LIPOSOME OF SAUSSUREA OBVALLATA AND ITS PROCESS OF PREPARATION

Abstract

The present disclosure relates to a liposome comprising: a leaf extract of Saussurea obvallata; and a phospholipid comprising cholesterol and other phospholipid (Soya PC), wherein weight ratio of the leaf extract of Saussurea obvallata to the phospholipid and cholesterol is in range of 1:3:1. The present disclosure also relates to its method of preparation.

Specification

Description:FIELD OF THE INVENTION:
[0001] The present disclosure relates to a liposome. More particularly, the disclosure relates to the liposomes of the leaf extract of Saussurea obvallata. The present disclosure also relates to its method of preparation.

BACKGROUND OF THE INVENTION
[0002] Currently, breast cancer represents 12.5% of all cancer diagnoses and a cumulative total of 2.3 million new cases in both males and females. Accounting for 25% of all cancer cases in females, it was the most prevalent cancer diagnosed in women. Medical interventions such as surgeries, chemotherapy, and radiotherapies can have harmful side effects and, as a result of multiple relapses, can lead to the spread of cancer in the body, known as metastasis. Extensive research has been conducted to investigate the therapeutic benefits of various plant-based bioactive compounds, such as flavonoids, terpenoids, saponins, carotenoids, phenolic acids, and organosulfur compounds, in the treatment of cancer. These compounds have been found to have protective effects by targeting multiple pathways involved in cancer development. They can inhibit the growth and spread of tumor cells, as well as reduce the invasiveness and formation of blood vessels in tumors, thereby slowing down the progression of cancer.
[0003] Saussurea obvallata is a plant with a history in ayurvedic medicine for its potential in cancer treatment, which has not been thoroughly explored in recent research. However, the plant extract shows lesser bioavailability.
[0004] Therefore, there is a need for a formulation which is biocompatible and shows cytotoxicity against breast cancer cells, making them a viable option for the treatment of breast cancer in a more natural, cost-effective, and convenient manner and help in overcoming the above-mentioned disadvantage.

OBJECTS OF THE INVENTION
[0005] Some of the objectives of the present disclosure, with at least one embodiment herein satisfied, are listed herein below:
[0006] It is the primary objective of the present disclosure to provide a liposomal composition of the leaf extract of Saussurea obvallata.
[0007] It is yet another objective of the present disclosure to enhance the bioavailability of the leaf extract of Saussurea obvallata
[0008] It is another objective of the present disclosure to improve therapeutic effectiveness: of leaf extract of Saussurea obvallata in anti-cancer applications by utilizing the liposomal formulation to target and kill cancer cells more efficiently.
[0009] It is further objective of the present disclosure to provide a liposomal composition for reducing toxicity and ensuring compatibility with the cells
[0010] It is yet another objective of the present disclosure to provide a ultrasound sensitive liposomal composition that increases the cytotoxicity of the cancer cells.
[0011] It is yet another objective of the present disclosure to provide a simple and cost-effective method for the preparation of liposomal composition.

SUMMARY OF INVENTION
[0012] The present disclosure relates to a liposome comprising:
a leaf extract of Saussurea obvallata; and
a lipid comprising phospholipid and cholesterol,
wherein weight ratio of the leaf extract of Saussurea obvallata to the phospholipid and the cholesterol is 1:3:1.
[0013] The present disclosure also relates to a method of preparing a liposome comprising leaf extract of Saussurea obvallata comprising:
a) providing the dried leaf extract of Saussurea obvallata;
b) dissolving the dried leaf extract and lipids (Soya PC and Cholesterol) in solvents (Chloroform and Methanol in 2:1 ratio) separately,
c) emulsifying the solution of dried leaf extract of Saussurea obvallata in the solution of phospholipids (Soya PC and Cholesterol); to form a mixture; in the ratio of 1:3:1 of plant extract, Soya PC and Cholesterol.
d) stirring continuously the mixture via a magnetic stirrer at a temperature range of 50- 75 °C to form liposome;
e) sonicating the liposomes at 40 W to 42 W with pulse ON for 2 to 3 seconds and OFF for 3 to 4 seconds done for 1 to 2 cycle of 5 to 10 minutes;
f) centrifuging the sonicated liposomes for 5 to 10 minutes at 6000 to 8000 rpm to obtain the liposome of Saussurea obvallata.

