PROPOSAL SUBMITTED TO BE SPONSORED TO ENGAGE IN MENTORED RESEARCH IN THE 2016/2017 (YEAR 2) ROUND OF THE STAMINA GRANT
TITLE: STUDIES ON THE POTENTIAL ANTICANCER ACTIVITY OF TWO PLANTS - ADENODOLICHOS PANICULATUS AND BOSWELLIA DALZIELII.
MENTOR
Name: Prof. ALEMIKA, Taiwo Emmanuel
Department: Pharmaceutical Chemistry
Area of Specialization: Natural Product Chemistry
Telephone: 08037013057
E-mail Address: alemikat@unijos.edu.ng OR alemikat2@yahoo.com
MENTEE / APPLICANT
Name: Mr. OJERINDE, Olalekan Stephen
Department: Pharmaceutical Chemistry
Area of Specialization: Natural Product Chemistry
Telephone: 08036135408
E-mail Address: ojerindeo@unijos.edu.ng OR lekstep2000@yahoo.com
ESTIMATED BUDGET FOR THE PROJECT = #1,959,321.00
STUDIES ON THE POTENTIAL ANTICANCER ACTIVITY OF TWO PLANTS - ADENODOLICHOS PANICULATUS AND BOSWELLIA DALZIELII.
ABSTRACT
Adenodolichos paniculatus Hua & Hutch (Leguminosae) is a plant whose root is used by traditional medicine practitioners in Nigeria to treat various ailments while Boswellia dalielii Hutch (Burseraceae) is a plant whose stem bark is being used by traditional medicine practitioners to treat early stage of prostrate cancer. Preliminary clinical evaluation and anticancer screening of these plants tend to support the healers' claims. Cancer has been a major public health burden in both developed and developing countries. Cancer is the leading cause of death in the United States where one in four deaths is due to cancer. The situation in Nigeria and other Sub-Saharan countries is not much different. The proposed study aims at isolating and characterizing the anticancer constituents of these plants. The plants will be extracted by percolation and decoction methods using 70% methanol & water respectively. Thereafter, the extracts will be subjected to a bioassay-guided fractionation using standard chromatographic procedures. Active compounds which are expected to be isolated will be characterized by chemical and spectroscopic techniques (UV, IR, NMR, MS). Anticancer activity screening test using Sulforhodamin B (SRB) assay which is a well established in vitro method for cytotoxicity against cell lines will serve as the main bioassay. The chemistry of the plants will also be investigated in greater detail, using the activity-guided approach, for which general bioassay methods such as the antioxidant (DPPH, ABTS radical cations, FRAP and hydroxyl radical scavenging assays) and Brine Shrimp lethality test (Cytotoxicity test) will be used. The study is therefore expected to provide potential drug compounds which could be useful in the treatment of cancer and also to contribute to the knowledge of the chemistry of the plants being investigated. Thus the isolation and characterization of bioactive compounds from these plants, leading to a greater understanding of their chemistry would constitute the short term goals of the study. Considering the unpleasant side-effects of most chemotherapeutic anticancer agents in current use, there is a need to source for newer compounds. Besides, plants have traditionally served as sources of new chemical templates, it is logical to look in this direction. Therefore, in the long term, this study is expected to contribute to the search for newer drug molecules of chemical templates that may modified to produce drugs useful in the treatment of cancer.
BUDGET FOR CHEMICALS REQUIRED FOR RESEARCH.
