Indian Journal of Traditional Knowledge Vol. 16 (1), January 2017, pp. 141-145
Evaluation of anticancer and antimicrobial activities of selected medicinal plants of Kashmir Himalayas, India Saleem Mushtaqa,b, Qazi Parvaiz Hassana,*, Rashmi Sharmac, Rabiya Majeedd, Abid Hamid Dard, Phalisteen Sultana, Inshad Ali Khanc, Sheikh Abid Alia & Md Niamat Alib a
Biotechnology Division, CSIR - Indian Institute of Integrative Medicine, Sanatnagar, Srinagar- 190005, India; b Centre of Research for Development (CORD), University of Kashmir, Srinagar- 190006, India; c Clinical Microbiology Division, CSIR- Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India; d Cancer Pharmacology Division, CSIR - Indian Institute of Integrative Medicine, Canal Road, Jammu-180001, India E-mail:
[email protected]. Received 03 February 2016, revised 09 August 2016 Medicinal plants are known for their multifarious use in folk medicine of Kashmir Himalayas, India. The present study was designed to evaluate the anticancer and antimicrobial activities of extracts of some medicinal plants. Three different types of extracts were prepared by cold maceration technique using methanol, hexane and water. Anticancer activity of extracts was determined by using Sulpharhodamine B assay on four human cancer cell lines. Antimicrobial activity was done by using microdilution method. The methanol extract of Euonymus hamiltonianus exhibited broad spectrum anticancer activity with IC50 value of 20, 14, 27 and 55µg/ml against Lung A-549, Colon HCT-116, Pancreatic MiaPaca and Breast MCF-7 cell lines, respectively. The lowest IC50 value of 14µg/ml was observed against Colon HCT-116 cancer cell line. Colorectal cancer is the second leading cause of cancer deaths worldwide. The methanol and aqueous extracts of Euphorbia wallichii specifically inhibited the growth of Staphylococcus aureus which is one of the foremost pathogens in the list of antibiotic resistant strains. The MIC values were found to be 128-256µg/ml. Euonymus hamiltonianus and Euphorbia wallichii with promising activities could act as important source of biologically active compounds. Keywords: Euonymus hamiltonianus, Euphorbia wallichii, SRB assay, Microdilution method IPC Int. Cl.8: A61K 36/00, A01D 4/04, C12P, C12M, C12N
Medicinal plants have been used since time immemorial for the treatment of different ailments and continue to provide front-line pharmacotherapy for millions of people worldwide. About 80% of the population in developing countries depends on medicinal plants for their primary health care needs1. Medicinal plants are an essential part of alternative and complementary medicine2. They have wellestablished history, unique chemical diversity and appealing biological properties3. Drugs derived from medicinal plants have an important advantage as they offer a stable market throughout the world4. With the rapid increase in drug resistance, there is an urgent need to discover new compounds with diverse chemical structures and novel mechanisms of action5,6. Drug discovery from medicinal plants has played a crucial role in the treatment of cancer. Anticancer agents from plants currently in clinical —————— *Corresponding author
use can be categorized into four main classes of compounds: vinca alkaloids, epipodophyllotoxins, taxanes, and camptothecins7. Natural products also continue to be the primary source for antibacterial drug discovery as 66% of all the drugs currently approved as antibacterial agents are natural products or natural product derivatives8. The high altitude areas of Kashmir Himalayas, India with their rich biodiversity harbor about 2000 plant species9. Many of these plant species are still used traditionally by rural people in the treatment of various diseases. These medicaments are used in different forms such as decoctions, pastes and powders. However, it is important to obtain a scientific validation for the possible use of these herbal drugs in the treatment of various diseases. Here, the anticancer and antimicrobial activities of ten selected medicinal plants of Kashmir Himalayas, India were evaluated. These belong to nine different families.
INDIAN J TRADIT KNOWLE, VOL. 16, NO. 1, JANUARY 2017
142 Materials and methods
Anticancer activity
Plant materials Different medicinal plants were collected from various high altitude regions of Kashmir Himalayas, India like Gulmarg, Gurez, Budgam, etc., situated at an altitude of around 5000-8000 feet. A summary of the ethno-botanical data of these plants is given in Table 1. Plant specimens were deposited in the Departmental herbarium. The plant names have been verified with the www.plantlist.org.