BRIEF DESCRIPTION OF DRAWINGS
[0014] The present disclosure contains the following drawings that simply illustrates certain selected embodiments of the nanocarrier composition and processes that are consistent with the subject matter as claimed herein, wherein:
[0015] Figure 1 depicts the (a) image of Saussurea obvallata leaf (b) image of dried leaf extraction (c) BF Bright-field and UV images of the Plant Extract.
[0016] Figure 2 depicts UV and BF images of PE soluble in water, methanol, acetone, and chloroform.
[0017] Figure 3 depicts characterization of PE-loaded Liposomes (PE@Lipos) (a) Size of the PE@Lipos. (b) UV-vis spectra of PE and PE@Lipos. (c) Fluorescence spectra of the PE@Lipos. (d) The Stability of the particle size of PE@Lipos. (e) The FT-IR spectra of PE and PE@Lipos. (f) Standard graph of PE.
[0018] Figure 4 depicts biocompatibility of PE and PE@Lipos on L929 cells.
[0019] Figure 5 depicts cytotoxicity of PE and PE@Lipos on 4T1 cells.
[0020] Figure 6 depicts (a) Cellular uptake of PE and PE@Lipos in 4T1 CELLS. (b) Quantitative analysis of cellular uptake of PE@Lipos in 4T1 cells.
[0021] Figure 7 depicts (a) Live/Dead assay in 4T1 cells treated with PE and PE@Lipos without ultrasound waves (b) Live/Dead assay in 4T1 cells treated with PE and PE@Lipos with ultrasound waves
DESCRIPTION OF THE INVENTION:
[0022] A detailed description of various exemplary embodiments of the disclosure is described herein. It should be noted that the embodiments are described herein in such detail as to communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
[0023] The terminology used herein is to describe particular embodiments only and is not intended to be limiting to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising", or "includes" and/or "including" or "has" and/or "having" when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.
[0024] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
[0025] The present disclosure relates to a liposome comprising:
a leaf extract of Saussurea obvallata; and
a lipid comprising a phospholipid and a cholesterol,
wherein weight ratio of the leaf extract of Saussurea obvallata to the phospholipid to the cholesterol is in range of 1:3:1.
[0026] In an embodiment of the present disclosure, the phospholipid is selected from the group consisting of soybean phosphatidylcholine (Soya PC), Egg phosphatidylcholine, Hydro Soy phosphatidylcholine, and spinach lipid.
[0027] In an embodiment of the present disclosure, an amount of the Saussurea obvallata is in range of 6-7.5mg.
[0028] In an embodiment of the present disclosure, an amount of the phospholipid is in range of 20-25mg of Soya PC and Cholesterol of 6-7.5mg.
[0029] In an embodiment of the present disclosure, the liposome is prepared by solvent evaporation method.
[0030] In an embodiment of the present disclosure, the leaf extract of Saussurea obvallata is encapsulated inside liposomes.
[0031] In an embodiment of the present disclosure, the liposome has a diameter in range of 140 to 145 nm.
[0032] In an embodiment of the present disclosure, leaf extract of Saussurea obvallata shows pinkish fluorescence as shown in figure 2, therefore it can be used as a bioimaging agent as well as anticancer agent.
[0033] The present disclosure relates to a method of preparing a liposome comprising leaf extract of Saussurea obvallata comprising:
a) providing the dried leaf extract of Saussurea obvallata;
b) dissolving the dried leaf extract, cholesterol and phospholipid in a solvent separately;
c) emulsifying the solution of dried leaf extract of Saussurea obvallata in the solution of phospholipid to form a mixture;
d) stirring continuously the mixture via a magnetic stirrer at a temperature range of 50- 75 °C to form liposome;
e) sonicating the liposomes at 40 W to 42 W with pulse ON for 2 to 3 seconds and OFF for 3 to 4 seconds done for 1 to 2 cycle of 5 to 10 minutes;
f) centrifuging the sonicated liposomes for 5 to 10 minutes at 6000 to 8000 rpm to obtain the liposome of Saussurea obvallata.
[0034] In an embodiment of the present disclosure, the solvent is chloroform and methanol in the ratio of a 2:1.
[0035] In an embodiment of the present disclosure, the process of preparing the dried leaf extract of Saussurea obvallata comprising:
a) drying leaves of Saussurea obvallata till the moisture evaporates to form dried leaves;
b) boiling the dried leaves in Milli Q water at a temperature range of 60 to 100° C for 15 to 30 minutes to obtain an extract containing phytochemicals;
c) filtering the extract and drying the leaf for about 1- 1.5 week to obtain the dried leaf extract of Saussurea obvallata.
Origin of Saussurea obvallata:
Kingdom Plantae
Phylum Tracheophytan
Class Magnoliopsida
Order Asterales
Family Asteraceae
Tribe Cynareae
Genus Saussurea
Species Saussurea obvallata
Brahma Kamal, scientifically designated as Saussurea obvallata or Sacred Saussurea, is a flowering plant belonging to the Asteraceae family. This distinctive and exotic plant is indigenous to the alpine meadows of the Himalayas and other mountainous regions of India, Bhutan, Southwest China, Nepal, and Tibet. It is located in the Himalayas at an altitude of around 3700-4600 m. It is the state flower of Uttarakhand and is also known as the 'Queen of Night', 'Night Blooming Cereus' and 'Lady of Night', since it blooms exclusively at night and takes about two hours to bloom entirely, measuring around eight inches. It is also considered the 'King of Himalayan Flowers' due to its prevalence in that area. The S. obvallata plant produces enticing white flowers due to insect-mediated pollination. The image of Saussurea obvallata leaf, dried leaf extract and BF and UV images of the Plant Extract dissolved in a mixture of chloroform and methanol is shown in Figure 1.

