Comparative in vitro cytotoxic, anti-inflammatory and anti-microbiological activities of two indigenous Venda medicinal plants
© The Author(s) 2016
Received: 3 November 2016
Accepted: 2 December 2016
Published: 9 December 2016
The Vhembe region of the Limpopo province has a rich tradition of medicinal plants use. Traditionally, boiled roots of Ziziphus mucronata are used in the treatment of boils, general swelling and other skin infections. A combination of leaf paste and root infusion treats measles, dysentery, chest complains, and gland swelling. Pterocarpus angolensis is famous for the treatment of menorrhagia, infertility in women, wounds and pain management. The purpose of the present study was to compare the cytotoxicity, anti-inflammatory potential and anti-microbial activities of Ziziphus mucronata and Pterocarpus angolensis from the Vhembe region.
U937, MeWo, Vero and RAW 264.7 cells were treated to various concentrations (50, 100, or 125 or 250 μg/ml depending on assays) of Ziziphus mucronata and Pterocarpus angolensis. Cytotoxicity assay was done using MTT; Anti-inflammatory activity was assessed using NO production; Anti-bacterial activity was done using the Micro-Broth dilution method and Anti-mycobacteria activity was determined using the Alamar Blue Method while RT activity was measured by ELISA.
Cytotoxicity results showed that Pterocarpus was more toxic than Ziziphus as observed in the Vero and MeWo cells; however both displayed toxicity towards a Human cancer cell line. Both extracts did not inhibit nitrate production but induced significant increase in macrophage activation. The plant extracts have shown anti-tuberculosis activity at concentrations >500 μg/ml and there was moderation inhibition of HIV replication.
The results obtained indicated that the extracts have pro-inflammatory properties, and the observed toxicity on malignant cell lines must be investigated further for promising anti-cancer drug therapy.
KeywordsMedicinal plants Ziziphus mucronata Pterocarpus angolensis Cytotoxicity Cancer
The use of medicinal plants has been recorded for centuries with consistency regarding their uses as well as effectiveness in the treatment of diseases . The use of traditional medicines by traditional healers has played an important role in the health care of millions of people. The diversity of about 24,000 indigenous plants in South Africa contributes an estimate of 10% of higher plants on globally . The majority of people in South Africa rely heavily on the traditional use of medicinal plants with an estimated 70% of the population using one or more of the approximate 3000 species of plants used as traditional medicines [24, 33, 35].
Ziziphus mucronata Willd is commonly known as buffalo thorn (Mukhalu), it belongs to the Rhamnaceae family and is found in most parts of the country [10, 29]. Traditionally, boiled roots concotions are used in the treatment of boils, general swelling and other skin infections. A combination of leaf paste and root infusion treats measles, dysentery, chest complains, and gland swelling. Bark infusions are known to treat coughs (; [35–37]; [14, 18]). The active ingredients are several alkaloids known as peptide alkaloids such as mucronine D .
Pterocarpus angolensis DC belongs to the family Fabaceae, it is commonly known as bleedwood tree (Mutondo). Bark infusions are dropped into aching ear to neutralize the pain, boiled extract can be taken orally for menorrhagia ; roots infusions are used as a remedy for infertility amongst women . Mutondo is also known for its popular use in treatment of eye infections , wounds and psoriasis . The value of its timber carries a lot of weight amongst the African cultures since it is easy to work with for furniture, implements and curios purposes. Mutondo is also employed in the construction of canoes because the wood does easily shrink or swell much. The African women mixes the red sap with fresh animal fat to make a cosmetic for faces and bodies to enhance their beauty. It is also believed to have magical properties with regards to cleansing human blood from different ailments and spiritual possessions’; the belief is due to the red sap close resemblance to blood.
In the current study, we evaluated the claim by traditional healers of these two plants’ biological activities.
Description of plant materials
Stem Barks of Ziziphus mucronata (MPT00123) and Pterocarpus angolensis (MPT00118) were collected in 2014 in their natural habitat in the Vhembe District of the Limpopo Province (South Africa). The collected plants were identified using their vernacular names and later confirmed by the taxonomic rank at the Department of Botany, University of Venda with reference to the international plant name index (Ziziphus mucronata Willd and Pterocarpus angolensis DC). The samples were deposited in the departmental herbarium.
