Antioxidant and anti-inflammatory activity of Ocimum labiatum extract and isolated labdane diterpenoid

BACKGROUND : Plants from the genus Ocimum are used as folk medicine for treating various diseases including inflammatory and immune-related diseases. Numerous reports have suggested plant extracts and their constituents as possible anti-inflammatory agents. Here, in vitro evidence of Ocimum labiatum’s immune-enhancing and antioxidant properties is presented for the first time. METHODS : The anti-inflammatory effect of O. labiatum ethanolic extract and an isolated diterpenoid was determined using a cytometric bead array (CBA) technique. The effect on phytohemagglutinin (PHA)-induced nitric oxide (NO) production in peripheral blood mononuclear cells (PBMCs) was also assessed. A battery of antioxidant assays were used for detecting antioxidant activity while the anti-inflammatory mechanism was evaluated using an ELISA-based activator protein (AP-1) (c-Jun) assay. Cytotoxicity was determined on TZM-bl and PBMCs using a tetrazolium dye and confirmed by a novel label-free real-time assay. RESULTS : A 25 μg/mL non-cytotoxic concentration of O. labiatum extract significantly (p < 0.05) inhibited the production of pro-inflammatory cytokines; IL-2, IL-4, IL-6 and IL-17A. Except for the dual acting pro- or anti-inflammatory cytokine, IL-6, which was upregulated, a non-cytotoxic 50 μM concentration of the isolated labdane diterpenoid compound significantly (p < 0.05) decreased the production of all the pro-inflammatory cytokines. In the anti-inflammatory pathway studies, the compound also inhibited AP-1 significantly (p < 0.05) at 50 μM. The extract demonstrated strong, dose dependent antioxidant activity with IC50 values ranging from 13 ± 0.8 to 54.86 ± 1.28 μg/mL while the terpene had no antioxidant property. The extract and diterpenoid decreased the production of the inflammatory mediator NO, at non-cytotoxic concentrations. The CC50 of the extract in TZM-bl and PBMCs was 62.6 ± 0.6 and 30.1 ± 0.4 μg/mL while that of the compound was 112.6 ± 0.2 and 70 ± 0.4 μM respectively. The real time studies confirmed tetrazolium dye assessed viability and also detected a unique growth pattern for the plant materials compared to untreated cells. CONCLUSIONS : O. labiatum extract demonstrated promising anti-inflammatory and antioxidant properties while the terpenoid showed anti-inflammatory but no antioxidant activity. The anti-inflammatory mechanism of the terpene was a result of inhibition of AP-1. These data represents promising first steps towards the development of naturally derived anti-inflammation drugs.Southern African Biochemistry and Informatics for Natural Products (SABINA), the Technology Innovation Agency (TIA, South Africa), Margaret McNamara Memorial Fund (MMMF), the Namibian Ministry of Education and the University of Pretoria.http://www.journal-inflammation.comhb2016Biochemistr

Inhibition of HIV-1 enzymes, antioxidant and anti-inflammatory activities of Plectranthus barbatus

Ethnopharmacological relevance: Plectranthus barbatus is widely used in African countries as an herbal remedy to manage HIV/AIDS and related conditions. Aim of the study: To investigate the HIV-1 inhibitory, anti-inflammatory and antioxidant properties of P. barbatus and thereby provide empirical evidence for the apparent anecdotal success of the extracts. Materials and methods: Ethanolic extract of P. barbatus's leaves was screened against two HIV-1 enzymes: protease (PR) and reverse transcriptase (RT). Cytotoxicity of the extract was determined through measuring tetrazolium dye uptake of peripheral blood mononuclear cells (PBMCs) and the TZM-bl cell line. Confirmatory assays for cytotoxicity were performed using flow cytometry and real-time cell electronic sensing (RT-CES). The free radical scavenging activity of the extract was investigated with 2,2- diphenyl-1-picrylhydrazyl while the anti-inflammatory properties of the plant extract were investigated using a Th1/Th2/Th17 cytometric bead array technique

Inhibition of HIV-1 enzymes, antioxidant and anti-inflammatory activities of Plectranthus barbatus

