University Department of Pharmaceutical Sciences, R.T.M. Nagpur University Nagpur, Mahatma Jyotiba Phule Shaikshanik Parisar, Amaravati road, Nagpur 440033 Maharashtra, India.
Email: prakashitankar@hotmail.com
Received: 25 Jun 2014 Revised and Accepted: 01 Aug 2014
ABSTRACT
Objective: This work aims to determine the in-vitroanti-cancer activity of biotransformed material (extract) of calendula officinalisflowers.
Methods: Various concentrations of the biotransformed material (extract) of calendula officinalis were tested. The MTT assay method was used using A549 cell lines, LN Cap-FGC cell linesand MDA-MB cell lines.
Results: The result shows promising effect of biotransformed material (extract) of calendula officinalis. The IC50 values were found to be less than10 µg against A549 cell lines, the activities against LN Cap-FGC cell lines were found to be 10 µg and IC 50 values were found to be 10 µg against MDA-MB cell lines
Conclusion: From this study it was evident that the cancer cell growth was significantly inhibited by treatment with biotransformed material (extract). Hence, in vitro studies of Calendula officinalis was found to have a potential anti-cancer activity.
Keywords: Calendula officinalis, MTT assay, A 549cell lines, LN Cap-FGC cell lines, MDA-MB cell lines, anti-cancer activity.
INTRODUCTION
A proper health care system can be established, supplying low cost medicine to population by using various medicinal plants. Medicinal plants are usually used for Ayurvedic, Unani and other treatments in rural areas. Recent discovery shows that these plants have fewer side effects than the Allopathic medicine. The medicinal importance of these plants is due to the presence of secondary metabolites present in them. Some of the important bioactive compounds are alkaloids, glycosides, triterpenoids, terpenoids flavonoids, polyphenols, reducing sugars, saponins, steroids and tannins etc. Among the plants, Calendula officinalishas been used in ayurvedic medicine and has a wide number of traditional uses including internal use for blepharitis, eczema, gastritis, minor burns including sunburns, warts and minor injuries such as sprains and wounds. Calendula flowers have been considered beneficial in reducing inflammation and wound healing. In homoeopathy, Calendula ointments are also used in the healing stages of second and third degree burns to stimulate regrowth of skin and to diminish scar formation [1].
Cancer is the abnormal growth of cells with uncontrolled division resulting in increased number of cells [2]. In spite of a number of new anti cancer drugs the prevalence of cancer has been found increased now-a-days. Cancer is considered as one of the serious disease which is characterized by abnormal or uncontrolled growth of tissues. It has become a major cause of death and there is an urgent need for its control. The most important drawback of the current cancer therapeutic practices such as chemotherapy and radiation therapy is the suppression of immune system. Since plants have been a prime source of natural products for the treatment of various diseases and they are also highly effective conventional drugs for the treatment of many forms of cancer [3-5].Calendula officinalis belonging to family Asteraceae has been widely used in homeopathic medicine for the treatment of many diseases [6]. It has been reported to possess many pharmacological activities, which include antioxidant[7], anti-inflammatory[8],antibacterial[9], antifungal[10] and antiviral activity[11]. It also possess cytotoxic as well as tumor reducing potential [12]. Literature survey revealed that Calendula officinalis, is used in traditional medicine all over the world for its antiseptic property as well as to treat certain skin disorders.
The process in which suitable modification of substrate structure is achieved by biological enzyme catalysis is termed as biotransformation.
There are the following three ways with the help of which the biotransformation process is carried out.
Ex. Bacteria or Fungi or Yeast
3. With isolated purified microbial or plant or animal cell enzyme.
In the current context, general goals of biotransformation can be listed as follows:
The first processes which were successfully accomplished by biotransformation were.
But, biotransformation gained its present status only after discovery of microbial transformations of steroids viz. Reduction of androstensdione to testosterone through yeast, 11-hydroxylation of progesterone by Rhizopusarrhizus( Peterson & Murray 1952).
In the aftermath, diverse transformations of substrates from many different groups and types of compounds were found, some of which were useful in chemosynthesis.
Very few biotransformation studies have been carried out regarding the chemical study of biotransformed products and their pharmacological evaluation. Biotransformation with alcohol, with water have been done however the biotransformation with cow urine has not been done yet. It was therefore envisaged to evaluate the biotransformation potential of Cow urine as an extraction medium and subsequent impact on the efficacy of its extract.