|
S/N |
Description |
|
Qty. |
Unit Price N |
Total Price N |
|
A |
Collection of plant materials |
|
|
|
20,000.00 |
|
B |
Chemicals for extraction |
|
|
|
76,000.00 |
|
C |
Chemicals for fractionation |
|
|
|
108,000.00 |
|
D |
Further fractionation via Chromatography |
|
|
|
|
|
01 |
Diaion HP - 20 |
|
1kg |
75,000.00 |
75,000.00 |
|
02 |
Sephadex LH - 20 |
|
50g |
152,900.00 |
152,900.00 |
|
03 |
Silica gel 60, 230 – 400 mesh |
|
1kg |
47,500.00 |
47,500.00 |
|
04 |
Reverse Phase TLC, 20 X 20 |
|
1 |
121,000.00 |
121,000.00 |
|
05 |
Normal Phase TLC, 20 X 20 |
|
1 |
34,000.00 |
34,000.00 |
|
06 |
Reverse Phase C18 Silica gel |
|
100g |
190,145 |
190,145.00 |
|
|
Sub - total |
620,545.00 |
|||
|
E |
Solvents required for fractionation via Chromatography |
|
|
|
165,800.00 |
|
F |
Reagents for TLC plates spray |
|
|
|
55,314.00 |
|
G |
In vitro anticancer assay |
|
|
|
|
|
01 |
Sulforhodamine B |
|
5g |
85,250.00 |
85,250.00 |
|
02 |
Other chemicals for the assay & cell lines |
|
|
|
275,000.00 |
|
03 |
96 well microtiter plates |
|
1 pkt |
148,580.00 |
148,580.00 |
|
|
Sub - total |
508,830.00 |
|||
|
H |
Antioxidant assays chemicals |
|
|
104,832.00 |
|
|
I |
Spectroscopic analysis (1 D & 2D NMR, HRMS, IR) |
|
|
|
300,000.00 |
|
|
GRAND TOTAL |
1,959,321.00 |
|||
Budget justification
Collection of plant materials: This is required for herbalists that would supply the plant materials, though I would accompany them to the bush for the harvesting of the materials.
Solvents and Chemicals: These are required for plant extraction, fractionation and isolation of compounds as well as for biological tests. 10 Litres of Methanol (#9500.00 per 2.5L, total is #38,000.00) and 10 Litres of Dichloromethane (#9500.00 per 2.5L, total is #38,000.00) are required for extraction while 10 Litres of hexane ( #9600.00 per 2.5L, total is #38,400.00),10 Litres of ethylacetate (#9600.00 per 2.5L, total is #38,400.00) and 10 Litres of butanol (#7,800.00 per 2.5 L, total is #31,200.00) are required for fractionation via solvent-solvent partitioning method. 10 Litres of Chloroform (#12,000.00 per 2.5L, total is #48,000.00), 10 Litres of ethylacetate (#9600.00 per 2.5L, total is #38,400.00), 10 Litres of methanol (#9500.00 per 2.5L, total is #38,000.00), 5 Litres of acetone (#6,500.00 per 2.5L, total is #13,000), 5 Litres of butanone (#7000.00 per 2.5 L, total is #14,000.00) and 5 Litres of toluene (#7,200.00 per 2.5L, total is #14,400.00) are required for the fractionation via chromatography (both column and TLC). Besides, sulforhodamine B, cell lines, buffers, 96 well microtiter plates, trichloroacetic acid, Foetal bovine serum, standards such as adriamycin, 5-fluorouracil, vinblastine are required for the in vitro anticancer assay while DPPH, ABTS, deoxy-2-ribose, TPTZ are required for the antioxidant assay.
Chromatography: Adsorbents and support of various types are needed for the chromatographic separations to be carried out. Considering the aqueous or polar nature of the extracts Diaion HP -20, Sephadex LH-20 and Reversed phase Silica gel C18 are required for the fractionation and isolation of the polar constituents from the extracts and fractions. TLC plates (Normal and reverse) are required for monitoring the separation of the compounds while silica 60, 230-400 mesh are required for some cleaning up and fractionation of non polar constituents from the hexane and ethyl acetate fractions.
Reagents for TLC plates spray: 100g of Vanillin (#13,272.00), 100g of Polyethylene glycol 4000 (#14,280.00) and 5g of 2-aminoethyl diphenylborinate (#27,762.00) are required to monitor the separation of compounds by forming colour for visualization because some compounds cannot be visualized without been derivatised.
Spectroscopic analyses: These Charges are required for acquiring NMR (1D & 2D – DEPT, COSY, HMBC, HSQC and HREIMS data of isolated compounds for elucidation of the isolated copmounds. The charges depend on the number of hour spent on each compound for all the assignments (DEPT, COSY, HMBC and HSQC)
Project Narrative
This work is expected to provide potential drug compounds or templates or lead which could be useful in the treatment of cancer. This project seeks to study the plants – Adenodolichos paniculatus and Boswellia dalzielii – with a view to isolating and characterizing the anticancer principles contained while also enhancing the knowledge of their chemistry.
