Claims:We Claim:

1. A method for extracting Guaianolide from Cyathocline purpurea, the method comprises steps of:
collecting raw Cyathocline purpurea plants;
cleaning and drying of collected Cyathocline purpurea plant;
grinding the dried Cyathocline purpurea plant to powder form;
mixing the grinded Cyathocline purpurea powder in a chemical solvent for obtaining a solvent soluble extract;
subjecting the obtained extract to a first stage and a second stage chromatographic separation processes consequently; and
separating Guaianolide from the first stage column fraction subjected to the second stage chromatographic separation process and separated Guaianolide is used for curing triple-negative breast cancer.

2. The method as claimed in claim 1, wherein the chemical solvent is methanol.

3. The method as claimed in claim 1, wherein the chemical solvent is petroleum- ether.

4. The method as claimed in claim 1, wherein the chemical solvent is ethyl acetate.

5. The method as claimed in claim 1, wherein the first stage chromatographic separation process is a column chromatographic method.

6. The method as claimed in claim 1, wherein 0.5kg of powdered Cyathocline purpurea is mixed in 2.0 litres of the chemical solvent.

7. The method as claimed in claim 1, wherein the first stage chromatographic separation process is carried by subjecting the solvent soluble extract to a chromatographic device on a silica gel column.

8. The method as claimed in claim 1, wherein the second stage chromatographic separation process is carried by subjecting the first stage column chromatographic fraction to a chromatographic device on a silica gel column with petroleum-ether and ethyl acetate solvent system.

9. The method as claimed in claim 1, wherein separated Guaianolide from the column fraction subjected to the second stage chromatographic separation process is concentrated in vacuum at 40 degree Celsius.

10. The method as claimed in claim 9, wherein concentrated Guaianolide is stored in a chemically non-reactive storage apparatus.
, Description:Field of the invention

The present invention relates to a method for extracting Guaianolide. More particularly, the present invention relates to a method for extracting Guaianolide from Cyathocline purpurea plant.

Background of the invention:

Sesquiterpene lactones are a class of chemical compounds. Sesquiterpene lactones are sesquiterpenoids (built from three isoprene units). Sesquiterpene lactones contains a lactone ring, hence they are called Sesquiterpenelactones. Sesquiterpene lactones are found in many plants and can cause allergic reactions and toxicity if overdosed, particularly in grazing livestock. Some are also found in corals such as Maasella edwardsi. Sesquiterpene lactones consists 15-carbon structure containing a-methylene moiety and other traditional functional groups.

Guaianolide is a Sesquiterpene lactone with unique structure. Nomenclature of Guaianolide is6 a-hydroxy-4[14], 10[15]-guainadien-8a, 12-olide [10] (SRCP1) having molecular weight of 246 gram and Molecular formula C15H18O3. Guaianolide has been isolated from several species of Asteraceae family. Moreover, sesquiterpene lactones from the plant source have been found to be potent in many cancers. 1-oxoeudesm-11[13]-eno-12,8a-lactone, (sinodielide-A) have been shown to be effective in glioblastoma and pancreatic cancer through the mechanism of the cell cycle arrest at G2/M phase. Cyathocline purpurea is a genus of flowering plants in the daisy family. Cyathocline purpurea species belongs to Asteraceae family.

Breast cancer is a type of cancer that develops from breast tissue. Triple negative breast cancer is a type of breast cancer. Triple negative breast cancer does not respond to hormonal therapy. According to FDA (Food and Drug Administration, USA), currently there is no treatment available for triple negative breast cancer [TNBC]. TNBC comprised of 10-15% of total breast cancer [Robles A. et al., Breast Cancer Res Treat. 2016]. The average progression free survival of TNBC is 12 months which is far less as compared to other subtype of breast cancer. Moreover, there are no specialised guidelines available for TNBC treatment.

Some studies show that a Sesquiterpene lactone is effective against wide range of human cancer cell lines including neuroblastoma especially in triple negative type of breast cancer. Specifically, Guaianolide shows more effective anti-cancer properties compared to other Sesquiterpene lactones. Presently there are no any available methods to extract Guaianolide from Cyathocline purpurea. Cyathocline purpurea plants are available in nature easily and affordably.

Therefore, there is need of method for extracting Guaianolide from Cyathocline purpurea which is used for treating triple negative breast cancer.

Objects of the invention

Object of the present invention is to provide a method for extracting Guaianolide from Cyathocline purpurea plant.

Another object of the present invention is to provide a method for isolation and purification of a Guaianolide from Cyathocline purpurea plant.

One more object of the present invention is to provide a method for isolation and purification of a Guaianolide from Cyathocline purpurea plant which is economical in operation.

