An in vitro assessment of pharmacodynamic interactions between selected herbal extracts and anticancer chemotherapeutic agents
- Hwehwe, Nyashadzashe, Swanepoel, Bresler
- Authors: Hwehwe, Nyashadzashe , Swanepoel, Bresler
- Date: 2024-04
- Subjects: Chemotherapy , Herbs -- Therapeutic use , Antineoplastic agents
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63506 , vital:73383
- Description: Cancer is a disease caused by uncontrolled growth and spreading of abnormal cells to distant body parts. Approximately 19.3 million and 10 million new cancer cases and cancer deaths, respectively were recorded in 2020. Despite advancements in prevention and therapeutic strategies, cancer remains a global health problem. Cancer patients are increasingly seeking complementary and alternative medicines, of which herbal medicines are the most common form of CAM used by patients. Numerous in vitro and in vivo studies of herbal medicines in cancer have shown that they have antioxidant and anticancer (antitumor and anti-proliferative) properties. While concurrent use with conventional cancer treatments may improve treatment efficacy, alleviate chemotherapy-related side effects, boost the immune system, or impede drug resistance, it can also prompt drug-herbal interactions, and this may affect the pharmacodynamics and pharmacokinetics of the chemotherapeutic drug. This study aimed to determine relevant pharmacodynamic interactions with chemotherapeutic drugs and investigate such interactions' mechanisms. The objectives of the study were to screen the cytotoxicity effects of drug compounds, herbal extracts, and drug-herb combinations, to determine the ability of treatments to induce apoptosis, and to determine the most beneficial treatment. The cytotoxic effects of cisplatin in HeLa cells, tamoxifen in MCF7 cells, 5-fluorouracil in Caco-2 cells, and grapeseed, green tea, fermented rooibos, and green rooibos in all the listed cell lines were evaluated individually and in combination using the bis-Benzamide H 33342 trihydrochloride/propidium iodide (Hoechst 33342/PI) dual staining method. CompuSyn 1.0 Software was used to quantify synergism and antagonism. The mechanism of apoptosis induction of the different synergetic combinations, drug compounds, and herbal extracts was illustrated by quantitative fluorescence image analysis, specifically cell cycle analysis, phosphatidylserine translocation, mitochondrial membrane potential analysis, caspase 3 activation, and reactive oxygen species production using the relevant contrast dyes. Grapeseed displayed cytotoxicity towards MCF7 and HeLa cells (IC50 57.98 and 83.28 μg/mL, respectively). Green tea was only cytotoxic against HeLa cells at an IC50 of 91,92 μg/mL. None of the extracts displayed cytotoxicity against Caco-2 cells (IC50 values > 200μg/mL). If the herbal extracts had inconclusive IC50 values in the three cell lines subsequent experiments were conducted using 100 μg/mL. The results showed that most of the combinations were antagonistic but, some combinations had synergistic or enhancement effects(1:3 for cisplatin with grape seed or green tea and tamoxifen with grapeseed or green tea, and 3:1 for cisplatin with green tea as well as for tamoxifen with fermented rooibos or green rooibos, and all the 1:1 combinations of 5- fluorouracil with all herbal extracts) with a combination index (CI) < 1. Grape seed and green tea were found to induce apoptosis in the three cell lines. Assays that were conducted to detect apoptosis induction showed positive staining for phosphatidylserine (PS), activated caspase 3, and reactive oxygen species (ROS), mitochondrial membrane depolarization. Analysis of the cell cycle showed two things; 1) that grape seed and green tea were apoptotic in HeLa and MCF7 cells only, and 2) the extracts of green tea and green rooibos, and the combinations of grape seed with all drug compounds arrested the cell in more than one phase of the cell cycle. The combinations of grapeseed and green tea potentially induced apoptosis in various manners but those with both rooibos extracts were unclear in all the cell lines. The results for combinations with grapeseed and green tea are promising and provide a basis for further research as combinations of chemotherapeutic drugs and herbal extracts may be effective therapeutic strategies. , Thesis (MPharm) -- Faculty of Health Sciences, School of Clinical Care & Medicinal Sciences, 2024
- Full Text:
- Date Issued: 2024-04
- Authors: Hwehwe, Nyashadzashe , Swanepoel, Bresler
- Date: 2024-04
- Subjects: Chemotherapy , Herbs -- Therapeutic use , Antineoplastic agents
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63506 , vital:73383
- Description: Cancer is a disease caused by uncontrolled growth and spreading of abnormal cells to distant body parts. Approximately 19.3 million and 10 million new cancer cases and cancer deaths, respectively were recorded in 2020. Despite advancements in prevention and therapeutic strategies, cancer remains a global health problem. Cancer patients are increasingly seeking complementary and alternative medicines, of which herbal medicines are the most common form of CAM used by patients. Numerous in vitro and in vivo studies of herbal medicines in cancer have shown that they have antioxidant and anticancer (antitumor and anti-proliferative) properties. While concurrent use with conventional cancer treatments may improve treatment efficacy, alleviate chemotherapy-related side effects, boost the immune system, or impede drug resistance, it can also prompt drug-herbal interactions, and this may affect the pharmacodynamics and pharmacokinetics of the chemotherapeutic drug. This study aimed to determine relevant pharmacodynamic interactions with chemotherapeutic drugs and investigate such interactions' mechanisms. The objectives of the study were to screen the cytotoxicity effects of drug compounds, herbal extracts, and drug-herb combinations, to determine the ability of treatments to induce apoptosis, and to determine the most beneficial treatment. The cytotoxic effects of cisplatin in HeLa cells, tamoxifen in MCF7 cells, 5-fluorouracil in Caco-2 cells, and grapeseed, green tea, fermented rooibos, and green rooibos in all the listed cell lines were evaluated individually and in combination using the bis-Benzamide H 33342 trihydrochloride/propidium iodide (Hoechst 33342/PI) dual staining method. CompuSyn 1.0 Software was used to quantify synergism and antagonism. The mechanism of apoptosis induction of the different synergetic combinations, drug compounds, and herbal extracts was illustrated by quantitative fluorescence image analysis, specifically cell cycle analysis, phosphatidylserine translocation, mitochondrial membrane potential analysis, caspase 3 activation, and reactive oxygen species production using the relevant contrast dyes. Grapeseed displayed cytotoxicity towards MCF7 and HeLa cells (IC50 57.98 and 83.28 μg/mL, respectively). Green tea was only cytotoxic against HeLa cells at an IC50 of 91,92 μg/mL. None of the extracts displayed cytotoxicity against Caco-2 cells (IC50 values > 200μg/mL). If the herbal extracts had inconclusive IC50 values in the three cell lines subsequent experiments were conducted using 100 μg/mL. The results showed that most of the combinations were antagonistic but, some combinations had synergistic or enhancement effects(1:3 for cisplatin with grape seed or green tea and tamoxifen with