Background
Hoodia is a genus of succulent plants belonging to the family Apocynaceae. It is widely used now and traditionally by the San Bushmen of Southern Africa, who believe that
Hoodia is their food, water and medicine [
1,
2].
Hoodia species are indigenous to the Kalahari Desert of Southern Africa, including Namibia, South Africa, Angola and Botswana. One of the popular
Hoodia species used is
Hoodia gordonii, a desert plant traditionally used by the San people as an appetite suppressant, thirst quencher and to treat severe abdominal cramps, haemorrhoids, tuberculosis, indigestion, minor infections, hypertension and diabetes [
2].
H. gordonii has been known by the indigenous populations of Southern Africa for a long time. For centuries this plant has been used to stave off hunger during long and tiring hunting trips or when food supplies were low [
2]. Despite its popular use and commercialization, the bioactivity of
H. gordonii has not been extensively studied.
A number of plants from the Apocynaceae family are considered to be potential sources of antioxidants which have been attributed to the high phenolic content in the phytochemical profile of some of these plants [
3‐
5]. Antioxidants may be defined as free radical scavengers which protect living organisms from damage caused by the accumulation of free radicals. Free radicals have been implicated in various pathological conditions such as ischemia, anaemia, asthma, arthritis, inflammation, neurodegeneration, as well as speeding up the ageing process and perhaps even causing certain dementias [
6‐
10]. Free radicals are produced by physiological and biochemical processes, or induced by environmental factors such as pollution and are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes, and other small molecules which result in cellular damage [
11].
Oxidative stress has also been implicated in the pathogenesis of HIV/AIDS since the virus replicates in a highly oxidized environment [
12]. There is ongoing search for better or alternative treatment that could also serve as adjuvant therapy to existing anti-HIV medicines. In addition to various severe side effects, antiretroviral (ARV) drugs reportedly increase oxidative stress [
13]; hence the need for antioxidants as adjuvant therapy for HIV therapy. In 2015, Tabe and colleagues administered
Hibiscus sabdariffa (Linnaeus) juice to HIV/AIDS patients on ARV therapy and reported an increase in white blood cells compared to the control group.
H. sabdariffa is a plant with high antioxidant capacity and is consumed as a leafy vegetable and herbal tea in many countries [
14,
15]. This study investigated the antioxidant and anti-HIV potential of
H. gordonii, a popular plant which has been commercialized as a diet suppressant to aid with weight loss [
2]. This data suggests potentially new applications for this plant in the future.
In vitro anti-HIV assays
HIV-1 reverse transcriptase colorimetric assay
The effect of H. gordonii crude extracts on HIV-1 reverse transcriptase (RT) was tested using an RT colorimetric ELISA kit from Roche Diagnostics (Mannheim, Germany). The assay was performed according to the manufacturer’s instructions. Extracts were tested at six different concentrations (50, 100, 200, 400, 800 and 1000 μg/mL). The enzyme was incubated for 1 h with extract at 37 °C. Subsequent 1 h incubations included addition of an antibody conjugated to peroxidase that binds to the digoxigenin-labeled DNA. In the final step, the ABTS substrate solution was cleaved by the peroxidase enzyme, producing a coloured reaction product. Doxorubicin, a known HIV-1 RT inhibitor was used as a positive control. The absorbance of the samples was read at 405 nm using a SpectraMax M2 plate reader.
HIV-1 integrase assay
The Xpress HIV-1 Integrase Assay Kit (Express Biotech International, USA) was used to measure the inhibitory effects of H. gordonii extracts (0.1, 0.2 and 0.4 mg/mL) on HIV-1 integrase activity. Streptavidin coated 96-well plates were coated with a double-stranded HIV-1 LTR U5 donor substrate oligonucleotide containing an end-labelled biotin. Full-length recombinant HIV-1 integrase protein was then loaded onto the oligo substrate. H. gordonii extracts or sodium azide (standard control) was added to the reaction plates together with a double-stranded target substrate (TS) oligo containing 3′-end modifications. The horseradish peroxidase (HRP)-labelled antibody was directed against the TS 3′-end modification and the absorbance due to the HRP antibody– tetramethylbenzidine peroxidase substrate reaction was measured at 450 nm using a SpectraMax M2 plate reader.