Microbial biotransformation employs microorganisms for achieving the desired conversion of various substrates. Microorganisms can catalyze the reactions which otherwise are difficult to carry out. The success of microbial biotransformation depends greatly on proper selection of microorganisms. The well known enrichment techniques are used for isolation of potent microbial agents. These often employ enrichment cultures obtained from soils, decomposing materials, sewage plants, river water or factory grounds etc. The sample is incubated in a growth medium containing substrate to be transformed as sole carbon source. Microorganisms employ both constitutive as well as inducible enzymes to degrade and synthesize a great variety of compounds. The discovery of the phenomenon of microbial biotransformation of complicated organic compounds provided impetus to employ microorganisms in the production of novel agents. Gradually, the methods of preparing new derivatives of known substrates were applied to modify organic compounds like Carbohydrates, steroids, sterols, terpenoids, flavonoids, alkaloids, antibiotics, amino acids etc. Such alteration encompasses many changes in the substrate moiety by the addition, degradation or modification. When basic structure of resultant molecule remains unaltered, the microbial biotransformation can be applied for formation of desired derivatives of precursor. Traditionally, cow urine itself has some medicinal properties and employing it in the process of extraction through biotransformation is envisaged to have positive improvement in the original potency of the herb under study.
MATERIALS AND METHODS
Plant Material
Procurement of Calendula officinalis flowers
Controlled dried flowers of Calendula officinalis were procured form the horticulture field at Nagpur.
Cow urine was procured from Govigyan Anusandhan Kendra, Dewlapar, Nagpur
Authentication
The flowers were authenticated by Dr. Dongarwar, Department of Botany, R.T.M. Nagpur University, Nagpur, with the herbarium voucher specimen number 9763.
Preparation of Extract
The collected flowers about 200 gm. were macerated in cow urine (1 Lit.) for time period of 28 days [13]. After the completion of this time period, the biotransformed material (Extract) was harvested, lyophilized, powdered (20 Gm.) which was then used for further studies.
Preparation of Aqueous Extract for comparative study
The collected flowers about 200 gm. were macerated in water (1 Lit.) for time period of 28 days [13]. After the completion of this time period, the biotransformed material (Extract) was harvested, lyophilized, powdered (20 Gm.) which was then used for further studies.
Maintenance of Cell culture
The A549 cell lines, LN Cap- FGC cell lines and MDA-MB cell lines were maintained at The Maratha Mandal Dental College, Belgaum, Karnataka. Cell lines were grown in Minimal essential medium (MEM) supplemented with 4.5 g/L glucose, 2 m ML-glutamine and 5% fetal bovine serum (FBS) (growth medium) at 37 0C in 5% CO2 incubator.
MTT Assay ( MICRO TITER ASSAY)
The in vitro MTT assay method was used to determine the inhibitory effects of test compounds on cell growth. The trypsinized cells from T-25 flask were seeded in each well of 96-well flat-bottomed tissue culture plate at a density of 5x103cells/well in growth medium and cultured at 37 0C in 5% CO2 to adhere. After 48 hr incubation, the supernatant was discarded and the cells were pre-treated with growth medium and were subsequently mixed with different concentrations of test samples (10, 20 and 30 μg) in triplicates to achieve a final volume of 100 μl and then cultured for 48 hr. The compound was prepared as 1.0 mg/ml concentration stock solutions in PBS. Culture medium and solvent were used as control and blank respectively. Each well then received 5 μl of fresh MTT (5mg/ml in PBS) followed by incubation for 2 hr at 37 0C. The supernatant growth medium was removed from the wells and replaced with 100 μl of DMSO to solubilise the colored Formosan product. After 30 min incubation, the absorbance (OD) of the culture plate was read at a wavelength of 492 nm on an ELISA reader [14].
The percentage inhibition of cancer cell lines was calculated with the following formula
% inhibition = 100-(At-Ab) / (Ac-Ab) × 100
Where, At: Absorbance of test,
Ab: Absorbance of blank,
Ac: Absorbance of control.
RESULTS AND DISCUSSION
The effect of biotransformed material (extract) of Calendula officinaliswas studied using MTT assay at a dose of 10, 20, 30 µg and Vincristine was used as a standard at a dose of 90 µg indicate the efficacy of microbial biotransformed material against cancerous cells.
Hence by
Cytotoxic properties of biotransformed material (extract) of Calendula officinalis against A549 cell lines
The IC50 value of Calendula officinalis against A549 cell lines was found to be Less than 10 µg and the results are provided in Table 1.
Cytotoxic properties of biotransformed material (extract) of Calendula officinalis against LN Cap-FGC cell lines
The IC50 value of Calendula officinalis against LN Cap-FGC cell lines was found to be 10 µg and the results are provided in Table 2.
