Anticancer Potential of Calotropis procera: A Contemporary Update Review

Root and root bark

The antitumor effects of water extract from the root bark of Calotropis procera against tumor cell lines were examined. The results indicated that the extract demonstrated cytotoxicity towards human colon adenocarcinoma Caco-2 and mouse neuroblastoma Neuro-2a cell lines. Moreover, the DNA fragmentation assay conducted on Caco-2 cell lines displayed DNA blurring, indicating nuclear material loss resulting in possible genetic toxicity.^[24]

A number of substances were isolated and tested for anticancer activity against prostate PC-3 cancer cell lines, glioblastoma U373, and non-small cell lung cancer A549 in a study on the root bark of Calotropis procera. Calotroposide K and Calotroposide M, two of the extracted compounds, hindered the growth of prostate PC-3 and glioblastoma U373 cancer cell lines.^[25] Nonetheless, the study did not state whether the inhibition effect was selective to the mentioned cancer cells or also harmed the normal cells, which is a critical factor in adapting these compounds for therapeutic uses.

Protein derived from the root bark of Calotropis procera was evaluated for cytotoxic properties in an in vitro and in vivo study. The results revealed that the protein suppressed growth and triggered programmed cell death of breast cancer cells by preventing the activity of nuclear regulator kappa B (NF-kB). Also, the set of rats receiving Calotropis procera protein had a strong elevation in vitamin C, potentially clarifying its potential anticancer characteristics.^[26]

The antitumor effect of the ethanolic extract of Calotropis procera roots against the canine mammary cancer CF41-Mg cell line. The extract exhibited remarkable cytotoxicity and triggered apoptosis. Consequently, Calotropis procera roots are considered a promising choice for anticancer therapy^[27]

Roots of Calotropis procera were extracted using ethanol. The extract was then fractioned into several fractions of chloroform, ethyl acetate, and water to evaluate the anticancer activity of these fractions on Artemia salina in an in vitro study. Ethyl and hexane extracts had the most cytotoxic activity of all fractions.^[28]

The roots of Calotropis procera were extracted using 95% alcoholic solvent. The extract was then evaluated for its antioxidant and anticancer effects on human prostate DU145 and breast MCF-7 cancer cells. The root extract of Calotropis procera exhibited the most inhibitory effect on both cell lines. The Soxhlet ethanolic extract of Calotropis procera showed significant apoptosis against breast MCF-7 and prostate DU145 cancer cell lines.^[29]

Another study was conducted to evaluate the growth inhibition activity of alcoholic, hydro-aqueous, and water extracts and extract separations of Calotropis procera roots on human oral KB and central nervous system (CNS) SNB-78 cancer cell lines. The outcomes of the study revealed that alcoholic extract inhibition activity was the most superior extract, On the other hand, the chloform and n-butanol fractions of the alchoholic extract shown antiproliferative effect on oral human oral cancer (KB) and brain central nervous system (CNS) cancer cell lines, respectively.^[30]

Stem

Various organic solvents were used to extract the stems of Calotropis procera. All extracts were investigated for cytotoxic effect in an in vitro cell lines and in vivo samples of mice with sarcoma. Ethyl acetate and acetone from all extracts were found to be most potent against multiple cell lines, such as human leukemia HL-60, murine melanoma BF16-10, and human colon cancer HCT-8. The inhibition potency was confirmed by the IC_50, which ranged from as low as 0.8 to 4.4 µg/mL across the tested cells. On the other hand, sarcoma 180-induced rats showed significant inhibition of the malignant cells after treatment with ethyl acetate and acetone extracts for a week. It was concluded that both extracts possess significant anticancer properties in both types of trials.^[31]

Latex

The milky sap of Calotropis procera is rich in anticancer compounds, both in pure and isolated forms. Among those compounds with cytotoxic activity are cardenolides, steroids, flavonoids, triterpenoids, and proteins, with various chitinase isoforms present in the latex.^[32]

The milky sap-derived proteins of Calotropis procera were evaluated for anticancer activity on induced sarcoma 180 in mice. The laticifer proteins have exhibited an inhibition effect of approximately 50%, prolonging the life of the mice by four days. The study revealed that proteins extracted from the milky sap of the shrub exhibited pharmacological ability to modulate cancer cell growth.^[33]