ADVANTAGES OF THE PRESENT INVENTION
[0036] In accordance with the present disclosure the liposome of leaf extract has the following advantages:
• The liposomal formulation enhances the solubility and bioavailability of BP, making it suitable for effective cellular uptake and targeted anti-cancer treatment.
• The liposomal formulation is sensitive to Ultrasonic waves that increase membrane permeability, and release plant extract-from the loaded liposomes.
• The formulation's ability to target cancer cells and induce apoptosis makes it more effective.
• Because liposome accumulate more in tumor tissues and less in healthy ones, they reduce off-target effects and minimize toxicity to healthy cells.
• Liposomal encapsulation can shield healthy tissues from the PE effects until it reaches the tumor site, potentially lowering side effects.
• Liposomes can release PE in a controlled manner, maintaining therapeutic levels over longer.
[0037] The present disclosure will be explained using the following examples:

EXAMPLE
Material:
Components involved in Liposome formulation are Soya PC and cholesterol procured from Sigma-Aldrich. Methanol, chloroform, ethanol, and diethyl sulfoxide (DMSO) were obtained from SRL Chemicals Limited, India.
Biological Source:
Saussurea obvallata plant has been obtained from the place Badrinath and the plant is grown under suitable conditions at home in hyderabad. The leaves used for all the experiments are obtained from the same plant.

Example 1:
Preparation of liposomes of fresh leaves extract of Saussurea obvallata
A. Preparation of the dried leaf extract of Saussurea obvallata:
[0038] Dried leaf extract of Saussurea obvallata has been used. The fresh leaves of Saussurea obvallata were taken and shade-dried until the moisture evaporates. The phytochemicals from the leaf were extracted by taking the leaves in MilliQ water and boiling them at 80 °C for 20 minutes. The leaves used for the extraction were dried for a week and made into powder using mortar-pestle. The powder (7.5mg/mL) was dissolved in a 2:1 ratio of chloroform and methanol and is used for further experimentation.
[0039] After that this extract was loaded into liposomes using solvent evaporation method. Briefly, 25 mg Soya PC, 7.5 mg of cholesterol and 7.5 mg/ml of PE, were mixed in a 2:1 ratio of chloroform and methanol separately. The solvent emulsification method was employed using the mixture (mentioned contents) and evaporated at 65 °C after placing it on a magnetic stirrer. To achieve a constant distribution of spherical nanoparticles, continuous stirring was maintained at 65 °C. After synthesis, the liposomes were sonicated at 40 W with a pulse ON for 2 seconds and OFF for 3 seconds, done for 1 cycle of 5 minutes each to ensure proper dispersion and stability. After that, liposomes were centrifuged for 5 minutes at 6000 rpm and, discarded the pellet & kept the supernatant to use for further characterization and other studies. The sample was stored at 4 °C for further analysis.
Example 2:
Characterisation of PE-loaded Liposomes
A. Dynamic Light Scattering (DLS):
[0040] The size of the liposomes was analysed by DLS using the instrument (Inc. Santa Barbara, California, USA)
[0041] . DLS data (Inc. Santa Barbara, California, USA) showed that the hydrodynamic diameter of PE@Lipos was 144.0 ± 2 nm, with a polydispersity index (PDI) of 0.343. The liposomes had an average size ranging from 120 to 140 nm (figure 3 (a)), and their homogeneity was validated by the polydispersity index (PDI).
[0042] The liposomes by varying the ratios of soya PC and cholesterol was tried and their hydrodynamic diameter is given in the table below:
Soya PC Cholesterol PE Size (in nm)
1 1 1 200
2 1 1 250
3 1 1 140