Yet another object the present invention is to provide a method for isolation and purification of a Guaianolide from Cyathocline purpurea plant which is easy in operation.

Summary of the invention:

According to present invention a method for extracting Guaianolide from Cyathocline purpurea is provided. The method comprises steps of collecting and cleaning raw Cyathocline purpurea plants. The cleaned Cyathocline purpurea plants are kept for drying. Dried Cyathocline purpurea plants are grinded to powder form and mixed in a chemical solvent for obtaining an extract. The obtained extract is subjected to a first stage and a second stage chromatographic separation processes consequently. The Guaianolide is separated from the column fractions obtained from the first stage of chromatography of the extract which is subjected to the second stage chromatographic separation process, which is used for curing triple-negative breast cancer.

Brief Description of drawings

Figure 1 shows a flowchart of a method for extracting Guaianolide from Cyathocline purpurea plant in accordance with present invention;

Figure 2 shows an image of a thin layer chromatography of different extracts of Cyathocline purpurea and pure Guaianolide (SRCP16 a-hydroxy-4[14], 10[15]-guainadien-8a, 12-olide [10] (SRCP1) extracted from the method shown in figure1;

Figure 3 shows an Infra-red (IR) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 4 shows a Proton nuclear magnetic resonance (1H-NMR) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 5 shows a carbon nuclear magnetic resonance (13C-NMR) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 6 shows a distortion less Enhancement by Polarisation transfer (DEPT) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 7 shows a correlation spectroscopy (COSY) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 8 shows a Heteronuclear single quantum coherence (HSQC) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 9 shows a Hetero-nuclear Multiple Bond Correlation (HMBC) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 10 shows a Nuclear over Hauser effect (NOESY) spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 11 shows a mass spectrum of extracted SRCP1 from the method shown in figure 1;

Figure 12 shows a microscopic image of MDA-MB-231 cells treated with SRCP1 extracted from the method shown in figure 1;

Figure 13 shows a microscopic image of MDA-MB-453 cells treated with SRCP1 extracted from the method shown in figure 1;

Figure 14 shows a microscopic image of morphological changes observed after treatment of SRCP1 on MCF-7 cells;

Figure 15 shows a microscopic image of MDA-MB-231 cells subjected to Wound scratch assay;

Figure 16 shows a microscopic image of MDA-MB-453 cells subjected to Wound scratch assay;

Figure 17 shows a graph of percent apoptosis observed in MDA-MB-231 cells after 96 hours of treatment;

Figure 18 shows a graph of dose response curve of SRCP1 on human breast cancer cells;

Figure 18a shows a graph cell viability of different types of cells;

Figure 18b shows the cell viability of Wharton's Jelly derived Mesenchymal Stem Cells (WJMSCs) after 24hrs of treatment respectively:

Figure 18c shows a graph of the cell viability of Mouse embryonic fibroblast cell line (3T3L1) cells (both non-cancerous) after 24hrs of treatment; and

Figure 19 shows a graph of percent of DNA content in MDA-MB-453 cells at different phases after drug treatment(SRCP1-11.35µM) in comparison with standard drug Tamoxifen(5µM) for 96hrs;

Detailed description of the invention

An embodiment of this invention, illustrating its features, will now be described in detail. The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and be open ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items.

The terms “first,” “second,” and the like, herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another, and the terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item.

The present invention provides a method for extracting Guaianolide from Cyathocline purpurea plant. The method isolates and purifies Guaianolide from Cyathocline purpurea plant. Further, the method is economical in operation. Furthermore, the method is easy in operation.

Referring now to Figure 1, a flow chart showing a method 100 for extracting Guaianolide from Cyathocline purpurea plants in accordance with the present invention is illustrated.

The method 100 starts at step 10.

At step 20, raw Cyathocline purpurea plants are collected. Cyathocline purpurea plant is a seasonal plant. Cyathocline purpurea plant is found in moist habitat such as watercourse and in rice fields of peninsular region. In the present embodiment, whole plant is collected from the fields. In an alternative embodiment, only a specific part of the plant such as flower, roots, stems, leaves may be collected. The collected Cyathocline purpurea plants are cleaned to remove dust and other particles from the plant.

At step 30, the cleaned Cyathocline purpurea plants are kept for drying. The drying is carried out in a drying apparatus. The usage of the drying apparatus for drying Cyathocline purpurea plants is obvious knowledge to person ordinarily skilled in the art.

At step 40, the dried Cyathocline purpurea plants are grinded to obtain powder form using any grinding machine. The grain size of the powder of Cyathocline purpurea plants is to be fine grain to obtain better results in the next set of operations.

