4 [+ or -] 0.1, while that of simulated intestinal fluid (SIF) was adjusted to pH 7.5 [+ or -] 0.1 (USP 1990) and for artificial saliva to pH 7.0 [+ or -] 0.1 (Boland et al. 2004). A concentration of 200[micro]M was used as the basis for both the pure P57 and P57 in the crude extract, which were added to the chambers as previously reported by Madgula et al. (2008) in an in vitro transport study across Caco-2 cell monolayers. Therefore pure P57 was added to each chamber in a concentration of 1.2 mg/5 ml. It was calculated that 2.4 g of plant material had to be extracted to provide approximately 1.2 mg of P57 per chamber based on the P57 content in the plant material as determined by LC-MS, namely 0.304%. Samples (200[micro]l) drawn from each of the original solutions before they were applied to the tissues in the transport studies were analysed for P57 content. The transport studies were initiated by adding 5 ml of each solution of different experimental groups as indicated in Table 1 to the donor chamber (viz. apical or basolateral chamber, respectively). Samples (200 ill) were drawn from the receiver chamber over a period of 2 hat 20min intervals and the volume of each sample was replaced with transport medium.
Table 1

Experimental groups used for ihe intestinal and buccal transport

Absorptive direction Secretory direction

Apical chamber Basolateral Apical Basolateral
 chamber chamber chamber


Pure P57 + butter Buffer Buffer PureP57+buffer
Extract + buffer Buffer Buffer Extract + buffer
Extract + SIF Buffer SIF Extract + buffer


Apical chamber Basolateral

Buccal transport
Pure P57 + buffer Buffer

Pure P57 + Buffer

Extract + buffer Buffer

Extract+ Buffer
artificial saliva

Quantification of P57

Quantification of P57 in the samples was performed with high performance liquid chromatography (HPLC). An HPLC system consisting of a Waters 2690 separation module and a Waters 996 photodiode array detector (Waters, MA, USA) was used and separation achieved using a Gemini C18 column (250 mm x 4.6 mm; 5[micro]m particle size; Phenomenex) equipped with a guard column (4 mm x 3.0 mm, Phenomenex). The mobile phase consisted of water containing 0.05% acetic acid (A) and acetonitrile (B). Gradient elution was used as follows: 100% A at 0 min, adjusted in the following 40 min to 20% A and 80% B. Each run was followed by a 5 min wash with 100% B and an equilibration period of 8 min. The flow rate was 1.0 ml/min, the column operated at a temperature of 30[degrees]C and the detection wavelength was 220 nm. The chromatographic data was collected and analysed with Empower software.


Data analysis and statistical evaluation

The transport results were corrected for dilution and the cumulative P57 transport was plotted as a function of time. The apparent permeability coefficient (Papp) values were calculated according to the following equation (Hansen and Nilsen 2009):

Papp=(dc/dt) (1/a.60.C0) (cm/s)

where dC/dtis the transport rate (slope), A is the surface area of the tissue (1.13 cm2) and C0 is the initial concentration of P57 applied. The P57 flux (J) values were calculated according to the following equation (Hansen and Nilsen 2009):

J = v(dc/dt)/A [([micro]g/cm.sup.2]/h)

where Vis the receiver chamber volume, dCis the receiver chamber P57 concentration ((xg/ml), dt is the transport time (h) and A is the tissue surface area [(cm.sup.2]).

The net flux of P57 (/net) was calculated as follows (Hansen and Nilsen 2009):

JNET = JB-A-JA-B [([micro]g/cm.sup.2]/h)

where Jb-a is the flux of P57 in the basolateral to apical direction andJA-B is the flux of P57 in the apical to basolateral direction.

Statistical analysis of the apparent permeability (PQpp) and flux (J) values for both the buccal and intestinal transport experiments was done by means of a repeated one-way analysis of variance (ANOVA) to indicate whether differences are significant (p leaa than 0.05) or not

Results and discussion

The Papp values are shown in Figs. 2 and 3 while all the flux (J) values for P57 across intestinal and buccal tissues are shown collectively in Table 2. In general, the in vitro transport of pure P57 in buffer across porcine intestinal tissue was very low, but it was significantly higher in the secretory direction (PGpp = 0.059 x [10.sup.-6]cm/s) than in the absorptive direction (Papp = 0.022 x [10.sup.-6]cm/s) indicating that P57 may be actively effluxed by intestinal membrane transporters. This is also shown by the positive net flux value (Table 2). These results are congruent with a previous study by Madgula et al. (2008) where the intestinal transport of pure P57 across Caco-2 cell monolayers revealed lower transport in the absorptive direction than in the secretory direction. In this Caco-2 cell study it was also shown that in the presence of inhibitors of two membrane transporters namely verapamil which inhibits P-glycoprotein (P-gp) and MK-571 which inhibits multidrug resistance-associated protein, the permeability of P57 increased in the absorptive direction indicating it is a substrate of these two active membrane transporters. In the absence of these efflux pump inhibitors, the P57 permeability was concentration-dependent indicating saturation of transporters (Madgula et al. 2008). The intestinal transport of P57 applied as a crude extract had a significantly higher Papp value of 74.479 x [10.sup.-6]cm/s compared to the Papp value of pure P57 of 0.022 x [10-.sup.6]cm/s in the absorptive direction as well as in the secretory direction with Papp values of 73.466 x [10.sup.-6] for the crude extractand 0.059 x [l0.sup.-6]cm/s for pure P57. Furthermore, P57 transport from the crude extract in buffer was slightly but not significantly higher in the absorptive direction (Papp = 74.479 x [10.sup.-6]cm/s) than in the secretory direction (Papp = 73.466 x [10.sup.-6]Gcm/s) indicating that phytochemicals in the crude extract influences P57 transport by means of efflux inhibition but other mechanisms such as changes to P57 solubility and membrane permeability cannot be excluded.The phenomenon that crude extracts are in some cases more permeable and biologically active than the pure compounds has been established before. For example, pure aspalathin was transported to a lower extent across Caco-2 cell monolayers compared to aspalathin from a crude Rooi-bos tea extract (Huang et al. 2008).
Table 2

Flux (J) and net flux Jnet) values [([mu]g/cm.sup.2]/h) for P57 across
porcine intestinal and buccal mucosa.