grapeseed or green tea, and 3:1 for cisplatin with green tea as well as for tamoxifen with fermented rooibos or green rooibos, and all the 1:1 combinations of 5- fluorouracil with all herbal extracts) with a combination index (CI) < 1. Grape seed and green tea were found to induce apoptosis in the three cell lines. Assays that were conducted to detect apoptosis induction showed positive staining for phosphatidylserine (PS), activated caspase 3, and reactive oxygen species (ROS), mitochondrial membrane depolarization. Analysis of the cell cycle showed two things; 1) that grape seed and green tea were apoptotic in HeLa and MCF7 cells only, and 2) the extracts of green tea and green rooibos, and the combinations of grape seed with all drug compounds arrested the cell in more than one phase of the cell cycle. The combinations of grapeseed and green tea potentially induced apoptosis in various manners but those with both rooibos extracts were unclear in all the cell lines. The results for combinations with grapeseed and green tea are promising and provide a basis for further research as combinations of chemotherapeutic drugs and herbal extracts may be effective therapeutic strategies. , Thesis (MPharm) -- Faculty of Health Sciences, School of Clinical Care & Medicinal Sciences, 2024
- Full Text:
- Date Issued: 2024-04
An in vitro evaluation of the anti-breast cancer activity of Nigella sativa extracts and its bioactive compound in combination with curcumin
- Authors: Botha, Susanna Gertruida
- Date: 2024-04
- Subjects: Herbs -- Therapeutic use , Radiation-protective agents , Breast -- Cancer -- Treatment
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63639 , vital:73571
- Description: Breast cancer constitutes 23% of all cancers in South African females. Curcumin and Nigella sativa have anti-cancer, anti-metastatic and antioxidant-properties and may be effective against breast cancer. This study focused on the effect of N. sativa extracts or thymoquinone and curcumin, individually and in combination, on breast cancer cells. An MTT assay showed that curcumin reduced cell viability by 50% (IC50) at 18 ± 2.63 μg/mL and thymoquinone (TQ) at 5 ± 0.95 μg/mL against the MDA-MB-231 cells. The IC50 values for curcumin and TQ were 35 ± 6.98 μg/mL and 4 ± 0.96 μg/mL against the MCF-7 cells, respectively. The IC50 value for the NSBE was determined to be 350 ± 55 μg/mL. The IC50 value of NSAE did not fall within the selected concentration range. Synergism was noted for combinations of NSBE with curcumin, and combinations of TQ with curcumin, against both MCF-7 and MDA-MB-231 cells. Two synergistic combinations per treatment per cell line, as determined by the combination index analysis, were chosen for further investigation. The combinations and individual treatments tested against the MCF-10A cells, were not significant, except for NSBE80:CURC20 combination. Curcumin had the most significant anti-oxidant activity; however, no link was noted between the anti-oxidant activity and the cytotoxicity of the combinations. The combination treatments induced apoptosis more effectively than the individual treatments. Caspase-3 dependent apoptosis was noted for NSBE10:CURC90 and TQ80:CURC20 combinations against the MDA-MB-231 cells, and the TQ60:CURC40 combination against the MCF-7 cells. The individual and combined treatments effectively reduced MDA-MB-231 cell adhesion to fibronectin, but not all reduced the cell adhesion to laminin. Based on these results, the combinations of curcumin with TQ or NSBE, have promising anticancer benefits against breast cancer. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-04
- Authors: Botha, Susanna Gertruida
- Date: 2024-04
- Subjects: Herbs -- Therapeutic use , Radiation-protective agents , Breast -- Cancer -- Treatment
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/63639 , vital:73571
- Description: Breast cancer constitutes 23% of all cancers in South African females. Curcumin and Nigella sativa have anti-cancer, anti-metastatic and antioxidant-properties and may be effective against breast cancer. This study focused on the effect of N. sativa extracts or thymoquinone and curcumin, individually and in combination, on breast cancer cells. An MTT assay showed that curcumin reduced cell viability by 50% (IC50) at 18 ± 2.63 μg/mL and thymoquinone (TQ) at 5 ± 0.95 μg/mL against the MDA-MB-231 cells. The IC50 values for curcumin and TQ were 35 ± 6.98 μg/mL and 4 ± 0.96 μg/mL against the MCF-7 cells, respectively. The IC50 value for the NSBE was determined to be 350 ± 55 μg/mL. The IC50 value of NSAE did not fall within the selected concentration range. Synergism was noted for combinations of NSBE with curcumin, and combinations of TQ with curcumin, against both MCF-7 and MDA-MB-231 cells. Two synergistic combinations per treatment per cell line, as determined by the combination index analysis, were chosen for further investigation. The combinations and individual treatments tested against the MCF-10A cells, were not significant, except for NSBE80:CURC20 combination. Curcumin had the most significant anti-oxidant activity; however, no link was noted between the anti-oxidant activity and the cytotoxicity of the combinations. The combination treatments induced apoptosis more effectively than the individual treatments. Caspase-3 dependent apoptosis was noted for NSBE10:CURC90 and TQ80:CURC20 combinations against the MDA-MB-231 cells, and the TQ60:CURC40 combination against the MCF-7 cells. The individual and combined treatments effectively reduced MDA-MB-231 cell adhesion to fibronectin, but not all reduced the cell adhesion to laminin. Based on these results, the combinations of curcumin with TQ or NSBE, have promising anticancer benefits against breast cancer. , Thesis (MSc) -- Faculty of Science, School of Biomolecular & Chemical Sciences, 2024
- Full Text:
- Date Issued: 2024-04
Synthesis and in vitro biological studies of ursolic acid-based hybrid compounds
- Authors: Khwaza, Vuyolwethu
- Date: 2022
- Subjects: Herbal medicine , Herbs -- Therapeutic use , Antineoplastic antibiotics
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27797 , vital:69689
- Description: Ursolic acid UA, a pentacyclic triterpenoid that is commonly found in many medicinal herbs and fruits, has been identified as a potential source of therapeutic agents because of its potent biological effects, which include its potential anticancer and antimicrobial activities. However, its limited solubility, rapid metabolism and poor bioavailability inhibit its clinical applications. Numerous UA derivatives have been prepared over the past years in an effort to mitigate the drawbacks associated with UA, as new chemical entities for the treatment of various infections. There is very little progress in the discovery of efficient UA derivatives. In this study, a class of ester and amide-linked ursolic acid-based hybrid compounds fused with selected pharmaceutical scaffolds were successfully synthesized using amidation and esterification reactions and tested for antibacterial and cytotoxicity activities. Hybridizing UA with other known pharmaceutical scaffolds has the potential of overcoming its drawbacks. FT-IR, Mass Spectroscopy, and 1H13C-NMR spectroscopy were used to confirm the structures of the synthesized hybrid compounds. Among the tested ester-linked hybrid compounds in Chapter three, compounds 3.14-3.19,3.21, 3.34, 3.31, and 3.30 demonstrated