In Vitro Antioxidant Activity of Crude Extracts of Harpagophytum Zeyheri and Their Anti-Inflammatory and Cytotoxicity Activity Compared With Diclofenac.

doi:10.21203/rs.3.rs-208753/v1

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In Vitro Antioxidant Activity of Crude Extracts of Harpagophytum Zeyheri and Their Anti-Inflammatory and Cytotoxicity Activity Compared With Diclofenac.

https://doi.org/10.21203/rs.3.rs-208753/v1

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Background

This study evaluated the in vitro antioxidant activity and comparison of anti-inflammatory and cytotoxic activity of Harpagopytum zeyheri with diclofenac.

Methods

In vitro assays were conducted using water, ethanol and ethyl acetate extracts of H.zeyheri. The antioxidant activity was evaluated using the 2,2'-diphenyl-1-picrylhydrazy (DPPH) and 2,2'- azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS)assays. The anti-inflammatory activity was determined by measuring the inhibition of nitric oxide (NO) on lipopolysaccharide (LPS)-induced RAW 264.7 mouse macrophages as well as cytokine (TNF-α and IL-10) expression on LPS-induced U937 human macrophages. For cytotoxicity, cell viability was determined using the 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.

Results

The ethyl acetate extract had the lowest IC50 values in the DPPH (5.91µg/ml) and ABTS (20.5µg/ml) assay compared to other extracts. Furthermore, the ethyl acetate extracts effectively inhibited NO and TNF-α and proved to be comparable to diclofenac at some concentrations. All extracts of H. zeyheri displayed dose dependent activity and were associated with low levels of human-IL-10 expression compared to quercetin. Furthermore, all extracts displayed low toxicity relative to diclofenac.

Conclusions

These findings show that H. zeyheri has significant antioxidant activity. Additionally, similarities exist in inflammatory activity of H. zeyheri to diclofenac at some concentrations as well as low toxicity in comparison to diclofenac.

Integrative & Complementary Medicine

Internal Medicine

antioxidant

cytotoxicity

cytokines

Harpagophytum zeyheri

inflammation

nitric oxide

Inflammation occurs in the body as a response to infection, injury and other harmful stimuli (Riciotti and Fitzgerald, 2011; Sagnia et al., 2014), which is related to the excess production of free radicals such as superoxide, hydroxyl, and peroxyl in the body resulting in damaging effects. Complex interactions occur between mediators of inflammation and inflammatory cells during inflammation (Turner et al., 2014) triggering the release of signalling molecules and enlistment of circulating leucocytes such as macrophages. These become stimulated at the area of inflammation, thereby releasing various types of mediators and cytokines with either pro- or anti-inflammatory action, such as nitric oxide (NO), Tumor Necrosis Factor (TNF-α), prostaglandins (PG), and interleukins IL-6, IL-10 and IL-1β (Zhang and An, 2007). Inflammation and inflammation cell interactions result in either a positive outcome of host-defence mechanism, or in uncontrolled cases, lead to tissue injury and chronic diseases (Mantovani et al., 2008) with studies showing that in almost 99% of cases, inflammations are intolerable if not treated properly (Beg et al., 2011).

TNF-α is an essential factor for the stimulation of the genetic expression of inducible nitric oxide synthase (iNOS) in various cell lines (Wolf et al., 2005). Nitric oxide produced in large amounts by one of the pro-inflammatory enzymes, iNOS, is known to be responsible for the vasodilation and hypotension observed during septic shock and inflammation (Yang et al., 2009). In addition, excessive NO production is found in many inflammation-related diseases such as asthma, arthritis, and multiple sclerosis. Signalling molecules released during inflammation may also interact with free radicals causing irreversible damage to cell membranes leading to cell death and tissue damage (Wang et al., 2004). All related inflammatory mediators play a role in the genesis and progression of the various inflammatory diseases (Sharma and Mohamed, 2006). Thus, because of the implications of inflammation in chronic diseases there is high demand for treatment, with Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) which are among the most used medications (Klein and Eliakim, 2010). Most drugs used for inflammatory diseases act by suppressing levels of pro-inflammatory cytokines, iNOS, prostaglandins, cyclooxygenases and lipoxygenases (Pohanka et al., 2011). Despite the effectiveness of these drugs, their safety is a cause for concern with studies linking almost 90% of them with related toxicities and side effects (Lanas and Sopeña, 2009), which has resulted in growing research on natural therapies for inflammation that are deemed safer but still provide the necessary relief.

