Antidiabetic Potential and Chemical Profiling of Chloroform Fraction from Euclea racemosa subsp. schimperi In Vitro

Hanan M. El-Tantawy ¹*

This study determined the potential therapeutic benefits of E. r. ssp. schimperi in managing insulin resistance and oxidative stress.

Abstract

Background: The chloroform fraction (CHCl3 Fr.) of Euclea racemosa subsp. schimperi has enormous interest for its potential therapeutic properties in managing diabetes and oxidative stress-related conditions. This study aimed to evaluate the chemical composition and biological activities of CHCl3 Fr. through LC-ESI-MS/MS and GC-MS analyses. Methods: The CHCl3 Fr. was analyzed using LC-ESI-MS/MS for identification of bioactive compounds, and its inhibitory effects on α-amylase and α-glucosidase were assessed in vitro. Additionally, the impact on insulin secretion, C-peptide levels, and oxidative stress markers (MDA and NO) were measured. Results: Nineteen bioactive compounds, including flavonoids, pentacyclic triterpenes, and monounsaturated fatty acids, were identified in the chloroform fraction (CHCl3 Fr.). It significantly inhibited α-amylase and α-glucosidase, with IC50 values of 21.16 ± 2.13 and 13.49 ± 0.83 µg/ml, respectively. In insulin-resistant Huh7 cells, CHCl3 Fr. reduced glucose levels and increased insulin and C-peptide levels (p < 0.05). While it decreased malondialdehyde and nitric oxide, it did not significantly enhance catalase or total antioxidant capacity (p > 0.05). Compared to metformin, CHCl3 Fr. showed less efficacy but may improve insulin resistance and reduce oxidative stress, likely due to its bioactive compounds. Conclusion: The presence of diverse bioactive compounds contributes to the antidiabetic effects of CHCl3 Fr. from E. r. ssp. shimperi, showed its potential as a natural therapeutic agent for diabetes management and oxidative stress reduction. Further investigations are warranted to explore its clinical applications and underlying mechanisms.

Keywords: E. r. ssp. schimperi, bioactive compounds, insulin resistance, α-amylase, oxidative stress

References

Abbirami, E.L., Kumar, D., Guna, R., Gayathr, N., Sivasudh, T. (2019). Alpha Amylase, Alpha Glucosidase Inhibition and Profiling of Volatile Compounds of Biologically Active Extracts from Momordica cymbalaria (Hook, Fenzl) Skin and Seeds, Asia Pac J Sci Technol 8(1): 67-73© https://doi.org/10.51983/ajeat-2019.8.1.1057.

Abd El-Kareem, M.S., Rabbih, M.A.E.F., Selim, E.T.M., Elsherbiny, E,A,E,M., El-Khateeb, A.Y. (2016). Application of GC/EIMS in combination with semi-empirical calculations for identification and investigation of some volatile components in basil essential oil. Int'l J. of Analytical Mass Spectrometry and Chromatography 4(1): 14-25. http://dx.doi.org/10.4236/ijamsc.2016.41002.

Abu-Reidah, I.M., Ali-Shtayeh, M.S., Jamous, R.M., Arráez-Román, D., Segura-Carretero, A. (2015). HPLC–DAD–ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits. Food Chem  166:179-191. https://doi: 10.1016/j.foodchem.2014.06.011.

Agus, S., Achmadi, S.S., Mubarik, N.R. (2017). Antibacterial activity of naringenin-rich fraction of pigeon pea leaves toward Salmonella thypi. Asian Pac J Trop Biomed 7(8): 725-728. https://doi. org/10.1016/j.apjtb.2017.07.019.

Ajala-Lawal,  R.A.,  Aliyu, N.O., Ajiboye, T.O., (2020). Betulinic acid improves insulin sensitivity, elevated blood glucose, antioxidants, inflammation and dyslipidaemia and oxidative stress. Arch Physiol Biochem  126(2):107-115. https://doi.org/10.1080/1381-3455. 2018.1498901.