significant antibacterial activities against some tested bacteria, with MIC values of 15.625 μgml. Furthermore, the in vitro cytotoxicity of these hybrids was determined using the MTT assay against three human tumor cell lines MCF7, MDA-MB-231, and HeLa cells. Compounds 3.19 and 3.34 were found to have better cytotoxic activity when compared to ursolic acid, with IC50 values of 46.99 and 48.18 μg ml respectively. Both compounds revealed more promising docking results, presenting favourable binding interactions as well as better docking energy against the MCF 7 protein target compared to the parent compound ursolic acid. In Chapter Four, among the tested amide-linked hybrid compounds, Compounds 4.17 and 4.24 demonstrated significant antibacterial activity against the majority of bacterial strains with MIC values of 15.625 gml. Compound 4.24 exhibited a MIC value of 15.625gmL against BS, SA, PV, KO, PM, and EC. Compound 4.23 was more cytotoxic to HeLa cells than ursolic acid. Furthermore, molecular docking calculations revealed that compound 4.16 strongly binds to the protein epidermal growth factor receptor while e compounds 4.17 and 4.24 showed a strong binding affinity for the methionyl-tRNA synthetase. In both cases, the hybrid compounds showed better conformational fittings in the active site of the targeted proteins as compared to the parent ursolic acid. , Thesis (MSc) -- Faculty of Science and Agriculture, 2022
- Full Text:
- Date Issued: 2022
- Authors: Khwaza, Vuyolwethu
- Date: 2022
- Subjects: Herbal medicine , Herbs -- Therapeutic use , Antineoplastic antibiotics
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/27797 , vital:69689
- Description: Ursolic acid UA, a pentacyclic triterpenoid that is commonly found in many medicinal herbs and fruits, has been identified as a potential source of therapeutic agents because of its potent biological effects, which include its potential anticancer and antimicrobial activities. However, its limited solubility, rapid metabolism and poor bioavailability inhibit its clinical applications. Numerous UA derivatives have been prepared over the past years in an effort to mitigate the drawbacks associated with UA, as new chemical entities for the treatment of various infections. There is very little progress in the discovery of efficient UA derivatives. In this study, a class of ester and amide-linked ursolic acid-based hybrid compounds fused with selected pharmaceutical scaffolds were successfully synthesized using amidation and esterification reactions and tested for antibacterial and cytotoxicity activities. Hybridizing UA with other known pharmaceutical scaffolds has the potential of overcoming its drawbacks. FT-IR, Mass Spectroscopy, and 1H13C-NMR spectroscopy were used to confirm the structures of the synthesized hybrid compounds. Among the tested ester-linked hybrid compounds in Chapter three, compounds 3.14-3.19,3.21, 3.34, 3.31, and 3.30 demonstrated significant antibacterial activities against some tested bacteria, with MIC values of 15.625 μgml. Furthermore, the in vitro cytotoxicity of these hybrids was determined using the MTT assay against three human tumor cell lines MCF7, MDA-MB-231, and HeLa cells. Compounds 3.19 and 3.34 were found to have better cytotoxic activity when compared to ursolic acid, with IC50 values of 46.99 and 48.18 μg ml respectively. Both compounds revealed more promising docking results, presenting favourable binding interactions as well as better docking energy against the MCF 7 protein target compared to the parent compound ursolic acid. In Chapter Four, among the tested amide-linked hybrid compounds, Compounds 4.17 and 4.24 demonstrated significant antibacterial activity against the majority of bacterial strains with MIC values of 15.625 gml. Compound 4.24 exhibited a MIC value of 15.625gmL against BS, SA, PV, KO, PM, and EC. Compound 4.23 was more cytotoxic to HeLa cells than ursolic acid. Furthermore, molecular docking calculations revealed that compound 4.16 strongly binds to the protein epidermal growth factor receptor while e compounds 4.17 and 4.24 showed a strong binding affinity for the methionyl-tRNA synthetase. In both cases, the hybrid compounds showed better conformational fittings in the active site of the targeted proteins as compared to the parent ursolic acid. , Thesis (MSc) -- Faculty of Science and Agriculture, 2022
- Full Text:
- Date Issued: 2022
Phytochemical analysis and antibacterial properties of aqueous and ethanol extracts of Brachylaena elliptica (Thurb.) dc. and Brachylaena ilicifolia (Lam.) Phill & Schweick
- Authors: Sagbo, Idowu Jonas
- Date: 2015
- Subjects: Medicinal plants , Traditional medicine , Herbs -- Therapeutic use
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11297 , http://hdl.handle.net/10353/d1021289 , Medicinal plants , Traditional medicine , Herbs -- Therapeutic use
- Description: Resistance of human pathogenic bacterial strains results in selective pressure against known antibiotic. However, plant derived compounds that possess antibacterial potential are currently being investigated for treatment of wound infections in diabetic patients as they are inexpensive and non-toxic. Hence, this dissertation was designed to evaluate two medicinal plants (Brachylaena elliptica and Brachylaena ilicifolia) traditionally used in the treatment of various diseases such as diabetes, and its secondary complications in diabetic patients. The in vitro antioxidant activity of both plants were evaluated using DPPH (1, 1-diphenylhydrazl), ferric reducing power, ABTS (2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), NO (nitric oxide) and H2O2 (hydrogen peroxide) techniques. The antibacterial test and Minimum inhibitory concentration (MIC) was determined by agar dilution method against 5 bacteria strains (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogene, Proteus vulgaris and Proteus mirabilis) infecting wounds in diabetic patients using amoxicillin and ciprofloxacin as positive control. The phytochemical analyses were assessed using standard published methods. Identification of bioactive components in essential oils of both plants were assessed using GCMS. The aqueous and ethanol extracts of both plants were also evaluated to identify bioactive components using LC-MS. The results of the phytochemical analysis revealed the presence of phenols, tannins, flavanoids, flavanols, proanthocyanidins, saponins and alkaloids in both plants. Both plants indicated strong antioxidant activities which might be due to the presence of bioactive compounds. The aqueous and ethanol leaf extracts of both plants demonstrated appreciable broad spectrum activities against these wound pathogens with MIC ranging between 5 and 0.3 mg/ml. The GC-MS analysis of the essential oils of both plants revealed the presence of monoterpenes, oxygenated sesquiterpenes, phenolics and esters. The LC-MS analysis of the aqueous and ethanol leaf extracts of both plants showed that both plants are rich in alkaloids, terpenes, terpenoids, monoterpernoids, and flavanoids. Conclusively, this study has partially justified the ethnomedicinal use of B. elliptica and B.licifolia leaves for the treatment of various diseases, including diabetes and wound infections caused by bacteria in diabetic patients. These may be attributed to the presence of antioxidant compound such as phenols, flavanoids, saponins, tannins, alkaloids and other phytochemical compounds.