Diclofenac is one such NSAID, which despite its effectiveness, notable side effects such as gastric irritation, ulceration, bleeding, renal failure, and hepatic failure among many others have been highlighted (Goci et al., 2013). Other indirect side effects of diclofenac include serious ecological impacts such as the catastrophic decline in vulture populations in the Indian subcontinent which has negative consequences on the environment (Shultz et al., 2004; Mundy & Ncube, 2014). Thus, studies on medicinal plants with similar properties as synthetic drugs, are becoming increasingly important as they are deemed safer alternatives, and research is focused on this topic worldwide (reviewed in Refs).

The Pedaliaceae family has the Harpagophytum genus which consists of two species: Harpagophytum procumbens (Burch. 1822) D.C. ex Meissn. and Harpagophytum zeyheri (Decne. 1865). Due to the close taxonomic similarities of the two species, they are both loosely referred to as Devil’s Claw or grapple plant, a common name derived from the hooked formation of the fruit giving it a claw-like appearance (Van Wyk et al., 1997). As an adaptation to the arid Kalahari sands, both plants consist of a tuberous taproot with secondary tubers developing on fleshy roots which grow from the primary tuber (Strohbach and Cole, 2007). The secondary tubers contain iridoiod glycosides, with harpagoside as the main active ingredient, which enable the use of the plants for medicinal purposes, particularly for treating rheumatism, arthritis, digestive disorders, sores, ulcers and boils (Van Wyk et al., 1997; Schneider et al., 2006). As a result, Devil’s Claw is commercially exploited for its medicinal properties which contribute significantly to the health, livelihoods and economies of rural communities in southern Africa (Grote, 2003; Simon et al., 2007). Most research on Devil’s Claw has focused on H. procumbens (Benito et al., 2000; Fiebich et al., 2001; McGregor et al., 2005; Grant et al., 2007; Abdelouahab and Heard, 2008) and less attention has been paid to H. zeyheri. According to Muzila et al. 2016, the medicinal value of H. zeyheri still remains uncertain despite its usage and commercialisation. This suggests the need for a detailed profiling of the medicinal properties of H. zeyheri if the medicinal potential of this species is to be fully realised.

To this end, we examined the biological activity of H. zeyheri subspecies sublobatum (hereafter referred to as H. zeyheri). The findings of this study have significant ramifications on the perceptions and commercial value as well as conservation of H. zeyheri. In Zimbabwe, H. zeyheri is patchily distributed in the western parts of the country, including Hwange district where it is commercially exploited by local communities (Stewart and Cole, 2005), which offers an opportunity to examine the biological activity of the plant under commercial exploitation. Therefore, the objectives of this study were to determine a) antioxidant activity, b) anti-inflammatory and c) cytotoxic activity of crude extracts of H. zeyheri. The anti-inflammatory and cytotoxic activity were further compared to diclofenac.

Collection of plant material

H. zeyheri tubers were collected in Hwange District, northwest Zimbabwe, in July 2016 after seeking permission from the Hwange Rural District Council and local community leadership. Plant identification was done by Anthony Mapaura, Senior Research Officer at the National Herbarium and Botanic Gardens of Zimbabwe, and a voucher specimen is deposited there (Voucher: CASSRGH108539).