Chemicals RPMI-1640 medium, Streptomycin, Fetal bovine serum, Mitomycin, Sodium bicarbonate, Phosphate buffer saline (PBS), Trypsin and 5-Fluorouracil (5-FU) were purchased from Sigma chemical Co (MO, USA). Glacial acetic acid was purchased from Fisher Scientific (Mumbai, India) and Trichloroacetic acid (TCA) from Merck specialties Private Ltd.
Preparation of extracts Different plant parts such as roots, shoots and leaves were shade dried and pulverized into powder using a mechanical grinder. Three different types of extracts, hexane, methanol and water were prepared using the cold maceration technique. The plant material was submerged in the respective solvents for 72 hrs at room temperature with regular shaking and filtered through Whatman No 1 filter paper. The residue was macerated twice with the same solvent overnight and filtered. The filtrates obtained from each extraction were mixed and concentrated under reduced pressure at 45–50 °C by using rotavapor. The extracts obtained were kept at 4 °C for further use.
Cell culture, growth conditions and treatment Different human cancer cell lines: breast cancer cell line (MCF-7), colon cancer cell line (HCT-116), pancreatic cancer cell line (MiaPaCa) and lung cancer line (A-549) were obtained from American Type Culture Collection (ATCC, Sigma). All the cell lines were grown in RPMI-1640/MEM medium containing 10% FCS, 100 U/ml Penicillin, 100 µg/ml Streptomycin and sterilized by filtering through 0.2 µm filter in laminar air flow hood. Cells were allowed to grow in CO2 incubator (Thermo Scientific USA) at 37 ºC with 98% relative humidity and 5% CO2 gas environment. Sulpharhodamine B assay for % growth inhibition The cytotoxic property of different extracts of eight medicinal plants was determined by Sulpharhodamine
Table 1—Ethnobotanical data of plant species screened for anticancer and antimicrobial activities Name of the plant species; Family; Local name Aconitum heterophyllum Wall. ex Royle; Ranunculaceae; Patis, Paewakh Artemisia maritima L.; Compositae; Murin Berberis lycium Royle; Berberidaceae; Kawdach Euonymus hamiltonianus Wall.; Celastraceae; Shoorkul, Sheelkul Euphorbia wallichii Hook.; Euphorbiaceae; Heerab Ficus carica L.; Moraceae; Anjeer
Ligustrum lucidum L.; Oleaceae; Privat kul Saussurea costus (Falc.) Lipsch.; Compositae; Kuth Viburnum grandiflorum Wall. ex DC.; Adoxaceae; Kulmanh Viscum album L.; Santalaceae; Ahul
Plant parts tested Root
Site of collection Gulmarg
Root
Gurez
Leaves
Budgam
Berries
Budgam
Root Aerial Fruit
Gurez Budgam
Berries
Srinagar
Root
Gurez
Berries
Gurez
Shoot
Budgam
Solvent used
Traditional uses
Methanol Water
Roots stored by nomadic tribes and used for different medicinal purposes17.
Hexane Methanol Water Hexane MethanolWater Methanol Water
The water extract is used against skin eruptions as external use17. Paste prepared from fresh fruits is used to heal wounds18. The plant species of this family have cytotoxic, antitumor and immunosuppressive activity19.
Hexane Methanol Water Methanol
Used as a folk medicine against skin problems17.
Hexane MethanolWater Water Hexane Methanol Water Hexane Methanol Water
Dried fruit decoction is given against abdominal pain, cold, asthma, constipation, jaundice, kidney and gallbladder stones, indigestion, liver enlargement and as a blood purifier20. Used against various infectious diseases in Chinese traditional medicine21. Bitter rhizome powder is used to treat joint pains, back pain, sole ulcers, dysentery and ever18. Used in the treatment of typhoid and whooping cough18. Used against hypertension and cancer17.
