Saravanakumar R, Karthik D, Kumar R, Mohan L, Rabeela R, Jerome N. Assessment of Apoptosis-inducing Potential of Cyclopeptide RA-V on Human COLO 205 Colon Cancer Cells. Biomed Pharmacol J 2025;18(1).

---

Manuscript received on :01-08-2024
Manuscript accepted on :20-11-2024
Published online on: 27-01-2025

---

Plagiarism Check: Yes
Reviewed by: Dr. Tarun kumar
Second Review by: Dr. Zena Sideeq Tawfeek
Final Approval by: Dr. em. Hans-Joachim Freisleben

---

How to Citeclose    |   Publication History close

 Views: 

Visited 82 times, 3 visit(s) today

 

 PDF Downloads: 

26

Rupachandra Saravanakumar^*, Dharshene Karthik, Raj Kumar, Lavanya Mohan, Rabeelaand Nithesh Jerome

Department of Biotechnology, School of Bioengineering, SRM Institute of Science and Technology, Kattankulathur, India

Corresponding Author E-mail: rupachas@srmist.edu.in

DOI : https://dx.doi.org/10.13005/bpj/3111

Abstract

The inadequacy of eliminating the entire population of tumor cell and the concomitant creation of chemoresistance is a major challenge for an effective colon cancer treatment. Many compounds which are natural have been shown to be optimistic, based upon their antitumor effects and a decrease in toxicity. Natural cyclopeptides are small peptides that are rich in disulfide bonds having a cyclic backbone being isolated from plants. The peptides are resistant to enzymatic, thermal and chemical conditions due to the presence of the cyclic cystine knot like structure. Plant derived cyclic peptides hold a very significant role in the field of cancer chemotherapy. However, a never-ending endeavor is a search to find novel therapeutic compounds for treating and controlling cancer. RA-V (deoxybouvardin), is a natural cyclopeptide that is extracted from the Bouvardia ternifolia roots belonging to the Rubiaceae family. RA-V cyclopeptide exhibits anti-angiogenesis, anti-inflammatory and anti-cancer activities. Apoptosis means a programmed cell death which is being characterized by membrane blebbing, nuclear fragmentation and chromosome condensation. This study focuses on evaluating the efficacy of cyclopeptide RA-V in human colon cancer cell lines.

Keywords

Apoptosis; Cell death; Chemotherapy; Colon Cancer; Cyclopeptide; Rubiaceae

Introduction

Colorectal cancer is among the most widespread and aggressively expanding cancers across global populations, ranking as one of the leading causes of cancer-related deaths.¹ This type of cancer often manifests sporadically, though it can also arise due to hereditary factors that promote inflammation within the bowel region.² Over the last few decades, the prevalence of colon cancer has been increasing, and this rise correlates strongly with the global surge in obesity rates.³ One of the primary preventative strategies to combat the onset and progression of colorectal cancer is through inhibiting tumor growth and carcinogenesis at the early stages. This approach, known as chemoprevention, involves the use of natural compounds that possess cancer-fighting properties, aiming to halt the progression of cancerous cells before they can advance.⁴

In recent years, bioactive peptides have garnered significant attention in cancer research due to their high tissue-binding properties, specificity, and overall effectiveness in promoting health.⁵ These peptides are known to perform a wide range of functions within human metabolism, making them particularly attractive candidates for drug development.⁶ Cyclopeptides, a subset of bioactive peptides, have emerged as promising targets for anticancer drug research. By evaluating current biomedical evidence, researchers have gained insights into the structure-activity relationship of cyclopeptides and examined their potential in therapeutic development.⁷ It has been favored that the natural compounds isolated from plants function with a lower level of toxicity when compared to the chemically derived drugs.⁸

RAs have fascinated a great interest due to their distinctive bicyclic structural featuring disulphide bridges and significant anti-cancer activities in vivo and in vitro.⁹ RA-V, also referred to as deoxybouvardin, is a well-known cyclic peptide initially isolated from the roots of Bouvardia ternifolia, possessing a molecular weight of 756.9 Daltons.¹⁰ RA-V has also been isolated from the roots of medicinal plants of other relevant species such as Rubia cordifolia and Rubia yunnanensis.^11–12 Research has shown that RA-V exhibits multiple cancer-suppressing properties, such as reducing inflammation,⁹ inhibiting tumor growth,¹¹ curtailing angiogenesis,¹³ and triggering apoptosis – the programmed cell death that prevents cancer cells from proliferating.¹⁴