Extraction

Preparation of the sample included washing, slicing and air drying of the tubers at room temperature (25°C) over a period of 5-8 days. Extraction was done guided by Do et al. (2014), with minor modifications. The dried tubers were ground into powder and 10 g of powder were placed in 100 ml of each of 3 different solvents (ethyl acetate, ethanol and water). The mixtures were left for 24 hours (h) at room temperature. The extracts were then filtered, and the filtrates were left standing at room temperature for 24 to 48 h to dry to a sticky or powdery substance (Handa et al. 2008). Water extracts were evaporated at 55°C in a ventilated oven for up to 4 days. All the crude extracts were then stored at 4°C in a cold room until use.

Antioxidant activity

The extracts of H. zeyheri were subjected to screening for antioxidant activity by two methods namely DPPH and ABTS free radical scavenging.

DPPH radical scavenging assay

The determination of the DPPH free radical scavenging activity of the crude extracts was done using the Shimada et al. (1992) method, with a few modifications. The crude extracts (40µL) were serially diluted in methanol in a 96-well plate followed by addition of a DPPH solution (160µL) freshly prepared at 25µg/ml in methanol. The plates were incubated at room temperature (25°C) in the dark for 30 minutes and the absorbance was measured on a microplate reader (Epoch, Biotek) at the wavelength of 517nm (Shimanda et al. 1992). The radical scavenging activity was calculated by the following equation:

DPPH radical scavenging (%) = equation 1.

where A₀ is the absorbance of the control and A₁ is the absorbance of the sample.

Ascorbic acid was used as the positive control while the mixture of methanol with DPPH solution was used as the negative control. The blanks were made of methanol and the extracts. The IC₅₀ values were calculated as the concentration of sample required to scavenge 50% of DPPH free radicals.

ABTS radical scavenging assay

The determination of ABTS radical scavenging activity was done using the ABTS cation decolourisation assay described by Re et al. (1999), with a few modifications. The ABTS radical cation (ABTS•+) was produced by reaction of 7mM stock solution of ABTS with 2.45mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. The ABTS•+ solution was diluted with methanol to give an absorbance of 0.70 ± 0.01 at 734nm (Re et al. 1999). Each plant extract (40µL) was serially diluted in a 96-well plate and then allowed to react with 160 µL of the ABTS•+ solution. After 6 min, the absorbance was measured at 714nm (Re et al. 1999). Trolox and ascorbic acid were used as positive controls while the mixture of methanol and ABTS•+ solution was used as the negative control. The blanks were made of methanol and the extracts. Percentage ABTS inhibition was expressed as a percentage following equation 1 above.

Anti-inflammatory activity

The anti-inflammatory activity of H. zeyheri extracts was examined by testing NO inhibition and cytokine expression of both TNF-α and IL-10 in mouse and human macrophage cells.

Nitric oxide production inhibitory assay in mouse RAW 264.7 macrophage cells

The mouse RAW 264.7 macrophage cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in a plastic culture flask in Dulbelcco’s Modified Eagle’s Medium (DMEM) containing L-glutamine (HycloneTM) and supplemented with 10% foetal bovine serum (FBS) (Capricorn Scientific Gmbh, South America) and 1% penicillin/streptomycin/fungizone (PSF) solution at 37°C with 5% CO₂. The cells were seeded (10 000 cells per well) in 96 well-microtitre plate and allowed to attach overnight. The cells were then treated simultaneously with LPS alone (control) and extracts at different concentrations. Quercetin and diclofenac were used as the positive controls.

The amount of nitrite produced was determined as described by Dzoyem et al. (2015). Then after 24 hours of incubation at 37°C with 5% CO₂, 100µL of cell supernatant from each well were transferred into a new 96-well microtiter plate and the same volume of Griess reagent was added. After 15 min of incubation in the dark at room temperature (25°C), the absorbance was recorded at 550nm on a microtiter plate reader (Epoch Biotek) (Dzoyem et al. 2015). Percentage of NO inhibition was calculated based on the ability of extracts to inhibit nitric oxide production by RAW 264.7 macrophage cells compared with the control (cells treated with LPS alone without samples).

