ANTICANCER EFFECT AGAINST MIA PA CA 2 and THP-1 CELL LINE STUDY OF MICHELIA CHAMPACA L

Abstract

ABSTRACT The present invention relates to the assessment of anti-cancer properties in a hydroalcoholic extract of Michelia champaca L. through in-vitro cytotoxicity evaluation. The MTT assay was employed to determine cell viability and growth inhibition, showing a concentration-dependent decrease in cell viability and increased growth inhibition. Notably, Michelia champaca exhibited IC50 values of 50 ug/ml and 139.83 ug/ml on pancreatic and leukemia cell lines, respectively, during a 24-hour incubation period within a concentration range of 50g to 150g. These results highlight the significant anti-cancer potential of the hydroalcoholic extract of Michelia champaca L., positioning it as a promising candidate for further anticancer research.

Specification

Description:FIELD OF INVENTION
The present invention relates to the cytotoxicity evaluation of Michelia champaca extract, shedding light on its potential medical applications, particularly in pharmacology and drug development.
BACKGROUND OF THE INVENTION
The background of the invention centers on Michelia champaca, a medicinal plant recognized for its traditional use in various cultures for its therapeutic properties. Michelia champaca, commonly known as "champaca " or " Sampige mara," has a history of application in folk medicine for treating a range of ailments. This herbaceous plant is native to many tropical and subtropical regions and is renowned for its diverse chemical composition. Its pharmacological properties have long been of interest to researchers, making it an intriguing candidate for further investigation.
Michelia champaca is known to contain various bioactive compounds, including alkaloids, flavonoids, tannins, and other phytochemicals, which contribute to its medicinal potential. The plant's extracts have shown promise in diverse medical applications, including anti-inflammatory, antioxidant, antimicrobial, and potential anticancer properties. The presence of these compounds in Michelia champaca has piqued the interest of researchers looking to harness its therapeutic potential for the development of new drugs or treatments.
In this context, the invention focuses on the cytotoxicity evaluation of Michelia champaca extract, with an emphasis on its potential in the realm of pharmacology and drug development. By assessing its impact on specific cell lines, the invention aims to provide valuable insights into the extract's medical applications, particularly its potential use in the treatment of diseases like cancer. This research aligns with the broader scientific interest in exploring natural compounds and their cytotoxic effects, which can ultimately lead to the development of novel pharmaceutical products with enhanced therapeutic benefits.
The following prior art is being reported:
The invention belongs to the technical field of medical biology, and particularly relates to a preparation method and an application of a Michelia champaca extract for resisting islet cell apoptosis. According to the preparation method, the Michelia champaca extract is prepared through the steps of heating reflux, extraction, silica gel column chromatography and the like. Cell experiments and animal experiments prove that the Michelia champaca extract prepared by the preparation method disclosed by the invention has a remarkable anti-islet cell apoptosis effect and has a bright application prospect in the aspect of preparing medicines for resisting islet cell apoptosis. The present invention is differed from this as it relates to an cytotoxicity evaluation of Michelia champaca extract, by evaluating the evaluating the test chemical's cytotoxicity against the MIA PA CA 2 and THP-1 cell lines
The invention discloses a scutellarin detoxified compound extract with an anti-cancer effect and a method for preparing the scutellarin detoxified compound extract, and relates to a compound traditional Chinese medicinal preparation and a method for preparing the compound traditional Chinese medicinal preparation. The method for preparing the scutellarin detoxified compound extract comprises the following steps of making bark, leaves and branches of fresh oriental alcohol M8 Magnolia champaca into powder through dry frying, atomization, detoxification and leaching; taking and making cordyceps, Michelia champaca, The smell of flowers is a good stimulant. The flowers are expectorant and useful in cough and rheumatism. The phytochemical analysis of the leaves and flowers of the plant showed the presence of alkaloids, tannins, glycosides, carbohydrates, amino acids, flavonoids, polyphenols and sterol in different solvent system. The alcoholic extract of the dried bark contained Palmitic acid , D - Galactose, Oleanolic acid L-Rhamnose, Chrysin-dimethyl ether D-Glucuronic acid. into powder through processing; taking wild honey, adding vinegar into the wild honey, and then stirring the powder into an obtained mixture, so as to obtain the detoxified compound extract. According to the scutellarin detoxified compound extract and the method, after the oriental alcohol M8 Magnolia champaca is subjected to detoxification and refining, beneficial substances of flavone, polyphenol and the like in the oriental alcohol M8 Magnolia champaca are all retained; further, the biological structures of effective components cannot be damaged; the content of multiple elements of scutellarin and the like in a Magnolia champaca extract obtained through water dissolution and leaching can be up to 97.9 percent or above at most; the increment of a cancer cell can be effectively prevented and inhibited; the curative effect is obvious. The present invention is differ from this as it relates to an cytotoxicity evaluation of Michelia champaca extract, by evaluating the evaluating the test chemical's cytotoxicity against the MIA PA CA 2 and THP-1 cell lines
MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on reduction of the yellow colored water soluble tetrazolium dye MTT to formazan crystals. Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple colour, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570nm.
OBJECTS OF THE INVENTION
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows.
It is an object of the present disclosure to ameliorate one or more problems of the prior art or to at least provide a useful alternative
An object of the present disclosure is to develop an efficient method for extracting bioactive compounds from Michelia champaca.
Another object of the present disclosure is to assess the cytotoxic effects of the Michelia champaca extract on specific cell lines.
Still another object of the present disclosure is to determine the IC50 values of the extract against human pancreatic cancer (MIA PA CA 2) cells.
Another object of the present disclosure is to determine the IC50 values of the extract against human acute leukemia (THP-1) cells
Still another object of the present disclosure is to evaluate the cytotoxicity of the extract in comparison to a standard control (1uM/ml of Doxorubicin).
Still another object of the present disclosure is to explore the potential pharmacological properties of the Michelia champaca extract.
Yet another object of the present disclosure is to measure the cell viability using the MTT assay.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.