ETHNOPHARMACOLOGICAL RELEVANCE : Plectranthus barbatus is widely used in African countries as an herbal remedy to manage HIV/AIDS and related conditions. AIM OF STUDY : To investigate the HIV-1 inhibitory, anti-inflammatory and antioxidant properties of P.barbatus and thereby provide empirical evidence for the apparent anecdotal success of the extracts. MATERIALS AND METHODS : Ethanolic extract of P. barbatus’s leaves was screened against two HIV-1 enzymes; protease (PR) and reverse transcriptase (RT). Cytotoxicity of the extract was determined through measuring tetrazolium dye uptake of peripheral blood mononuclear cells (PBMCs) and the TZM-bl cell line. Confirmatory assays for cytotoxicity were performed using flow cytometry and real-time cell electronic sensing (RT-CES). The free radical scavenging activity of the extract was investigated with 2,2-diphenyl-1- picrylhydrazyl while the anti-inflammatory properties of the plant extract were investigated using a Th1/Th2/Th17 cytometric bead array technique.Results P. barbatus extract inhibited HIV-1 PR and the 50% inhibitory concentration (IC50) was 62.0 μg/ml. The extract demonstrated poor inhibition of HIV-1 RT. Cytotoxicity testing presented CC50 values of 83.7 and 50.4 μg/ml in PBMCs and TZM-bl respectively. In addition, the extract stimulated proliferation in HIV negative and positive PBMCs treated. RT-CES also registered substantial TZM-bl proliferation after extract treatment. The extract exhibited strong antioxidant activity with an IC50 of 16 μg/ml and reduced the production of pro-inflammatory cytokines indicating anti-inflammatory potential. CONCLUSION : This is the first demonstration of the in vitro anti HIV-1 potential of P. barbatus including direct activity as well as through the stimulation of protective immune and inflammation responses. The low cytotoxicity of the extract is also in agreement with the vast anecdotal use of this plant in treating various ailments with no reported side-effects.This research was supported by the Southern African Biochemistry and Informatics for Natural Products (SABINA), the Technology Innovation Agency (TIA) of South Africa), Margaret McNamara Memorial Fund (MMMF), the Namibian Ministry of Education and the University of Pretoria.http:// www.elsevier.com/locate/jephb201

Triterpenoids from ocimum labiatum activates latent HIV-1 expression in vitro : potential for use in adjuvant therapy

Abstract: Latent HIV reservoirs in infected individuals prevent current treatment from eradicating infection. Treatment strategies against latency involve adjuvants for viral reactivation which exposes viral particles to antiretroviral drugs. In this study, the effect of novel triterpenoids isolated from Ocimum labiatum on HIV-1 expression was measured through HIV-1 p24 antigen capture in the U1 latency model of HIV-1 infection and in peripheral blood mononuclear cells (PBMCs) of infected patients on combination antiretroviral therapy (cART). The mechanism of viral reactivation was determined through the compound’s effect on cytokine production, histone deacetylase (HDAC) inhibition, and protein kinase C (PKC) activation. Cytotoxicity of the triterpenoids was determined using a tetrazolium dye and flow cytometry. The isolated triterpene isomers, 3-hydroxy-4,6a,6b,11,12,14b-hexamethyl-1,2,3,4,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydrop icene-4,8a-dicarboxylic acid (HHODC), significantly (p < 0.05) induced HIV-1 expression in a dose-dependent manner in U1 cells at non-cytotoxic concentrations. HHODC also induced viral expression in PBMCs of HIV-1 infected patients on cART. In addition, the compound up-regulated the production of interleukin (IL)-2, IL-6, tumour necrosis factor (TNF)-_, and interferon (IFN)- but had no effect on HDAC and PKC activity, suggesting cytokine upregulation as being involved in latency activation. The observed in vitro reactivation of HIV-1 introduces the adjuvant potential of HHODC for the first time here

Lamiaceae plant extracts and isolated compounds demonstrate activity against HIV/AIDS