Determination of cytokine expression in human U937 macrophage cells

To prepare the cells, the method described by Passmore et al. (2001) with minor modifications was used. The U937 macrophage cells from the American Type Culture Collection (ATCC, Rockville, MD, USA) were maintained in a humidified atmosphere at 37°C with 5% CO₂ in Roswell Park Memorial Institute (RPMI-1640) medium containing L-glutamine (Lonza, SA) and supplemented with 10% foetal bovine serum (FBS) (Capricorn Scientific Gmbh, South America) and 1% penicillin/streptomycin/fungizone (PSF) solution. The cells were seeded (500 000 cells per well) in a 6-well microtitre plate and treated with LPS alone (control) and extracts at different concentrations (Passmore et al. 2001). Quercetin, a flavonoid found in plants with known anti-inflammatory properties and diclofenac were used as the positive controls.

Determination of the cell viability

The cell viability was determined in order to establish whether the inhibition of NO production, inhibition of TNF-α and expression of IL-10 by extracts was not due to their cytotoxic effects. The cytotoxicity of crude extracts was determined using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described by Mosmann (1983). After each of the assays, the culture medium was removed from the plates and after washing with 200µL of phosphate buffered saline (PBS), 200µL of fresh culture medium and 30µL of MTT solution (5mg/mL) were added to all wells and the plates were incubated at 37°C with 5% CO₂ for 4 h. After incubation, the culture medium was carefully aspirated using a suction pump (Integra, USA), and 50µL of dimethylsulphoxide (DMSO) was added to all wells. The absorbance was read using a microplate reader (Biotek Synergy, USA) at a wavelength of 570nm and a reference wavelength of 630nm. The percentage of cell viability was calculated by comparing the absorbance of the samples to the negative control (cells treated only with LPS considered as 100% viability) (Mossman 1983).

Determination of inhibitory concentration (IC₅₀) and statistical analysis

All experiments were carried out in triplicate and results are presented as mean ± standard deviation. The IC₅₀ value of tested samples, which represent the concentration of the sample required to inhibit 50% of activity compared to the negative control, was determined by using a non-linear regression curve of percentage of inhibition against the logarithm of concentrations. The extracts with lower IC₅₀ values show high activity of the extract or compound. A one-way analysis of variance (ANOVA) was used to test for differences in the measured variables between extracts. All statistical calculations were conducted using R version 3.5.3 with an accepted significance of p < 0.05 in all tests.

Antioxidant activity of crude extracts

Crude extracts of Harpagophytum zeyheri had varying antioxidant activities with significant differences (p < 0.001) between samples (Table 1). The highest DPPH radical-scavenging activities of extracts were displayed by the ethyl acetate extract closely followed by the ethanol extract (IC₅₀ value < 32 µg/ml; Table 1). The water extracts had no activity in both assays (IC₅₀ value > 100µg/ml; Table 1). The extracts showed concentration dependent activity, with a gradual increase in activity in the water extract relative to the rapid activity of the other extracts (Fig. 1).

The IC₅₀ values of crude extracts in the ABTS assay ranged from 12.56µg/ml to 127.89µg/ml. Good radical scavenging activity in the ABTS assay was displayed by the ethyl acetate and ethanol extracts which had the lowest IC₅₀ values of 12.56µg/ml and 19.59µg/ml, respectively (Table 1). The water extract had an IC₅₀ value greater than 100µg/ml showing no antioxidant activity in the ABTS assay (Table 1). There was a rapid increase in activity in the ABTS assay with increase in extract concentration (Fig. 2). Like the DPPH assay, the water extract showed a gradual increase in activity with increase in concentration (Fig. 2).

