Secondary metabolites, also called specialized metabolites, toxins, natural products, are organic

compounds produced by bacteria, fungi, or plants which are not directly involved in the growth,
development, or reproduction of an organism. Secondary metabolites is accumulated during the later
stage of fermentation known as the idiophase which follows the active growth phase called the
trophophase. Compounds produced within the idiophase don't have any direct relationship to the
synthesis of cell material and normal growth of the microorganisms (Singh, 2014). Different analytical
methods can be used for the separation and detection of the secondary metabolites. The usual methods
involves the use of thin layer chromatography, high performance liquid chromatography (HPLC) , gas
chromatography, flow injection electrospray mass spectrometry, micellar capillary electrophoresis,
ultraviolet diode array detection and nuclear magnetic resonance detection.

Detailed chemical studies on the secondary metabolites of the fruit C. cujete are scarce; a literature
journal revealed that there are a few studies addressing its phytochemical composition such as the
presence of tannins, phenols, flavonoids, saponins, alkaloids and hydrogen cyanide (Ejelonu et al., 2011).
Additional studies showed the presence of iridoids (such as aucubin and ningpogenin), n-alkyl
glycosides, lignan glycosides (acanthoside D), and benzoyl derivatives. A phytochemical study by
Ogbaugu. (2008) using the African variety of the fruit revealed that there is the presence of flavonoids,
sapnonins, phenolic compounds, tannins, cyanogenic glycoside, minerals (Na, K, Ca, P and MG), and
vitamins A, C, E, thiamin, niacin, and riboflavin. Flavonoids like quercetin, apigenin and naphthoquinone
are found in Crescentia cujete fruit and also anthraquinone can be found in the fruit which has an
antiangiogenic property (He et al., 2008).

Flavonoids are a group of natural substances with a mix of phenolic structure which are found in fruits,
vegetables and plants. Flavonoids are attributed because of their anti-oxidative, anti-inflammatory, anti-
mutagenic and anti-carcinogenic properties coupled with their capacity to modulate key cellular enzyme
function. According to (Panche, 2016), flavonoids can also inhibit angiogenic effect. Several studies
document the ability of flavonoids to inhibit the proliferation and migration of endothelial cells by
interfering with key angiogenesis signaling cascades such as the mitogen activated protein kinase
(MAPK) and phosphoinositide 3-kinase (PI3K) pathways (Khater, 2020).

Quercetin is a flavonoid which is found in fruits and vegetables and it has a unique property which
improves physical performance and it reduce infection risk. Quercetin is also known to have an anti-
carcinogenic, anti-inflammatory, antiviral, antioxidant, and psychostimulant activities, as well as the
ability to inhibit lipid peroxidation, platelet aggregation and capillary permeability, and to stimulate
mitochondrial biogenesis (Li et al., 2016). Quercetin is a potent inhibitor of angiogenesis in vitro, ex vivo
and in vivo. Quercetin treatment inhibited the activation of VEGF-R2 and thereby suppressed the
AKT/mTOR/P70S6K mediated angiogenesis signaling pathways (Song et al., 2017)

Anthraquinone is a polycyclic aromatic hydrocarbon which contains two opposite carbonyl groups at
9,10 position which is yellow or light gray to gray-green crystal powder and it is insoluble in water.
Anthraquinone is a starting material for the production of coloring compounds, antioxidants, and
polymerization inhibitors. It also has different biological effects including antimicrobial, anticancer, and
anti-inflammatory. A study on rhubarb shows that anthraquinone has an antiangiogenic property like
quercetin (He et al., 2008).

Benzene and its derivatives such as toluene and o-xylene are known contaminants in industrial areas,
although natural benzene and benzenoids are important precursors in the synthesis of secondary
metabolites. Exposure to benzene and its derivative compounds is associated with a range of acute and
long-term adverse effects, which includes narcosis, decreased host resistance to infection, cancer, and
aplastic anemia (IARC 2009).

Khater, M., et al. (2020). Antiangiogenic Activity of Flavonoids: A Systematic Review and Meta-Analysis.
Retrived from http://www.mdpi.com/molecules-25-04712-v2.pdf

Singh, N. (2014). METABOLIC PATHWAYS | Production of Secondary Metabolites – Fungi. Retrieved from
https://www.sciencedirect.com/science/article/pii/B9780123847300002020

Ejelonu, B.C., Lasisi, A.A., Olaremu, A.G., Ejelonu, O.C., 2011. The chemical constituents of calabash
(Crescentia cujete). Afr. J. Biotechnol. 10, 19631–19636. https://doi.org/ 10.5897/ajb11.1518.

He Z., He M., Ma S., & But P. (2008). Antiangiogenic Effects of Rhubarb and its Anthraquinone
Derivatives. Retrieved from https://pubmed.ncbi.nlm.nih.gov/19061946/

Kaneko, T., Ohtani, K., Kasai, R., Yamasaki, K., Neguyen, M.D., 1998. n-Alkyl glycosides and p-
hydroxybenzoyloxy glucose from fruits of Crescentia cujete. Phytochemistry 47, 259–263.
https://doi.org/10.1016/S0031-9422(97)00409-3.

Wang, G., Yin, W., Zhou, Z.Y., Hsieh, K.L., Liu, J.K., 2010. New iridoids from the fruits of Crescentia cujete.
J. Asian Nat. Prod. Res. 12, 770–775. https://doi.org/10.1080/ 10286020.2010.503189

[IARC] International Agency for Research on Cancer (in preparation). A Review of Human Carcinogens.—
Part F: Chemical Agents and Related Occupations. Lyon, International Agency For Research on Cancer.
IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. 100p. [Summary in Baan R, et al.
2009. A Review of Human Carcinogens—Part F: Chemical Agents and Related Occupations. The Lancet
Oncology 10(12): 1143–44. Retrieved from http://www.thelancet.
com/journals/lanonc/article/PIIS1470-2045(09)70358- 4/fulltext]

Panche, A. N., Diwan, A. D., & Chandra, S. R. (2016). Flavonoids: an overview. Journal of nutritional
science, 5, e47. https://doi.org/10.1017/jns.2016.41

Zhang, Y.J., Gan, R.Y., Li, S., Zhou, Y., Li, A.N. & Xu, D.P. 2015. Antioxidant phytochemicals for the
prevention and treatment of chronic diseases. Molecules 20: 21138-21156.
Song, W., Zhao, X., Xu, J., & Zhang, H. (2017). Quercetin inhibits angiogenesis-mediated human
retinoblastoma growth by targeting vascular endothelial growth factor receptor. Oncology letters, 14(3),
3343–3348. https://doi.org/10.3892/ol.2017.6623

Li, Y., Yao, J., Han, C., Yang, J., Chaudhry, M. T., Wang, S., Liu, H., & Yin, Y. (2016). Quercetin,
Inflammation and Immunity. Nutrients, 8(3), 167. https://doi.org/10.3390/nu8030167