Ali, M.S., Jahangir, M., Hussan, S.S.,  Choudhary, M.I. (2002). Inhibition of α-glucosidase by oleanolic acid and its synthetic derivatives. Phytochem60:295–299. https://doi: 10.1016-/s00319422(02)-00104-8.

Asres, K., Gibbons, S., Bucar, F. (2006). Radical scavenging compounds from ethiopian medicinal plants. Ethiop Pharm J 24 (1): 23-30. https://doi:10.4314/epj.v24i1.-35095.

Attia, R,A., El-Dahmy, S.I., Abouelenein, D.D., Abdel-Ghani, A.E. (2022). LC-ESI-MS profile, cytotoxic, antioxidant, insecticidal and antimicrobial activities of wild and in vitro propagated Tanacetum sinaicum Del. ex DC. Zagazig J. Pharm. Sci. 31(2): 8- 21. https:// doi. 10.21608/ZJPS.2022.-157581.1041.

Avula, B., Sagi, S., Masoodi, M.H., Ji-Yeong, B., Wali, A.F., Khan, I.A. (2020). Quantification and characterization of phenolic compounds from Northern Indian Propolis extracts and dietary supplements. J AOAC Int 103(5): 1378–1393. https://doi.org/10.1093/jaoacint-/qsaa-032.

Banerjee, S., Bose, S., Mandal, M.S.C., Dawn, S.M., Sahoo, U., Ramadan. M.A., Mandal, S.K.   (2019). Pharmacological property of pentacyclic triterpenoids. Egypt J Chem 62(1): 13 – 35. https://doi.org/10.21608/ejchem.-2019.16055.1975.

Birgani, G.A., Ahangarpour, A., Khorsandi, L., Moghaddam, H.F.  (2018). Anti-diabetic effect of betulinic acid on streptozotocin-nicotinamide induced diabetic male mouse model. Braz J Pharm Sci  54 (2): e17171- 7. https://doi.org/10.1590/s217597902018-000217171.

Boritnaban, D.A., Karomah, A.H., Septaningsih, D.A., Majiidu, M., Dwiyanti, F.G., Siregar, I.Z., Rafi, M. (2022). Metabolite Profiling of Ebony (Diospyros celebica Bakh) leaves and wood extracts using LC-MS/MS. Indones J Chem 22 (2): 352 – 360. http://dx.doiorg/10.22146/ijc.-68529.

Botha, E. (2016).  "Investigating the production of secondary metabolites effective in lowering blood glucose levels in Euclea undulata Thunb. Var Myrtina (Ebenaceae)". Env Sci., p 120. https://api.semanticscholar-.org/Corpus-ID:99557343.

Buzgaia, N., Soo. Y.L., Rukayadi, Y., Abas. F., Shaari. K.  (2021). Antioxidant Activity, α-Glucosidase Inhibition and UHPLC–ESI–MS/MS Profile of Shmar (Arbutus pavarii Pamp). Plants (Basel) 10(8): 1659. https://doi: 10.3390/plants10081659.

Chaudharya, A., Kaura, P., Kumara, N., Singha, B., Awasthia, S., Lalb, B. (2011). Chemical fingerprint analysis of phenolics of Albizia chinensis based on ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry and antioxidant activity. Nat Prod Commun 6 (11): 1617 – 1620. https://doi:10.1177/1934578x-110060-1115.

Chen, Q., Zhang, Y., Zhang, W., Chen, Z. (2011). Identification and quantification of oleanolic acid and ursolic acid in Chinese herbs by liquid chromatography–ion trap mass spectrometry. Biomed Chromatogr 25 (12): 1381–1388. https://doi: 10.1002/bmc.1614. 

 Draper, M.H.H., Hadley, M. (1990). Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol. 186:421-31. https://doi: 10.1016/0076-6879(90)86135-i.