RESEARCH PLAN
Specific aims
Since plants have traditionally served as sources of new chemical templates, and their therapeutic potential for the treatment of cancer has been established in Ayurvedic and other traditional medicines, it is logical to look in this direction for new cancer therapies. Therefore, the study aims at isolating, from the root extract of Adenodolichos paniculatus, and stem bark extracts of Boswellia dalzielii, compounds that would be active for cancer. Standard chromatographic methods will be employed to isolate the compounds which would then be characterized and their structures elucidated using chemical and spectroscopic techniques. Antioxidant test (DPPH, ABTS radical cations, FRAP and hydroxyl radical scavenging assays) and sulforhodamin B assays will be used to monitor the activity of extracts, fractions and isolated compounds. The entire process will be bioassay-guided. This is expected to contribute to the search for newer drug molecules or chemical templates that may be modified to produce drugs useful in the treatment of cancer.
Background and Significance
Cancer has been a leading cause of death all over the world and is still a major public health burden in both developed and developing countries. Cancer is the leading cause of death in developed countries such as United States and the second leading cause of death in developing countries[1, 2], even in Nigeria alongside with other Sub-Saharan countries, where one in four deaths is due to cancer. The burden of cancer is increasing in Nigeria and other Sub-Saharan countries due to population aging and growth as well as, increasingly, an adoption of cancer-associated lifestyle choices including smoking, physical inactivity, and “westernized diets”[3]. It was estimated that there were 14.9 million new cases, 8.2 million deaths, and 32.6 million persons living with cancer around the world in 2013 [4]. Several anticancer agents are in clinical use all over the world; different approaches have been employed and are still in use, individually or in combination, in the treatment of cancer [2] and a number of promising agents are in clinical or preclinical stages of development. These drugs are without side effects and thus searching for a new class of compound is essential to overcome these problems. Management of cancer without any side effect is still a challenge to the medical community [2]. Therefore, it is prudent to look for options in herbal medicines for cancer as well. Medicinal plants, either through systematic screening programs or by serendipity, possess desirable bioactivity. Thus they occupy an important position in the drug discovery [5]. Indeed many modern drugs have their origin in traditional medicine of different cultures [6]. Hence, despite the advantages of synthetic and combinatorial chemistry, as well as molecular modelling, medicinal plants remain an important source of new drugs, new drug leads and new chemical entities [6]. Therefore, this study will address the need for newer drugs and drug templates useful in the treatment of cancer.
Although, herbal medicine have long been used effectively in treating diseases in African communities and throughout the world, there has been increasing demand for the use of plant products with anticancer activity due to low cost, easy availability and lesser side effects. Therefore, plant materials are continuously scrutinized and explored for their effect as anticancer agents. Two of such plants are Adenodolichos paniculatus and Boswellia dalzielii the root and stem bark extracts respectively of which were found to be effective in the treatment of cancer. The study of the bioactive compounds from these plants can potentially lead to a sustainable exploitation of this natural resource.
Preliminary data
The extracts, fractions, sub-fractions, flavonoids mixture and saponins mixture had been screened both for their cytotoxicity against Brine shrimp and antioxidant activity using DPPH, ABTS radical cations, FRAP and hydroxyl radical scavenging assays and the screening tends to support the healers’ claims.
Experimental Design and Methods
Collection and Preparation of Plant Material:- The roots of Adenodolichos paniculatus and the stem bark of Boswellia dalzielii will be collected from mature shrubs in bushes around the Jos plateau, Nigeria between September and November. The material will be air-dried, chopped to small pieces and ground to coarse powder. The dried ground root and stem bark will be stored in cool, dry conditions. About 10 kg of the dry material of the plants will be required. Herbarium specimens of these plants will be prepared and deposited at the Faculty of Pharmaceutical Sciences, University of Jos and Forestry Research Institute of Nigeria for proper authentication.
Extraction:- Two solvent systems (70% aqueous methanol and water) will be used for the extraction. First the dried powdered roots and stem bark of the plants will be separately extracted by percolation with 70% aqueous methanol at room temperature and the extracts concentrated to dryness in vacuo on a rotary evaporator. This would yield crude methanolic extracts of the plants. Again portions of the dried powdered roots and stem bark of the plants will be extracted by decoction, this time by boiling in water for 60 min. and then left standing for 24 h. This is to simulate the method of application employed in tradtional medical practice. The extracts will be lyophilized and stored for further work.Thus there would be two different types of extract namely: aqueous methanolic extracts of the plants and aqueous lyophilized extracts of the plant. The different extracts will be compared for activity in the bioassay systems that would be employed. Further fractionation will be carried out on the most active type of extracts.