 Intestinal transport

Group J(A-B) J(A-B)
 [[mu]g/cm.sup.2]/h) [[mu]g/cm.sup.2]/h)

Pure P57 + 0.174 ** 0.501 **

Extract + 10.523 10-476

Extract+ 1.357 1356

Pure P57 + N/A N/A

Extract + N/A N/A

 Buccal transport

Group J (NET) J (A-B)
 [flux([mu]g7cm.sup.2]/h) [B([mu]g/cm.sup.2]/h)
Pure P57 + 0.3265 0

Extract + -0.0469 2.220

Extract+ -0.0009 N/A

Pure P57 + N/A 0

Extract + N/A 2.204

A-B: apical to basolateral; B-A; basolateral to apical; N/A: not
* Flux values between all groups for A-B intestinal transport are
statistically significantly different (p [less than or equal to] 0.05).
** Flux values between A-B and B-A intestinal transport for pure P57
were statistically significantly different (p [less than or equal to]


The transport of P57 from crude extract dissolved in SIF was significantly decreased in both the absorptive and secretory directions (Papp = 10.617x [10.sup.-6]; 14.608 x [10.sup.-6] cm/s) compared to the P57 transport from extract dissolved in buffer(Papp = 74.479x [10.sup.-6]; 73.466 x [10.sup.-6]cm/s). The transport in SIF was significantly higher in the secretory direction compared to the absorptive direction (Fig. 2), indicating that phytochernicals in the crude extract were probably degraded and therefore did not inhibit efflux of P57 as in the case when dissolved in buffer. The lower transport results of P57 can be explained by the fact that the stability of P57 was significantly affected by SIF as shown with a stability study by Madgula et al. (2010b) where it was determined that 8.6% of the initial concentration of pure P57 applied was degraded within 180min when exposed to SIF. In addition, 100% of pure P57 was degraded in simulated gastric fluid (SCF) after 60 min. As the simulated intestinal fluid is used to mimic in vivo conditions, these results indicate that the bioavailability of orally administered P57 may be significantly affected by enzymatic degradation and/or hydrolysis. An in vivo study by Madgula et al. (2010a) in CD1 female mice revealed a peak plasma level of P57 after 0.6 h and moderate bioavailability of 47.5% after oral administration of a methanolic extract of H. gor-donii (equivalent to a dose of 25mg/kg P57). The moderate oral bioavailability is likely due to degradation in gastric and intestinal fluids. The buccal route of administration may be considered as an alternative route of administration to avoid the harsh conditions in the gastrointestinal tract that degrades P57.

Buccal administration of compounds has distinct advantages over oral administration such as avoidance of the deleterious effects of the gastrointestinal tract (degradation) and bypassing first-pass hepatic metabolism (Nicolazzo and Finnin 2008). In this study, no buccal transport of pure P57 in either buffer or artificial saliva was obtained at the concentration tested. However, when crude extract was dissolved in buffer, the Papp value was 19.149 x [10.sup.-6] cm/s (Fig. 3), indicating that phytoconstituents present in the crude extract significantly enhanced the transport of P57 across the buccal tissue. Interestingly, the transport was significantly higher when the crude extract was dissolved in artificial saliva (Papp =22.081 x [10.sup.-6] cm/s) as compared to the buffer indicating no degradation of P57. This can possibly be explained by components of the saliva improving transport by mechanisms such as changes to the solubility of P57 and membrane permeability.


It was shown with the in vitro transport experiments across both porcine intestinal and buccal tissue that the transport of P57 from a crude extract was significantly higher than the transport obtained for pure P57. This indicates that the crude extract contains phytoconstituents that influence the transport of P57 across both intestinal and buccal tissues. The results obtained reinforce the credibility of the traditional use of the plant as a whole as opposed to administration of isolated P57. It can further be concluded that buccal transport of P57 from plant material may contribute to the overall bioavailability and therefore the pharmacological effect of H. gordonii.


The authors would like to thank the National Research Foundation (NRF) and Tshwane University of Technology (TUT) for financial support.

0944-7113/$ - see front matter[C] Elsevier GmbH. All right reserved.

doi: 10.1016/j.phymed.2011.01.017


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I. Vermaak, A.M. Viljoen, W. Chen, J.H. Hamman *

Department of Pharmaceutical Sciences, faculty of Science, Tshwane University of Technology, Private BagX680, Pretoria 0001, South Africa

* Corresponding author. Tel: +27 12 382 6397; fax: +27 12 382 6243. E-mail address: (J.H. Hamman).
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Author:Vermaak, I.; Viljoen, A.M.; Chen, W.; Hamman, J.H.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:6SOUT
Date:Jun 15, 2011
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