Indian Journal of Traditional Knowledge Vol. 12 (4), October 2013, pp. 682-687
Evaluation of some plant extracts for standardization and anticancer activity SN Gaidhani1*, Arjun Singh1, Suman Kumari1, GS Lavekar1, AS Juvekar2, S Sen2 & MM Padhi1 1
Central Council for Research in Ayurvedic Sciences, 61-65, Institutional area, Opposite ‘D’ Block, Janakpuri, New delhi; 2 Advance Centre for Treatment, Research and Education in Cancer (ACTREC), Kharger, Navi Mumbai-410 210, India E-mail:
[email protected] Received 17.11.10, revised 04.07.13
In recent times, the trend in cancer research is shifting towards identifying new medicines from natural resources for management of cancer. Medicinal plants such as Sthauneyaka (Taxus baccata L.) and compound formulations like Triphala ghrita, Khadirarista, Madhusnuhi rasayana, Maha triphaladya ghrita, Panchatikta guggulu ghrita are indicated in the Ayurvedic texts for management of cancer/ tumour. The anti-proliferative activities of hydro-alcoholic extracts of some standardized plant materials were screened against a panel of 14 human cancer cell lines representing different tissues (lung, pancreas, colon, cervix, oral, bladder, prostate, breast, leukaemia, etc.) through Sulforhodamine-B (SRB) assay. The findings revealed that Cedrus deodara (Roxb.) ex Lamb. and Berberis aristata (Roxb.) ex DC. have maximum anticancer activity against 3 cell lines while Withania somnifera Dunal. showed activity against two cell lines. In addition to these, Picrorhiza kurroa Royle ex Benth. and Piper longum L. were found active against only one cell line. These results indicate the potential of Ayurvedic medicinal herbs as anti-neoplastic agents mentioned in the Ayurvedic texts. However, further studies are needed for evaluating their mechanism of action and to isolate the active anticancer compounds responsible for this activity. Keywords: Medicinal plants, Standardization, Anticancer activity IPC Int. Cl.8: A61K 36/00, A01D 4/04, A01D 4/34
In Present scenario, Cancer is one of the greatest killers worldwide and is spreading promptly. The study of Ayurvedic classics has revealed that the symptomatology of the disease entity Arbuda can be correlated to that of tumor or cancer. The most out standing symptom is that of a swelling which continuously goes on increasing in size but never reaches to the stage of suppuration until and unless complicated by superimposed infection. This swelling is circular, immovable, slightly painful, slowly growing and broad based1. According to Ayurvedic literature three humors, Vata (air), Pitta (fire) and Kapha (water), mutually coordinate to perform the normal function of the body. In benign tumour (Vataja, Pittaja or Kaphaja) one or two of the three humors/ systems are out of control and therefore not too harmful. Malignant tumours are very harmful because all the three major humors/ systems lose mutual coordination and therefore a failure of regulation is exhibited which results into a deadly morbid condition2. In medical science the methods available to treat a cancer patient mainly includes surgery, chemotherapy and radiotherapy, etc. As these ______________ *Corresponding author
known methods are very costly and have side effects with limitations of their use, there is need of effective and acceptable cancer therapeutics agents that should be non-toxic, highly efficacious against multiple cancers, palatable, cost effective and acceptable by human population. In India, records indicate that herbs have been used for treating disease since ancient times. Some of popularly used plants such as Withania somnifera Dunal., Picrorhiza kurroa Royle ex Benth., Cedrus deodara (Roxb.) ex Lamb., Berberis aristata (Roxb.) ex DC. and Piper longum L. have been used in the Indian traditional medicine system for centuries for the treatment of various ailments including cancer. Withania somnifera is an erect, evergreen, tomentose shrub found throughout the drier parts of India in waste places and sometimes cultivated. Roots of Withania somnifera are stout, fleshy and whitish brown in colour. It is mainly prescribed for hiccough, female disorders, asthma, ulcer, scabies, cough, rheumatism and dropsy. It can be used as a sedative in the cases of senile debility3. Withanolides, the active constituents of Withania somnifera may act as immunomodulator having anticancer activity. Withaferin-A showed marked tumour inhibitory
GAIDHANI et al.: EVALUATION OF PLANT EXTRACTS FOR STANDARDIZATION AND ANTICANCER ACTIVITY