Fractionation and Isolation:- The most active extract will be fractionated using a modified form of the scheme developed by Gunnar Samuelsson [7]. The crude extract will be fractionated by: 1) Solvent-solvent partitioning; and 2) Gel filtration. Solvent-solvent partitioning of the extracts will be carried out in successive fashion using hexane, ethyl acetate and n-butanol. This would yield fractions of low polarity (hexane), medium polarity (ethyl acetate) and high polarity (n-butanol). The aqueous mother liquor will also be preserved and tested. Using lipophilic gel, such as Sephadex LH-20, Diaion HP 20, the extracts will be separated into major fractions depending on molecular size.
Thereafter chromatographic separation (TLC and column) will be carried out to further fractionate and isolate compounds from active fractions. Column chromatography will entail the use of Baeckstrom Separo equipment for Accelerated Gradient Chromatography (AGC) which is a form of Medium Pressure Liquid Chromatography (MPLC) on silica adsorbent/ support (normal/ RP-18 bonded). Judicious use of this in combination with thin-layer chromatography – (both analytical and preparative) is expected to yield pure compounds. The entire fractionation process will be bioactivity-driven, the extract, fractions, subfractions and isolated compounds will be tested for biological activity.
Characterisation and Structure Elucidation:- The compounds isolated will be characterised and their chemical structures elucidated by joint application of spectroscopic techniques - UV, IR, NMR (proton & C-13; 1-D & 2-D, DEPT, HMBC, HSQC, COSY & NOESY) and HREIMS.
Pharmacology Studies:- The extracts, fractions, sub-fractions and isolated compounds will be evaluated for their cytotoxicity against A549 (non small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer cells) and HCT15 (colon cancer cells) cells in vitro using the sulforhodamin B (SRB) method [8] as well as Brine shrimp lethality assay. In addition, considering the perceived relationship between oxidative stress and various pathological conditions, antioxidant activity of extracts, fractions, sub-fractions and isolated compounds will be screened using DPPH, ABTS radical cations, FRAP and hydroxyl radical scavenging assays. The results obtained in this study will be displayed as mean ± SEM. Data analysis will be done using one-way ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism software. Values will be considered significant at P < 0.05.
- In vitro cytotoxicity assay
- Sulforhodamine B Assay (SRB)
The in vitro cytotoxicity of the extracts, fractions and isolated compounds will be determined by semiautomated assay using Sulforhodamine B (SRB). The human cancer cell lines, A549 (non small cell lung adenocarcinoma), SK-OV-3 (ovarian cancer cells) and HCT15 (colon cancer cells) cells will be seeded in 96-well culture plates at a concentration of 2 x 104 cells per well and cultured at 37oC in 5% CO2 and 90% relative humidity. After 24h of incubation, the cells will be washed twice with the growth medium, RPMI – 1640 medium which will be bufferd with bicarbonate, supplemented with 10% heat inactivated foetal bovine serum (HIFBS), pH 7.4. Thereafter, the cells will be challenged with the following concentrations of the extracts, fractions and isolated compounds (1000, 500, 250, 125, 62.5, 31.25 and 15.625μg/ml) using twofold serial dilution in 5% DMSO which will be diluted with the growth medium. The positive control or reference drug (vinblastine or adriamycin) will be employed to challenge the cells at the following concentrations 160, 80, 40, 20, 10 and 5μg/ml serially diluted using growth medium and 5% DMSO.
The plates will be incubated for 48h at 37oC in an atmosphere of 5% CO2 and 90% relative humidity. Thereafter, 50µl of chilled 50% TCA (Trichloroacetic acid) will be added gently to each well of the plates, making a final concentration of 10%. The plates will be incubated at 4oC for 1h to fix the cells attached to the bottom of the wells. The plates will then be washed 5 – 6 times with distilled water and thereafter air-dried. To each well, 100μl of SRB dye (0.4% wt/vol in 1% acetic acid) will be added and left at room temperature for 30 min. Thereafter, the plates will be washed with 1% acetic acid. The plates will be air-dried again and 100µl of Tris base solution (10mM, pH 10.5) will be added to each well. The plates will be shaken gently for 10 -15min on a mechanical shaker for 5min. Blank wells will contain growth medium with no cells or test samples; the negative control wells will contain growth medium and cells while the test samples blank wells will contain growth medium with test sample but no cells. All these experiments will be done in triplicates. The optical density (OD) of the plates will be recorded with a microplate reader at 540nm. The percentage of cell – growth inhibition will be calculated using the expression below. Thereafter, IC50 of the test samples will be determined by using a plot of a dose-response curve between the test samples concentrations and percent growth inhibition. The IC50 values will be derived using curve-fitting methods with statistical analysis software, GraphPad Prism software.