Cytotoxic properties of biotransformation material (extract) of Calendula officinalis against MDA-MB cell lines
The IC50 value of Calendula officinalis against MDA-MB cell lines was found to be
10 µg and the results are provided in Table 3.
Table 1: Cytotoxicity effect against A549 cell lines
| S. No. | Name of drug | Concentration (µg) | O.D. at 492 nm | %of cell lysis | IC50 |
| 1. | Calendula officinalis Biotransformation material (extract) | 10 | 0.719 | 75% | <10 µg |
| 20 | 0.782 | >75% | |||
| 30 | 1.226 | 100% | |||
| 2. | Control | - | 0.240 | No lysis | - |
| 3. | Vincristine | 90 | 1.117 | 90% | - |
Table 2: Cytotoxicity effect against LN Cap-FGC cell lines (LN Cap-FGC - Human carcinoma Prostate)
| S. No. | Name of drug | Concentration (µg) | O.D. at 492 nm | %of cell lysis | IC50 |
| 1. | Calendula officinalis Biotransformation material (extract) | 10 | 0.578 | 50% | 10 µg |
| 20 | 0.768 | 75% | |||
| 30 | 1.084 | 100% | |||
| 2. | Control | - | 0.321 | No lysis | - |
| 3. | Vincristine | 90 | 1.117 | 90% | - |
Table 3: Cytotoxicity effect against MDA-MB cell lines
| S. No. | Name of drug | Concentration (µg) | O.D. at 492 nm | %of cell lysis | IC50 |
| 1. | Calendula officinalis Biotransformation material (extract) |
10 | 0.622 | 50% | 10 µg |
| 20 | 0.764 | 75% | |||
| 30 | 0.940 | 100% | |||
| 2. | Control | - | 0.334 | No lysis | - |
| 3. | Vincristine | 90 | 1.117 | 90% | - |
Comparative results for Aqueous Extracts
Table 1: Cytotoxicity effect against A549 cell lines
| S. No. | Name of drug | Concentration (µg) | O.D. at 492 nm | %of cell lysis | IC50 |
| 1. | Calendula officinalis Aqueous extract |
10 | 0.719 | No Lysis | --------- |
| 20 | 0.782 | No Lysis | |||
| 30 | 1.226 | No Lysis | |||
| 2. | Control | - | 0.240 | No lysis | - |
| 3. | Vincristine | 90 | 1.117 | 90% | - |
Table 2: Cytotoxicity effect against LN Cap-FGC cell lines (LN Cap-FGC - Human carcinoma Prostate)
| S. No. | Name of drug | Concentration (µg) | O.D. at 492 nm | %of cell lysis | IC50 |
| 1. | Calendula officinalis Aqueous extract |
10 | 0.578 | No lysis | ------- |
| 20 | 0.768 | No lysis | |||
| 30 | 1.084 | No lysis | |||
| 2. | Control | - | 0.321 | No lysis | - |
| 3. | Vincristine | 90 | 1.117 | 90% | - |
Table 3: Cytotoxicity effect against MDA-MB cell lines
| S. No. | Name of drug | Concentration (µg) | O.D. at 492 nm | %of cell lysis | IC50 |
| 1. | Calendula officinalis Aqueous extract |
10 | 0.622 | No lysis | -------- |
| 20 | 0.764 | No lysis | |||
| 30 | 0.940 | No lysis | |||
| 2. | Control | - | 0.334 | No lysis | - |
| 3. | Vincristine | 90 | 1.117 | 90% | - |
CONCLUSION
The anti-cancer studies of the extract of Calendula officinalisobtained after biotransformation using cow urine as an extraction medium on A549 cell lines, LN Cap-FGC cell lines and MDA-MB cell lines was carried out by using MTT cell growth inhibition assay. The results showed that the maximum percentage inhibition of cancer cell lines by Calendula oficinalis biotransformed extract was found to be 50% at a dose of less than 10 μg for A549 cell lines, 50% at a dose of 10 μg for LNCap-FGC cell linesand 50% at a dose of 10 μg for MDA-MB cell lines.
Hence biotransformed material (extract) of Calendula officinalis obtained by using cow urine as medium for extraction can be used as a potent anti-cancer agent whereas the aqueous extract of same plant material doesn’t show any activity.
CONFLICT OF INTERESTS
Declared None
ACKNOWLEDGEMENTS
We wish to thank the Govigyan Anusandhan Kendra, Dewlapar, Nagpur, for providing fresh cow urine and Maratha Mandal Dental college, Belgaum, Karnataka, who gave support to carry the in vitro anti-cancer studies.
REFERENCES