- Full Text:
- Date Issued: 2015
- Authors: Sagbo, Idowu Jonas
- Date: 2015
- Subjects: Medicinal plants , Traditional medicine , Herbs -- Therapeutic use
- Language: English
- Type: Thesis , Masters , MSc (Biochemistry)
- Identifier: vital:11297 , http://hdl.handle.net/10353/d1021289 , Medicinal plants , Traditional medicine , Herbs -- Therapeutic use
- Description: Resistance of human pathogenic bacterial strains results in selective pressure against known antibiotic. However, plant derived compounds that possess antibacterial potential are currently being investigated for treatment of wound infections in diabetic patients as they are inexpensive and non-toxic. Hence, this dissertation was designed to evaluate two medicinal plants (Brachylaena elliptica and Brachylaena ilicifolia) traditionally used in the treatment of various diseases such as diabetes, and its secondary complications in diabetic patients. The in vitro antioxidant activity of both plants were evaluated using DPPH (1, 1-diphenylhydrazl), ferric reducing power, ABTS (2, 2’-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid), NO (nitric oxide) and H2O2 (hydrogen peroxide) techniques. The antibacterial test and Minimum inhibitory concentration (MIC) was determined by agar dilution method against 5 bacteria strains (Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogene, Proteus vulgaris and Proteus mirabilis) infecting wounds in diabetic patients using amoxicillin and ciprofloxacin as positive control. The phytochemical analyses were assessed using standard published methods. Identification of bioactive components in essential oils of both plants were assessed using GCMS. The aqueous and ethanol extracts of both plants were also evaluated to identify bioactive components using LC-MS. The results of the phytochemical analysis revealed the presence of phenols, tannins, flavanoids, flavanols, proanthocyanidins, saponins and alkaloids in both plants. Both plants indicated strong antioxidant activities which might be due to the presence of bioactive compounds. The aqueous and ethanol leaf extracts of both plants demonstrated appreciable broad spectrum activities against these wound pathogens with MIC ranging between 5 and 0.3 mg/ml. The GC-MS analysis of the essential oils of both plants revealed the presence of monoterpenes, oxygenated sesquiterpenes, phenolics and esters. The LC-MS analysis of the aqueous and ethanol leaf extracts of both plants showed that both plants are rich in alkaloids, terpenes, terpenoids, monoterpernoids, and flavanoids. Conclusively, this study has partially justified the ethnomedicinal use of B. elliptica and B.licifolia leaves for the treatment of various diseases, including diabetes and wound infections caused by bacteria in diabetic patients. These may be attributed to the presence of antioxidant compound such as phenols, flavanoids, saponins, tannins, alkaloids and other phytochemical compounds.
- Full Text:
- Date Issued: 2015
Biological activities and mechanisms of action of two ethnobotanically selected South African medicinal plants on some bacteria associated with gastrointestinal infections
- Olajuyigbe, Olufunmiso Olusola https://orcid.org/0000-0002-7889-0416
- Authors: Olajuyigbe, Olufunmiso Olusola https://orcid.org/0000-0002-7889-0416
- Date: 2012-08
- Subjects: Medicinal plants , Herbs -- Therapeutic use , Gastrointestinal system
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/25439 , vital:64249
- Description: In this study, 36 plant species representing 24 families were found to be commonly used for the treatment of a variety of gastrointestinal disorders in Eastern Cape, South Africa. The family Fabaceae had the highest number of species. Out of these, 47.06percent were used in the treatment of dysentery alone while 46.15percent were used in the treatment of diarrhoea. Acacia mearnsii De Wild and Ziziphus mucronata subsp. mucronata Willd were selected for this research because they are extensively used in folkloric medicine in South Africa and there was lack of scientific reports that documented their biological activities. The phytochemical screening, antioxidant activities, in vitro antimicrobial activities, cytotoxicity, the synergistic potentials and mechanisms of actions of these plants were investigated. The phytochemical screening and the antioxidant activities of the two species showed that the quantity of the phenolic compounds, flavonoids and proanthocyanidins detected differ significantly in the various extracts. Of the aqueous, acetone, ethanolic and methanolic extracts of A. mearnsii, the ethanolic extract had the highest flavonoids while the acetone extract had the highest phenolic contents. The proanthocyanidins were highest in the methanol extract while aqueous extracts had the least phytochemicals. Aqueous extract showed the least ferric reducing power but methanol extract indicated the highest reducing power. The reducing power of the extracts was lower than those obtained from the reference standard such as butylated hydroxytoluene (BHT), rutin and ascorbic acid. 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) diammonium salt showed that ethanol extract exhibited the highest antioxidant activity at the highest concentration tested. Also, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay indicated that ethanol extract had the highest radical scavenging activity at the lowest concentration and the activities of all the extracts decreased with increase in their concentrations. In Z. mucronata subsp. mucronata, the phenolics were significantly higher than the flavonoids and proanthocyanidin contents in all the extracts investigated. The ethanol extract had the highest antioxidant activity, followed by the acetone extract while the aqueous extract was the least active. Reacting with ABTS, the 50percent inhibitory concentrations (IC50) were (0.0429 ± 0.04 mg/ml) for aqueous, (0.0317 ± 0.04 mg/ml) for acetone and (0.0306 ± 0.04 mg/ml) for ethanol extracts while they inhibited DPPH radical with 50percent inhibitory concentration (IC50) values of 0.0646 ± 0.02 mg/ml (aqueous), 0.0482 ± 0.02 mg/ml (acetone) and 0.0422 ± 0.03 mg/ml (ethanol). The investigation showed that a positive linear correlation existed between the total phenolic content and antioxidant activity of the extracts and that these plants have strong antioxidant property and free radical scavenging capability. The in vitro antibacterial activities of Acacia mearnsii and Z. mucronata subsp. mucronata showed that their minimum inhibitory concentrations ranged between 0.039 mg/ml and 1.25 mg/ml. With the exception of acetone extract of A. mearnsii having MICs greater than 1.0 mg/ml for Enterococcus faecalis ATCC 29212 and Bacillus subtilis KZN, all other isolates had MICs less than 0.7 mg/ml. In all the bacteria treated with Z. mucronata subsp. mucronata extracts, Enterobacter cloacae ATCC 13047 had MIC greater than 1 mg/ml in methanol extract, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 6538 had MICs greater than 1 mg/ml in acetone extract while all other isolates were highly susceptible to the different extracts of Z. mucronata subsp. mucronata and had MICs less than 0.7 mg/ml. While aqueous extract was as active as the alcoholic extracts in A. mearnsii, that of Z. mucronata had no effect. The ethanol extracts exhibited the highest degree of antibacterial activity in both plants. This study, also, showed