All background absorbances will be subtracted from all wells.
% of control cell growth = 
% growth inhibition = 100 - % of control cell growth.
Therefore, the following outcomes will be measured during the experiments
The absorbances of the blank wells, test sample blank wells, negative control wells and test samples concentrations (1000 – 15.625μg/ml) will be recorded on microplate reader and their means will be determined for each experiment and this will be done for the three experiments (experiment in triplicate). The percent growth inhibition will be determined for each experiment and IC50 values will be expressed as Mean ± SEM for all the experiments. The same procedures will be adopted for the positive control in this experiment.
ii. Brine Shrimp lethality assay
The cytotoxicity screening of the extracts, fractions and isolated compounds will be carried out against a simple zoological organism, brine shrimp nauplii. Artemia salina leaches (brine shrimp eggs) will be placed in a small tank containing 3.8% noniodized NaCl solution (sea water) for two days to hatch the shrimp and to be matured as nauplii. 40mg of each extract, fractions and isolated compounds will be dissolved in 200ml of DMSO (dimethylsulfoxide). Then 100ml of solution will be taken into a test tube containing 5ml of sea water and 10 shrimp nauplii. Thus, final concentration of the first test tube solution will be 400mg/ml. then a series of solutions of varying concentrations will be prepared from the stock solution by serial dilution. Thus, the concentrations of the obtained solution in each test tube will be 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.778125mg/ml for 10 dilutions. 2.5mg of vincristine, the positive control will be dissolved in DMSO to get an initial concentration of 40mg/ml from which series dilutions will be made using DMSO to get 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 and 0.078125mg/ml. The positive control solutions will contain 10 living brine shrimp nauplii in 5ml sea water.
For negative control, 100ml of DMSO will added to each of the premarked test tubes containing 5ml of sea water and 10 shrimp nauplii. The vials will be maintained under illumination. Survivors will be counted after 24hours and the percentage mortality of each vial and control will be determined using the equation: % mortality = (No. of dead nauplii / initial No. of live nauplii) x 100. The concentration – mortality data will be analyzed statistically by using probit analysis in GraphPad Prism for the determination of LC50 values and linear regression for the samples. All the experiments will be done in triplicate and LC50 values will be expressed as Mean ± SEM, (n = 3)
- In vitro antioxidant activity.
The antioxidant activity (free radical scavenging activity) of the extracts, fractions and isolated compounds on the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) will be determined according to the method described in (Brand-Williams, et al., 1995). The following concentrations of test samples will be prepared 500, 250, 125, 62.50, 31.25, 15.62, 7.8125, 3.91, 1.95 and 0.98µg/mL. All the solutions will be prepared with methanol. 2mL of each prepared concentration will be mixed with 4mL of 50µM DPPH solution in methanol. Experiment will be done in triplicate. The mixture will be vortexed for 10s to homogenise the mixture and test tubes will be incubated for 30min at room temperature in the dark, after 30min of incubation the absorbance will be measured at 515nm using UV-vis spectrophotometer (Shimadzu, 1620 Japan). Gallic acid, ascorbic acid and rutin will be used as standards with the following concentrations 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.7812, 0.391 and 0.195µM. Blank solution will be prepared by mixing 2mL of methanol with 4mL of 50µM DPPH solution in methanol.