At step 50, Cyathocline purpurea powder is mixed in a chemical solvent for obtaining a solvent soluble extract. In the present embodiment, the Cyathocline purpurea powder is mixed in methanol, petroleum- ether and ethyl acetate independently. In the present embodiment, the chemical solvents are methanol, petroleum- ether and ethyl acetate. Methanol, petroleum- ether and ethyl acetate is mixed with the Cyathocline purpurea powder individually. In the present embodiment 0.5kg of Cyathocline purpurea powder is mixed in 2.0 lit of methanol by cold percolation for 12hours in a 5 Litre flat bottom flask at room temperature. The process of mixing is repeated three times with methanol.

Each time the soluble extract was concentrated under vacuum at 40ºC and pooled together to obtain 37.2 grams of concentrated solvent soluble extract. Similarly, 0.5kg of Cyathocline purpurea powder is mixed in 2.0 Litre of petroleum ether by cold percolation for 12hr in a 5Litre flat bottom flask at room temperature. The solution of petroleum- ether is concentrated under vacuum at 40ºC together to obtain12.4 grams greenish sticky concentrated petroleum ether extract of Cyathocline purpurea powder. Similarly, 0.5kg of Cyathocline purpurea powder is mixed in 2.0 litre of ethyl acetate by cold percolation for 12hr in a 5 Litre flat bottom flask at room temperature. The solution of ethyl acetate is concentrated under vacuum at 40ºC and to obtain 21.75grams of concentrated ethyl acetate extract of Cyathocline purpurea powder.

The concentrated extract of methanol, petroleum ether and ethyl acetate of Cyathocline purpurea powder is subjected to a testing process for finding anticancer properties. Usage of the testing process for finding anticancer properties of the extracts may be obvious knowledge to person who is ordinarily skilled in the art. After analysing the anticancer properties, the extract having favourable anticancer properties are selected for further operations.

At step 60, the extract is subjected to a first stage chromatographic separation process. The first stage chromatographic separation process is a thin layer chromatography method. It may be obvious to person skilled in the art to use the column chromatography (CC) method for separation. The first stage chromatographic separation process is carried by subjecting the extract to a chromatographic device on a silica gel column. The first stage chromatographic separation process is eluted with a stepwise gradient of petroleum-ether and ethyl acetate. The fractions of filtrate were collected separately and concentrated under vacuum at 40ºC. The fractions after thin layer chromatography (TLC) pooled together the similar fractions to obtain five fractions. The fraction having highest weight (percentage) of Guaianolide(SRCP1) is selected.

Further, the selected fraction having highest weight (percentage) of Guaianolide (SRCP1) is subjected to a second stage chromatographic separation process. The second stage chromatographic separation process is also the column chromatography method. The second stage chromatographic separation process is carried out by subjecting the highest weight fraction on to a chromatographic device using a silica gel column for fractionation with petroleum-ether and ethyl acetate solvent system. The fractions obtained from the second stage chromatographic separation process were concentrated under vacuum at 40ºC. The fraction having highest weight of SRCP1 is purified by the Preparative TLC (PTLC) using ethyl acetate (7:3). The Purified SRCP1 is stored in a chemically non-reactive storage apparatus. The Purified SRCP1 is used for treatment of triple negative breast cancer.

A sample of Guaianolide (SRCP1) is extracted using the method 100. The Cyathocline purpurea plants are material collected from rice fields of Pirangut area, near Pune, Maharashtra, India in summer season. Authentication of plant was carried out at Botanical Survey of India, Pune and voucher specimen [BSI/WRC/Tech/2013/1094] was deposited with the Institute. The collected Cyathocline purpurea plants are subjected to steps 30, 40, 50.
The extract of methanol, petroleum- ether and ethyl acetate with Cyathocline purpurea powder are obtained. The solvent soluble extracts are subjected to testing processes for analyzing anticancer properties. The testing procedures are carried out as follows.

Different solvent extract and SRCP1 were dissolved in Dimethyl sulfoxide (DMSO) at the concentration of 100mg/ml and 100mM respectively. Stock solution of 100mg/ml is prepared in DMSO filtered through 0.22 µ filter [HiMedia Labs]. Drugs dilutions were prepared freshly every time in appropriate medium before use. Stock solution was stored at -80 °C for further use.

Human triple negative breast cancer cell lines procured from National Centre for cell science, Pune, India. MCF-7 cells were maintained in EMEM [HiMedila Labs Pvt. Ltd.], MDA-MB-453 and MDA-MB-231 were maintained in Leibovit’z medium [Invitrogen]. NCI-H23 and K-562 cells were maintained in RPMI 1640 medium. Hep G2 cells were cultured in high glucose Dulbecco's Modified Eagle Medium. SH-SY-5Y cell was cultured in DMEM/Ham’s F12 medium.