Apoptosis is a dominant mechanism which eliminates cancer cells without causing damage such as morphological changes and rupture to normal cells and the tissues surrounding the tumor cells. The thought process of apoptosis provoked the development of therapies that are targeted through novel drugs that induce apoptosis or enhance the sensibility of tumor cells to authorized agents that are cytotoxic in nature. Previous studies have shown that RA-V significantly and effectively blocked the proliferation of tumor cells in human breast cancer cells.¹⁴ This study is aiming at the analysis of the effect of RA-V on the proliferation of cells and apoptotic activity against human COLO 205 colorectal cancer cells.

Materials and Methods

Chemicals and Reagents

RPMI-1640 (Roswell Park Memorial Institute medium1640), fetal bovine serum (FBS), Hoechst 33342, penicillin and streptomycin were acquired from Hi-Media (Chennai, Tamil Nadu, India). acridine orange, ethidium bromide, 2′,7′-dichlorofluorescein diacetate, MTT (2,5-diphenyltetrazolium bromide), resazurin were purchased from SRL (Sisco Research Laboratories) (Chennai, Tamil Nadu, India). The Real-time cell viability assay kit and the flow cytometry assay kit were acquired from Promega (Chennai, Tamil Nadu, India) and Bio-vision (Chennai, Tamil Nadu, India) respectively. The cyclopeptide RA-V was purchased from Chem-Faces (Chennai, Tamil Nadu, India). This entire study was performed in Sri Ramaswamy Memorial Institute of Science and Technology. (Chennai, Tamil Nadu, India)

Cell proliferation inhibition

Cell culture

The human COLO 205 colon cancer cell lines were procured from NCCS (National Centre for Cell Science), Pune, India. The cells were cultured with RPMI 1640 medium along with 5% FBS (Fetal Bovine Serum), 1% Streptomycin (100µg/mL) incubated at a temperature of 37˚C and humidity of 5% CO_2.

Cell viability assays

MTT assay

The inhibition of viability of cells was determined by MTT Assay.¹⁴ The process is carried out by incubating the COLO 205 cells (1×10⁵cells/mL) seeded in 96-well plate treated with positive control doxorubicin and different concentrations of RA-V (50, 75, 100 and 125µM) for the various time periods (24 and 48 h). After the incubation of a specific time interval, the MTT solution (7 mg/mL) was added and incubated for 2 h in a cell culture incubator. The media was discarded from all the wells and the formazan crystals were dissolving by adding DMSO (100 µl). At the end of 30 mins of incubation, the absorbance was read at 570 nm using a microplate reader.

Resazurin Cell viability assay

The proliferation of cells is inhibited and the percentage of cell viability was analyzed by Resazurin Cell Viability Assay¹⁵ in which the colon cancer cells with a density of 1×10⁵cells/mL were seeded in a 96-well plate and treated with different concentrations of RA-V (50, 75, 100 and 125µM) to determine the viability of cells. 200 µl of resazurin solution (500 µg/mL) was added to all the wells at the end of incubation of different time intervals (24 and 48 h) and further incubated for about 4 hours. The absorbance was measured using a microplate reader at 560 nm and 590 nm.

Real-time cell viability assay

The inhibition of cell proliferation was performed by RealTime-Glo™ MT Cell Viability Assay.¹⁶ In a 96-well plate, the cells were seeded, incubated and treated for distinct time intervals at different concentrations of RA-V (50, 75, 100 and 125µM) to determining the viability of cells after the treatment. The Luminescence at 570 nm was measured after incubating the cells with 100 µl of RealTime-Glo™ reagent for 1 hour by using a microplate reader and the results are expressed statistically in terms of mean±SEM.

Apoptotic effect on RA-V treated human colon cancer cell lines

Acridine Orange/Ethidium Bromide Staining

The determination of apoptotic activity on cells was performed by co-staining of acridine orange/ethidium bromide (AO/EtBr) staining.¹⁷ The COLO 205 cells were seeded in a 6-well plate upon coverslips and treated with optimum concentrations of RA-V (100 and 125µM) for different time intervals (24 and 48 h). The fixation of cells with 4% paraformaldehyde was performed; 100µl and 30µl of acridine orange (1 mg/mL) and ethidium bromide (1 mg/mL) respectively were added on to all the wells under dark followed by PBS wash to remove the excess stain. The cells were mounted and visualized using a fluorescent microscope.