MUSHTAQ et al.: ANTICANCER AND ANTIMICROBIAL ACTIVITIES OF SELECTED MEDICINAL PLANTS
B (SRB) assay10. In the present study, all the cell lines seeded in flat bottomed 96-well plates were allowed to adhere overnight in a CO2 incubator set at 37 ºC. Different plant extracts were added to the wells at 50 µg/ml concentration followed by incubation for 48 hrs. After incubation period of 48 hrs, the cell growth was stopped by adding 50 µl of chilled 50% TCA to each well and plates were further incubated at 4 ºC for an hour to fix the cells attached to bottom of the wells. The plates were washed five times with distilled water and air dried. The cells were stained with 100 µl per well SRB dye (0.4% w/v SRB in 1% acetic acid) and the plates left at room temperature for 30 min. Then plates were washed with 1% acetic acid to remove unbound SRB and allowed to dry overnight. 100 µl of 10mM Tris base was added to each well to solubilize the SRB dye and stirred for 5 min at room temperature. The optical density was measured on ELISA reader at 570 nm. Further the IC50 values of the cancer cells were determined by non-linear regression analysis using Graph Pad Software (2236 Avenida de la Playa La Jolla, CA 92037, USA). Antimicrobial assays Bacterial strains and culture conditions Antibacterial and antifungal activities of different extracts of ten medicinal plants were performed using microdilution method as per the guidelines of Clinical and Laboratory Standards Institute11 against two bacterial strains (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213) and two fungal strains (Candida albicans ATCC 90028 and Aspergillus fumigatus MTCC 1811).
Minimum inhibitory determination
concentration
(MIC)
Antibacterial testing was performed in Muller Hinton Broth (MHB) whereas for antifungal testing Roswell Park Memorial Institute (RPMI) supplemented with 0.165 M MOPS was used. The Minimum Inhibitory Concentration (MIC) of the plant extracts was determined by serial two fold diluting the compounds in the above mentioned media in 100µl volume in a 96 well U bottom microtitre plate. The bacterial and fungal suspension of the overnight grown bacterial and fungal culture was prepared in sterile normal saline and the density was adjusted to 0.5 and 0.8 Mcfarland, respectively. The bacterial cultures were further diluted and added in 100µl volume at a final inoculum of 1 x105 CFU/ml. For
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fungal cultures 1 x 103 CFU/ml was used. The plates were incubated at 37 oC for 24 hrs for bacterial cultures and 28 oC for 48 hrs for fungal cultures. The plates were read visually and the minimum concentration of the plant extracts showing no turbidity was recorded as MIC. Results and discussion Anticancer activity
Different extracts of eight medicinal plants evaluated for anticancer activity varied in their efficiency and selectivity against the four human cancer cell lines tested, viz. Lung A-549, Breast MCF-7, Pancreatic MiaPaca and Colon HCT-116. The primary screening results revealed that different extracts of Aconitum heterophyllum, Ligustrum lucidum, Ficus carica, Euphorbia wallichii and Euonymus hamiltonianus exhibited greater than 50% inhibition at 50 µg/ml concentration against one or more cancer cell lines tested as shown in Table 2. Rest of the extracts were found to be inactive. The methanol extract of the berries of Euonymus hamiltonianus Wall showed highly significant anticancer activity against the tested cancer cell lines. The IC50 values for the methanol extract of Euonymus hamiltoninaus were found to be 20, 14, 27 and 55µg/ml against Lung A-549, Colon HCT-116, Pancreatic MiaPaca and Breast MCF-7, respectively as shown in Fig. 1. 5-FU and adriamycin were used as the positive controls. According to US National Cancer Institute, a crude extract is generally considered to possess in vitro cytotoxic activity if its IC50 value is less than 20µg/ml12. Therefore, the methanol extract of Euonymus hamiltonianus Wall with IC50 value of 14 µ g/ml against colon cancer cell line HCT-116 can be considered as a promising anticancer botanical. Colorectal cancer is the third leading cause of cancer related deaths in both men and women and the second leading cause of cancer deaths when men and women are combined13. Euonymus species are well known for various biological activities, such as antitumor, antidiabetic, insecticidal, anti-inflammatory and antimicrobial14. However, no data is available on the anticancer constituents of Euonymus hamiltonianus Wall. Antimicrobial activity
Out of the ten medicinal plants evaluated for antimicrobial activity, only the methanol and aqueous extracts of Euphorbia wallichii Hook showed
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Table 2—Anticancer activity (% growth inhibition) of different samples at 50 µg/ml concentration against various human cancer cell lines Plant species Aconitum heterophyllum Wall. ex Royle
Berberis lycium Royle Euonymus hamiltonianus Wall. Euphorbia wallichii Hook. Ficus carica L. Ligustrum lucidum L.