activity against cells derived from human carcinoma of nasopharynx (KB)4 and also acts as a potent antiproliferative activity against pancreatic cancer cell5. Many reports indicate that Withania somnifera decreases the levels of the nuclear factor kappa B, suppresses the intercellular tumor necrosis factor and potentiates apoptotic signalling in cancerous cell lines6. Similarly Piper longum, used as an ingredient of Ayurvedic anticancer formulations, has antioxidant potency in both in-vitro and in-vivo conditions7. Piper longum is a slender aromatic climber with perennial woody roots occurring in the hotter parts of India, from central Himalayas to Assam, lower hills of Bengal and evergreen forests of western Ghats. Fruits of Piper longum attributed with numerous medicinal uses for diseases of respiratory tract like cough, bronchitis, asthma, epilepsy and insomnia. It is generally used as tonic and haematinic8. Many reports also suggested the anticancer potential of Piper longum9,10. Cedrus deodara is a large evergreen tree upto of 60 m height and 10mm girth, found throughout the western Himalayas. It is the strongest of Indian coniferous woods and about as strong as Teak. The wood is oily, strong and characteristically scented, light, straight, fairly even grained and medium fine textured. It possesses diuretic, diaphoretic and carminative properties especially useful in fever, pulmonary and urinary disorder. It is used in many Ayurvedic preparations like Mandoor Vataka and Bhadradarvadi taila prescribed for anorexia, oedema, piles, diabetes, leprosy and sciatica8. The lignan mixture from Cedrus deodara stem wood has cytotoxic potential in-vitro, against human cancer lines from different tissues such as breast, cervix, colon, liver and prostate. It was also found to induce tumor regression in-vivo, induction of intracellular DNA fragmentation and DNA cell cycle analysis11. Picrorrhiza kurroa is a more or less hairy perennial herb with an elongated stout, creeping rootstock abundantly found in the alpine Himalayas from Kashmir to Sikkim. Roots of the Picrorrhiza kurroa have wide range of medicinal properties and considered as a bitter tonic. Roots consist of cylindrical, deep greyish-brown, longitudinally wrinkled with annulations at the tip. It is antiperiodic, cholagogoue, stomachic, laxative in small doses and cathartic in large doses8. Picrorrhiza kurroa and Withania somnifera may be used as immunomodulator along with therapeutic agents as a
683
supportive therapy. Berberis aristata is a shrub of 1.8-2.4 m in height and armed with trifid spines. It is commonly found in Himalayas from Himachal Pradesh to Assam, Bihar and some parts of Madhya Pradesh. The roots of Berberis aristata are mildly laxative and reported to possess anticancer activities12. Since all these plants were showed some anticancer activity with different extracts and in different cell lines as per the references and based on Ayurvedic literature, these plants were chosen for the present study. There are many works on isolates of these plants. However, no study on the standard hydroalcoholic extracts of plants has been reported. In order to identify the anecdotal claims, the anticancer activity of these plant extracts by using different cell lines has been investigated. In Ayurvedic treatment some drugs such as Asva/Arista are used which is aqueous/alcoholic extraction by fermentation. On this view, as per Ayurvedic Pharmacopoeia of India in recent trend, extracts of medicinal plants are recommended to enhance the potency and reduction in bulk of dosage form, convenience in administration of doses and acceptability by improving palatability13. Further, it is observed that major of phyto-chemical content may be available in hydro-alcoholic extract (60:40) as compared to the other solvents so that it has been selected for the study13. Materials and methods Drug procurement and authentication
The hydro-alcoholic extracts were prepared from botanically identified and authenticated raw plant materials, i.e. roots/rhizome of Withania somnifera Dunal. , Picrorhiza kurroa Royle ex Benth. , Berberis aristata (Roxb.) ex DC. , wood of Cedrus deodara (Roxb.) ex Lamb. and fruits of Piper longum L. Preparation of standardization
hydro-alcoholic
extract
(60:
40)
and
Fourty per cent ethanol was added to the coarse powdered samples in a ratio of 4:1 and was macerated the mixture for 4 hrs. Then, the mixtures were heated for 2 hrs at 80°C and the steps were repeated 3 times. Each time sufficient quantity of 40% ethanol was added when required. Then, the extract was filtered and concentrated under vacuum. After vacuum tray drying at 70-80°C for 14-16 hrs the dried lumps were milled up to the particle size of 40 mesh. The extracts were analyzed and standardized as per API/WHO guidelines.