SUMMARY OF THE INVENTION
The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.
The present invention is generally directed to the phytochemical screening and cytotoxicity evaluation of Michelia champaca extract.
An embodiment of the present invention is the MTT assay is employed to assess cytotoxicity on two cell lines, MIA PA CA 2 and THP-1 .
This colorimetric method measures cell proliferation and cytotoxicity by converting the MTT dye into formazan crystals.
Another embodiment of the invention is MIA PA CA 2 cells are cultivated in a 96-well plate, with the initial setup involving the growth of cells for 24 hours without the introduction of the test agent. The test agent is then added, and the MTT reagent is used to measure cell viability. The process is conducted under controlled conditions to ensure consistency.
Yet another embodiment of the invention is the THP-1 cells are grown in a 96-well plate. The test agent is introduced after 24 hours, and cell viability is measured
using the MTT assay. The IC50 value is determined from the viability graph, allowing for the assessment of cytotoxicity.
Yet another embodiment of the invention is the study involves evaluating the test chemical's cytotoxicity against the MIA PA CA 2 and THP-1 cell lines.
Yet another embodiment of the invention is

BRIEF DESCRIPTION OF THE DRAWINGS
Fig.1: - MIA PA CA2- 12.5ug (1)
Fig.2: - MIA PA CA2- 12.5ug (2)
Fig.3: - MIA PA CA2- 25ug (1)
Fig.4: - MIA PA CA2- 25ug (2)
Fig.5: - MIA PA CA2- 50ug (1)
Fig.6: - MIA PA CA2- 50ug (2)
Fig.7: - MIA PA CA2- 100ug (1)
Fig.8: - MIA PA CA2- 100ug (2)
Fig.9: - MIA PA CA2- 200ug (1)
Fig.10: - MIA PA CA2- 200ug (2)
Fig.11: - MIA PA CA2- Standard Control (1)
Fig.12: - MIA PA CA2- Standard Control (2)
Fig.13: - MIA PA CA2- Untreated (1)
Fig.14: - MIA PA CA2- Untreated (2)
FIg.15: - THP-1 - 12.5 ug (1)
FIg.16: - THP-1 - 12.5 ug (2)
FIg.17: - THP-1 - 25 ug (1)
FIg.18: - THP-1 - 25 ug (2)
FIg.19: - THP-1 - 50 ug (1)
FIg.20: - THP-1 - 50 ug (2)
FIg.21: - THP-1 - 100 ug (1)
FIg.22: - THP-1 - 100 ug (2)
FIg.23: - THP-1 - 200 ug (1)
FIg.24: - THP-1 - 200 ug (1)
FIg.25: - THP-1 - Standard Control (1)
FIg.26: - THP-1 - Standard Control (2)
FIg.27: - THP-1 - Untreated (1)
FIg.28: - THP-1 - Untreated (2)
Fig 29: Overlaid Bar graph depicted the % cell viability values of MIA PA CA 2 cells treated with various concentrations of MC1 after the incubation period of 24hours.
Fig 30: Overlaid Bar graph depicted the % cell viability values of THP-1 cells treated with various concentrations of MC1 after the incubation period of 24hours.
Fig 31: Comparative Bar graph depicted the IC50 values of MC1 against the MIA PA CA 2 and THP-1 cells after the incubation period of 24hours.
DETAILED DESCRIPTION OF THE INVENTION
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
BIOLOGICAL SOURCE
Magnolia was obtained from the bark of Michelia champaca Linn L. (Plantaginaceae)