All the Human triple negative breast cancer cells were supplemented with 10% fetal bovine and serum 1% antimycotic-antibiotic solution. Cells were maintained at 37°C in a 5% CO2 atmosphere except MDA-MB-231 and were grown at least 80% confluency and then harvested using trypsinising and used for the assay. Cell viability was determined by trypan blue dye exclusion method.

The MTT assay [3-[4, 5-dimethylthiazol-2-yl] 2, 5-diphenyltetrazolium bromide] was performed. 180µl of Cells were seeded at the density 1x105 cells/ml in 96 well-plate per well. Cells were incubated overnight for adherence. Furthermore, 20µl of drug were added per well. Cells were treated with different concentration of drugs for different time interval. After treatment medium was replaced with 20 µl of 5mg/ml MTT [HiMedia Labs]. Plate was incubated at same conditions for another 4 hrs. Afterwards, the formed formazan crystals were dissolved in 100µl of DMSO [HiMedia]. Absorbance was measured at 620 nm using Dynex microplate reader. Cell Viability was determined as compare to the untreated sample. Using a best curve fit equation and IC 50 values calculated.

Cells were treated with different concentrations of drug and images were taken at different time interval using compound microscope. Cells were grown at 60-70% confluency. Wound were scratched using 200µl tip [Tarson], further cells were washed with 1X PBS twice. Drugs were added and images were taken at different time interval.

For cell cycle analysis after drug treatment of 96hrs cells were harvested and washed using 1X PBS twice. Cells were centrifuges at 1500 rpm for 3 min. 50µl of propidium iodide[5mg/ml] [Invitrogen], 50µl of Pure link RNase A[20mg/ml][Invitrogen] and 2mM MgCl2 were added and cells were immediately analysed using FACS analyser[BD Biosciences]. As Per DNA content, cells were categorised into different phases. Experiments were performed in duplicate.

For apoptosis analysis, after treatment of 96 hrs cells were harvested and washed using 1X PBS twice. Cell pellet were re-suspended in 1X PBS. Apoptosis analysis was carried out as per manufacture’s protocol (Molecular Probes).
A Student’s t test was used to calculate significant value of cell percent Inhibition. For cell cycle analysis one way ANOVA was performed followed by Tukey’s multiple comparison tests used to obtain significance value. P less than 0.05 were considered as significant. Each experiment was performed at least in triplicate.

Referring to figure 12 an image showing a microscopic view of MDA-MB-231 cells were treated for SRCP1 and standard drug Tamoxifen for different time interval and images were taken at different time points like 24hrs, 48hrs and 96hrs. Referring to figure 13 an image showing a microscopic view of MDA-MB-453 cells were treated for SRCP1 and standard drug Tamoxifen for different time interval and images were taken at different time points like 24hrs, 48hrs (40X). Refer figure 14 showing an image from a microscopic view of Morphological changes observed after treatment of SRCP1 and Tamoxifen at 24hrs on MCF-7 cells. Images were taken using Carl Zeiss Avert 40CFL Inverted microscope at [20X].

Referring now to figure 15, showing an image from a microscopic view of MDA-MB-231 cells subjected to Wound scratch assay. Referring figure 16 an image showing a microscopic view of MDA-MB-453 cells subjected to Wound scratch assay. Referring to figure 17 showing a graph of percent apoptosis observed in MDA-MB-231 cells after 96 hrs of treatment are: Control 43%, SRCP1 59% and Tamoxifen 46%. Refer figure 18a shows a graph cell viability of different types of cells.

Referring to figure 18b, a graph of the cell viability of Wharton's Jelly derived Mesenchymal Stem Cells (WJMSCs) after 24hrs of treatment respectively is illustrated. Referring figure 18c, a graph of the cell viability of Mouse embryonic fibroblast cell line (3T3L1) cells (both non-cancerous) after 24hrs of treatment is illustrated. Referring to figure 19, a graph of percent of DNA content in MDA-MB-453 cells at different phases after drug treatment (SRCP1-11.35µM) in comparison with standard drug Tamoxifen(5µM) for 96hrs is illustrated.

As per the data we can see that cell growth is arrested at G2/M Phase significantly as compared to the positive control Tamoxifen. In S phase, also the cells are decreased in SRCP1 treated cells but it is not significant. In Apoptotic phase the SRCP1 treated cells at the concentration 11.35µM shows higher percent 57-60% of total cells. While Tamoxifen shows less percent of apoptotic cells as compare to control as well as SRCP1 treated cells significantly. Referring to figure 18 showing a graph of Dose response curve of SRCP1 on MDA-MB-231, MDA-MB-453 and MCF-7 Cells.