DCFDA Staining

The generation of reactive oxygen species which indicates apoptosis was determined by DCFDA staining.¹⁸ COLO 205 cells with a density of 1×10⁶ were grown on coverslips in a 6-well plate and the cells were treated for different time intervals at optimized concentrations of RA-V (100 and 125µM). 100 µl of DCFDA solution (25 mg/mL HBSS buffer) was added to all the wells after fixing the cells and was incubated for 15 mins. The cells were visualized to determine the oxidative stress under a confocal microscope.

Hoechst Staining

The condensation of chromosomes in apoptotic cancer cells was visualized by Hoechst staining.¹⁹ 30 µl of Hoechst 33342 staining solution (10µg/mL) prepared from 10 mg/mL of stock solution was added on to all the wells consisting of coverslips seeded with COLO 205 cells treated with optimum concentrations of RA-V (100 and 125µM) for 24 and 48 h. The cells were incubated for 15 mins and were photographed with confocal microscopy.

Results and Discussion

RA-V inhibits cell viability of human COLO 205 colon cancer cell lines

The morphological changes of COLO 205 cells upon the treatment of RA-V were examined using phase contrast microscopy (Fig.1). The cell viability upon the action of RA-V on COLO 205 cells was found to be constantly decreasing with the increase in the concentrations of RA-V by MTT assay which proved that RA-V possesses anti-cancerous activity inhibiting the proliferation of cells in human colon cancer (fig 2). The cell viability was found to be lowest at the concentrations of 100 µM, and 125µM for 48 h treatment when compared to 24 h treatment. The results obtained were analogous to the effect of RA-XII against breast cancer with the inhibition of cell proliferation.²⁰ Further, the examination of the effect of RA-V on cell viability by Resazurin Cell Viability (fig 3) and RealTime-Glo™ MT Cell Viability Assays (fig 4) showed a drastic reduction in the percentage of viability of cells as the concentration of RA-V increases which is relative to the results obtained from MTT assay. The results of the Resazurin Cell Viability assay obtained in this study is comparative to the action of peptides isolated from various medicinal plants against breast cancer cells showing a decrease in the proliferation of cells.²¹ The bio-active peptide from the residual protein of Juglans regia inhibits the proliferation of cancer cells, demonstrating the cytotoxic activity of peptide.²² Further, the effect of RA-V against colon cancer cells determined from RealTime-Glo™ MT Cell Viability Assay depicted the inhibition of cell proliferation and tumor growth with a decrease in the number of viable cells, which are similar to the results obtained in the study of the protein hydrolysates of Brassica napus against sarcoma cancer cells.²³ The morphological changes, membrane damage and the stages of apoptosis were observed in RA-V treated COLO 205 colon cancer cells by Acridine Orange and Ethidium Bromide co-staining method (fig 5).  The binding of Acridine Orange to the live cells makes the control cells appear green in colour. The apoptosis is induced after the treatment of RA-V for appropriate time intervals expressing the early apoptotic (reddish-orange) and late apoptotic (orange) stages, as dead cells take up the Ethidium Bromide stain. The similar results showing morphological changes were obtained with the bioactive protein isolated from Abies webbiana inducing apoptotic activity of lung cancer cells.¹⁷ The beta-amyloid peptide isolated from Juglans regia induced apoptosis in PC12 cells associated with morphological changes.²⁴ Further the stages of apoptosis and changes in the morphology of cells were also observed in liver cancer cell lines upon the action of MussaeninA extracted from Mussaenda glabrata belonging to the Rubiaceae family ²⁵ stained with Acridine Orange and Ethidium Bromide. The chromosomal condensation being a characteristic feature of apoptosis was studied by Hoechst33342 staining in RA-V treated COLO 205 cells which revealed the nuclear changes and chromosomal condensation with the increasing concentrations of RA-V in the treated cells which are characteristics of apoptosis (fig 6). The nuclear changes and chromosomal condensation were observed in the previous studies which revealed that β-Purothionin, a peptide induced cell death ²⁶ showing an enhanced apoptotic activity. The protein isolated from the tubers of Corydalis cava exhibited apoptotic activity against HeLa cervical cancer cells by disrupting the cell membrane associated with nuclear changes²⁷ aiding the chromosomal condensation which is also a characteristic feature of apoptosis in RA-V treated colon cancer cell lines observed by Hoechst 33342 staining. Further, the oxidative stress evaluation was correlated with the production of ROS (reactive oxygen species) in the colon cells treated with multiple concentrations of RA-V was performed by DCFDA staining method (fig 7) which were compared with the earlier studies of antioxidant properties of the peptides isolated in soybean and lunasin.²⁸ The peptides isolated from the protein samples of Juglans Sigillata Dode and Juglans regia L also showed antioxidant activities, thereby inhibiting the production of reactive oxygen species.^29-30