The milky sap of Calotropis procera was used to extract laticifer proteins. The isolated proteins were then tested against an array of malignant cell lines, including promyelocytic HL-60, bowel HCT-8, human breast MDA-MB-435, and brain SF295 cancer cell lines, in an in vitro study. The isolated latex proteins were found to show strong cytotoxic effects. Values of IC₅₀ varied between 0.42 and 1.36 μg/mL, confirming the activity of these isolated species against cancerous cells. Notably, the latex-isolated proteins showed preferential toxicity on malignant cells with minimal harm on normal cells. Nonetheless, the need for in vivo studies, which is ongoing, is critical to support the cytotoxic effect of the proteins further.^[22] Nonetheless, in vivo confirming studies of these findings are a critical step before utilizing the proteins in human trials, given the known toxicity of the milky sap of Calotropis procera. Until proven under in vivo conditions, the potential of these derived proteins as an anticancer agent remains unestablished.

The milky sap of Calotropis procera was fractioned into several organic fractions. The fractions were then investigated for anticancer activity on leukemia HL-69, colon cancer HCT-116, ovarian malignancy Ovcar-8, and glioblastoma SF-295 cell lines. Of the organic portions used in this study, hexane and ethyl acetate had the most anticancer activity against all malignant cell lines tested. The IC₅₀ values reported according to the study varied between 0.05 and 6.5 µg/mL.^[34]

Another study used the latex from Calotropis procera to isolate cardiac glycosides and tested them on the breast MCF-7 cancer cell line. The latex extract restrained MCF-7 cells from growing significantly, and the effect increased in a direct relationship with concentration. Also, there was an increase in the levels of reactive oxygen species (ROS) inside cells, which is thought to be caused by the extract of cardiac glycosides. The extract of cardiac glycosides caused MCF-7 cells to die, endure programmed cell death, and autophagy.^[35]

The milky sap of Calotropis procera was utilized to extract an alkaloid called T-undecalactone. The compound exhibited cytotoxic activity, and the cytotoxic effect of T-undecalactone increased proportionally with the concentration against oral and oesophageal cell cancer.^[36]

Methanolic extract of Calotropis procera latex was evaluated against bowel cancer. The treatment with dried latex extract resulted in normalization of levels of glutathione, superoxide dismutase, nitrite, myeloperoxidase, prostaglandin E2, tumor nercosis factor α (TNF-a), and interleukin-1 beta in the rats diagnosed with bowel cancer.^[37]

The milky sap of Calotropis procera was used to isolate enzymes specifically chitinase enzymes in a study. The enzymes were evaluated for anticancer activity on colon cancer HCT-116, ovarian cancer OVCAR-8, and brain cancer SF-295 cell lines. The study observed that the chitinase enzyme LPp1-P4 kept all anticancer activities of LPp1 against cancer cell lines, which was notable in IC₅₀ found in values ranging between 1.2 μg/mL and 2.9 μg/mL. A conclusion was reached that chitinase enzyme isoforms LPp1-P4 isolated from the plant possess significant anticancer properties against cancerous cells.^[38]

One of the cardenolides present in the milky sap of Calotropis procera has shown significant properties in fighting cancer cells, including brain, breast, lung, liver, and colon cancer cells.^[39]

Leaves

The methanol extract of Calotropis procera leaf was evaluated to assess its extractability in fighting breast cancer in MCF-7 breast cancer cells. The extract has been found to exhibit a significant cytotoxic effect on breast cancer cell lines.^[40]

One of the constituents of Calotropis procera leaves was isolated and found to be colchicine. The isolated compound was evaluated for its anticancer activity on different cell lines, namely breast MCF-7 and human esophageal cancer cell lines. The extracted compound exhibited marked activity on both MCF-7 and human esophageal carcinoma SK-GT-4 cell lines with values of IC₅₀ of 55.33 µg/mL and 522 µg/mL respectively.^[41]