Anti-inflammatory activity through nitric oxide inhibition

The findings showed that nitric oxide inhibition was exhibited only by the ethyl acetate and ethanol extracts (all with IC₅₀ values < 100µg/mL), in contrast to the water extract which showed no inhibition (Table 2). Overall, the inhibitory effect of the extracts was weaker relative to diclofenac and the positive control, quercetin (p < 0.05; Table 2). All extracts displayed a dose dependent inhibition, with high NO inhibition at high concentrations (100, 50 and 25) not significantly differing with diclofenac and quercetin in the case of the ethyl acetate and ethanol extract (p > 0.05; Table 3).

Modulatory effect of crude extracts on cytokine expression and cell viability on human U937 macrophage cells

The inhibition of pro-inflammatory cytokines is a good trait for a sample targeted for reducing inflammation. The highest inhibition of the TNF-α cytokine was displayed by the ethyl acetate extract while the water and ethanol extracts showed limited inhibition (Table 4). A comparison of the ethyl acetate extract against the synthetic drug diclofenac showed no significant differences in their inhibitory effect (p > 0.05; Table 4). However, the ethyl acetate extract showed better inhibition of TNF-α than the positive control, quercetin (Table 4). The expression of the cytokine IL-10, an anti-inflammatory cytokine, was also examined and only the positive control quercetin encouraged expression of this cytokine (Table 4). Extracts of H. zeyheri and diclofenac had very low levels of human IL-10 expression (Table 4).

Cytotoxicity

The extracts generally had limited toxicity relative to diclofenac and quercetin (Table 3). The extract with generally the highest inhibitory effect, ethyl acetate, displayed the highest viability of cells (60%) at the highest concentration in comparison with diclofenac (1.12%) and quercetin (28%). In general, diclofenac had the highest cell toxicity even at the lowest concentrations (Table 3). Consequently, the cells which were subjected to the crude extracts are more viable compared to those subjected to diclofenac at lowest concentrations.

However, despite having a low expression of human IL-10, the plant extracts had limited toxicity on the cells relative to diclofenac and quercetin (Table 4). Cell viability at 50 µg/ml sample concentration was > 90% in the plant extracts compared to 25% and 50% with diclofenac and quercetin, respectively.

In the present study, H. zeyheri had significant antioxidant, anti-inflammatory and less cytotoxic properties relative to diclofenac and quercetin. These findings are consistent with findings by other studies (Baghdikian et al., 1997; Grant et al., 2007) which indicated that H. zeyheri has significant antioxidant and anti-inflammatory properties like the well-studied H. procumbens. In this regard, our findings contradict assertions by Chantre et al. (2000), on stating H zeyheri as not having ethnobotanical relevance and only good as food for insect larvae!

To the best of our knowledge, there is limited work that has been conducted on H. zeyheri. Focus has mainly been on H. procumbens which has been deemed as the more medicinally active species in the Harpagophytum genus. Related studies on crude extracts of H. procumbens showed significant antioxidant activity of H procumbens and good radical scavenging demonstrated by the methanolic extract (Georgiev et al. 2012) and by aqueous extracts containing 2.6% harpagoside (European Herbal Monograph, 2016). The results of this study showed strong antioxidant activity for ethyl acetate extracts of H. zeyheri in both the DPPH and ABTS assay (IC₅₀ value < 27µg/ml) but was lower to that of trolox, a potent antioxidant. In the ABTS assay, both the ethyl acetate and ethanol extracts of H. zeyheri had an IC₅₀ value (< 20µg/ml) comparable to methanol extracts of H. procumbens (19.8µg/ml; Frum and Viljoen, 2006) in DPPH assay, which suggests that the former has better antioxidant properties than previously reported (Chantre et al., 2000; Mcwangi et al., 2012). The similarity in antioxidant activity of H. procumbens to this study’s results can be attributed to common glycosides such as harpagoside that are found in both H. zeyheri and H. procumbens.

