Abstract
Diterpenoids, a class of bioactive compounds with significant pharmacological properties, have shown
promising potential in cancer treatment due to the_ir cytotoxic and apoptotic effects. This study focuses
on the extraction, characterization, and evaluation of diterpenoids from Euphorbia tirucalli (pencil
cactus) for anticancer applications. The plant material was subjected to solvent extraction, followed by
chromatogrdphic and spectroscopic techniques for compound isolation and identification .. The
anticancer activity of the diterpenoid-rich extract was assessed using in vitro assays on selected cancer
cell lines, evaluating cytotoxicity, apoptosis induction, and oxidative stress modulation. Preliminary
findings indicate significant anticancer potential, suggesting that Euphorbia limcalli could serve as a
valuable natural source for developing novel cancer therapeutics. Further investigations into its
molecular mechanisms and potential clinical applications are warranted.

Field of Invention
The present invention relates to the field of pharmaceutical and biomedical sciences, specifically to
natural product-based drug discovery. More particularly, it focuses on the extraction. characterization,
and application of diterpenoids from Euphorbia tirucalli (pencil cactus) for potential anticancer
applications. This invention encompasses methods for isolating bioactive diterpenoid compounds,
evaluating their cytotoxic effects on cancer cells, and exploring their potential use in developing novel
plant-derived anticancer therapeutics. Additionally, it extends to nutraccutical and pharmaceutical
formulations where these bioactive compounds may be utilized for cancer prevention and treatment.

Methodology
I. CoUection and Preparation of Plant Material
Fresh Euphorbia timcalli (pencil cactus) stems were collected from a verified botanical source.
The plant material was thoroughly washed with distilled water to remove dirt and contaminants.
The stems were shade-dried for 7-10 days at room temperature and then ground into a fine powder
using a mechanical grinder.
The powdered sample was stored in an airtight container for further processing.
2. Extraction of Diterpcnoids
The powdered plant material was subjected to solvent extraction using ethanol, methanol, or an ethyl
acetate system.
The Soxhlet extraction method was employed to optimize diterpenoid yield.
The extract was filtered using Whatman filter paper and concentrated under reduced pressure using
a rotary evaporator.
The crude extract was stored at 4°C for further analysis.
3. Phytochemical Screening nod Chnmcterizntion
Qualitative Anal>•sis: Standard phytochemical tests (e.g., Sulkowski test) were performed to confirm
the presence of diterpenoids.
Quantitative Analysis: The diterpenoid content was estimated usmg colorimetric methods and
spectrophotometric analysis.
Chromatographic Techniques:
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Thin Layer Chromatography (TLC): Used for preliminary compound separation .
High-Performance Liquid Chromatography (HPLC): Used for purification and quantification
of diterpenoids.
Spectroscopic Characterization:
Fourier Transform Infrared Spectroscopy (FTIR): To identify functional groups .
Gas Chronmtography-Mass Spc;ctrometry (GC-MS): To determine molecular
structure.
weight and
Nuclear Magnetic Resonance (NMR) Spectroscopy: For detailed stmctural elucidation .
4. Evaluation of Anticancer Activity
Cell Culture: Selected human cancer cell lines (e.g., MCF-7 for breast cancer, A549 for lung cancer,
Hela for cervical cancer) were cultured in DMEM or RPMI-1640 medium supplemented with IQ%
fetal bovine serum (FBS).

Cytotoxicity Assay (MTT Assay): Cell viability was determined by treating cancer cells with different
concentrations of the diterpenoid extrdct and measuring absorbance at 570 nm.
Apoptosis Assay:
Annex in V/PJ staining was performed to detect apoptosis induction.
Reactive Oxygen Species (ROS) Assay:
The intracellular ROS generation was analyzed using DCFH-DA fluorescence assay.
Gene and Protein Expression Analysis: Rtwerse Transcription~ Polymerase Chain Reaction (RT-PCR)
and Western blotting were used to analyze the expression ofapoptotic and cancer-related genes (e.g .•
Bax, Bcl-2, Caspase-3).
5. Statistical Analysis:
All experiments were conducted in triplicates.
Data were expressed as mean ± standard deviation (SO) and analyzed using GraphPad Prism
sofiware.Statistical significance was determined using ANOVA or Student's t-test, with a significance
level set at p < 0.05.
Reference can be made to KR I 0 JJ97873B I Enhancing of biological activity in mixture of flower
extracts from Camellia species and pharmaceutical composition for treating and preventing cancer
disease: The invention provides the anticancer composition including the antioxidant activity and these
extracts the anticancer activity is confirmed of the thea Sinensis Linn petal and the camellia petal
extract which are the anticancer composition, more specifically, the tea family plant comprising the
petal extract of the tea family plant. The effect of the etc. which shows the antioxidant activity which
is higher than in that case, it independently uses in case of the anticancer composition included mixing
especially, the thea Sinensis Linn petal and camellia petal and using and anticancer activity the tea
family plant petal ext met of the invention and therefore can be applied to the fimctional food indicative
of the cancer prevention and antioxidant effect it is used together and functional cosmetic material etc.
with the various diseases of thempeutic agents and anti-cancer and cancer-preventive effect. The
antioxidant effect is expressed arc expected .
Reference can be made to CN I 0113405 7 A Compound medicnment of natural plant ·extract
containing paclitaxelind anticancer application thereof: The present invention is the application
of yew branch and leaf extract containing taxol in preparing natural compound medicine and orally
taken anticancer medicine. The natural compound medicine consists of yew branch and leaf extract
with taxol content of 1-6 % as the main anticancer component, and epimedium extract with icariin
content of I 0-40 %or Huoba tree root extinct with total alkaloid content of2-15 % for inhibiting the
outward medicine exhaust of cell to mise the bioavailability and anticancer effect of taxol. Animal
experiment shows that the orally taken compound taxol medicine has obvious inhibition on animal's
oophororna, breast cancer, lung cancer, etc.

Claims:
I. A method for extracting diterpenoids from Euphorbia timcalli (pencil cactus), comprising:
• Collecting and drying the plant material under controlled conditions,
• Grinding the dried plant material into a fine powder,
• Extracting diterpenoids using a solvent-based method such as Soxhlet extraction or maceration,
• Filtering and concentrating the ext met to obtain a diterpenoid-rich fraction, and
• Storing the extract for further phannaceutical applications.
2. A diterpenoid-rich extract derived from Euphorbia tirucalli, characterized by its potential
anticancer activity, wherein the extract exhibits cytotoxic, apoptotic, and oxidative stress-modulating
effects on cancer cells.
3. A pharmaceutical composition comprising diterpenoids extracted from Euphorbia tirucall~ in
combination with a pharmaceutically acceptable carrier, wherein the composition is formulated for
treating or preventing cancer by inducing apoptosis and inhibiting cancer cell proliferation.
4. A method for inhibiting cancer cell proliferation using a diterpenoid extract from Euphorbia
tirucalli, comprising:
5.
• Administering an effective amount of the diterpenoid extract to cancer cells,
• Inducing cytotoxicity as determined by in vitro MTT assays,
• Activating apoptosis pathways as confirmed by Annex in V staining and flow cytometry,
and
• Modulating key apoptotic and oxidative stress-related biomarkers, including Bax, Bcl-
2, and Caspase-3.
A nutraceutical or cosmetically formulation comprising diterpenoids from Euphorbia tirucalli,
when:in the formulation provides antioxidant,
administered orally or applied topically.