Gold nanoparticles produced from Calotropis procera leaves extract were assessed for cytotoxic activities against MCF-7 cell lines. The generated gold nanoparticles turned the cells less viable, with an IC₅₀ value of 0.312 µg/mL.^[42]

The methanolic extract of the leaves of Calotropis procera was evaluated for cytotoxic effect on breast MCF-7 cancer cell lines. It was concluded that the alcoholic leaves extracts showed antiproliferative properties on breast MCF-7 cancer cells. Also, the leaves extract showed a potent antibacterial effect.^[20,43]

The extract was then evaluated for anticancer activity against prostate cancer cells. The extract has blocked the proliferation and movement of prostate tumor cells by modulating the autophagic process and diminishing intracellular ROS concentrations. Moreover, analysis using liquid chromatography-mass spectrometry (LC-MS) of the extract and the cells treated with extract showed that the major compounds present inside prostate cancer cells were terpene derivatives, flavonoid glycosides, and cardenolides indicating possible explanation that those compounds are responsible for anticancer activity on prostate cancer cells.^[44]

Pure alcoholic solvent was used to extract the leaves of Calotropis procera. The extract was then evaluated against human colon cancer HCT-116, breast malignant tumor MCF-7 cell lines. The blocking effect of the extract on cell growth was found to be proportional to concentration. Values of IC₅₀ were 50 μg/mL and 55 μg/mL on breast malignancy MCF-7 and colon tumor HCT-116, respectively. The measurement IC₅₀ showed that the extract had potent anticancer activity.^[45]

Methanol was used to extract Calotropis procera leaves. The leaves extract was tested on H1299 lung malignant cells was investigated. The article showed that the methanol extract of Calotropis procera leaves exhibits antioxidant and antigrowth properties. The methanol extract of leaves decreased cluster of differentiation 146 (CD146) expression on blood cells after 24 h. The study revealed that Calotropis procera leaf extract exhibits antiproliferative properties by reducing cell viability and possesses the potential to scavenge OH and radicals.^[46]

A further investigation assessing the cytotoxicity of Calotropis procera leaf extract revealed significant cytotoxic effects in the brine shrimp mortality bioassay, attributed to the existence of quercetin derivatives.^[47]

The anticancer effect of Calotropis procera leaf extract was investigated against different human glioblastoma cell line types. The extract showed potent efficacy in reducing the proliferation of all three glioblastoma multiform (GBM) cell line types, exhibiting heightened cytotoxicity in human glioblastoma U251 GM cells, with an IC₅₀ value of 2.63 ± 0.23 μg/mL, probably by stopping the cell cycle at the G2 to M progression. Nevertheless, the extract demonstrated no harmful impacts in zebrafish eggs, even at doses reaching 1000 μg/mL. The study indicated that Calotropis procera leaf extract is a proposed effective treatment for glioblastoma with minimum side effects.^[48]

Water was used to extract the leaves of Calotropis procera. The extract was evaluated for the survival rate of human colon HT-29 cells. The extract showed moderate anticancer activity as shown in IC₅₀ value of 236.87 µg/mL. Moreover, the survival time of cells diminished with increasing concentration of the extract.^[49]

Aqueous solvent was used to extract the dried leaves of Calotropis procera. Soxhlet-assisted extraction was employed to obtain a polyphenol-rich extract. The extract was then investigated for cytotoxic activity on human glioblastoma cancer LN-18 cell lines. It is believed that the polyphenol content of the extract may contribute to the anticancer activity on LN-18 cells.^[50]

A crude methanolic solvent was used to extract the leaves of Calotropis procera. The extract was then investigated for its anticancer properties on breast cancer MCF-7 cell lines. The crude extract exhibited antigrowth effect on MCF-7 cells.^[20,51]

Calotropis procera leaves were extracted with ethanol. The butanol fraction of the extract was then divided by column chromatography to get different elutes. The elutes were then evaluated on liver cancer HEPG2 cell lines using the Sulforhodamine B (SRB) method. Chloroform: methanol 9:1 fraction exhibited significant anticancer activity, reducing the cell growth by half. It was concluded that the flavonoid content of the fraction may be responsible for cytotoxic activity.^[52]