that the antifungal activity of A. mearnsii ranging 0.3125 – 5.0 mg/ml was higher than those of the different extracts of Z. mucronata subsp. mucronata ranging 1.25 – 10.0 mg/ml. It is evident from the results of the brine shrimp lethality assay that the crude extracts of A. mearnsii with the LC50 equaled 112.36 µg/ml and having the highest levels of toxicity (100percent) death at 500 μg/ml was non toxic (LC50 > 100 μg/ml) while the LC50 for Z. mucronata subsp. mucronata equaled 90.27 µg/ml indicated a low level of toxicity. The effects of combining the crude extracts of these plants with eight antibiotics were investigated by means of checkerboard and agar diffusion methods. On using the methanol extract of A. mearnsii, the agar diffusion assay showed that extract-kanamycin combination had zones of inhibition ≥ 20 ± 1.0 mm in all the bacteria tested (100percent), followed by extract chloramphenicol (90percent) > extract-ciprofloxacin = extract-tetracycline (70percent) > extract amoxicillin (60percent) > extract-nalidixic acid (50percent) > extract-erythromycin (40percent) > extract metronidazole (20percent). The checkerboard showed synergistic interaction (61.25percent), additivity/indifference (23.75percent) and antagonistic (15percent) effects. I, therefore, concluded that the antibacterial potentials of the antibiotics were improved and combining natural products with antibiotic could be a potential source of resistance-modifying agents useful against multi-drug resistant bacteria. The influences of these extracts on the ultrastructures, elemental components, protein and lipid leakages of five different bacteria were determined as the possible mechanisms of action of the extracts investigated. The scanning electron microscopy indicated varied ultrastructural changes in the morphology of bacterial cells treated with the extracts. The X-ray microanalysis showed significant differences between the elemental contents of extract-treated and untreated bacteria while lipids and proteins were leaked to a great extent from the extract-treated bacterial strains in comparison with the untreated ones. The possible mechanisms of action of the extracts may include inhibition of a significant step in peptidoglycan assembly, inhibition of metabolic processes, disruption of cell wall and cell membranes resulting in the efflux of lipid and protein in all the bacteria tested. The possible mechanism of action involved in the lipid and protein leakages in the bacterial cells could be attributed to lipid peroxidation and protein oxidation owing to the antioxidant activities of the extracts that were active beyond the protective levels. I concluded that the morphological changes and the observed leakages showed rapid killing, significant membrane depolarization resulting in leakages and efflux of disintegrated cellular materials. In general, this study has justified the ethnotherapeutic importance of A. mearnsii and Z. mucronata subsp. mucronata in the treatment of microbial infections by indicating the possible mechanisms of action of the crude extracts on the tested bacteria. , Thesis (PhD) -- Faculty of Science and Agriculture, 2012
- Full Text:
- Date Issued: 2012-08
- Authors: Olajuyigbe, Olufunmiso Olusola https://orcid.org/0000-0002-7889-0416
- Date: 2012-08
- Subjects: Medicinal plants , Herbs -- Therapeutic use , Gastrointestinal system
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10353/25439 , vital:64249
- Description: In this study, 36 plant species representing 24 families were found to be commonly used for the treatment of a variety of gastrointestinal disorders in Eastern Cape, South Africa. The family Fabaceae had the highest number of species. Out of these, 47.06percent were used in the treatment of dysentery alone while 46.15percent were used in the treatment of diarrhoea. Acacia mearnsii De Wild and Ziziphus mucronata subsp. mucronata Willd were selected for this research because they are extensively used in folkloric medicine in South Africa and there was lack of scientific reports that documented their biological activities. The phytochemical screening, antioxidant activities, in vitro antimicrobial activities, cytotoxicity, the synergistic potentials and mechanisms of actions of these plants were investigated. The phytochemical screening and the antioxidant activities of the two species showed that the quantity of the phenolic compounds, flavonoids and proanthocyanidins detected differ significantly in the various extracts. Of the aqueous, acetone, ethanolic and methanolic extracts of A. mearnsii, the ethanolic extract had the highest flavonoids while the acetone extract had the highest phenolic contents. The proanthocyanidins were highest in the methanol extract while aqueous extracts had the least phytochemicals. Aqueous extract showed the least ferric reducing power but methanol extract indicated the highest reducing power. The reducing power of the extracts was lower than those obtained from the reference standard such as butylated hydroxytoluene (BHT), rutin and ascorbic acid. 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) diammonium salt showed that ethanol extract exhibited the highest antioxidant activity at the highest concentration tested. Also, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay indicated that ethanol extract had the highest radical scavenging activity at the lowest concentration and the activities of all the extracts decreased with increase in their concentrations. In Z. mucronata subsp. mucronata, the phenolics were significantly higher than the flavonoids and proanthocyanidin contents in all the extracts investigated. The ethanol extract had the highest antioxidant activity, followed by the acetone extract while the aqueous extract was the least active. Reacting with ABTS, the 50percent inhibitory concentrations (IC50) were (0.0429 ± 0.04 mg/ml) for aqueous, (0.0317 ± 0.04 mg/ml) for acetone and (0.0306 ± 0.04 mg/ml) for ethanol extracts while they inhibited DPPH radical with 50percent inhibitory concentration (IC50) values of 0.0646 ± 0.02 mg/ml (aqueous), 0.0482 ± 0.02 mg/ml (acetone) and 0.0422 ± 0.03 mg/ml (ethanol). The investigation showed that a positive linear correlation existed between the total phenolic content and antioxidant activity of the extracts and that these plants have strong antioxidant property and free radical scavenging capability. The in vitro antibacterial activities of Acacia mearnsii and Z. mucronata subsp. mucronata showed that their minimum inhibitory concentrations ranged between 0.039 mg/ml and 1.25 mg/ml. With the exception of acetone extract of A. mearnsii having MICs greater than 1.0 mg/ml for Enterococcus faecalis ATCC 29212 and Bacillus subtilis KZN, all other isolates had MICs less than 0.7 mg/ml. In all the bacteria treated with Z. mucronata subsp. mucronata extracts, Enterobacter cloacae ATCC 13047 had MIC greater than 1 mg/ml in methanol extract, Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 6538 had MICs greater than 1 mg/ml in acetone extract while all other isolates were highly susceptible to the different extracts of Z. mucronata subsp. mucronata and had MICs less than 0.7 mg/ml. While aqueous extract was as active as the alcoholic extracts in A. mearnsii, that of Z. mucronata had no effect. The ethanol extracts exhibited the highest degree of antibacterial activity in both plants. This study, also, showed