El-Sayed, M.A., Abbas, F,A., Refaat, S., El-Shafae, A.M., Fikry, E.  (2021). UPLC-ESI-MS/MS profile of the ethyl acetate fraction of aerial parts of Bougainvillea 'Scarlett O'Hara' cultivated in egypt. Front Ecol Evol 64 (2): 793 – 806. https://doi.10.21608-/EJCHEM.2020.45694.2933.

El-Tantawy. H.M., Hassan. A.R., Taha. H.E. (2022). Anticancer mechanism of the non-polar extract from Echium angustifolium Mill. aerial parts in relation to its chemical content. Egypt J Chem 65 (10):17-26.   https://doi.10.21608/EJCHEM.2022.130795.5757.

Esteban, M.A., Sánchez-Hernández, S., Samaniego, S.C., Olalla, H.M. (2020). Differences in the phenolic profile by UPLC coupled to high resolution mass spectrometry and antioxidant capacity of two Diospyros kaki varieties. Antioxidants (Basel) 10(1): 31-40. https://-doi: 10.3390/antiox10010031.

 Fernández-Aparicio, Á., Correa-Rodríguez, M.,  Castellano, J.M., Valle, J.S.R., González-Jiménez, E. (2022). Potential Molecular Targets of Oleanolic Acid in Insulin Resistance and Underlying Oxidative Stress: A Systematic Review. Antioxidants (Basel),   11(8): 1517.   https://doi.org/10.3390/-antiox11081517.

Gawel, S., Wardas, M., Niedworok, E., Wardas, P. (2004). Malondialdehyde (MDA) as a lipid peroxidation marker. Wiad Lek 57 (9-10): 453-5. PMID: 15765761.

Gong, L., Feng, D. Wang, T. Ren, Y., Liu, Y., Wang, J.  (2020). Inhibitors of α-amylase and α-glucosidase: Potential linkage for whole cereal foods on prevention of hyperglycemia. Food Sci Nutr 8(12): 6320–6337. https://doi.org/10.1002/fsn3.1987.

Goufo, P., Singh, R.K., Cortez, I. (2020), A reference list of phenolic compounds (including stilbenes) in grapevine (Vitis vinifera L.) roots, woods, canes, stems, and leaves. Antioxidants (Basel). 9(5): 398- 435. https://doi: 10.3390/antiox9050398.

Grover, J.K., Yada,. S. Vats, V. (2002). Medicinal plants of India with anti-diabetic potential. J. Ethnopharmacol 81(1):81–100. https:// doi: 10.1016/s0378-8741(02)00059-4.

Haque. MdE., Shekhar. H.U., Mohamad. A.U., Rahman. H., Mydul. I.A.M., Hossain. M.S. (2005). Triterpenoids from the stem bark of Avicennia officinalis. . Pharm Sci 5(1-2):53-57. https://doi:10.3329/dujps.v5i1.229.

Hassan, A.R. (2022). Chemical profile and cytotoxic activity of a polyphenolic-rich fraction from Euphorbia dendroides aerial parts. S Afr J Bot. 147, 332-339. https://doi.org/10.-1016/j.-sajb.2022.01.035.

Hassan, W.H.B., Abdel-aziz, S., Al Yousef, H.M. (2018). Chemical composition and biological              activities of the aqueous fraction of Parkinsonea aculeata L. growing in saudi arabia. Arab J Chem 12(3):377-387. https://doi.org/10.1016/j.arabjc.2018.08.003.

Ipav, S.S., Igoli, J.O., Tor-Anyiin, T.A. (2022). Isolation and characterization of alpha and beta amyrins from Propolis obtained from benue state. J Chem Soc Nigeria 47(2): 250 –261. https://doi.org/10.46602/jcsn.v47i2.723.

Jakovljevic, N.K. Pavlovic, K. Zujovic, T, Kravic-Stevovic, T. Jotic, A. Markovic, I. Lalic, N.M. (2021). In vitro models of insulin resistance: Mitochondrial coupling is differently affected in liver and muscle cells. Mitochondrion 61: 165-173. https://doi: 10.1016/j.mito.-2021.10.001.