In this study, the ethyl acetate and ethanol extract inhibited production of NO in LPS stimulated mouse macrophages as demonstrated by the IC₅₀ values below 100 µg/ml. Further, although the inhibition of NO in the extracts was weaker relative to diclofenac and quercetin, the crude extracts of H. zeyheri showed the ability to inhibit inflammatory mediators, an indication of potentially good anti-inflammatory properties. However, in dose dependence tests, inhibition of NO by the ethyl acetate extract was comparable to that of diclofenac and quercetin at concentrations of 100 µg/ml and 50 µg/ml. In addition, the inhibition of TNF-α by the ethyl acetate extract was like diclofenac. Diclofenac has been shown to suppress pro-inflammatory mediators such as NO and TNF- α as part of its mode of action (Al Armin et al., 2013). In this study, the ability of H. zeyheri to inhibit the pro-inflammatory cytokine in a similar way to diclofenac shows the potential of the extract for further exploration as an alternative. These findings also correspond with in vitro studies on the anti-inflammatory action of H. procumbens, which showed inhibition of inflammation mediators such as LOX, TNF, IL-6, IL-1β, prostaglandin PGE2 in LPS induced human and animal cell lines (Benito et al., 2000; Fiebich et al., 2001; Jang et al., 2003; McGregor et al., 2005; Abdelouahab and Heard, 2008). Indeed, H. zeyheri and H. procumbens share common active ingredients which include harpagoside, harpagide and verbascoside (McGregor et al., 2005) hence the similarities in activity between the two species. In addition, the very low levels of human IL-10 expression in extracts of H. zeyheri relative to quercetin but like diclofenac, together with the strong correlation between these findings and the medicinal use of the plant by local communities in inflammatory-related disorders, confirm the possible mode of action of H. zeyheri that is similar to diclofenac.

Relative to the other extracts, the water extract showed no antioxidant activity in both the DPPH and ABTS assay. However, Devil’s Claw extracts are particularly rich in water-soluble antioxidants (Betancor-Fernandez et al., 2003), and the roots are generally administered orally in powdered form in traditional medicine. Further, Zhang et al. (2011) showed that hydrolysis of the glycosidic bonds of harpagide and harpagoside products of Devil’s Claw was a prerequisite step for activity. In their study, anti-inflammatory activity was significant in hydrolysed products of the iridoid glycosides, harpagide and harpagoside, relative to the unhydrolysed compounds (Zhang et al., 2011). Thus, the lack of activity in water extracts may suggest problems associated with interference in the assays but may also confirm that the ethyl acetate extract was a superior extractant as compared to water.

Medicinal plants, due to their natural origin, are receiving growing attention as they are deemed to be much safer than many conventional drugs and are often used without medical supervision. Indeed, we found that H. zeyheri extracts had limited toxicity on the cells relative to diclofenac and quercetin. Thus, inhibitory effect of H. zeyheri extracts in both the mouse and human macrophages assays were not due to cell death. Likewise, studies on H. zeyheri’s taxonomic ally H. procumbens showed low cell toxicity in animal experiments (Chrubasik et al., 2003). In contrast, diclofenac had significant cell death suggesting that the inhibitory effect of diclofenac may also be due to cell death. That H. zeyheri had lower cell toxicity levels than diclofenac even at high concentrations, not only suggest that low toxicity may be a general property of Harpagophytum extracts, but also lends support to the notion that natural products derived from plants may be safer than NSAIDs (Chrubasik et al., 2002; 2005). Nonetheless, some studies have reported Harpagophytum to cause adverse effects such as mild gastrointestinal upset such as bloating, dyspepsia, and loss of taste (Brendler et al., 2006), but at recommended doses Harpagophytum seems to be well-tolerated (Chrubasik et al., 2005). However, given the limited amount of comparable data in the literature regarding H. zeyheri, there is need to remain conservative in the interpretation of these findings, and suggest the need for more research on this species. Nonetheless, our findings have important implications for the medicinal value and conservation of this plant.