EXAMPLE 1: Preparation of Extract
To prepare the extract, the drug substance is first placed in a filter paper shaped like a thimble, which is then carefully inserted into a glass thimble. This thimble is equipped with both a siphon tube and an inlet tube, and a water condenser is attached to the top of a cylindrical apparatus. The entire setup is then inserted into the neck of a round-bottom flask containing a Hydroalcoholic solution (in a 70:30 ratio).
The flask is then gently heated either using a water bath or a heating mantle. As the solvent is heated, its vapors travel up through the inlet tube and condense when they reach the condenser. These condensed solvent droplets come into contact with the drug substance contained in the thimble, causing the drug to dissolve and mix into the solution. This mixture then moves up the siphon tube.
This process ensures a continuous supply of solvent vapors within the thimble, and any liquefied drug returns to the flask. Once the desired extraction has taken place, the heating is stopped, and the solution in the flask is filtered to collect the extract.
EXAMPLE 2: MTT Assay
A cytotoxicity assessment was performed on two cell lines using the MTT assay. The sample details for this study are as follows:
Sample Name/Code: MC1
Concentrations: 5 (12.5, 25, 50, 100, and 200 µg/ml)
Cell Lines: MIA PA CA 2 and THP-1
The MTT assay is a colorimetric assay used for the determination of cell proliferation and cytotoxicity, based on reduction of the yellow colored water soluble tetrazolium dye MTT to formazan crystals. Mitochondrial lactate dehydrogenase produced by live cells reduces MTT to insoluble formazan crystals, which upon dissolution into an appropriate solvent exhibits purple colour, the intensity of which is proportional to the number of viable cells and can be measured spectrophotometrically at 570nm
The MIA PA CA 2 (Human pancreatic adenocarcinoma cell line) was purchased from NCCS, Pune, India. The cells were maintained in DMEM media with high glucose supplemented with 10 % FBS along with the 1% antibiotic-antimycotic solution in the atmosphere of 5% CO2, 18-20% O2 at 370C temperature in the CO2 incubator and sub-cultured for every 2 days. Passage number 38 was used for the present study.
The THP-1 (Human acute leukemia cell line) was purchased from NCCS, Pune, India. The cells were maintained in RPMI-1640 media supplemented with 10 % FBS along with the 1% antibiotic-antimycotic solution in the atmosphere of 5% CO2, 18-20% O2 at 370C temperature in the CO2 incubator and sub-cultured for every 2 days. Passage number 24 was used for the present study.
EXAMPLE 3: MIA PA CA 2 Cells
The assessment with MIA PA CA 2 cells began by placing a Seed 200μl cell suspension in a 96-well plate at required cell density (20,000 cells per well), without the test agent. Allow the cells to grow for about 24 hours. Add appropriate concentrations of the test agent (Mentioned in the results - Excel sheet). Incubate the plate for 24hrs at 37°C in a 5% CO2 atmosphere. After the incubation period, takeout the plates from incubator, and remove spent media and add MTT reagent to a final concentration of 0.5mg/mL of total volume. Wrap the plate with aluminium foil to avoid exposure to light. Return the plates to the incubator and incubate for 3 hours. (Note: Incubation time varies for different cell lines. Within one experiment, incubation time should be kept constant while making comparisons.) Remove the MTT reagent and then add 100μl of solubilization solution (DMSO). Gentle stirring in a gyratory shaker will enhance dissolution. Occasionally, pipetting up and down may be required to completely dissolve the MTT formazan crystals especially in dense cultures.