[0043] The liposomes with ratio of leaf extract to soya PC to Cholesterol 1:1:1 and 1:2:1 shows high liposome size and thus PE@lipos of ratio 1:3:1 is selected for further analysis.
B. UV-visible spectroscopy:
[0044] The UV-visible spectra of the synthesized PE@Lipos is done by UV-1800, Japan.
[0045] The UV-visible spectra of the synthesized PE@Lipos exhibited a prominent peak within the wavelength range of 274-276 nm (figure 3 b). The results suggest that PE has been effectively incorporated into the liposome, resulting in PE@Lipos that exhibit higher absorbance in the UV spectrum compared to PE alone.
C. Fluorescence spectroscopy:
[0046] The emission of PE@Lipos is recorded on RF 6000, Japan.
[0047] The fluorescence spectrophotometer recorded distinctive peak wavelengths of 363 nm for excitation and 492 nm for emission of PE@Lipos (figure 3c).
D. Standard calibration curve:
[0048] The calibration curve was prepared by taking various concentrations of PE (ranging from 10 to 50 µl/ml and the readings were collected using UV Spectrophotometer (UV-1800, Japan) and the graphs are plotted using Graph pas prism software which revealed a linear equation of y = 0.0012x for PE, with correlation coefficients (R2) of 0.9507 (figure 3f). This indicates a high level of accuracy in detecting PE concentrations.
E. The FT-IR Analysis
[0049] The FTIR spectra was analyzed using Shimadzu FT/IR-4600 type A, Japan instrument to identify the specific functional groups in the leaf extract of S. obvallata and the synthesized PE@Lipos. The plant extract comprises tannins, saponins, glycosides, flavonoids, terpenoids, phenols, alkaloids, proteins, and steroids. The plot represents the FTIR spectra of plant extract and PE@Lipos. The characteristic band stretching of plant extract at 3359 cm-1 corresponds to the O-H stretching vibrations of phenol or carboxylic acid, and the band at 1603 cm-1 corresponds to the C=O of phenyl rings of flavonoids. The band at 1058cm-1 corresponds to the C-O stretching vibration of the carboxylic acids alcohols (figure 3 e).
[0050] The FTIR spectra of the leaf extract of S. obvallata and PE@Lipos exhibit comparable peaks, with the exception that the spectra of the latter exhibit slight shifts in peak positions and are characterized by sharper and more intense peaks. This shows the role of phytochemicals in the leaf extract on the synthesized PE@Lipos.
Example 4:
The Stability of the particle size of PE@Lipos.
[0051] The synthesized PE@Lipos were kept 4 ºC for 1 month period with a gap of 7 days and checked for stability. The size of liposomes was checked by DLS.
[0052] There is not much difference observed in the size of liposomes over a month with (144.0 ± 2 nm) as shown in Figure 3 (d), indicating the no aggregation and high stability.
Example 3:
Biocompatibility of PE and PE@Lipos on L929 Cells.
[0053] The PE@Lipos were tested for biocompatibility with L929 cells (mouse fibroblast cells).
[0054] The cells exhibited 80 ± 3 % cell viability as shown in figure 4, representing excellent bio-compatibility of the synthesized LPs on normal cell lines uptill 0.01-1000 µg/ml concentration
Example 4:
Cytotoxicity of PE and PE@Lipos on 4T1 cells.
[0055] The viable cell count was calculated upon 4T1 breast cancer cells treated with PE and PE@Lipos for 24 hours. Untreated cells were maintained as a negative control. PE and PE@Lipos had IC50 values of 0.5 mg/ml and 1 mg/ml towards 4T1 cell lines (Figure 5).
Example 5:
Cellular uptake of PE and PE@Lipos in 4T1 CELLS
[0056] The cellular internalization of the PE and PE@Lipos was estimated in 4T1 cell lines. Due to low cellular internalization, the hydrophobic compounds cannot meet the effective threshold. Therapeutic efficacy and high cellular internalization are possible with optimal hydrophilicity drugs. The cell nucleus was stained using DAPI. The most prominent and intense red fluorescence (Figure 6) was detected in 4T1 cells with PE@Lipos compared to other control groups, indicating the uptake of the synthesized LPs into the cell cytoplasm.
Example 6: Ultrasound Assay:
[0057] Method: Live/Dead Assay was carried out in 4T1 cells. 4T1 cells (4×104 cells/well) were seeded and cultured for 24 hours in a 6-well plate for adhesion of the cells. The cells were treated with PE and PE@Lipos at IC50 values. Following the treatment, the cells were incubated in a CO2 incubator for 3 hours. After that we treated the cells with ultrasound waves, treated each well with 5 minutes. Following the treatment, the cells were incubated in a CO2 incubator for 24 hours. The live and dead cell groups were visible using FDA (green fluorescence for live cell staining) and PI (red fluorescence for dead cell staining). FDA and PI were utilized at doses of 20 μM concentrations each. The images of live and dead cells were captured with a Zoe fluorescent microscope.