In conclusion, crude extracts of H. zeyheri exhibited strong antioxidant (radical scavenging of DPPH and ABTS) and anti-inflammatory (inhibition of TNF-α and NO) properties. Results obtained on H. zeyheri were comparable to an extensively studied close taxonomic species, H. procumbens. More specifically, the ethyl acetate extract proved to be the most effective radical scavenger extract in the DPPH and ABTS assays, thus a potentially attractive antioxidant. The ethyl acetate extract further displayed highest inhibition of the production of NO and TNF-α, respectively and displayed comparability in inhibition to diclofenac at some concentrations. However, extracts of H. zeyheri were associated with low levels of human-IL-10 expression. All the crude extracts of H. zeyheri had low cell toxicity compared to diclofenac. Overall, these results provide evidence of the beneficial medicinal properties of H. zeyheri against inflammation, although further studies are required, given the limited research conducted to date on this species.

2,2’-diphenyl-1-picrylhydrazy (DPPH); 2,2’-12 azino-bis (3- ethylbenzothiazoline-6-sulphonic acid) (ABTS); 15-lipoxygenase (15-LOX); nitric oxide (NO); lipopolysaccharide (LPS); 3-(4, 5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT)

Acknowledgements

The National University of Science and Technology provided the funding for the project (Grant RB/58/16). The Hwange Rural District council, Hwange community leadership and community members granted permission to collect samples in the district and assisted. Anthony Mapaura, Senior Research officer at the National Herbarium and Botanic Garden in Zimbabwe did the formal identification of the plant and a voucher specimen is deposited there (Voucher: CASSRGH108539). The University of Pretoria’s Phytomedicine Programme availed the laboratory facilities to conduct the experimental work.

Funding

This manuscript is part of a PhD project supported by a grant (Grant: RB/58/16) from the Research Board of the National University of Science and Technology, Zimbabwe.

Availability of data and materials

All data generated and used to support findings of this study are adequately contained within the manuscript.

Ethics Approval and consent to participate

Permission to collect tuber samples was sought from the Hwange Rural District council, as well as the local leadership (Chief, Headman and the Kraal heads) in the specific ward. The collection of the wild plant samples complied with the Forest Resources and Wildlife department’s guidelines at the National University of Science and Technology. The procedure for sample collection, preservation and laboratory analysis was reviewed by the department to ensure that there was compliance.

Consent for publication

Not applicable.

Competing interest

The authors declares that they have no financial or competing interest that may have influenced the writing of this manuscript.

Author’s contribution

S. N participated in the design of the study, carried out the field and laboratory work, and prepared the manuscript. L. M participated in supervision of the laboratory work and manuscript revision. E. N participated in assistance in conducting the laboratory work and manuscript revision. H. N, P. M and S. S participated in the overall supervision of design of the study, providing technical advice and revision of the manuscript.

Author information

S. N is a PhD student in the Forest Resources and Wildlife Management Department (FRWM) at the National University of Science and Technology (NUST). L. M is a Professor and leader of the Phytomedicine Program at the University of Pretoria. E. N was a Post-Doctoral Fellow at the Phytomedicine Department during the time of the study and is currently a Senior Lecturer at the University of Yaoundé 1 in Cameroon. H. N and P.M are both in the Forest Resources and Wildlife Department at NUST, with the former being an Associate Professor and the latter a full Professor. S.S is Associate Professor in the Applied Chemistry Department at NUST.

Author Details

^1.Department of Forest Resources and Wildlife Management, Faculty of Applied Science, National University of Science and Technology, P.O Box AC939, Ascot,

Bulawayo, Zimbabwe

^2.Phytomedicine Programme, Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110,

South Africa

^3.School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Private Bag 3, Wits 2050, South Africa

^4.Department of Applied Chemistry, Faculty of Applied Science, National University of Science and Technology, P. O Box AC939, Ascot, Bulawayo, Zimbabwe

Department of Biochemisty, Faculty of Sciences, University of Yaoundé 1, Cameroon

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Table 1 Mean (± standard deviation) IC₅₀ values of antioxidant activity of crude extracts of H. zeyheri compared to their respective positive controls.