Kerebba, N., Oyedeji, A.O., Byamukama, R., Kuria, S.K., Oyedeji, O.O. (2022).   UHPLC- UHPLC-ESI-QTOF-MS/MS characterisation of phenolic compounds from <i>Tithonia diversifolia</i> (Hemsl.) A. Gray and Antioxidant Activity. ChemistrySelect 7(16): e202104406-22. https://doi.org/10.1002/slct.202104406.

Lachowicz, S., Oszmianski, J., Rapak, A., Ochmian, I.M. (2020). Profile and content of phenolic compounds in leaves, flowers, roots, and stalks of Sanguisorba officinalis L. determined with the LC-DAD-ESI-QTOF-MS/MS analysis and their in vitro antioxidant, antidiabetic, antiproliferative potency. J Pharm 13(8): 191-214. https://doi: 10.3390/ph-13080191.

Leighton, E., Sainsbury, C.A., Jones, G.C. (2017). A Practical review of C-Peptide testing in diabetes. Diabetes Ther 8(3):475-487.  https://doi: 10.1007/s13300-017-0265-4.

Li, M., Pu, Y., Yoo, C.G., Ragauskas, A.J. (2016). The occurrence of tricin and its derivatives in plants. Green Chem18(6):1439-1454. . DOI: 10.1039/x0xx00000x

Li. S., Zhang, Y., Sun, Y., Zhang, G., Bai, J., Guo. J., Su, X., Du, H., Cao, X., Yang, J., Wang, T.  (2019).  Naringenin improves insulin sensitivity in gestational diabetes mellitus mice through AMPK. Nutr Diabetes. 9 (28): 10. https://doi: 10.1038/s41387-019-0095-8

Liu, Y., Peterson, D.A., Kimura, H., Schubert, D. (1997). Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem 69(2):581-93. https://doi: 10.1046/j.1471-4159.1997.69020581.x.

Liu. X., Fu. Y., Ma. Q., Yi., J., Cai. S., (2021). Anti-diabetic effects of different phenolic-rich fractions from Rhus chinensis Mill. fruits in vitro. eFood. (1):37–46. https:// doi:10.2991-/efood-.k.210222.002.

López-Gómez, C., Santiago-Fernández, C., García, S., García-Escobar, E. Gutiérrez-Repiso, C. Rodríguez-Díaz, C, Ho-Plágaro, A. Martín-Reyes, F. Garrido-Sánchez, L., Valdés, S., Rodríguez-Cañete, A., Rodríguez-Pacheco, F., García-Fuentes, E. (2021).  Oleic acid protects against insulin resistance by regulating the genes related to the PI3K signaling pathway. J Clin Med9(8): 2615- 2629.  https://doi.org/10.3390/jcm9082615.

Lourenço, A., Marques, A.V., Gominho, J. (2021). The identification of new triterpenoids in Eucalyptus globulus wood. Mol 26(12): 3495.-3508. https://doi.org/10.3390%-Fmolecules-26123495.

Mechchate, H.,  Es-safi, I., Louba, A., Alqahtani, A.S.,  Nasr, F.A. Noman, O,M., Farooqm M.,  Alharbi, M.S.,  Aziz, A., Bari, A., Hicham, B., Bousta, D. (2021). In vitro alpha-amylase and alpha-glucosidase inhibitory activity and in vivo antidiabetic activity of Withania frutescens L. foliar Extract. Mol 26 (2): 293 https://doi.org/10.3390/molecules-26020293.

Mekonnen, A., Atlabachew, M. Kassie, B. (2018). Investigation of antioxidant and antimicrobial activities of Euclea schimperi  leaf extracts. Chem biol technol Agric  5): 16( https://doi. org/10.-1186/s40538-018-0128-x

Mihailovic, M., Dinic, S., Jovanovic, J.A., Uskokovic, A., Grdovic, N., Vidakovic, M. (2021). The influence of plant extracts and phytoconstituents on antioxidant enzymes activity and gene expression in the prevention and treatment of impaired glucose homeostasis and diabetes complications. Antioxidants (Basel) 10(3): 480-505. https://doi.org/10.3390/antiox-10030480.