[0058] The ultrasound assay was carried out using Plant extract liposomes (PE@Lipos) formulated using two phospholipids Soya PC and cholesterol in the ratio of 1:3:1 of plant extract (PE), Soya PC and cholesterol.
[0059] It was observed that the cells treated with PE@Lipos exhibited considerable cell death with ultrasound waves. Still, negligible death of cells was observed in the untreated groups without ultrasound (Figure 7). Fig 7 (a) Live/Dead assay in 4T1 cells treated with PE and PE@Lipos without ultrasound waves (b) Live/Dead assay in 4T1 cells treated with PE and PE@Lipos with ultrasound waves.
[0060] This qualitative analysis revealed that the synthesized complexes possess anti-cancer properties and can be used as an anti-carcinogenic agent.
[0061] Physical features of the liposomal bilayer and interactions within it make phospholipid formulations like soybean phosphatidylcholine (PC) and cholesterol sensitive to ultrasonic vibrations. Effects of ultrasonic waves on this formulation include the following:
1. Ultrasonic waves temporarily increase liposomal membrane permeability. This greater permeability boosts plant extract release.
2. Ultrasonic waves cause shear stresses and cavitation in the medium. Energy from bubble collapse breaks the lipid bilayer, potentially releasing liposome-encapsulated plant extract.
3. Ultrasonication breaks down liposomes into smaller, more homogeneous vesicles. Reduced liposome size may improve chemical stability and release.
4. Soya PC and cholesterol form a strong bilayer that may endure minor physical stress.
In conclusion, ultrasonic waves can change liposome structure, increase membrane permeability, and release plant extract-loaded liposomes. Liposomal composition and ultrasonic parameters determine sensitivity.
, Claims:
WE CLAIM
1. A liposome comprising:
i) a leaf extract of Saussurea obvallata; and
ii) a lipid comprising phospholipid and cholesterol,
wherein weight ratio of the leaf extract of Saussurea obvallata to the phospholipid to cholesterol is in range of 1:3:1.
2. The liposome as claimed in claim 1, wherein the phospholipid is selected from the group consisting of soybean phosphatidylcholine, Egg Phosphatidylcholine, Hydro Soy Phosphatidylcholine, and spinach lipid.
3. The liposome as claimed in claim 1, wherein an amount of the Saussurea obvallata is in the range of 6-7.5 mg.
4. The liposome as claimed in claim 1, wherein an amount of cholesterol is in the range of 6-7.5 mg
5. The liposome as claimed in claim 1, wherein an amount of the phospholipid is in the range of 20-25 mg.
6. The liposome as claimed in claim 1, wherein the liposome is prepared by solvent evaporation method.
7. The liposome as claimed in claim 1, wherein the leaf extract of Saussurea obvallata is encapsulated inside liposomes.
8. The liposome as claimed in claim 1, wherein the liposome has a diameter in range of 140 to 145 nm.
9. The liposome as claimed in claim 1, wherein the leaf extract Saussurea obvallata is the dried leaf extract
10. A method of preparing a liposome comprising leaf extract of Saussurea obvallata comprising:
a) providing the dried leaf extract of Saussurea obvallata;
b) dissolving the dried leaf extract and lipids in a solvent separately;
c) emulsifying the solution of dried leaf extract of Saussurea obvallata in the solution of phospholipid to form a mixture;
d) stirring continuously the mixture via a magnetic stirrer at a temperature range of 50- 75 °C to form liposome;
e) sonicating the liposomes at 40 W to 42 W with pulse ON for 2 to 3seconds and OFF for 3 to 4 seconds done for 1 to 2 cycle of 5 to 10 minutes.
f) centrifuging the sonicated liposomes for 5 to 10 minutes at 6000 to 8000 rpm to obtain the liposome of the leaf extract of Saussurea obvallata.
11. The process as claimed in claim 10, wherein the solvent is chloroform and methanol in the ratio of a 2:1.
12. The process of preparing the dried leaf extract of Saussurea obvallata comprising:
a) drying leaves of Saussurea obvallata till the moisture evaporates to form dried leaves;
b) boiling the dried leaves in Milli Q water at a temperature range of 60 to 100° C for 15 to 30 minutes to obtain an extract containing phytochemicals;
c) filtering the extract and drying the leaf for about 1- 1.5 week to obtain the dried leaf extract of Saussurea obvallata.

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