Extract	Antioxidant
	DPPH	ABTS
Ethyl acetate	27.40 ± 0.29^a	12.56 ± 0.09^a
Ethanol	32.09 ± 0.73^b	19.59 ± 0.49^b
Water	143.20 ± 0.26^c	127.89 ± 0.39^c
Ascorbic Acid	19.67 ± 0.67^d	7.16 ± 0.33^d
Trolox	3.18 ± 0.42^e	10.69 ± 0.58^e

Values with different superscript letters in each column are significantly different at p < 0.05

Table 2 Mean (± standard deviation) IC50 values of anti-inflammatory activity of crude extracts of H. zeyheri compared to their respective positive controls.

Extract	Anti-inflammation
	NO
Ethyl acetate	72.80 ± 4.53^a
Ethanol	92.70 ± 3.26^a
Water	184.53 ± 4.45^b
Diclofenac	10.32 ± 0.54^c
Quercetin	18.10 ± 0.16^c

Values with different superscript letters in each column are significantly different at p < 0.05

Table 3 Dose dependent activities of the three extracts of H. zeyheri on nitric oxide production inhibition and cell viability in RAW 246 mouse macrophages

	Concentration	Ethyl acetate	Ethanol	Water	Diclofenac	Quercetin
NO	100	62.84 ± 2.66^a	53.87 ± 2.56^b	27.01 ± 4.23^c	68.26 ± 0.81^a	69.05 ± 1.46^a
	50	41.68 ± 3.22^a	29.34 ± 2.05^b	9.24 ± 2.05^c	65.31 ± 0.31^a	68.59 ± 0.51^a
	25	24.67 ± 5.81^a	17.92 ± 5.73^b	8.03 ± 3.29^c	60.09 ± 1.15^a	57.27 ± 0.98^a
	12.5	14.45 ± 7.48^a	14.10 ± 6.39^a	7.38 ± 5.63^b	49.77 ± 1.08^c	40.62 ± 0.01^c
Cell viability	100	60.65 ± 3.02^a	85.90 ± 4.26^a	102.97 ± 3.40^a	1.12 ± 0.38^b	27.97 ± 2.08^c
	50	90.91 ± 5.85^a	96.68 ± 0.73^a	111.17 ± 4.75^a	2.98 ± 0.02^b	34.72 ± 0.17^c
	25	88.95 ± 3.50^a	96.72 ± 1.33^a	90.79 ± 3.16^a	7.57 ± 1.16^b	59.15 ± 6.52^a
	12.5	89.98 ± 6.56^a	98.32 ± 3.48^a	95.95 ± 4.26^a	15.57 ± 1.09^b	86.98 ± 0.03^a

Values with different superscript letters in each row are significantly different at p < 0.05

Table 4 Inhibitory activities of three extracts of H. zeyheri on expression of cytokines (TNF-α, IL10) and cell viability in human U937 macrophages at 50 µg/mL concentration

Extract	TNF-α (pg/mL)	IL-10 (pg/mL)	Cell viability (%)
Ethyl acetate	47.92 ± 0.69^a	7.95 ± 0.05^a	98.96 ± 2.16^a
Ethanol	164.07 ± 0.74^b	8.63 ± 0.37^a	99.27 ± 4.02^a
Water	217.13 ± 3.24^c	8.06 ± 0.48^a	99.97 ± 2.48^a
Diclofenac	44.49 ± 6.62^a	8.42 ± 0.30^a	25.11 ± 1.24^b
Quercetin	73.06 ± 1.94^d	31.55 ± 2.69^b	49.48 ± 5.51^c

Values with different superscript letters in each column are significantly different at p < 0.05

No competing interests reported.

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In Vitro Antioxidant Activity of Crude Extracts of Harpagophytum Zeyheri and Their Anti-Inflammatory and Cytotoxicity Activity Compared With Diclofenac.

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