Mirmiran, P., Esfandyari, S., Moghadam, S.K., Bahadoran, Z., Azizi, F. (2018). Fatty acid quality and quantity of diet and risk of type 2 diabetes in adults:Tehran Lipid and Glucose Study. J Diabetes Complications 32(7): 655–659. https://doi.org/10.1016/j.jdiacomp.2018.-05.003.

Mishra, S., Mishra, B.B. (2017) Study of lipid peroxidation, nitric oxide end product, and trace element status in type 2 diabetes mellitus with and without complications. Int J Appl Basic Med Res 7(2): 88–93. https://doi: 10.4103/2229-516X.205813

Nazaruk, J., Kluczyk, B.M. (2015). The role of triterpenes in the management of diabetes mellitus and its complications. Phytochem Rev 14(4): 675–690. https://doi.org/10.1007/-s111-01-014-9369-x.

Nguyen, H.T.,  Pandey, R.P., Thuy, T.T.T, Park, J,W., Sohng, J.K, (2013).  Improvement of Regio-Specific Production of Myricetin-3-O-α-L-Rhamnoside in Engineered Escherichia coli.  Appl Biochem Biotechnol 171(8): 1956–1967. https:// doi 10.1007/s12010-013-0459-9

Oboh, M., Govender, L., Siwela, M., Mkhwanazi, B.N., (2021). Anti-diabetic potential of plant-based pentacyclic triterpene derivatives: progress made to improve efficacy and bioavailability. Mol 26(23): 7243-7264. https://doi.org/10.3390%2Fmolecules-26237243.

Odukoya, J.O., Odukoya, J.O., Mmutlane, E.M., Ndinteh, D.T. )2022(. Ethnopharmacological Study of Medicinal Plants Used for the Treatment of Cardiovascular Diseases and Their Associated Risk Factors in sub-Saharan Africa. Plants (Basel) 11 (10): 1387. doi: 10.3390/plants-11101387

Okba, M.M., El-Shiekh, R.A., Abu-Elghait, M., Sobeh, M., Ashour. R.M.S. (2021). HPLC-PDA-ESI-MS/MS profiling and anti-Biofilm potential of Eucalyptus sideroxylon Flowers. Antibiot 10(7):761-778. https://doi: 10.3390/antibiotics10070761.

Ormazabal, V., Soumyalekshmi, N. Elfeky, O., Aguayo, C. Salomon, C., Zuñiga, F.A. (2018). Association between insulin resistance and the development of cardiovascular disease. Cardiovasc Diabetol 17:122 https://doi.org/-10.1186/s12933-018-0762-4

Oršolic, N., Damir, S., Odeh. D., Gajski, G., Balta, V., Šver, L., Jembrek, M.J.  (2021). Efficacy of Caffeic Acid on Diabetes and Its Complications in the Mouse. Mol 26 (11): 3262. https://doi.org/10.3390/molecules26113262.

 Pereira, P., Cebola, M., Oliveira, M.C.,  Gil, G.B.M.  (2017). Antioxidant capacity and identification of bioactive compounds of Myrtus communis L. extract obtained by ultrasound-assisted extraction. J Food Sci Technol. 54(13):4362-4369. https://doi: 10.1007/s13197-017-2907-y. 1

 Prieto, P., Pineda, M. Aguilar, M. (1999). Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: Specific application to the determination of vitamin E. Anal Biochem 269 (2):337-41. https://doi.org/10.1006/-abio.1999.4019

Quintão, N.L.M., Rocha, W., Silva, G.F., Reichert, S., Claudino, V.D., Lucinda-Silva. R.M., Malheiros, A., De Souza, M.M., Filho, V.C., Bresolin, T.M.B., Machado, M.d.S., Wagner, T.M., Meyre, S.C. (2014). Contribution of α,β-amyrenone to the anti-inflammatory and antihypersensitivity effects of Aleurites moluccana (L.) willd. Biomed Res Int 636839-50. https://doi: 10.1155-/2014/636839.