Bark samples for each plant were air dried and ground to fine powder using a Buchi mixer (Buchi Labortechnik AG, Flawil, Switzerland). The powder was carefully stored in a cool dry place until further use. Water soluble extracts were prepared by soaking 50 g of each samples in hot water (900 ml) and allowed to stand overnight. The homogenate was filtered using Whatman filter paper (110 mm, Sigma Aldrich) then freeze dried (FTS systems, Stone Ridge, NY; USA) to obtain 20 g of crude extracts. Unless indicated, plant extracts were solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 50 mg/ml and stored at 4 °C until required.
Ethical clearance was obtained from the University of Venda, Research and Ethics committee. The human macrophage cell line U937 (ATCC® CRL1593.2), Mewo (ATCC® HTB 65), Vero (ATTC® CCL 81) and the Murine macrophage RAW 264.7 cells (ATTC® TIB 71) were maintained in Roosevelt Park Memorial Institute medium (RPMI, Sigma Aldrich®; St Louis, MI; USA) or Dubelco’s modified Eagle Media (DMEM; Sigma Aldrich®; St Louis, MI; USA) respectively. The media were supplemented with 10% foetal bovine serum and 0.01% Gentamycin Sulfate (Sigma Aldrich, St Louis, MI, USA).
The method described by Klos et al.  was used. Three cell lines (U923, Mewo and Vero Cells) were used in this assay. The cells were seeded at a density of 10 000 cells/well (U937) or 6000 cells/well (MeWo/Vero) in 96-well plates (NUNC, Rochester, NY, USA) and incubated for overnight at 37 °C in a humidified 5% CO2 incubator (Thermofisher, Waltham, MA, USA). Samples were tested in two concentrations, 125 μg/ml and 250 μg/ml for U937cells and 50 μg/ml and 100 μg/ml for the MeWo/Vero cells for optimal results as obtained from Prof Van Der Venter’s Lab (Bioassaix, Nelson Mandela Metropolitan University, PE, South Africa). After adding the plant samples, the plates were further incubated for 48 h. Melphalan (Glaxo-smithkline, Paris, France) was used a positive known toxic chemical.
RAW 264.7 cells were seeded at a density of 25 000 cells/wells in a 96-well microtitre plate (NUNC, Rochester, NY, USA) and allowed to attach overnight. Plant extracts (50 and 100 μg/ml) in complete DMEM medium were added to replenish the spent culture medium in each well. In order to stimulate macrophages, 50 μl of lipopolysaccharide (LPS; 10 μg/ml) containing medium was added to specific wells whereas for unstimulated macrophages, 50 μl of complete medium without LPS was added to the other wells. A well-known inhibitor of nitric oxide (Aminoguanidine, [1 × 106]; Sigma Aldrich, St Louis, Missouri, USA) served as a positive control. To assess the pro-inflammatory potential. The plates were further incubated for an hour. To quantify the production of nitric oxide (NO) in both stimulated and non-stimulated cells, 50 μl of the spent culture medium was transferred to new 96 well plates and an equal amount of Griess reagent (Roche Diagnostics, Risch-Rotkreuz, Switzerland) added. Absorbance was measured at 510 nm and results compared to the respective controls. Cell viability was also assessed using the MTT assay to rule out toxicity as a contributory factor to NO production.
This section describes the following tests done: Anti-bacterial, anti-mycobacterial and anti-HIV activities.
Five clinical strains Enterococcus faecalis, Escherichia coli, Klebsellia Pneumoniae, Salmonella enterica and Streptococcus agalactiae were grown in Mueller-Hinton (MH) broth while the media for S. agalactiae was supplemented with 10% horse serum. The strains were inoculated in the MH broth (20 ml) and allowed to grow overnight for 16 h in an orbital incubator (EcoTherm, Hartkirchen, Austria) 37 °C. Gentamicin sulfate (2 mg/ml; Sigma Aldrich) and vancomycin hydrochloride (2 mg/ml; Sigma Aldrich) were used against both Gram negative and Gram positive bacteria respectively for controls. Due to Salmonella resistance to gentamicin sulfate, imipinem dehydrate, penicillin G, chloramphenicol and ampicillin were tested as additional antibiotics.
The two plant extracts were dissolved in DMSO at a stock concentration of 50 mg/ml. From the stock solutions, a working concentration (4 mg/ml) was prepared in MH broth and filter sterilized (Merck, Darmstadt, Germany). The experiment was carried out as previously described by Eloff .