The difference in absorbance between the test and the control (DPPH in methanol) will be calculated and expressed as % scavenging of DPPH radical. The capability to scavenge the DPPH radical was calculated by using the following equation
% inhibition = 
Finally, the IC50 value, defined as the concentration of the sample leading to 50% reduction of the initial DPPH concentration, will be calculated from the separate linear regression of plots of the mean percentage of the antioxidant activity against concentration of the test extract (µg/mL) using GraphPad Prism software. The same procedures will be employed for the other antioxidant assay (ABTS radical cations, FRAP and hydroxyl radical scavenging) by determining the absorbances of the concentrations of the test samples and thereafter, their IC50 values will be expressed as Mean ± SEM, (n =3)
Outcomes Measurements and Data analysis
The mean absorbances of the different concentrations of the extracts (aqueous and methanolic), fractions and isolated compounds, the mean absorbances of the negative control, the mean absorbances of the positive control and the mean absorbances of the blank will be determined. Thereafter, these values will be employed to calculate the % growth inhibition of test samples on the cell lines by the various concentrations of test samples. The % growth inhibition values obtained will be plotted against the concentrations of the extracts, fractions and isolated compounds. Thereafdter, IC50 (50% inhibitory concentration) will be obtained from the graph using GraphPad Prism software. The same procedures will be employed for the positive control. Therefore, the mean IC50 of the extracts, fractions and isolated compounds obtained (experiment done in triplicate) will be compared with the mean IC50 of the positive control. The Analysis of Variance (ANOVA) will be employed to analyse the data at 95% level of confidence (P < 0.05 will be considered significant).This will be necessary because different extracts (aqueous and methanolic), fractions and isolated compounds will be compared with the positive control. In summary, the effects of different extracts, fractions and isolated compounds at different concentrations against cell lines will be studied in comparison with the standard drug (positive control).
For the antioxidant activity of the extracts, fractions and isolated compounds, the mean absorbances of the various concentrations of the test samples will be obtained and these values will be employed to calculate the % inhibition of the test samples. The % inhibition of test samples will be plotted against the various concentrations of the test samples whereby the mean IC50 values will be derived, the same will be compared with mean IC50 of the positive control. The Analysis of Variance (ANOVA) will be employed to analyse the data at 95% level of confidence (P < 0.05 will be considered significant). All the results will be expressed as Mean ± SEM.
Proposed experiments and laboratory Methods
The preliminary information on these plants was done by this team at the Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, University of Jos.
The research team can perform all the proposed experiments and laboratory methods at the Department of Pharmaceutical Chemistry except for the following:
- In vitro cytotoxicity assay using Sulforhodamine B
- Structural elucidation of the isolated compounds using NMR (proton & C-13; 1-D & 2-D, DEPT, HMBC, HSQC, COSY & NOESY) and HREIMS.
The techniques required to carry out the in vitro cytotoxicity assay using Sulforhodamine B of the fractions as well as isolated compounds are not well-established in my home institution and we do not have the capacity to carry out the structural elucidation and characterization of the isolated compounds using NMR (proton & C-13; 1-D & 2-D, DEPT, HMBC, HSQC, COSY & NOESY) and HREIMS. Therefore, these aspects of the study will require the intervention of the foreign laboratories to carry out these aspects of the study. Moreso, this has been built into the budget.
Future directions:- It is expected that at the end of this project, compounds possessing anticancer activity from these plants would have been isolated and characterised. Such compounds will serve as potential drug candidates or serve as templates for further modification to useful drug compounds.
References for the study.
- Park SU. Anticancer compounds from plants, EXCLI J. 2012; 11:386–389.
- Akindele AJ, Wani ZA, Sharma S, Mahajan G, Satti NK, Adeyemi OO, et al. In vitro and in vivo anticancer activity of Root extracts of Sansevieria liberica Gerome and Labroy (Agavaceae), Evid Based Complement Alternat Med. 2015, 1-11.
- Jemal A, Bray F, Center MM, Ferlay F, Ward E, and Forman D. Global cancer statistics, CA Cancer J Clin. 2011; 61(2):69–90.
- Naghavi M. The global burden of cancer 2013, Global burden of disease cancer collaboration, JAMA Oncol. 2015; 1(4): 505-527.
- Cragg GM and Newman DJ. Medicinals for the millennia: the historical record, Ann N Y Acad Sci, 2001; 953: 3–25.
- Newman DJ, Cragg GM and Snader KM. The influence of natural products upon drug discovery, Nat Prod Rep. 2000; 17(3): 219–220.
- Samuelsson G, Kyerematen G and Farah MH. Preliminary chemical characterization of pharmacologically active compounds in aqueous plant extracts, J Ethnopharmacol. 1985; 14(2-3), 193-201.
- Lee IK, Kim DO, Lee SY, Kim KR, Choi SU, Hong JK, et al. Triterpenoic Acids of Prunella vulgaris var. lilacina and their cytotoxic activities in vitro, Arch Pharm Res. 2008; 31(12): 1578-1583.