Figure 18b and figure 18c shows the cytoxicity of SRCP1 on non-cancerous cells of human and mice origin respectively after 24hrs
Sr. No. Cancer type Cell line IC50(µM)
1 Neuroblastoma SH-SY-5Y 9.28
2 Hepatocellular carcinoma Hep-G2 31.40
3 Triple negative breast cancer MDA-MB-231 22.78
4 Her2 positive breast cancer MDA-MB-453 35.87
5 Hormone receptor positive breast cancer MCF-7 38.51
Table 1
Table 1 shows IC50 Values of cells after 24 hrs of treatment.
Sr. No. SRCP Viability Percentage Inhibition
1 100µm 100 0
2 10µm 99 1
3 1µm 100 0
4 0.1µm 97 3
5 0.01µm 98 2
6 0.001µm 99 1
Table 2
Table 2 shows cytotoxicity of SRCP1 towards human PBMCs after 24hrs.
Based on obtained results, petroleum ether extract shows highest favourable anti-cancer properties. The extract of petroleum-ether of Cyathocline purpurea is subjected to the first stage chromatographic separation process at step 60. The column fractions after TLC examination is pooled together to obtain five fractions: 1.A (0.28gm), 2.A (0.56gm), 3.A (0.50gm), 4.A (0.46gm), and 5.A (0.19gm). The fraction 2.A showed presence of SRCP1 in substantial quantity. Referring now to figure 2, an image of Thin layer chromatography (TLC) of different extracts of Cyathocline purpurea and pure SRCP1 (CPME: Methanol extract, TPE: Total petroleum ether extract, Mefr: Methanol fraction, CP-1: Pure isolated SRCP1) in accordance with the present invention is illustrated.

Fraction 2.A having 0.56gm of Guaianolide (SRCP1) is subjected the second stage chromatographic separation process at step 70. Fractions obtained from the second stage chromatographic separation process were pooled together to obtain 1.2A (0.20gm), 2.2A (0.11gm), 3.2 A (0.05gm), 4.2A (0.10gm), 5.2 A (0.02gm) of SRCP using the TLC. The fraction 3.2A and 4.2A are fractions having highest weight of SRCP1. The pure SRCP1 is purified from the fractions 3.2A and 4.2 A by repeated preparative TLC using petroleum-ether and ethyl acetate (7:3) solvent system used for the purification. Purified SRCP1 is obtained. The purity of SRCP1 was confirmed by different types of spectrum like 1H-NMR,13C-NMR (nuclear magnetic resonance), DEPT (Distortion-less enhancement by polarization transfer) and TLC. The structure of SRCP1 was confirmed by (1D,2D) 1H-NMR, 13C-NMR, DEPT, COSY, HSQC, HMBC, NOESY, IR and Mass spectral data.

Referring to figure 3 showing an IR spectrum of extractedSRCP1. Refer figure 4 showing a 1HNMR spectrum of extracted SRCP1. Refer figure 5 showing a 13C-NMR spectrum of extracted SRCP1. Refer figure6 showing a DEPT spectrum of extracted SRCP1. Referring figure 7 showing a COSY spectrum of extracted SRCP1. Refer Figure 8 showing a HSQC spectrum of extracted SRCP1. Referring figure 9 showing a HMBC spectrum of extracted SRCP1. Referring figure 10 showing a NOESY spectrum of extracted SRCP1. Referring figure 11 showing a Mass spectrum of extracted SRCP1. The extracted SRCP1 is stored in the chemical non-reactive storage apparatus.

The method ends at step 80.

The advantage of the present invention is to provide a method 100 of extracting Guaianolide from Cyathocline purpurea in a very less time. The method 100 does not require costly equipment, chemical and biological materials. Hence, the method 100 is cost effective. The Guaianolide extracted using the method 100 is used in the treatment of triple cell negative breast cancer. The method 100 utilizes Cyathocline purpurea for extracting Guaianolide. Cyathocline purpurea is abundantly available in nature. Hence, the cost of medicines prepared from naturally extracted Guaianolide is economically affordable. The method 100 involves in usage of simple operations such as chromatographic separation, concentration under vacuum and the like, thereby making the method 100 simple and easy in operation. The method 100 provides a medicine to cure triple negative breast cancer.

The foregoing descriptions of specific embodiments of the present invention have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed, and obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated. It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the spirit or scope of the claims of the present invention.