The absorbance at 570nm and 630nm was then measured as reference wavelengths using a spectrophotometer or an ELISA reader.
To calculate cell viability, the formula [OD of treated cells/OD of untreated cells] * 100 was used. Furthermore, the IC50 value was calculated using the linear regression equation: Y = Mx + C, with values Y = 50, M, and C being determined from the viability graph.
EXAMPLE 4: THP-1 Cells
The analysis with THP-1 cells involved placing 100 μl of cell suspension into the wells of a 96-well plate without the test agent. The desired cell density was set at 10,000 cells per well, and the cells were allowed to grow for approximately 24 hours. Following this growth period, the appropriate concentrations of the test agent, as indicated in the results (Excel sheet), were added to the respective wells.

The plates were then incubated at 37°C for 24 hours in a 5% CO2 environment. After incubation, the plates were removed from the incubator, and centrifugation at 1000 rpm for 2 minutes at room temperature was performed to remove the spent medium. The MTT reagent was added to achieve a final concentration of 0.5 mg/mL in the total volume. To prevent exposure to light, the plates were covered with aluminum foil and placed back in the incubator for 3 hours.

After this incubation period, the MTT reagent was removed, and 100 μl of solubilization solution (DMSO) was added to each well. Similar to the procedure with MIA PA CA 2 cells, gentle shaking on a gyratory shaker and occasional pipetting up and down may be required to ensure the complete dissolution of MTT formazan crystals, especially in thick cell colonies.

The absorbance at 570nm and 630nm was then measured as reference wavelengths using a spectrophotometer or an ELISA reader. To calculate cell viability, the formula [OD of treated cells/OD of untreated cells] * 100 was used. The IC50 value was calculated using the linear regression method, with values Y = 50, M, and C being determined from the viability graph.
In this invention, a specific test chemical was assessed for cytotoxicity against two cell lines, MIA PA CA 2 and THP-1. The concentrations of the test chemical utilized to treat the cells were as specified in the results.

Table 1: Details on medication administration to cell lines utilized in the study
S. No Test Cell Line Concentration treated to cells
1 Untreated MIA PA CA 2 and THP-1 No treatment
2 Std Control MIA PA CA 2 and THP-1 1uM/ml
3 Blank - Only Media without cells
4 MC1 MIA PA CA 2 and THP-1 12.5, 25, 50,100 and 200µg/ml
In this study, given Test compound was evaluated to measure the cytotoxicity study against the 2 cell lines namely, MIA PA CA 2 and THP-1. The used concentrations of the compound used to treat the cells are as follows:


Table 2: MTT Assay-Summary-Cell Viability Values
MTT ASSAY-SUMMARY-CELL VIABILITY VALUES
Drug conc (ug/ml) MIA PA CA2 THP-1
Untreated 100.00 100.00
Dox-1uM 39.37 47.14
MC1 -12.5ug 83.74 96.60
MC1 -25ug 69.12 91.03
MC1 -50ug 52.02 80.30
MC1 -100ug 33.20 62.66
MC1 -200ug 12.49 29.65
IC50 conc (ug/ml) 50.23 139.83
Table showed the % cell viability values and IC50 values of the given test compound against the Human pancreatic cancer (MIA PA CA 2) cell line and Human acute leukemia cell line (THP-1) after the treatment period of 24hrs and represented in bar graphs as below
Figure 1 and Figure 2 illustrates an overlaid bar graph illustrating the percentage of cell viability values of MIA PA CA 2 cells, THP-1 cells after treatment with various concentrations of MC1 during a 24-hour incubation respectively. Wherein the figure 3 represents an comparative bar graph displaying the IC50 values of MC against MIA PA CA 2 and THP-1 cells following a 24-hour incubation.
EXAMPLE 5: Cytotoxicity Study of Michelia champaca against MIA PA CA 2 and THP-1 Cell Lines
The Observations in Statistical data of cytotoxicity study by MTT assay suggesting us that against MIA PA CA2 and THP-1 cells, given test compound namely MC1 showed effective toxicity with IC50 concentration at 50.23ug/ml and moderate toxicity with IC50 value of 139.83ug/ml after the incubation period of 24hrs. Doxorubicin was used as a std control for the study.
The Observed absorbance readings with calculations were enclosed in the separate folder of the report in MS Excel format. Direct microscopic observations of drug treated images of MIA PA CA 2 and THP-1 cell line captured at the 20x magnification are enclosed in separate folder along with this report.
The MTT assay results suggested us that the given test compound; MC1 have effective cytotoxic potential capacity against Human pancreatic cancer cells with IC50 value of 50ug/ml and effective toxicity on Human acute leukemia cells with IC50 value of 140ug/ml respectively. MC1 may have effective toxic potential due to its moderate IC50 value on MIA PA CA 2 cells. Further mechanistic studies related to the anticancer potential need to be performed to evaluate the mechanism of action behind the anticancer activity of MC1 against the pancreatic cancer cell line in in-vitro condition.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
 