Raspotnig, G., Schaider, M., Föttinger, P., Schönhofer, A. (2017). A model for phylogenetic chemosystematics: Evolutionary history of quinones in the Scent Gland secretions of Harvestmen. Nat Ecol Evol 17(5): 139. https://doi.org-/10.3389%2Ffevo.2017-.00139.

Rigler, R., Pramanik, A., Jonasson, P., Kratz, G., Jansson, O.T., Nygren, P., Stâh, S., Ekberg, K., Johansson, B., Uhlén, S., Uhlén, M., Jörnvall, H. Wahren, J.  (1999). Specific binding of proinsulin C-peptide to human cell membranes. Proc Natl Acad Sci USA 96(23):13318-23.  https://doi: 10.1073/pnas.96.23.13318.

Salih, E.Y.A., Fyhrquist, P., Abdalla, A.M.A., Abdelgadir, A.Y., Kanninen, M., Sipi, M., Luukkanen, O., Fahmi, M.K.M., Elamin, M.H., Ali, H.A. (2017). LC-MS/MS Tandem mass spectrometry for analysis of 3 Phenolic compounds and pentacyclic triterpenes in antifungal extracts of Terminalia brownii (Fresen). Antibiotics 6 (4):37-59. https://doi.org/10.3390%2-Fantibiotics-6040037.

Santos, F.A., Frota, J.T., Arrud, B.R., de Melo, T.S., da Silva, A.C.A., Brito, G.A.C., Chaves, M.H., Rao, V.S. (2012). Antihyperglycemic and hypolipidemic effects of α, β-amyrin, a triterpenoid mixture from Protium heptaphyllum in mice. Lipids Health Dis11: (98): 1476-511. https://doi.org/10.1186/1476-511X-11-98.

Shai LJ, Magano SR, Lebelo SL, Mogale AM (2011) Inhibitory effects of five medicinal plants on rat alpha-glucosidase: Comparison with their effects on yeast alpha-glucosidase. J Med Plant Res 5(13):2863-2867. http://www.academi cjournals.org/-JMPR.

Shamsudin, N.F., Ahmed, Q.U., Mahmood, S., Shah, S.A.A., Sarian, M.N., Khattak, M. A.K., Khatib, A., Sabere, A.S.M, Yusoff, Y.M.D., Latip, J. (2022). Flavonoids as antidiabetic and anti-Inflammatory agents: A review on structural activity relationship-based studies and meta-analysis. Int J Mol Sci 23(20): 12605. https://doi.org/10.3390-/ijms232012605.

Spínola, V., Pinto, J., Castilho, P.C, (2015). Identification and quantification of phenolic compounds of selected fruits from Madeira Island by HPLC-DAD–ESI-MSn and screening for their antioxidant activity. Food Chem 173:(14-30). https://doi: 10.1016/j.food-chem. 2014.09.163.

Steiner, D.F., Cunningham, D., Spigelm, L., Aten, B. (1967). Insulin biosynthesis: evidence for a precursor. Sci 157(3789):697-700. https://doi: 10.1126/science.157.3789.697.

Tamil, I.G., Dineshkumar, B., Nandhakumar, M., Senthilkumar. M., Mitral, A. (2010). In vitro study on α-amylase inhibitory activity of an Indian medicinal plant. J Pharmacol 42(5): 280–282.  https:// doi: 10.4103/0253-7613.70107.

Taye, A.D., Bizuneh, G.K. Kasahun, A.E. (2023). Ethnobotanical uses, phytochemistry and biological activity of the genus Euclea: A review. Front pharmacol 14: 1170145. doi: 10.3389/-fphar.2023.1170145. 62.