Background: HIV/AIDS remains a major health concern worldwide and the number of people infected in Sub-Saharan Africa continues to increase. This despite increased awareness and availability of HIV drugs in most countries. The success of current HIV-1 drugs is overshadowed by the emergence of drug resistant viral strains and the adverse side-effects they may cause. It is these limitations and many more that drives the continuous search for better HIV treatments. Research into drug discovery and development using natural products is becoming better established. With natural products, there are endless opportunities for discovering novel compounds which either ends up as final drugs or as backbones of drug leads. Methods: In this thesis, sixteen Lamiaceae (mint) plants were investigated for inhibitory properties against HIV-1 as well as for beneficial immune enhancing effects. This family of plants is commonly used in traditional medicine preparations for the treatment of various ailments including those that are virus induced. Cytotoxicity of the plant material was determined using tetrazolium dyes and the results subsequently confirmed with flow cytometry and real-time cell analysis. Direct enzyme assays were used to determine the inhibitory properties of the extracts and isolated compounds against HIV-1 protease (PR), reverse transcriptase (RT) and integrase (IN). The effect of the plant materials was also evaluated in an in vitro model of chronic and latent infection by measuring HIV-1 p24 protein secretion of an infected cell line (U1) following treatment. Most HIV-infected individuals only seek treatment during the chronic stages of disease and latent reservoirs of the virus perpetuate treatment. The immune modulating properties were determined by quantitating the effects of plant extracts/compounds on Th1/2/17 cytokine production in human mononuclear cells using the cytometric bead array technology. Finally, anti-oxidant and anti-inflammatory properties were also assessed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide colorimetric assays respectively. Results and discussion: The 50% cytotoxic concentration (CC50) of the extracts was between 4.2 and 100 μg/ml. Of the sixteen, extracts from six plants (Ocimum labiatum, Ocimum serratum, Plectranthus barbatus, Plectranthus neochilus, Salvia apiana and Stachys byzantina) were active against HIV-1. Four plants (P. neochilus, O. serratum, S. apiana and S. byzantina) demonstrated moderate inhibitory properties against HIV-1 PR, RT and IN (40-49%) and three of these plants (O. serratum, S. apiana and S. byzantina) significantly (p<0.05) suppressed HIV-1 replication in U1 cells. The most exciting data was obtained from extracts of P. barbatus and O. labiatum which demonstrated inhibition classified as good (>50%) against HIV-1 PR (IC50s 62 ±0.2 and 49.8 ±0.4 μg/ml), reduced the production of pro-inflammatory cytokines at non-cytotoxic concentrations and demonstrated strong antioxidant properties (IC50 values 13 ±0.8 and 15.8 ±0.3 μg/ml). O. labiatum extract also suppressed HIV-1 expression in U1 cells, significantly (p<0.05). In addition, one of the extracts (P. ciliatus) had anti-cancer potential with CC50 values <10 μg/ml. O. labiatum extract was purified to yield a chlorophyll derivative, pheophytin-a (phy-a); triterpene isomers (3-hydroxy-4,6a,6b,11,12,14b-hexamethyl- 1,2,3,4,6,6a,6b,7,8,8a,9,10,11,12,12a,14,14a,14b-octadecahydropicene-4,8adicarboxylic acid), amyrin and a labdane diterpenoid (labda-8(17),12E,14-triene-2R,18- diol). Phy-a inhibited HIV-1 PR with an IC50 value of 44.4 ±1.5 μg/ml. The triterpenes activated latent HIV-1 (a serious obstacle in the eradication of the virus) while the diterpenoid reduced the production of pro-inflammatory cytokines. These activities were observed at non-toxic concentrations of these compounds. There is an ongoing search for novel compounds that are able to activate latent HIV-1 to use in conjunction with HAART. If the triterpenes were to progress to clinical use, their use would be in activating latent virus for eradication by existing treatments. Conclusion: The findings presented in this thesis provide some scientific explanation for the anecdotal success of some Lamiaceae plants used traditionally to manage HIV/AIDS. The findings also conform to recommendations by the scientific community regarding the validation of the beneficial effects of plant products used traditionally.Thesis (PhD)--University of Pretoria, 2013.gm2014Biochemistryunrestricte