that the antifungal activity of A. mearnsii ranging 0.3125 – 5.0 mg/ml was higher than those of the different extracts of Z. mucronata subsp. mucronata ranging 1.25 – 10.0 mg/ml. It is evident from the results of the brine shrimp lethality assay that the crude extracts of A. mearnsii with the LC50 equaled 112.36 µg/ml and having the highest levels of toxicity (100percent) death at 500 μg/ml was non toxic (LC50 > 100 μg/ml) while the LC50 for Z. mucronata subsp. mucronata equaled 90.27 µg/ml indicated a low level of toxicity. The effects of combining the crude extracts of these plants with eight antibiotics were investigated by means of checkerboard and agar diffusion methods. On using the methanol extract of A. mearnsii, the agar diffusion assay showed that extract-kanamycin combination had zones of inhibition ≥ 20 ± 1.0 mm in all the bacteria tested (100percent), followed by extract chloramphenicol (90percent) > extract-ciprofloxacin = extract-tetracycline (70percent) > extract amoxicillin (60percent) > extract-nalidixic acid (50percent) > extract-erythromycin (40percent) > extract metronidazole (20percent). The checkerboard showed synergistic interaction (61.25percent), additivity/indifference (23.75percent) and antagonistic (15percent) effects. I, therefore, concluded that the antibacterial potentials of the antibiotics were improved and combining natural products with antibiotic could be a potential source of resistance-modifying agents useful against multi-drug resistant bacteria. The influences of these extracts on the ultrastructures, elemental components, protein and lipid leakages of five different bacteria were determined as the possible mechanisms of action of the extracts investigated. The scanning electron microscopy indicated varied ultrastructural changes in the morphology of bacterial cells treated with the extracts. The X-ray microanalysis showed significant differences between the elemental contents of extract-treated and untreated bacteria while lipids and proteins were leaked to a great extent from the extract-treated bacterial strains in comparison with the untreated ones. The possible mechanisms of action of the extracts may include inhibition of a significant step in peptidoglycan assembly, inhibition of metabolic processes, disruption of cell wall and cell membranes resulting in the efflux of lipid and protein in all the bacteria tested. The possible mechanism of action involved in the lipid and protein leakages in the bacterial cells could be attributed to lipid peroxidation and protein oxidation owing to the antioxidant activities of the extracts that were active beyond the protective levels. I concluded that the morphological changes and the observed leakages showed rapid killing, significant membrane depolarization resulting in leakages and efflux of disintegrated cellular materials. In general, this study has justified the ethnotherapeutic importance of A. mearnsii and Z. mucronata subsp. mucronata in the treatment of microbial infections by indicating the possible mechanisms of action of the crude extracts on the tested bacteria. , Thesis (PhD) -- Faculty of Science and Agriculture, 2012
- Full Text:
- Date Issued: 2012-08
Validating the use of Hippobromus pauciflorus (L.f) Radlk for the treatment of eye infections in the Eastern Cape South Africa
- Authors: Pendota, Srinivasa Chary
- Date: 2010
- Subjects: Medicinal plants -- South Africa -- Eastern Cape , Herbs -- Therapeutic use , Eye -- Infections -- South Africa -- Eastern Cape , Sapindaceae
- Language: English
- Type: Thesis , Doctoral , PhD (Botany)
- Identifier: vital:11303 , http://hdl.handle.net/10353/d1001052 , Medicinal plants -- South Africa -- Eastern Cape , Herbs -- Therapeutic use , Eye -- Infections -- South Africa -- Eastern Cape , Sapindaceae
- Description: An ethnobotanical survey of plants used for the treatment of eye infections was carried out in the Eastern Cape Province of South Africa. The study revealed that 12 plant species are commonly used with Hippobromus pauciflorus (L.f) Radlk being the most frequent. The crude extracts of the leaves, stem bark and roots of the plant were investigated for antimicrobial activity against 10 bacterial and four fungal strains. The methanol extracts of the plant parts were the most active and showed appreciable activity against Gram-positive and Gram-negative bacteria. The methanol extracts of the leaves and stem bark inhibited the growth of fungi with activities ranging from 78.70 percent to 100 percent on Aspergillus niger and Penicillium notatum. The acetone extracts of the leaves and stem bark were active against A. niger (51.76 percent) and P. notatum (77.22 percent). The aqueous extract of Hippobromus pauciflorus leaves at 50, 100 and 200 mg/kg body weight doses were evaluated for anti-inflammatory, analgesic and antipyretic activities in male Wistar rats. Anti-inflammatory activity was studied by using carrageenan-and histamine induced oedema right hind paw volume while the analgesic effect was evaluated using formalin-induced pain and tail flick nociception response. The brewer’s yeast-induced pyrexia model was used for the antipyretic investigation. The extract at all the doses used significantly inhibited both the carrageenan- and histamine-induced inflammation in a manner that was not dose dependent. The extract reduced the formalin-induced pain licking as well as prolonged the reaction time in the tail flick-induced pain. The effects of the aqueous extract of the plant leaves at 50, 100 and 200 mg/kg body weight doses were investigated for 14 days on some biochemical parameters of male Wistar rats. The extract at all the doses tested did not significantly alter the levels of white blood cells, red blood cells, mean corpuscular volume, platelets, neutrophils, monocytes, lymphocytes and large unstained cells. While the levels of haemoglobin, packed cell volume and basophils increased at specific doses, those of mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and eosinophils decreased. In vitro antiviral activities of the plant against herpes simplex virus type 1(HSV-1) and coxsakie virus B6 were investigated. Cytotoxicity was evaluated by MTT assay in Vero cells. At concentrations ranging from 165-270/μg/ml there was significant antiviral activity against HSV-1, but toxicity was also noted. There was no antiviral activity against coxsakie virus B6. Bioactivity-guided fractionation of the leaves of H. pauciflorus yielded three known compounds. From the ethyl acetate fraction, two compounds were isolated (epicatechin and -sitosterol ) and from the n- hexane fractions, one compound (lupeol) was isolated. They were isolated and identified using various techniques. The antimicrobial, anti-inflammatory, analgesic and anti-pyretic activity of these compounds have been reported in literature. The structure and distribution of foliar appendages on the leaves of H. pauciflorus were examined by scanning electron microscope. The leaves have long unicellular nonglandular trichomes which were distributed over the mid rib and densely populated at the edges of the adaxial and abaxial surfaces. In general, the experiments and tests conducted in this study appear to have justified the use of Hippobromus pauciflorus for the treatment of eye infections and make a substantial contribution to the knowledge base of the use of herbal medicine for the treatment of the eye infections.