684
INDIAN J TRADITIONAL KNOWLEDGE, VOL. 12, No. 4, OCTOBER 2013
SRB assay14
The anticancer activities of extracts were studied at Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Mumbai where 14 cell lines were maintained in ideal laboratory conditions. The cell lines were grown in RPMI 1640 medium containing 10% fetal bovine serum and 2mM L-glutamine. For present screening experiment, cells were inoculated into 96 well microtiter plates 90 µL/well at appropriate plating densities, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37°C, in 5% CO2, 95% air and 100% relative humidity for 24 hrs prior to addition of experimental drugs. After 24 hrs, cells from one plate of each cell line were fixed in- situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental extracts were solubilised in appropriate solvent at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to 10 times the desired final maximum test concentration with complete medium containing test article at a concentration of 100, 200, 400 and 800 µg/ml. Aliquots of 10 µl of these different dilutions were added to the appropriate micro-titer wells already containing 90 µl of cell suspension, resulting in the required final drug concentrations of 10, 20, 40 and 80 µg/ml. For each of the experiments, a known anticancer drug Adriamycin (Doxorubicin) was used as a positive control. Endpoint measurement
After compound addition, plates were incubated at standard conditions for 48 hrs and assay was terminated by the addition of cold TCA. Cells were fixed in-situ by the gentle addition of 50 µl of cold 30% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant was discarded; the plates were washed 5 times with tap water and air dried. Sulforhodamine B (SRB) solution (50 µl) at 0.4% (w/v) in 1% acetic acid was added to each of the wells, and plates were incubated for 20 minutes at room temperature. After staining, unbound dye was recovered and the residual dye was removed by washing 5 times with 1% acetic acid. The plates were air dried. Bound stain was subsequently eluted with 10 mM Trizma base, and the absorbance was read on an Elisa Plate Reader at a wavelength of 540
nm with 690 nm reference wavelength. Per cent growth was calculated on a plate-by-plate basis for test wells relative to control wells. Per cent growth was expressed as the ratio of average absorbance of the test well to the average absorbance of the control wells * 100. Using the 6 absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug at the 4 concentration levels (Ti)]; the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition was calculated as: [(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz (Ti-Tz) positive or zero [(Ti-Tz)/Tz] x 100 for concentrations for which Ti80 >80 >80 51 >80 18
44 >80 >80 59 >80 80 >80 >80 77 >80 43
>80 >80 75 46 52 10
54 >80 >80 72 46 80 >80 >80 >80 >80 30
75 >80 70 34 79 80 >80 >80 >80 >80 35
>80 >80 >80 57 >80 26
>80 >80 >80 >80 41 38
Table 4 Lethal concentration values (LC50) (µg/ml) Name of the drugs B. aristata P. longum P. kurroa C. deodara W. somnifera Adriamycin (Doxorubicin) positive control
Colo 205 Hop 62 HT 29 SiHa MIA-PA-CA -2 DWD T24 PC3 >80 >80 >80 >80 >80 38
>80 >80 >80 >80 >80 48
>80 >80 >80 >80 >80 >80
>80 >80 >80 >80 >80 49
>80 >80 >80 74 >80 50
concentration of plant extract17. In the present investigation, Adriamycin (Doxorubicin) served as positive control and showed 100% anticancer activity against all the 14 cell lines. M. Soriful Islam, et al. (2009) has also observed similar phenomenon18. Our findings support the reported therapeutic use of these plant extracts as a anticancer agent in Ayurveda which will be useful to integrate for improving modern cancer care. Further experimental analyses of these plants are needed, to obtain more detail mechanism of action for development of new drug which may be useful in the treatment and/or prevention of cancer which may be boon for the society. This study will also be useful to the other researchers to take