Anti-HIV Activity of Ocimum labiatum Extract and Isolated Pheophytin-a

Ocimum plants are traditionally used to manage HIV/AIDS in various African countries. The effects of Ocimum labiatum extract on HIV-1 protease (PR) and reverse transcriptase (RT) is presented here along with characterization of an identified bioactive compound, achieved through 1H- and 13C-NMR. The extract’s effect on HIV-1 replication was assessed by HIV-1 p24 antigen capture. Cytotoxicity of samples was evaluated using tetrazolium dyes and real-time cell electronic sensing (RT-CES). Ocimum labiatum inhibited HIV-1 PR with an IC50 value of 49.8 ± 0.4 μg/mL and presented weak inhibition (21%) against HIV-1 RT. The extract also reduced HIV-1 replication in U1 cells at a non-cytotoxic concentration (25 μg/mL). The CC50 value of the extract in U1 cells was 42.0 ± 0.13 μg/mL. The HIV-1 PR inhibiting fraction was purified using prep-HPLC and yielded a chlorophyll derivative, pheophytin-a (phy-a). Phy-a inhibited HIV-1 PR with an IC50 value of 44.4 ± 1.5 μg/mL (51 ± 1.7 μM). The low cytotoxicity of phy-a in TZM-bl cells was detected by RT-CES and the CC50 value in U1 cells was 51.3 ± 1.0 μg/mL (58.9 ± 1.2 μM). This study provides the first in vitro evidence of anti-HIV activity of O. labiatum and isolated phy-a, supporting further investigation of O. labiatum for lead compounds against HIV-1

Antibacterial and anticandidal activity of Tylosema esculentum (marama) extracts

Bean and tuber extracts of Tylosema esculentum (marama) – an African creeping plant – were obtained using ethanol, methanol and water. Based on information that T. esculentum is used traditionally for the treatment of various diseases, the antibacterial and anticandidal effects of tuber and bean extracts were investigated. The antimicrobial activity of the extracts was tested on methicillin-resistant Staphylococcus aureus (MRSA, ATCC 6538), Mycobacterium terrae (ATCC 15755), Corynebacterium diphtheriae (clinical) and Candida albicans (ATCC 2091). We performed the broth microdilution test for the determination of the minimum inhibitory concentration (MIC) and a method to determine survival of microorganisms after in vitro co-incubation with the highest concentrations of T. esculentum extracts, followed by assessment of colony counts. Ethanol and methanol (phenolic) bean extracts exhibited higher potency against bacteria and yeast than aqueous extracts. Marama bean seed coat crude ethanolic extract (MSCE) and seed coat polyphenolic fractions, especially soluble-bound fraction (MSCIB), were highly antimicrobial against M. terrae, C. diphtheriae and C. albicans. All marama bean polyphenolic fractions, namely cotyledon acidified methanol fraction (MCAM), seed coat acidified methanol fraction (MSCAM), cotyledon insoluble-bound fraction (MCIB), seed coat insoluble-bound fraction (MSCIB), cotyledon-free polyphenolic fraction (MCFP) and seed coat free polyphenolic fraction (MSCFP) had high antimicrobial effects as shown by low respective MIC values between 0.1 mg/mL and 1 mg/mL. These MIC values were comparable to those of control antimicrobials used: amphotericin B (0.5 mg/mL) and cesfulodin (0.1 mg/mL) against C. diphtheriae, streptomycin (1.0 mg/mL) and gentamicin (0.4 mg/mL) against M. terrae, and amphotericin B (0.05 mg/mL) against C. albicans. Marama seed coat soluble-esterified fraction (MSCS) had closer activity to that of cefsulodin against M. terrae. High amounts of phenolic substances, such as gallic acid, especially in the seed coats, as well as high amounts of phytosterols, lignans, certain fatty acids and peptides (specifically protease inhibitors) in the cotyledons contributed to the observed antibacterial and anticandidal activities. Marama extracts, especially phenolic and crude seed coat extracts, had high multi-species antibacterial and anticandidal activities at concentrations comparable to that of some conventional drugsthese extracts have potential use as microbicides