The Mycobacterium tuberculosis H37 strain was obtained from the National Health Laboratory services (NHLS; Port Elizabeth; South Africa) and was grown in a culture broth containing DifcoTM Middlebrook 7H9 broth (Difco, Franklin Lakes, NJ; USA), glycerol (Sigma Aldrich; St Louis, MI, USA), Tween®80 (Sigma Aldrich, St Louis, MI; USA) and Middlebrook albumin-dextrose-catalase growth supplement (Sigma) at 37 °C for 10 d. Rifampicin (1 mg/ml) and ethambutol dihydrochloride (2 mg/ml) were dissolved at stock concentration of 1 mg/ml and 2 mg/ml respectively; and served as positive controls. The working concentrations obtained were 16 μg/ml and 1 mg/ml prepared in culture broth without Tween®80 and then filter sterilized (Merck, Darmstadt, Germany). The plant extracts were dissolved at working concentrations of 4 mg/ml prepared in culture broth without Tween®80. The assay was performed as described by Franzblau et al.  with slight modifications.
Reverse transcriptase inhibition
Reverse transcriptase enzyme is a potential therapeutic target against retrovirus infection since it plays a vital role in the conversion of viral ribonucleic acid (RNA) to complementary deoxyribonucleic acid (cDNA) . The assay is based on the incorporation of dioxigenin and biotin labelled nucleotides into the new DNA synthesis. A commercially available kit was used and the assay was conducted as per the manufacturer’s instructions (Roche Diagnostics; Risch-Rotkreuz, Switzerland).
All data were captured in Microsoft® Excel spreadsheets (Office XP, 2010), where descriptive statistics such as the average, standard deviation, and coefficient of variance were preformed and for generating graphs. Student t-test was used for comparison of results obtained for Ziziphus mucronata and Pterocarpus angolensis Differences in the data were considered significant when P < 0.05.
MIC values of antibiotics and aqueous extracts of Z. mucronata and P. angolensis
Gentamicin sulfate/ imipenem dehydrate/ amicillin/ penicillin G/ chloramphenicol
MIC values of antibiotics and aqueous extracts of Z. mucronata and P. angolensis
Reverse transcriptase inhibition
For medicinal plants to be useful in clinical applications, the preparations must be selectively toxic to the targeted microorganisms or must at least interfere directly with a particular reaction pathway with minimal toxic effects to the host cells. The present study was aimed at comparing the in vitro activities of Z. mucronata and P. angolensis.
Our results demonstrated the selective toxicity effect of the two plants on cancerous cells. As observed in Fig. 1, cytotoxicity was more pronounced in U937 (P < 0.005) cells for both extracts seen as a decrease in viability. The observation suggests some degree of selectivity towards the U937 cells, thus it can be concluded that cytotoxicity of these extracts might be cell specific. The cytotoxic effects observed also in Mewo cells was also highly statistically significant (P < 0.005). Z. mucronata displayed the best inhibition on cancerous cells and stimulatory effect on normal cells. A study done by Bessong et al.,  showed no cytotoxicity effects observed in Z. mucronata extract at the highest tested concentration (400 μg/ml) in HeLaP4 cells. Brine shrimp toxicity/lethality results as observed by McGaw et al.  showed Z. mucronata with minimal toxicity at 0.9 mg/ml. P. angolensis hexane leaf extract showed toxicity of 3.8 mg/ml with increased toxicity as compared to Z. mucronata. Previous reports revealed that P. angolensis extracts possess anti-inflammatory activity . Z mucronata also exhibited cytotoxic effect on brine shrimp at a concentration of 90.27 μg/ml .
This study has demonstrated firstly that Z. mucronata and P. angolensis do not inhibit NO production and secondly they do induce activation of macrophages. Macrophages are known to produce an extremely great number pro-inflammatory signals which can change the functionality of the surrounding cells. These includes production of nitric oxide which has been implicated in the ability to participate in diverse regulatory and cytotoxic action, it plays major role in mediating the human innate immunity . The production of NO has been previously demonstrated to be of great importance in the regulation of both acute and chronic inflammation as well as host defense mechanisms against various pathogens [6, 21], though it might also induce toxic reactions against host tissues if produced in higher levels [1, 31]. Our results indicate that the extracts have pro-inflammatory properties; however, it is necessary to confirm the absence of endotoxins which might present a similar effect. The nature of the extracts, in terms of their polysaccharide content, may also provide an indication as to their pro-inflammatory potential, as many plant polysaccharides are known to have immunomodulatory activity. A study done by Samie et al.,  showed that the seven compounds isolated from bark of P. angolensis were less toxic when tested against human cells (HCT-8) with an inhibitory concentration of 175–375 μg/ml.