Figure 1: Photo micrographs (10x) of the morphology of the COLO 205 cells treated with RA-V using phase contrast microscope (A)Control (B)50µM (C)75µM (D)100µM (E)125µM for 48hr. Click here to view Figure

Figure 2: Determination of viability of RA-V treated colon cancer cells using MTT assay. The values are expressed as mean±SEM and were statistically significant (****P<0.0001), compared with control (A) 24hr (B) 48hr. Click here to view Figure

Figure 3: Determination of viability of RA-V treated colon cancer cells using Resazurin cell viability assay. The values are expressed as mean±SEM and were statistically significant (****P < 0.0001), compared with control (A) 24hr (B) 48hr. Click here to view Figure

Figure 4: Determination of viability of RA-V treated colon cancer cells using Real-time cell viability assay. The values are expressed as mean±SEM and were statistically significant (****P <0.0001), compared with control (A) 24hr (B) 48hr. Click here to view Figure

Apoptotic study of RA-V treated human COLO 205 colon cancer cell lines 

Figure 5: Representative of fluorescent microscopy images(40X) of Colo 205 cells after treatment with RA-V using Acridine orange and Ethidium bromide stain – 24 hr :(A) Control, (B)100µM, (C)125µM; 48 hr (D) Control, (E)100µM, (F)125µM. Click here to view Figure

Figure 6: Representative of confocal microscopy images (40X) of Colo 205 cells after treatment with RA-V using 2,4, Dichlorofluorescein diacetate stain – 24 hr :(A) Control, (B)100µM, (C)125µM; 48 hr :(D) Control, (E)100µM, (F)125µM. Click here to view Figure

Figure 7: Representative of confocal microscopy images(40X) of Colo 205 cells after treatment with RA-V using Hoechst 33342 stain – 24 hr :(A) Control, (B)100µM, (C)125µM; 48 hr :(D) Control, (E)100µM, (F)125µM. Click here to view Figure

Conclusion

RA-V, a cyclic peptide exhibits antitumor and apoptotic activities against human COLO 205 colon cancer cell lines. In a series of in vitro assays, RA-V demonstrated the ability to inhibit cell viability, induce apoptosis-related morphological alterations, and enhance oxidative stress, corroborating its efficacy as a potent antitumor agent. The noted reduction in cell proliferation, as demonstrated by MTT and Resazurin assays, highlights the cytotoxic effectiveness of RA-V, particularly at elevated concentrations and extended exposure durations. The findings of this study are consistent with earlier research on analogous cyclopeptides, demonstrating that RA-V interferes with cellular mechanisms essential for the survival of cancer cells while preserving normal cells. The findings indicate that RA-V shows considerable potential as a targeted therapy for colorectal cancer, warranting further development in clinical contexts to enhance its efficacy and reduce toxicity.

The apoptotic activity has been studied using several fluorescent and confocal microscopy techniques such as Acridine Orange, DCFDA staining and Hoechst staining. Staining elucidated the phases of apoptosis, ranging from chromatin condensation to membrane disruption, thereby affirming RA-V’s capacity to induce apoptosis.  Therefore, our results confirm that RA-V is a novel anticancer agent that prevents the growth of colorectal tumor cells and induces apoptosis.

Acknowledgement

We would like to express our deepest gratitude to the Department of Biotechnology, School of Bioengineering, Faculty of Engineering & Technology, SRM Institute of Science and Technology, Kattankulathur 603203, Tamil Nadu, India for helping with the laboratory facilities and also thank SRM-DBT for providing the facility to use the fluorescent microscopy.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article

Conflict of Interests

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required

Clinical Trial Registration

This research does not involve any clinical trials

Author contributions

Dharshene Karthik: Literature findings, data and result collection, and manuscript writing. Raj Kumar: Literature findings, data and result collection. Lavanya Mohan: Literature findings, data and result collection. Rabeela: Literature findings, data and result collection. Nithesh Jerome: Formal analysis and editing. Rupachandra S: Conceptualization and supervision of the experiment.