HIV-1 protease fluorogenic assay
A SensoLyte 490 HIV-1 Protease (PR) kit from AnaSpec (San Jose, CA, USA), was used to assay H. gordonii extracts against HIV-1 PR. Due to the limited number of reactions of the kit, samples were tested at five concentrations, namely; 25, 50, 100, 200 and 400 μg/mL. Acetyl pepstatin (AP) was used as a known standard for HIV-1 PR inhibition. Briefly, test samples were incubated at room temperature with HIV-1 PR enzyme and substrate for 45 min. Stop solution (50 μl) was added to each reaction then the fluorescence intensity was measured at Excitation/Emission = 340/490 nm using a SpectraMax M2 plate reader.
Discussion
A number of glycosides have been isolated from
H. gordonii [
2] and amongst these glycosides is the popular P57 glycoside attributed to the appetite suppressant properties of
H. gordonii. The attention on
H. gordonii species was elicited by the discovery of hunger suppressing glycosides. Despite its popular use, minimal reports are available on biological studies conducted on
H. gordonii. However, the safety profile of
H. gordonii extracts has already been determined in a number of in vivo studies [
25].
The only antioxidant study conducted on
H. gordonii was on glycosides isolated from the plant which did not demonstrate antioxidant property [
2]. The presence of other phytochemicals in the crude extracts such as phenolics, alkaloids, tannins and terpenes could be attributed to the antioxidant potential observed in this study. The
H. gordonii ethanol extract exhibited the highest reducing activity and these results were in agreement with the high DPPH scavenging activity observed in ethanol extracts. The total phenolic content of the ethanol extract was relatively high compared to that of the ethyl acetate extract and the presence of tannins in the ethanol extract but not in ethyl acetate extract could all be responsible for the high antioxidant potential observed in the ethanol extract. Phenolic compounds and tannins are widely reported as natural antioxidants [
19,
20] and the present study revealed that
H. gordonii could be a potential source of useful natural antioxidants.
H. gordonii is among the most popular anti-obesity products on the market [
26]. Obesity is a chronic disease and amongst other morbidities, it is associated with an increase in oxidative stress [
27]. The role of oxidative stress in the pathogenesis of various ailments, such as psychiatric, inflammatory and infectious diseases has been well documented [
6,
9,
12,
28]. The antioxidant activity observed in the present study could contribute to the scavenging of accumulated free radicals in mostly obese individuals that consume
H. gordonii products.
In addition to being associated with obesity, oxidative stress has also been linked to the progression of HIV [
29] which is supported by a study that reported the promotion of HIV replication by oxidizing agents as compared to antioxidants [
30]. Before the present study, there was no literature reporting on the in vitro anti-HIV-1 properties of
H. gordonii. The extracts demonstrated good inhibition against HIV-1 reverse transcriptase and protease which are two of the three HIV enzymes that play a major role in the replication of the virus in host cells. Current HIV therapy targets various steps of the HIV life cycle, which includes HIV enzymes [
31]. However, this antiretroviral therapy is often limited by adverse side effects leading to patients discontinuing treatment and in the process contributing to the development of HIV drug resistant strains [
31]. The search for better HIV therapy is ongoing and the in vitro anti-HIV data from the present study is a valuable contribution towards this search.
H. gordonii is already regarded as a complementary and alternative medicine for the treatment of obesity [
26]. Further in vivo validation of this research could support the use of this commercial product as a supplement for HIV therapy as well as a natural antioxidant.
Conclusion
The in vitro anti-HIV and antioxidant data obtained in this study suggests new potential uses of H. gordonii, which is currently commercialized and mainly used as an anti-obesity supplement. The chemistry of H. gordonii has been reported as well as the isolation and characterization of the glycosidic compounds. However, based on the results of this work, future investigations will also research the isolation and characterization of H. gordonii compounds which are responsible for the above mentioned in vitro anti-HIV-1 and antioxidant activity.
Acknowledgments
The authors would like to thank Farm Vredelus for the donation of H. gordonii plant material. This research was supported by the University of Namibia’s Research and Publication Unit (Grant no. URPC/2014/184).
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