Viburnum grandiflorum Wall. ex DC.
Viscum album L.
Standard drugs
Plant Parts tested Root Root Aerial
Extract Methanol Water Methanol Methanol Aerial Water Hexane Methanol Berries Water Aerial Methanol Aerial Water Root Methanol Root Water Root Methanol Hexane Berries Water Methanol Methanol Berries Hexane Water Water Aerial Hexane Methanol 5-FU (20µM) Adriamycin (1µM)
Lung A-549 35 12 36 4 9 0 81 22 75 27 61 62 36 87 0 0 0 5 0 0 0 0 67 -
Breast MCF-7 29 0 0 26 27 34 46 0 10 84 34 28 67 0 25 0 31 0 0 19 0 14 85
Pancreatic MiaPaca 65 16 0 5 36 51 73 47 16 40 53 27 28 16 34 0 23 1 0 0 9 26 -
Colon HCT-116 56 0 0 0 20 72 0 0 50 24 0 33 52 1 0 32 0 0 0 5 21 71 -
Table 3—Minimum inhibitory concentrations (µg/ml) of different extracts of Euphorbia wallichii Hook against bacterial and fungal pathogens determined by micro dilution method. Plant parts used
Type of solvent used
S. aureus E. coli C. albicans A. fumigates ATCC 29213 ATCC 25922 ATCC 90028 MTCC 1811 Root Hexane NA NA NA NA Methanol 128 NA NA NA Water 256 NA NA NA Aerial Hexane NA NA NA NA Methanol NA NA NA NA Water 256 NA NA NA Ciprofloxacin 0.125 0.007 Amphotericin B 0.5 0.5 Note: S. aureus = Staphylococcus aureus ATCC 29213, E.coli = Escherichia coli ATCC 25922, C. albicams = Candida albicans ATCC 90028 and A. fumigatus = Aspergillus fumigatus MTCC 1811. NA= Not active.
promising antimicrobial activity. Both the aerial and underground parts of Euphorbia wallichii inhibited the growth of Staphylococcus aureus. The MIC values were found to be in the range of 128-256 µg/ml. The antimicrobial activities of plant extracts were compared with standard antibiotics, viz. Ciprofloxacin and Amphotericin B, which were used as positive controls. The MIC values are given in Table 3. The results of this study are encouraging as Staphylococcus aureus is one of the foremost
pathogens in the list of antibiotic resistant strains causing both community and hospital-acquired infections. The emergence of S. aureus resistance to even last-line antibiotics heightens the need for the development of new drugs with novel targets15. In this context, the antimicrobial activity of Euphorbia wallichii is interesting as the MIC values of the active samples in the present study lie well below 1mg/ml which can be considered as significant in view of the development of
MUSHTAQ et al.: ANTICANCER AND ANTIMICROBIAL ACTIVITIES OF SELECTED MEDICINAL PLANTS 4
5
6
Fig. 1—Anticancer activity (% growth inhibition) of Euonymus hamiltonianus Wall at 50 µg, 30 µg, 20 µg, 10 µg and 5 µg concentration against different human cancer cell lines
strong antimicrobials from plants16. Although, several macrocyclic diterpenoids with antibacterial, antimultidrug-resistant, prolyl endopeptidase inhibitory, antifeedant, anti-HIV and analgesic activities have been isolated from different Euphorbia species17, there are hardly any reports on the antimicrobial constituents of this particular species, i.e., Euphorbia wallichii Wall. Conclusion The above results show that Euonymus hamiltonianus and Euphorbia wallichii with promising biological activities could act as important source of bioactive compounds. The anticancer activity of Euonymus hamiltonianus and antimicrobial activity of Euphorbia wallichii could be due to the presence of diterpenoid compounds. However, this requires further phytochemical and pharmacological studies. Acknowledgement The first author is grateful to University Grants Commission (UGC), New Delhi for providing him fellowship. The authors are thankful to Dr. Ram A. Vishwakarma (Director) Indian Institute of Integrative Medicine, for providing the necessary facilities during this period. References 1
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