- Full Text:
- Date Issued: 2010
- Authors: Pendota, Srinivasa Chary
- Date: 2010
- Subjects: Medicinal plants -- South Africa -- Eastern Cape , Herbs -- Therapeutic use , Eye -- Infections -- South Africa -- Eastern Cape , Sapindaceae
- Language: English
- Type: Thesis , Doctoral , PhD (Botany)
- Identifier: vital:11303 , http://hdl.handle.net/10353/d1001052 , Medicinal plants -- South Africa -- Eastern Cape , Herbs -- Therapeutic use , Eye -- Infections -- South Africa -- Eastern Cape , Sapindaceae
- Description: An ethnobotanical survey of plants used for the treatment of eye infections was carried out in the Eastern Cape Province of South Africa. The study revealed that 12 plant species are commonly used with Hippobromus pauciflorus (L.f) Radlk being the most frequent. The crude extracts of the leaves, stem bark and roots of the plant were investigated for antimicrobial activity against 10 bacterial and four fungal strains. The methanol extracts of the plant parts were the most active and showed appreciable activity against Gram-positive and Gram-negative bacteria. The methanol extracts of the leaves and stem bark inhibited the growth of fungi with activities ranging from 78.70 percent to 100 percent on Aspergillus niger and Penicillium notatum. The acetone extracts of the leaves and stem bark were active against A. niger (51.76 percent) and P. notatum (77.22 percent). The aqueous extract of Hippobromus pauciflorus leaves at 50, 100 and 200 mg/kg body weight doses were evaluated for anti-inflammatory, analgesic and antipyretic activities in male Wistar rats. Anti-inflammatory activity was studied by using carrageenan-and histamine induced oedema right hind paw volume while the analgesic effect was evaluated using formalin-induced pain and tail flick nociception response. The brewer’s yeast-induced pyrexia model was used for the antipyretic investigation. The extract at all the doses used significantly inhibited both the carrageenan- and histamine-induced inflammation in a manner that was not dose dependent. The extract reduced the formalin-induced pain licking as well as prolonged the reaction time in the tail flick-induced pain. The effects of the aqueous extract of the plant leaves at 50, 100 and 200 mg/kg body weight doses were investigated for 14 days on some biochemical parameters of male Wistar rats. The extract at all the doses tested did not significantly alter the levels of white blood cells, red blood cells, mean corpuscular volume, platelets, neutrophils, monocytes, lymphocytes and large unstained cells. While the levels of haemoglobin, packed cell volume and basophils increased at specific doses, those of mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration and eosinophils decreased. In vitro antiviral activities of the plant against herpes simplex virus type 1(HSV-1) and coxsakie virus B6 were investigated. Cytotoxicity was evaluated by MTT assay in Vero cells. At concentrations ranging from 165-270/μg/ml there was significant antiviral activity against HSV-1, but toxicity was also noted. There was no antiviral activity against coxsakie virus B6. Bioactivity-guided fractionation of the leaves of H. pauciflorus yielded three known compounds. From the ethyl acetate fraction, two compounds were isolated (epicatechin and -sitosterol ) and from the n- hexane fractions, one compound (lupeol) was isolated. They were isolated and identified using various techniques. The antimicrobial, anti-inflammatory, analgesic and anti-pyretic activity of these compounds have been reported in literature. The structure and distribution of foliar appendages on the leaves of H. pauciflorus were examined by scanning electron microscope. The leaves have long unicellular nonglandular trichomes which were distributed over the mid rib and densely populated at the edges of the adaxial and abaxial surfaces. In general, the experiments and tests conducted in this study appear to have justified the use of Hippobromus pauciflorus for the treatment of eye infections and make a substantial contribution to the knowledge base of the use of herbal medicine for the treatment of the eye infections.
