- 5.1: Case Study 1: Oralmat and Asthma
Current page is 5.2: Case Study 2: Oralmat and Cancer
- 5.4: Case Study 3: Oralmat and CNS
- 5.5: Case Study 4: Oralmat and HIV
- 5.6: Case Study 5: Oralmat and HSV
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Case Study 2: Oralmat and Cancer
Synopsis of the study conducted by Indres Moodley and Ntyiso Shengwenyana of the Department of Pharmacy, Faculty of Health Sciences, University of the Witwatersrand, South Africa.
The study was conducted to investigate the effects of Oralmat, an extract of rye grass (Secale Cereale) on five different cancer lines by using the MTT assay. It was noted that the Rye extract inhibited cell proliferation in all the cancer lines tested.
The inhibitory effect of rye grass extract on neo plastic cell lines, even at low doses, was comparable to common chemotherapeutic drugs.
The following is an abstract & summary of the following article:
Evaluation of an extract of Rye Grass, Secale cereale (Oralmat™) for anti-neoplastic activity in vitro using 5 cancer cell lines
By Indres Moodley and Ntyiso Shengwenyana. Department of Pharmacy, Faculty of Health Sciences, University of the Witwatersrand, South Africa.
ABSTRACT
The anti-neoplastic properties of an extract of rye grass (Secale cereale) on 5 different cancer cell lines were examined using the MTT assay. The Rye extract inhibited cell proliferation in all the cancer cell lines tested. The inhibitory response to the Rye extract differed according to the cell line tested. The response was most marked (>75%) in the PLC, MCF7 and K562 cell lines and approximately 50% in A498 and K562 cells. The concentrations of the extract and the magnitude of the inhibition in each cell line are indicated in the table below. The inhibitory effect of rye grass extract on neoplastic cells lines, even at low doses, was comparable to common chemotherapeutic drugs.
|
Cell Line |
% Inhibition |
Concentration of Rye Extract |
|
PLC MCF7 A498 K562 Hep 3B |
89.30 89.00 52.30 78.13 55.00 |
0.10% 1.00% 1.00% 0.10% 0.10% |
INTRODUCTION
Anecdotal evidence suggests that an extract of Rye (Oralmat™) may have a wide range of in vivo effects including promoting wound-healing, improved immune responses and an improved sense of general well-being. These anecdotes are supported by an increasing number of scientific studies suggesting several different effects of the extract including an immunomodulatory action (Rubin & Levine (2001)), an anxiolytic or sedative effect (Stough et al (2002) and a beneficial effect on asthma (Cooper et al (2002)). The wide range of effects found and the number of anecdotal reports prompted this study of the in vitro effects of the extract on cancer cell lines.
Rye extract was supplied to the Department of Pharmacy by Schumacher Pharmaceuticals. The extract was tested for anti-neoplastic activity by the MTT assay in a number of cell lines. The activity of the Rye extract was compared to the well characterised cytotoxic drugs vincristine, vinblastine and cycloheximide.
Methods & Test Extract
The MTT-microculture tetrazolium assay by Mossmann (1983) was adapted to study the anti-neoplastic activity of the Rye extract. The Rye extract contained 89.64% plant (Secale cereale) extract in a mixture of 9.96% ethanol and 0.40% Ascorbic acid.
The 5 Cancer Cell Lines That Were Tested Were As follows:
1. Liver Carcinoma (PLC)
2. Chronic Myelogenous Leukaemia (K562)
3. Renal Cancer (A498)
4. Breast Cancer (MCF7, 1, 2)
5. Liver Cancer (Hep 3B)
Controls
The positive controls used consisted of 10 l cells with 25l of a 1g/ml solution of Vincristine (1g/ml), Vinblastine (1g/ml) and Cycloheximide (1g/ml). Further controls were 100l cells only and 100 l cells with 25l of a 0.01%, 0.03%, 0.06%, 0.10%, 0.30% and 1.00% mixture of 9.96% and 0.40% of ethanol and ascorbic acid respectively.
The test solutions consisted of 100l cells with 25 l of a 0.01%, 0.03%, 0.06%, 0.10%, 0.30% and 1.00% Rye extract solution supplied.