Telagari, M., Hullatti,  K. (2015). In-vitro α-amylase and α-glucosidase inhibitory activity of Adiantum caudatum Linn. and Celosia argentea Linn. extracts and fractions., Indian J Pharmacol 47(4): 425–429. https://doi:10.4103/0253-7613.161270.

Torun, A.N., Kulaksizoglu. S., Kulaksizoglu, M., Pamuk, B.O., Isbilen, E., Tutuncu, N.B. (2009). Serum total antioxidant status and lipid peroxidation marker malondialdehyde levels in overt and subclinical hypothyroidism J Clin Endocr70(3):469-74. https://doi.org/-10.1111/j.1365-2265.2008.03348.x

Tundis, R., Loizzo, M.R., Menichini, F. (2010). Natural products as alpha-amylase and alpha-glucosidase inhibitors and their hypoglycaemic potential in the treatment of diabetes: an update. Mini Rev Med Chem 10(4):315-31. https://doi: 10.2174/13895571079133-1007.

Viet, T.D., Xuan. T.D., Anh, L.H. (2021). α-Amyrin and β-Amyrin isolated from Celastrus hindsii leaves and their antioxidant, anti-xanthine oxidase, and anti-tyrosinase potentials. Mol 26(23): 7248-7262. https://doi: 10.3390/molecules26237248.

Vijayan, K.P.R., Raghu, A.V. (2019). Tentative characterization of phenolic compounds in three species of the genus Embelia by liquid chromatography coupled with mass spectrometry analysis. An Macromol Rapid Commun 52(10): 1532-2289. https://doi.org/10.1080/-00387010.2019-.1682013.

Villalva-Pérez, J.M., Ramírez-Vargas, M.A., Serafín-Fabían, J.I., Ramírez, M., Moreno-Godínez, M.E., Espinoza-Rojo, M. Flores-Alfaro, E. (2020). Characterization of Huh7 cells after the induction of insulin resistance and post-treatment with metformin. Cytotechnology 72(4): 499–511. https://doi: 10.1007/s10616-020-00398-4. 

Weir, N.l., Johnson, L., Guan, W., Steffen, B., Djousse, L., Mukamal, K.J., Tsai, M.Y, Wozniak, A., Paduch, R. (2012).  Aloe vera extract activity on human corneal cells. Pharm Biol. 2012; 50(2): 147–154. https://doi.org/3A10.3109-/13880209.2011.579980

         Wozniak, A., Paduch, R. (2012). Aloe vera extract activity on human corneal cells. Pharm Biol. 50(2): 147–154. http:// doi: 10.3109/13880209.2011.579980.  

Wu. J., Wang. Z, Cheng. X., Lian. Y., An. X., Wu. D. (2024). Preliminary study on total component analysis and in vitro antitumor activity of Eucalyptus leaf residues. Mol 29(2): 280-298. https://doi: 10.3390-/molecules29020280.

 Wube, A.A., Streit, B., Gibbons, S., Asres, K., Bucara., F. (2005). In vitro 12(S)-HETE inhibitory activities of naphthoquinones isolated from the root bark of Euclea racemosa ssp. Schimperi. J Ethnopharmacol 102(2): 191-196. https:// doi: 10.1016/j.jep.2005.06.009.

Yang, H., Jin, X., Lam, C.W., Yan, S.K. (2011). Oxidative stress and diabetes mellitus. Clin Chem Lab Med 49 (11):1773-82. https://doi: 10.1515/CCLM-.2011.250.

Yang, J.Y., Park, J.H., Chung, N., Lee, H.S. (2017). Inhibitory Potential of Constituents from Osmanthus fragrans and Structural Analogues Against Advanced Glycation End Products, α-Amylase, α-Glucosidase, and Oxidative Stress. Sci Rep 7:45746. https://doi: 10.1038/srep45746.

Yang, X., Li, D. (2023). Tricin attenuates diabetic retinopathy by inhibiting oxidative stress and angiogenesis through regulating sestrin 2/Nrf2 signaling. Hum Exp Toxicol 42: 1-10. https://doi.org/10.1177/09603271231171642.