References

1. Siegel, R. L., Miller, K. D., Wagle, N. S., & Jemal, A. Cancer statistics, 2023. CA a Cancer Journal for Clinicians., 2023; 73(1): 17–48.
   CrossRef
2. Zhao Y, Hu X, Zuo X, Wang M. Chemopreventive effects of some popular phytochemicals on human colon cancer: A review. Food & function., 2018; 9(9): 4548-68.
   CrossRef
3. Kim DH, Shin EA, Kim B, Shim BS, Kim SH. Reactive oxygen species‐mediated phosphorylation of p38 signaling is critically involved in apoptotic effect of Tanshinone I in colon cancer cells. Phytotherapy Research., 2018; 32(10): 1975-82.
   CrossRef
4. Lemieszek MK, Ribeiro M, Marques G, Nunes FM, Pożarowski P, Rzeski W. New insights into the molecular mechanism of Boletus edulis ribonucleic acid fraction (BE3) concerning antiproliferative activity on human colon cancer cells. Food & function., 2017; 8(5): 1830-9.
   CrossRef
5. Daliri EB, Oh DH, Lee BH. Bioactive peptides. Foods., 2017; 6(5): 32.
   CrossRef
6. Su LY, Shi YX, Yan MR, Xi Y, Su XL. Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway. Acta Pharmacologica Sinica., 2015; 36(12): 1514-9.
   CrossRef
7. Zhang, J., Xia, Y., & Zhang, H. Natural Cyclopeptides as Anticancer Agents in the Last 20 Years. International Journal of Molecular Sciences., 2021; 22(8): 3973.
   CrossRef
8. Ranjbarnejad T, Saidijam M, Moradkhani S, Najafi R. Methanolic extract of Boswellia serrata exhibits anti-cancer activities by targeting microsomal prostaglandin E synthase-1 in human colon cancer cells. Prostaglandins & other lipid mediators., 2017; 131: 1-8.
   CrossRef
9. Zha, M., Lin, P., Yao, H., Zhao, Y., & Wu, C. A phage display-based strategy for the de novo creation of disulfide-constrained and isomer-free bicyclic peptide affinity reagents. Chemical Communications., 2018; 54(32): 4029–4032.
   CrossRef
10. Balachandran, P., Ibrahim, M. A., Zhang, J., Wang, M., Pasco, D. S., & Muhammad, I. Crosstalk of Cancer Signaling Pathways by Cyclic Hexapeptides and Anthraquinones from Rubia cordifolia. , 2021; 26(3): 735.
    CrossRef
11. Qiao, Z., Zhang, D., Hou, C., Zhao, S., Liu, Y., Gao, Y., . . . Wang, H. A pH-responsive natural cyclopeptide RA-V drug formulation for improved breast cancer therapy. Journal of Materials Chemistry B., 2015; 3(22): 4514–4523.
    CrossRef
12. Tan NH, Zhou J. Plant cyclopeptides. Chemical reviews., 2006; 106(3): 840-95.
    CrossRef
13. Yue GG, Fan JT, Lee JK, Zeng GZ, Ho TW, Fung KP, Leung PC, Tan NH, Lau CB. Cyclopeptide RA‐V inhibits angiogenesis by down‐regulating ERK1/2 phosphorylation in HUVEC and HMEC‐1 endothelial cells. British journal of pharmacology., 2011; 164(7): 1883-98.
    CrossRef
14. Fang XY, Chen W, Fan JT, Song R, Wang L, Gu YH, Zeng GZ, Shen Y, Wu XF, Tan NH, Xu Q. Plant cyclopeptide RA-V kills human breast cancer cells by inducing mitochondria-mediated apoptosis through blocking PDK1–AKT interaction. Toxicology and Applied Pharmacology., 2013; 267(1): 95-103.
    CrossRef
15. Borra RC, Lotufo MA, Gagioti SM, Barros FD, Andrade PM. A simple method to measure cell viability in proliferation and cytotoxicity assays. Brazilian oral research., 2009; 23: 255-62.
    CrossRef
16. Benedetto A, Bocca C, Brizio P, Cannito S, Abete MC, Squadrone S. Effects of the rare elements lanthanum and cerium on the growth of colorectal and hepatic cancer cell lines. Toxicology in Vitro., 2018; 46: 9-18.
    CrossRef