Flowers

Methanolic solvent was used to extract the flowers of Calotropis procera using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on laryngeal cancer Hep2 and mammalian kidney cell line Vero cells. The extract exhibited complete cytotoxicity against Hep2 cell lines at a dosage of 12.5 mg, while Vero cells showed 83% at the same concentration.^[53]

Ethanolic solvent was used to extract the flowers of Calotropis procera. The extract was then examined in a chromatographic evaluation by aqueous and a mixture of aqueous/alcohol solvents. Six compounds were isolated from the extract, which were then tested for antitumor properties on a panel of human cancer cell lines, including breast MCF-7, colon HCT-16, liver HepG-2, and lung A549 cancers. Among the isolated compounds, methyl ferulate was found to be the most active against all cancer cells, with an IC₅₀ ranging from 4.99 to 12.6 µg/mL.^[21]

An ethanolic solvent with 70% was used to extract the flowers of Calotropis procera using the MTT assay. The extract was then evaluated for anticancer activity on breast tumor MCF-7 and cervical tumor HeLa cell lines. It was concluded that the flower extract possesses significant anticancer properties on both malignant cells.^[54]

Distilled aqueous solvent was used to extract the flowers of Calotropis procera. The extract has been used to synthesize silver nanoparticles that have been incorporated as a gel for use on skin. The formulated nanogel composed of silver nanoparticles has shown anticancer activity on skin cancer A-431 cells. It was concluded that the nanoparticles derived from the shrub are a potential skin cancer candidate to produce skin cancer drugs^[55]

Aerial parts

Another study evaluated the effects of Cathepsin L (CTSL) inhibition on cancer cell migration and invasion. SnuCalCpI15, a proprietary derived from the I29 domain of a CTSL-like cysteine protease isolated from the aerial parts of Calotropis procera, was evaluated for its inhibitory activity. The findings showed that SnuCalCpI15 selectively inhibited CTSL in a reversible, slow-binding manner. Its activity was supported by a low inhibitory constant (Ki) of 1.38 ± 0.71 nM. Furthermore, the propeptide was detected within MDA-MB-231 human breast cancer cells, where it suppressed malignant cell migration and invasion.^[56]

Ethanolic and water extracts from the aerial parts of Calotropis procera were used to synthesize environmentally friendly (green) nanoparticles containing iron or zinc. The most potent antitumor activity on A-431 human epidermoid malignant cells was demonstrated by zinc nanoparticles made from the aqueous extract, with an IC₅₀ value of 188.97 µg/mL. The result highlights the significant potential of sustainable nanomaterials as anticancer agents.^[57]

A summary of cytotoxicity studies on different components of Calotropis procera has been summarized in Table 2.^[58-61]

Cardenolides

Eleven cardenolides were extracted through fractionation guided by bioassay of the effective chloroform extract of Calotropis procera latex. The antiproliferative properties of all isolated cardenolides from Calotropis procera were investigated using the MTT colorimetric assay on human lung A549 and HeLa cervix cancer cell lines. Calactin showed the highest cytotoxicity of these substances on both cell lines, with IC₅₀ measurements of 0.036 μM for A549 and 0.083 μM for HeLa, respectively. A tentative structure-function connection within these compounds indicates that the doubly connected six-membered cyclic sugar system offers a further positive impact [Figure 2].^[58] However, the structure-activity relationship is based on a few compounds. A larger study could solidify the hypothesis.

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Figure 2:

Doubly linked six-membered ring moiety.^[58]

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The ethyl acetate fraction was fractioned from the methanolic extract of Calotropis procera root bark during a study. The process of fractionation resulted in the identification of three cardenolides, namely procera A, frugoside, and calotropin. The separated compounds were then tested for cytotoxic effect on lung cancer A549, prostate cancer PC-3, and glioblastoma U373 cell lines. Calotropin has been proven to possess the most active properties among the three compounds on all cancer cell lines, with IC₅₀ values of 0.005 µg/mL on all tested cancer cells.^[23]