MTT Assay
The plates were incubated at 370C in an atmosphere containing 5% CO2 in a humidified incubator. After 48 hours the plates were centrifuged and the supernatants were aspirated from the wells and 100l of culture medium with 10 l of a 5mg/ml solution of MTT was added to each well. The plates were incubated for a further 4 hours and then centrifuged for 10 minutes at the end of the four-hour period. The supernatants were removed and 180 l of DMSO was added to each well. After addition of the DMSO the plates were incubated for another 48 hours then the optical density (absorption) in each well was read at 550nm on a Labsystems Multiskan RC microplate reader.
The inhibition of proliferation was calculated as follows:
%Inhibition = (mean absorption. of control - mean absorption. of sample) X 100
mean absorption of control
Each test was conducted in triplicate and the results averaged. The tests were repeated on five different days by two different researchers and the results are reported as means (+SEM) of the five replicate experiments.
RESULTS
Maximal inhibition by vincristine was observed at 1g/ml. For comparison concentrations of 1g/ml cycloheximide, vincristine and vinblastine were studied for inhibitory activity on the same cancer cell lines.
The known anti-neoplastic agent, vincristine, caused a concentration-related inhibition of cell proliferation in all five cell lines (PLC, MCF7, A498, K562 and Hep 3B cells) over the concentration range 0.1g/ml to 2g/ml (Fig. 1). Comparison of the inhibition of cell growth caused by cycloheximide, vincristine and vinblastine at 1g/ml, showed that all three anti-neoplastic agents were approximately equally active against all the cell lines tested causing inhibition of cell growth, in the range 43% to 65%.
The rye extract exhibited similar inhibitory activity to the reference anti-neoplastic agents on all the cell lines tested. For the liver carcinoma (PLC) cell line, the Rye extract caused a bell-shaped response over the concentration range 0.01% to 1.00% with the greatest activity (89% inhibition) at a concentration of 0.10%. At concentrations higher than 0.10% a decline in the anti-proliferative activity was observed. A similar bell-shaped response was found with the chronic myelogenous leukaemia cells (K562), with the highest activity (78% inhibition) being observed at a concentration of 0.10% of the extract.
The Rye extract also caused a bell-shaped concentration response curve on the Hep 3B cells similar to that observed against the PLC cells. However, the extract had lower activity in the Hep 3B cells when compared to the PLC cells. The extract exhibited the highest activity of 55% at a concentration level of 0.10%.
On the breast cancer cells (MCF7), the Rye extract exhibited a steady increase of anti-neoplastic activity with increasing concentration with the highest activity of 89% observed at a concentration level of 1.0%.
When tested against the renal cancer cells (A498) the Rye extract exhibited a pattern similar to that observed in the breast cancer cells. However, the highest activity observed in the A498 of 53% was less than that observed in the breast cancer cells.
The maximal inhibitory effects the Rye extract on the different cancer cell lines are summarised in Table 1 on page 1.
DISCUSSION
The Rye extract caused a concentration related inhibition of cell proliferation in all the cancer cell lines. This inhibition was equal to or greater than that produced by commonly used cytotoxic drugs. These results demonstrate that the Rye extract has inhibitory actions comparable to the commonly used anti-neoplastic drugs. This effect is found across diverse types of cancerous cells. However, the bell-shaped concentration response characteristics observed with the Rye extract suggests that the mechanism of action differs from the reference anti-neoplastic agents. Furthermore, the observation also suggests that the agent is not simply cytotoxic to the cancer cell lines. Further studies are required to understand the mechanism of action. Initially, characterisation of the composition of this extract in all likelihood will provide whether the inhibitory activity resides in a single or a combination of compounds within the extract.
The lack of any known side effects of taking this natural remedy suggest that its potential use as an anti-neoplastic therapy is worthy of further study.
Reference
Rubin, D & Levine, S. (2001). Oralmat and HIV Disease: A report of five cases. J. Orthomolecular Medicine 16: 183-7.
Stough, C., Lloyd, J., Ryan, J. & Nathan, P. (2002) The central nervous system effects of an extract of the rye plant, Secale cereale (Oralmat): preliminary indication of an anxiolytic/sedative mode of action. Phytomedicine (submitted).
Cooper, D. M., Rogers, K. M. & Falconer, J. (2002) A double-blind placebo controlled study of a Rye based herbal extract (Oralmat™) in adult asthma; evidence of improvement demonstrated. Phytomedicine (submitted).
Mosmann, T (1983). Rapid colorimetric assay of cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55-63.
1. J. Natl. Cancer Inst (Bethesda), 51, 1409-1416, 1973.
2. Int. J. Cancer, 68, 535-564, 1996.