- Full Text:
- Date Issued: 2010
Pharmaceutical analysis and quality of complementary medicines : sceletium and associated products
- Patnala, Satya Siva Rama Ranganath Srinivas
- Authors: Patnala, Satya Siva Rama Ranganath Srinivas
- Date: 2007
- Subjects: Alternative medicine , Herbs -- Therapeutic use
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3872 , http://hdl.handle.net/10962/d1018263
- Description: There has been an upsurge in the use of Complementary and Alternate Medicines (CAMs) in both developed and developing countries. Although herbal medicines have been in use for many centuries, their quality, safety and efficacy are still of major concern. Many countries are in the process of integrating CAMs into conventional health care systems based on the knowledge and use of traditional medicines. The quality control (QC) of herbal products usually presents a formidable analytical challenge in view of the complexity of the constituents in plant material and the commercial non-availability of appropriate qualified reference standards. Sceletium, a genus belonging to the family Aizoaceae, has been reported to contain psychoactive alkaloids, specifically mesembrine, mesembrenone, mesembrenol and some other related alkaloids. Sceletium is marketed as dried plant powder and as phyto-pharmaceutical dosage forms. Sceletium products and plant material marketed through health shops and on the internet are associated with unjustified claims of specific therapeutic efficacy and may be of dubious quality. Validated analytical methods to estimate Sceletium alkaloids have not previously been reported in the scientific literature and the available methods have focused only on qualitative estimation. Furthermore, since appropriate markers were not commercially available for use as reference standards, a primary objective of this study was to isolate relevant compounds, qualify them as reference standards which could be applied to develop appropriate validated qualitative and quantitative analytical methods for fingerprinting and assay of Sceletium plant material and dosage forms. The alkaloidal markers mesembrine, mesembrenone and ∆⁷ mesembrenone were isolated by solvent extraction and chromatography from dried plant material. Mesembranol and epimesembranol were synthesised by hydrogenation of the isolated mesembrine using the catalyst platinum (IV) oxide and then further purified by semi-preparative column chromatography. All compounds were subjected to analysis by ¹H, ¹³C, 2-D nuclear magnetic resonance and liquid chromatography-tandem mass spectroscopy. Mesembrine was converted to hydrochloride crystals and mesembranol was isolated as crystals from the hydrogenation reaction mass. These compounds were analysed and characterised by X-ray crystallography. A relatively simple HPLC method for the separation and quantitative analysis of five relevant alkaloidal components in Sceletium was developed and validated. The method was applied to determine the alkaloids in plant material and dosage forms containing Sceletium. An LCMS method developed during the study provided accurate identification of the five relevant Sceletium alkaloids. The method was applied for the quantitative analysis and QC of Sceletium plant material and its dosage forms. This LCMS method was found to efficiently ionize the relevant alkaloidal markers in order to facilitate their detection, identification and quantification in Sceletium plant material as well as for the assay and QC of dosage forms containing Sceletium. The chemotaxonomy of some Sceletium species and commercially available Sceletium dosage forms were successfully studied by the LCMS method. The HPLC and LCMS methods were also used to monitor the bio-conversion of some of the alkaloids while processing the plant material as per traditional method of fermentation. Additionally a high resolution CZE method was developed for the separation of several Sceletium alkaloids in relatively short analysis times. This analytical method was used successfully to fingerprint the alkaloids and quantify mesembrine in Sceletium and its products. Sceletium species grown under varying conditions at different locations, when analyzed, showed major differences in their composition of alkaloids and an enormous difference was found to exist between the various species with respect to the presence and content of alkaloids. Sceletium and its products marketed through health shops and the internet may thus have problems with respect to the quality and related therapeutic efficacy. The QC of Sceletium presents a formidable challenge as Sceletium plants and products contain a complex mixture of compounds. The work presented herein contributes to a growing body of scientific knowledge to improve the QC standards of herbal medicines and also to provide vital information regarding the selection of plant species and information on the specific alkaloidal constituents to the cultivators of Sceletium and the manufacturers of its products.
- Full Text:
- Date Issued: 2007
- Authors: Patnala, Satya Siva Rama Ranganath Srinivas
- Date: 2007
- Subjects: Alternative medicine , Herbs -- Therapeutic use
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3872 , http://hdl.handle.net/10962/d1018263
- Description: There has been an upsurge in the use of Complementary and Alternate Medicines (CAMs) in both developed and developing countries. Although herbal medicines have been in use for many centuries, their quality, safety and efficacy are still of major concern. Many countries are in the process of integrating CAMs into conventional health care systems based on the knowledge and use of traditional medicines. The quality control (QC) of herbal products usually presents a formidable analytical challenge in view of the complexity of the constituents in plant material and the commercial non-availability of appropriate qualified reference standards. Sceletium, a genus belonging to the family Aizoaceae, has been reported to contain psychoactive alkaloids, specifically mesembrine, mesembrenone, mesembrenol and some other related alkaloids. Sceletium is marketed as dried plant powder and as phyto-pharmaceutical dosage forms. Sceletium products and plant material marketed through health shops and on the internet are associated with unjustified claims of specific therapeutic efficacy and may be of dubious quality. Validated analytical methods to estimate Sceletium alkaloids have not previously been reported in the scientific literature and the available methods have focused only on qualitative estimation. Furthermore, since appropriate markers were not commercially available for use as reference standards, a primary objective of this study was to isolate relevant compounds, qualify them as reference standards which could be applied to develop appropriate validated qualitative and quantitative analytical methods for fingerprinting and assay of Sceletium plant material and dosage forms. The alkaloidal markers mesembrine, mesembrenone and ∆⁷ mesembrenone were isolated by solvent extraction and chromatography from dried plant material. Mesembranol and epimesembranol were synthesised by hydrogenation of the isolated mesembrine using the catalyst platinum (IV) oxide and then further purified by semi-preparative column chromatography. All compounds were subjected to analysis by ¹H, ¹³C, 2-D nuclear magnetic resonance and liquid chromatography-tandem mass spectroscopy. Mesembrine was converted to hydrochloride crystals and mesembranol was isolated as crystals from the hydrogenation reaction mass. These compounds were analysed and characterised by X-ray crystallography. A relatively simple HPLC method for the separation and quantitative analysis of five relevant alkaloidal components in Sceletium was developed and validated. The method was applied to determine the alkaloids in plant material and dosage forms containing Sceletium. An LCMS method developed during the study provided accurate identification of the five relevant Sceletium alkaloids. The method was applied for the quantitative analysis and QC of Sceletium plant material and its dosage forms. This LCMS method was found to efficiently ionize the relevant alkaloidal markers in order to facilitate their detection, identification and quantification in Sceletium plant material as well as for the assay and QC of dosage forms containing Sceletium. The chemotaxonomy of some Sceletium species and commercially available Sceletium dosage forms were successfully studied by the LCMS method. The HPLC and LCMS methods were also used to monitor the bio-conversion of some of the alkaloids while processing the plant material as per traditional method of fermentation. Additionally a high resolution CZE method was developed for the separation of several Sceletium alkaloids in relatively short analysis times. This analytical method was used successfully to fingerprint the alkaloids and quantify mesembrine in Sceletium and its products. Sceletium species grown under varying conditions at different locations, when analyzed, showed major differences in their composition of alkaloids and an enormous difference was found to exist between the various species with respect to the presence and content of alkaloids. Sceletium and its products marketed through health shops and the internet may thus have problems with respect to the quality and related therapeutic efficacy. The QC of Sceletium presents a formidable challenge as Sceletium plants and products contain a complex mixture of compounds. The work presented herein contributes to a growing body of scientific knowledge to improve the QC standards of herbal medicines and also to provide vital information regarding the selection of plant species and information on the specific alkaloidal constituents to the cultivators of Sceletium and the manufacturers of its products.
- Full Text:
- Date Issued: 2007
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