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Abstract 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 (5.91 μg/ml) and ABTS (20.5 μg/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 the inflammatory activity of H. zeyheri to diclofenac at some concentrations as well as low toxicity in comparison to diclofenac.

• Devil's claw • Harpagophytum procumbens • Harpagophytum zeyheri • BRONX algorithm • DNA barcoding • UHPLC-MS • HPTLC Devil's claw is a popular natural anti-inflammatory phytomedicine. The preparation of Devil's claw products is complicated by the similarity between the morphotypes of Harpagophytum procumbens and H. zeyheri. This study investigated the potential of a validated ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) method to investigate the harpagoside content of methanol extracts of the secondary tubers of both species. In addition, DNA barcoding was used to determine the identity of the Harpagophytum species included in commercial Devil's claw products. Herbal reference material of H. procumbens and H. zeyheri, originally harvested in southern Africa, was morphologically authenticated and commercial products containing Harpagophytum were purchased on the internet using the search terms " Harpagophytum " or "Devil's claw." High-performance thin-layer chromatography (HPTLC) and UHPLC-MS was used for the qualitative and quantitative determination of harpagoside content in commercial products. The UHPLC-MS method developed had a limit of detection (LOD) of 0.165 µg/mL and a limit of quantification (LOQ) of 0.499 µg/mL. Quantitative UHPLC-MS analysis revealed high variability in the harpagoside content for both species; H. procumbens (0.17-4.37%) and H. zeyheri (0.00-3.07%). Only 41% of H. procumbens and 17% of H. zeyheri raw materials contained harpagoside. Using HPTLC, 90% of the commercial products tested were determined to contain harpagoside. The results were congruent with the UHPLC-MS analysis where the harpagoside content was out of specification for 10% of analyzed commercial products. The harpagoside content in commercial products varied from not detected to 3.50%. PCR amplification followed by comparative sequencing analysis of trnL-F, and ycf1 plastid gene amplicons indicated specific sites in trnL-F at positions 118 and 146 and at position 538 in ycf1 amplicons suitable to distinguish the two Harpagophytum species. These sites were used to barcode the herbal products and Character-based Barcode Recognition Obtained with Nucleotide eXposés (BRONX) analyses verified the taxonomic identity of the Harpagophytum in the samples. The results revealed that 76% (16/21) of the commercial products tested were incorrectly labelled as H. procumbens and, instead, contained H. zeyheri. Our study suggests that DNA barcoding in combination with the UHPLC-MS and HPTLC could significantly support herbal product authentication. This study contributed knowledge and methods that can be implemented to enhance the quality of commercial Devil's claw products. [ABSTRACT FROM AUTHOR]

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 (5.91 μg/ml) and ABTS (20.5 μg/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 the inflammatory activity of H. zeyheri to diclofenac at some concentrations as well as low toxicity in comparison to diclofenac. [ABSTRACT FROM AUTHOR]

Devil’s claw is the vernacular name for a genus of medicinal plants that occur in the Kalahari Desert and Namibia Steppes. The genus comprises two distinct species: Harpagophytum procumbens and H. zeyheri. Although the European pharmacopeia considers the species interchangeable, recent studies have demonstrated that H. procumbens and H. zeyheri are chemically distinct and should not be treated as the same species. Further, the sale of H. zeyheri as an herbal supplement is not legal in the United States. Four markers were tested for their ability to distinguish H. procumbens from H. zeyheri: rbcL, matK, nrITS2, and psbA-trnH. Of these, only psbA-trnH was successful. A novel DNA mini-barcode assay that produces a 178-base amplicon in Harpagophytum (specificity = 1.00 [95% confidence interval = 0.80–1.00]; sensitivity = 1.00 [95% confidence interval = 0.75–1.00]) was used to estimate mislabeling frequency in a sample of 23 devil’s claw supplements purchased in the United States. PCR amplification failed in 13% of cases. Among the 20 fully-analyzable supplements: H. procumbens was not detected in 75%; 25% contained both H. procumbens and H. zeyheri; none contained only H. procumbens. We recommend this novel mini-barcode region as a standard method of quality control in the manufacture of devil’s claw supplements.