Methanol and butanol were used to extract the aerial portions of Calotropis procera, while methanol only was used to extract the milky latex of the shrub. The process of extraction resulted in the identification of eight cardenolides. Of the isolated compounds, calotropin and calotoxin were found to possess anticancer effects on breast cancer MDA-MB-231 and human lung A549 malignant cells. The activity of calotoxin was confirmed by the IC₅₀ values, which were found to be 0.046 and 0.057 µg/mL on breast and lung cancer cells, respectively.^[63]

Evaluation of Calotropis procera root bark led to the detection of a cardenolide, 2ʺ-oxovoruscharin. The compound was used to hemi-synthesize another cardenolide exhibiting significant cytotoxicity against several cancer cell lines. The hemi-synthesized compound showed considerable cytotoxicity, with IC₅₀ values of 3, 9, 24, 10, and 8 µM against human glioma Hs683, glioblastoma U373, colon HCT-15, colon LoVo, and A549, respectively.^[64]

Uzaigenin and calotropagenin, two elements of Calotropis procera, are natural compounds that have exhibited an ability to suppress cancer cells both in vitro and in vivo, with a specific emphasis on mitochondrial activity.^[65]

Oxypregnanes

An evaluation of the normal butanol extract fraction from the root bark of Calotropis procera resulted in the identification of a set of novel oxypregnane oligoglycosides. The antiproliferative activity of the normal butanol fraction and extracted chemicals was tested on human lung A549, glioblastoma U373, and prostate PC-3 tumor cell lines. Two substances revealed notable cytotoxicity, with IC₅₀ values between 0.5 and 0.7 µM on U373 and PC-3 cell lines.^[25]

Flavonoids

Evaluation of Calotropis procera leaf extract resulted in the separation of 13 compounds. These compounds were evaluated for anticancer effects on brine shrimp. The brine shrimp mortality bioassay test revealed that of the isolated compounds, quercetin, isoharmentin, and azaleatin exhibited cytotoxic effects on brine shrimp with IC₅₀ values of 10, 22, and 21 µg/mL, respectively. The existence of free OH substituents on the A- or B-cycles is likely essential for the antitumor effect of quercetin, because complete methyl substitution of quercetin hydroxyl substituents totally deactivated it. Additionally, 3-etherification entirely removed the cytotoxicity, confirming the significance of the three hydroxyl groups substitution of quercetin for its activity [Figure 3].^[47]

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Figure 3:

Key free hydroxy groups for quercetin cytotoxicity.^[47]

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The ethanolic extract of Calotropis procera stems was used to isolate five components. Of these compounds, uzarigenin exhibited moderate cytotoxicity. The findings revealed that uzarigenin reduced the biochemical effect of HT29 and HepG2 cells by 59% and 35%, respectively, at a concentration of 50 µM.^[66]

Triterpenes

Root bark of Calotropis procera hexane fraction was used to separate calotroprocerol A, a novel triterpene [Figure 4]. The compound was evaluated for inhibition activity using the MTT assay method on three cancer cell lines. The results revealed that calotroprocerol A had cytotoxic efficacy, with IC₅₀ values of 11.84, 11.84, and 21.98 µg/mL in A549, U373, and PC-3 cell lines, respectively.^[67]

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Figure 4:

Compounds with cytotoxic activity in Calotropis procera.^[23,58,63]

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Other compounds

The fruits, flowers, and leaves of Calotropis procera were utilized to extract methyl ferulate. The chemical was assessed for cytotoxic efficacy on MCF-7, HCT-116, HepG-2, and A549 human cancer cell lines.^[68] Methyl ferulate demonstrated considerable cytotoxicity, with IC₅₀ values of 12.60 ± 2.0 µg/mL for MCF-7, 6.80 ± 0.46 µg/mL for HCT-116, 4.99 ± 0.60 µg/mL for HepG-2, and 6.03 ± 0.52 µg/mL for A549, respectively.^[21]

Compounds with cytotoxic potential isolated from Calotropis procera have been summarized in Table 3, Figure 5 and referenced in table 4.

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Figure 5:

Summary of cytotoxic compounds isolated from different parts of Calotropis procera.^{[23,58,63,68]}

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