The two plants tested did not have any activities on selected clinical bacterial species. The percentage for plant extract inhibition was not calculated as there was no observable microbial growth comparing the test and control wells. The results observed contradict the findings by Olajuyigbe and Afolayan . The bacterial isolates (E. faecalis, E. coli, K. pneumoniae) exhibited varied degree of susceptibility with zones of inhibition ranging between 17 and 27 ± 1.0 mm, though the difference in results can be attributed to the extraction solvent they used. A study report published by Samie et al.,  showed that ethanolic extracted compounds from P. angolensis inhibited bacterial growth of Staphylococcus aureus with median inhibitory concentration of 25 μg/ml. The results obtained in this study are not surprising because previous reports have shown that certain plant water extracts do not possess any biological activity .
However, the same plant extracts tested against M. tuberculosis H37 demonstrated strong anti-TB activities at concentrations >500 μg/ml. Tuberculosis is regarded as one of the major health problem especially in developing countries and is listed as a leading cause of death worldwide. An estimation of about 80% individuals are diagnosed yearly [7, 8]. Due to increase in multi drug resistance (MDR) and extensively drug resistance TB (XDR) strains of M. tuberculosis, the need of discovering newer anti-mycobacterial agents to eradicate the infections has increased. A study by Ibekwe et al.,  showed that Pterocarpus osun Craib exhibited an MIC value of 1225 μg/ml against strain H37.
Furthermore, Z. mucronata and P. angolensis displayed anti-HIV replication activity (from 20 to 60% inhibition) with the activity of P. angolensis similar to that of Niverapine at 10 μM. Mulaudzi et al.  have classified four levels of RT enzyme inhibition for medicinal plant extracts; with activities below 20% classified as insignificant, 20–40% considered low activity, 40–70% moderate, and 70–100% high activity. According to this classification the activity of P. angolensis is considered moderate and may thus warrant further dose response determination and calculation of an IC50 value. A study done by Bessong et al., 2005 showed RDDP IC50 (77.5 and 81.5) and RNase H IC50 (>100 and 75) activities of Z. mucronata for aqueous and methanol respectively; however the plant extracts were prepared from roots and leaves.
Natural products have been considered the first form of treatment in the treatment of human diseases. The use of such products has been in practice for centuries. The use of modern medicine has developed over the years through scientific evaluations and observational efforts of researchers. However, the basis of its development ought to be attributed to traditional medicine and therapies . Nevertheless, ancient wisdom passed on from generation to generation will remain the basis of modern medicine and will forever be considered as one important source of future medicine and therapeutics.
Results obtained showed that both plants have selective anti-tumor activities with P. angolensis being toxic to normal cells at higher concentrations but displayed profound anti-HIV activity compared to Z. mucronata. The observed toxicity on malignant cell lines is to be investigated further for promising anti-cancer therapy drugs. The activities observed for the two extracts shows the importance role they can display in treatment of HIV/AIDS thus reducing the burden of disease, with the sky rocketing pandemic of the infections in Sub-saharan Africa natural interventions are urgently needed to help alleviate this burden of disease.
Acquired immune deficiency syndrome
Complementary deoxyribonucleic acid
Dulbeco’s modified eagle medium
Enzyme-linked immunosorbent assay
Human immunodeficiency virus
Multi drug resistant
Minimal inhibitory concentration
National health laboratory services
Nelson Mandela Metropolitan University
Roosevelt Park Memorial Institute Medium
Extensively drug resistant
The authors would like to acknowledge the work done by Prof M Van Der Venter and her Team at the NMMU in Port Elizabeth We also acknowledge funding contribution by the Research & Innovation Directorate of the University of Venda. The assistance from the traditional healers (Mr Maphiri and Mr Munyai) is highly appreciated.
This work was supported by the University of Venda, Research and Innovation Directorate (S/15/MBY10) grants provided to Dr AN Traore a researcher in the Microbiology Department.
Availability of data and material
Data sharing not applicable.
MTS and MPT conducted interviews with traditional healers and collected the plant materials. ANT and MTS carried out laboratory work with assistance from Prof van de Venter and her team in NMMU. The remaining co-authors were involved in the write up based on their area of expertise. All the authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent of publication
Ethics approval and consent to participate
The experimental procedure were approved by the ethics committee of the University of Venda (SMNS/14/MBY/30/1210) and were accordance with international guidelines.
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