Harpagophytum procumbens (Pedaliaceae) and its close taxonomical ally Harpagophytum zeyheri , indigenous to southern Africa, are being harvested for exportation to Europe where phytomedicines are developed to treat inflammation-related disorders. The phytochemical variation within and between natural populations of H. procumb ens ( n = 241) and H. zeyheri ( n = 107) was explored using proton nuclear magnetic resonance ( 1 H-NMR) and ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in combination with multivariate data analysis methods. The UHPLC-MS results revealed significant variation in the harpagoside content: H. procumbens (0.17–4.37%); H. zeyheri (0.00–3.07%). Only 41% of the H. procumbens samples and 17% of the H. zeyheri samples met the pharmacopoeial specification of ⩾1.2%. Both principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) indicated separation based on species (UHPLC-MS data OPLS-DA model statistics: R 2 X = 0.258, R 2 Y (cum) = 0.957 and Q 2 (cum) = 0.934; 1 H-NMR data OPLS-DA model statistics: R 2 X = 0.830, R 2 Y = 0.865 (cum) and Q 2 (cum) = 0.829). It was concluded that two species are not chemically equivalent and should not be used interchangeably. [ABSTRACT FROM AUTHOR]

Abstract: Devil''s claw is the common name for two species in the genus Harpagophytum. Their root extracts contain the iridoid glycoside, harpagoside, which has been found to be effective in the treatment of degenerative rheumatoid arthritis, osteoarthritis, tendonitis, kidney inflammation, and heart disease. Most of the world''s supply comes from Namibia, with lesser amounts from South Africa and Botswana. In 2002, the peak year of export, 1018tonnes of dried tubers were exported from southern Africa, representing the harvest of millions of plants. In 2001, sales in Germany were estimated at 30M euros, accounting for 74% of the prescriptions for rheumatism. Harvest has improved income levels in marginalized communities but it has also raised questions of sustainability. In 2000, recommendations were made to the Convention on the International Trade in Endangered Species (CITES) to add devil''s claw to Appendix II. In 2004, the proposal was formally withdrawn due to the efforts of the range states to address sustainability issues. Replacing wild collection with cultivation has generated a debate on the positive and negative effects on harvester income and rural farmers. Successful cultivation efforts have involved micropropagation techniques and growing the plant without water or fertilizers. The governments of the main range states are working with local communities to develop policies and regulations to protect the species and to determine a sustainable harvest. [Copyright &y& Elsevier]

Devil's claw is the vernacular name for a genus of medicinal plants that occur in the Kalahari Desert and Namibia Steppes. The genus comprises two distinct species: Harpagophytum procumbens and H. zeyheri. Although the European pharmacopeia considers the species interchangeable, recent studies have demonstrated that H. procumbens and H. zeyheri are chemically distinct and should not be treated as the same species. Further, the sale of H. zeyheri as an herbal supplement is not legal in the United States. Four markers were tested for their ability to distinguish H. procumbens from H. zeyheri: rbcL, matK, nrITS2, and psbA-trnH. Of these, only psbA-trnH was successful. A novel DNA mini-barcode assay that produces a 178-base amplicon in Harpagophytum (specificity = 1.00 [95% confidence interval = 0.80–1.00]; sensitivity = 1.00 [95% confidence interval = 0.75–1.00]) was used to estimate mislabeling frequency in a sample of 23 devil's claw supplements purchased in the United States. PCR amplification failed in 13% of cases. Among the 20 fully-analyzable supplements: H. procumbens was not detected in 75%; 25% contained both H. procumbens and H. zeyheri; none contained only H. procumbens. We recommend this novel mini-barcode region as a standard method of quality control in the manufacture of devil's claw supplements. [ABSTRACT FROM AUTHOR]