17. Ramachandran S, Menon DB. A novel protein fraction isolated from the leaves of Abies webbiana Lindl. Induces apoptosis in lung cancer cells via the intrinsic pathway. International Journal of Herbal Medicine., 2016; 4(6): 26-33.
18. Shinohara A, Imai Y, Nakagawa M, Takahashi T, Ichikawa M, Kurokawa M. Intracellular reactive oxygen species mark and influence the megakaryocyte-erythrocyte progenitor fate of common myeloid progenitors. Stem Cells., 2014; 32(2): 548-57.
    CrossRef
19. Arora S, Tandon S. Achyranthes aspera Root Extracts Induce Human Colon Cancer Cell (COLO‐205) Death by Triggering the Mitochondrial Apoptosis Pathway and S Phase Cell Cycle Arrest. The Scientific World Journal., 2014; 2014(1): 129697.
    CrossRef
20. Leung HW, Wang Z, Yue GG, Zhao SM, Lee JK, Fung KP, Leung PC, Bik-San Lau C, Tan NH. Cyclopeptide RA-V inhibits cell adhesion and invasion in both estrogen receptor positive and negative breast cancer cells via PI3K/AKT and NF-κB signaling pathways. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research., 2015; 1853(8): 1827-40.
    CrossRef
21. Tepkeeva II, Moiseeva EV, Chaadaeva AV, Zhavoronkova EV, Kessler YV, Semushina SG, Demushkin VP. Evaluation of antitumor activity of peptide extracts from medicinal plants on the model of transplanted breast cancer in CBRB-Rb (8.17) 1Iem mice. Bulletin of Experimental Biology and Medicine., 2008; 145: 464-6.
    CrossRef
22. Ma S, Huang D, Zhai M, Yang L, Peng S, Chen C, Feng X, Weng Q, Zhang B, Xu M. Isolation of a novel bio-peptide from walnut residual protein inducing apoptosis and autophagy on cancer cells. BMC Complementary and Alternative Medicine., 2015; 15: 1-4.
    CrossRef
23. Xue Z, Yu W, Wu M, Wang J. In vivo antitumor and antioxidative effects of a rapeseed meal protein hydrolysate on an S180 tumor-bearing murine model. Bioscience, biotechnology, and biochemistry., 2009; 73(11): 2412-5.
    CrossRef
24. Muthaiyah B, Essa MM, Chauhan V, Chauhan A. Protective effects of walnut extract against amyloid beta peptide-induced cell death and oxidative stress in PC12 cells. Neurochemical research., 2011; 36: 2096-103.
    CrossRef
25. Sahadevan LD, Menon DB. Mussaenin A isolated from Mussaenda glabrata induces apoptosis in the liver cancer cells via mitochondrial pathway. Int. J. Pharmacogn. Phytochem. Res., 2017; 9: 1266.
    CrossRef
26. Latrasse, D., Benhamed, M., Bergounioux, C., Raynaud, C., & Delarue, M. Plant programmed cell death from a chromatin point of view. Journal of Experimental Botany., 2016; 67(20): 5887–5900.
    CrossRef
27. Nawrot R, Wolun-Cholewa M, Bialas W, Wyrzykowska D, Balcerkiewicz S, Gozdzicka-Jozefiak A. Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells. BMC Complementary and Alternative Medicine., 2010; 10: 1-7.
    CrossRef
28. Hernández-Ledesma B, Hsieh CC, de Lumen BO. Antioxidant and anti-inflammatory properties of cancer preventive peptide lunasin in RAW 264.7 macrophages. Biochemical and biophysical research communications., 2009; 390(3): 803-8.
    CrossRef
29. Gu MU, Chen HP, Zhao MM, Wang XI, Yang B, Ren JY, Su GW. Identification of antioxidant peptides released from defatted walnut (Juglans Sigillata Dode) meal proteins with pancreatin. LWT-Food Science and Technology., 2015; 60(1): 213-20.
    CrossRef
30. Chen N, Yang H, Sun Y, Niu J, Liu S. Purification and identification of antioxidant peptides from walnut (Juglans regia L.) protein hydrolysates. Peptides., 2012; 38(2): 344-9.
    CrossRef