We Claim,
1. A method for efficiently preparing Michelia champaca extract, comprising the following steps:
a. immersing Michelia champaca plant material in a Hydroalcoholic solution with a ratio of 70:30;
b. heating the solution using a water bath or heating mantle;
c. condensing solvent vapors for continuous extraction by utilizing a glass thimble equipped with a siphon tube and an inlet tube;
d. ensuring the return of any liquefied plant material to the solution; and
e. concluding the method by filtration of the solution, resulting in the extraction of high-quality Michelia champaca extract.
2. The method as claimed in claim 1, wherein the Hydroalcoholic solution comprises 70% alcohol and 30% water.
3. The method as claimed in claim 1, wherein the heating of the solution is performed at a temperature range between 40°C and 60°C.
4. The method as claimed in claim 1, wherein the glass thimble is designed to maintain a continuous supply of solvent vapors during the extraction process.
5. The method as claimed in claim 1, wherein the prepared extract exhibits cytotoxic activity.
6. The method as claimed in claim 1, wherein the cytotoxic activity of the prepared Scoparia dulcis extract is determined by subjecting it to MTT assay.
7. The method as claimed in claim 1, wherein the Michelia champaca extract exhibits cytotoxicity against human pancreatic adenocarcinoma cells.
8. The method as claimed in claim 1, wherein the Michelia champaca extract demonstrates cytotoxicity against human acute leukemia cells.
9. The method as claimed in claim 1, wherein the Michelia champaca extract displays an IC50 value of 50ug/ml against human pancreatic adenocarcinoma cells.
10. The method as claimed in claim 1, wherein the Michelia champaca extract exhibits an IC50 value of 139.83ug/ml against human acute leukemia cells.



 
TITLE: ANTICANCER EFFECT AGAINST MIA PA CA 2 and THP-1 CELL LINE STUDY OF MICHELIA CHAMPACA L

ABSTRACT

The present invention relates to the assessment of anti-cancer properties in a hydroalcoholic extract of Michelia champaca L. through in-vitro cytotoxicity evaluation. The MTT assay was employed to determine cell viability and growth inhibition, showing a concentration-dependent decrease in cell viability and increased growth inhibition. Notably, Michelia champaca exhibited IC50 values of 50 ug/ml and 139.83 ug/ml on pancreatic and leukemia cell lines, respectively, during a 24-hour incubation period within a concentration range of 50g to 150g. These results highlight the significant anti-cancer potential of the hydroalcoholic extract of Michelia champaca L., positioning it as a promising candidate for further anticancer research.


, Claims:We Claim,
1. A method for efficiently preparing Michelia champaca extract, comprising the following steps:
a. immersing Michelia champaca plant material in a Hydroalcoholic solution with a ratio of 70:30;
b. heating the solution using a water bath or heating mantle;
c. condensing solvent vapors for continuous extraction by utilizing a glass thimble equipped with a siphon tube and an inlet tube;
d. ensuring the return of any liquefied plant material to the solution; and
e. concluding the method by filtration of the solution, resulting in the extraction of high-quality Michelia champaca extract.
2. The method as claimed in claim 1, wherein the Hydroalcoholic solution comprises 70% alcohol and 30% water.
3. The method as claimed in claim 1, wherein the heating of the solution is performed at a temperature range between 40°C and 60°C.
4. The method as claimed in claim 1, wherein the glass thimble is designed to maintain a continuous supply of solvent vapors during the extraction process.
5. The method as claimed in claim 1, wherein the prepared extract exhibits cytotoxic activity.
6. The method as claimed in claim 1, wherein the cytotoxic activity of the prepared Scoparia dulcis extract is determined by subjecting it to MTT assay.
7. The method as claimed in claim 1, wherein the Michelia champaca extract exhibits cytotoxicity against human pancreatic adenocarcinoma cells.
8. The method as claimed in claim 1, wherein the Michelia champaca extract demonstrates cytotoxicity against human acute leukemia cells.
9. The method as claimed in claim 1, wherein the Michelia champaca extract displays an IC50 value of 50ug/ml against human pancreatic adenocarcinoma cells.
10. The method as claimed in claim 1, wherein the Michelia champaca extract exhibits an IC50 value of 139.83ug/ml against human acute leukemia cells.

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