forward the references for further scientific evaluation of anticancer activity. Conclusion Five plant extracts for studying anti-cancer activity based on available literature have been selected randomly and found that all the plants having activity against specific cell lines, viz. prostrate (PC3), ovary (A2780), breast (MCF7), oral (DWD), lungs (Hop62) and colon (Colo205). Present study implicit the observation that P. kurroa and P. longum showed a promising anti-cancer activity against only one cell line and may be active against the other cell lines. However, other plants extracts showed their activity
71 >80 >80 >80 >80 42
>80 >80 >80 >80 >80 >80 >80 77 >80 79 >80 44
A549 ZR-75-1 A-2780 DU-145 MCF7 K562 >80 >80 >80 >80 88 37
>80 >80 >80 >80 >80 65
>80 >80 >80 63 >80 39
>80 >80 >80 >80 >80 57
>80 >80 >80 >80 >80 59
>80 >80 >80 >80 76 67
more than one cell lines by inhibiting the specific cancer indicating need to investigate underlying mechanism and responsible active principle by which this activity was exhibited. Further, all these plants extract need to be screened against different cell lines apart from the selected cell lines to confirm the activity. References 1 Shastri AD, Sushrut Samhita,Nidan Sthan, Part- I, 11th edn, (Choukhamba Sanskrit Sansthan , Varanasi), 1997, 272-273. 2 Bhishagrantha KL, Sushruta Samhitha, (Choukhamba Orientalia, Varanasi), 1991. 3 Wealth of India-Raw material series, Vol. X, (Publication & Information directorate, CSIR, New Delhi), Reprint 1982, 581-585 4 Rao GV, Kumar S, Islam M & Mansour SE, Folk medicines for anticancer therapy-a current Status, Cancer Therapy, 6 (2008) 913-922. 5 Yu Y, Hamza A, Zhang T , Gu M, Zou P, Newman B, Li Y, Gunatilaka AA, Zhan CG & Sun D, Withaferin-A targets heat shock protein 90 in pancreatic cancer cell, Biochem Pharmacol, 79(4) (2010) 542-551. 6 Ichikawa H, Takada Y, Shishodia S, Jayaprakasam B, Nair MG & Aggarwal BB, Withanolides potentiate apoptosis, inhibit invasion, and abolish osteoclastogenesis through suppression of nuclear factor-kappaB (NF-kappaB) activation and NF-kappaB-regulated gene expression, Molecular Cancer Therapeutics, 5(6) (2006) 1434-1445. 7 Koul IB & Kapil A, Evaluation of the liver protective potential of Piperine, Planta Med, 59 (1993) 413-417.
GAIDHANI et al.: EVALUATION OF PLANT EXTRACTS FOR STANDARDIZATION AND ANTICANCER ACTIVITY
8 Wealth of India-Raw material series, Vol. III, (Publication & Information directorate CSIR, New Delhi), Reprint 1982, 49, 96-98, 400-408. 9 Sunila ES & Kuttan G, Immunomodulatory and antitumor activity of fruits of Piper longum L. and Piperine, J Ethnopharmacol, 90(2-3) (2004) 339-346. 10 Selvendiran K & Sakthisekaran D, Chemopreventive effect of piperine on modulating lipid peroxidation and membrane bound enzymes in benzo (a) pyrene induced lung Carcinogenesis, Biomed Pharmacother, 58(4) (2004) 264-267. 11 Singh SK, Shanmugavel M, Kampasi H., Singh R, Mondhe DM., Rao JM, Adwankar MK, Saxena, AK & Qazi GN, Chemically standardized isolates from Cedrus deodara stem wood having anticancer activity, Planta Med, 73 (2007) 519-526. 12 Wealth of India-Raw material series, Vol. II, (Publication & Information directorate CSIR, New Delhi), 1988, 117.
687
13 The Ayurvedic pharmacopoeia of India, Ministry of health and family welfare, Govt. of India, New Delhi, 1st edn, Part III, 2011. 14 P Skehan, R Storeng, D Scudiero, A Monks, J McMahon, D Vistica, JT Warren, H Bokesch, S Kenney & M R Boyd, New Colorimetric Cytotoxicity Assay for Anticancer-Drug Screening, J Nat Cancer Inst, 82 (1990) 1107. 15 The Ayurvedic pharmacopoeia of India, Ministry of health and family welfare, Govt. of India, New Delhi, 1st edn, Part 1, 1990. 16 Quality control methods of plant material, WHO Geneva, 1998. 17 Hussain, Cytotoxic and antitumor potential of Fagonia cretica L., Turkish J Biol, 31 (2007) 19-24. 18 Islam MS, Antitumor and phytotoxic activities of leaf methanol extract of Oldenlandia diffusa (Willd.) Roxb., Global J Pharmacol, 3(2) (2009) 99-106.
