Wound healing mechanisms of Couroupita guianensis fruit pulp: An ethnomedicine used by traditional healers in India

Abstract In connection to search for safe and alternative plant-based drugs, the wound healing mechanisms of an Indian ethnomedicine Couroupita guianensis fruit pulp was analyzed in this project work. Gas chromatography coupled with mass spectrometer (GC-MS) analysis revealed the existence of phytochemicals such as 2-furoic acid, 2,4-heptadienal, pyrazole and 8-hydroxyquinoline in the methanol extract. Methanol extract of C. guianensis exhibited remarkable radical scavenging activity against 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (89.88%), superoxide (91.51%), hydrogen peroxide (24.25%) and hydroxyl radicals (73.62%). Further, it showed remarkable anti-inflammatory (24.09-62.16%) and anti-bacterial activity (zone of inhibition, ZOI: 13.00 mm, minimum inhibitory concentration, MIC: 6.25 mg/mL and minimum bactericidal concentration, MBC: 12.51 mg/mL) and also controlled the growth rate of methicillin resistant Staphylococcus aureus (MRSA) within 30 min of treatment. The angiogenic potential of C. guianensis was proved in chick chorioallantoic membrane (CAM) model and it does not exhibit any toxicity in peripheral blood monocyte cells (PBMC) model. Graphical Abstract


Introduction
Overproduction of free radicals can cause delayed and impaired wound healing and therefore antioxidants play a crucial role in the healing process of skin injuries (Schafer and Werner 2008).Inflammation plays a significant role in wound healing because it helps to clear cell debris and pathogens through macrophages and neutrophils (ellis et al. 2018) and alteration of this key phase can result in improper healing.Pathogens like Staphylococcus aureus can infect the wound and produce chemotaxis molecules that impair neutrophil extravasation from blood to the wound site (Kobayashi et al. 2018).Several bacterial pathogens can inhibit free radical production (Caldwell 2020) and also inactivate degrading enzymes that are released by neutrophils and macrophages and thus affect inflammatory response (Lei et al. 2019).In addition, blood vessel damage leads to hypoxic state in the wound region as tissue oxygen decreases.During re-modeling phase of wound healing, growth factors can bind to the receptors of endothelial cells in the damaged blood vessel and resulting in the activation of signaling pathways in angiogenesis (Honnegowda et al. 2015).
While a handful of synthetic wound healing agents exist such as povidone-iodine, hansaplast ointment, burnol-cream, silver sulfadiazine, silverx gel and neosporin, they all seem to have several adverse effects with prolonged usage such as allergy, irritation, hyperosmolality and methemoglobinemia (Fuller 2009;Singh et al. 2021).Alternatively, few plant-based drugs such as phytoseptic cream, goheal, B & W ointment, calendula gel, healeviate cream, scortchnil ointment, orgado cream and panchavalkala cream were emerged to overcome the side effects caused by the synthetic drugs.However, there is still a need to discover newer and better natural products to heal chronic wounds.In this juncture, certain herbal drugs used traditional healers can serve as potential source to develop medicine for chronic wounds.One such herbal material used by traditional healers in Neyveli, Cuddalore, Tamil Nadu, India is Couroupita guianensis fruit pulp.It is commonly known as cannon ball tree in english and Nagalingam in Tamil that belongs to the family Lecythidaceae.It is a popular ornamental plant in Caribbean and South-east Asian botanical gardens and listed as a rare tree in India (Shekhawat and Manokari 2016).Leaves, flowers, bark, or fruits of the cannonball tree are used to treat hypertension, tumors, colds, stomach-aches, tooth-aches, malaria, pain, skin diseases and wound (Pinheiro et al. 2013).even though wound healing activity of C. guianensis fruit was investigated in rat model (Umachigi et al. 2007), mechanism of action is not yet studied.So, the current project was designed to analyze wound healing mechanisms such as antioxidant, anti-inflammatory, anti-bacterial and angiogenic activities of C. guianensis fruit pulp using in vitro assays with a view to develop drug to treat chronic wounds.

Anti-inflammatory activity
The result shows that the methanol extract of C. guianensis can inhibit the denaturation of albumin (29.46%), proteinase activity (24.09%) and red blood cells (RBC) membrane damage (62.16%) at a concentration of 10 mg/mL (Supplementary Figure S5A-C).The C. guianensis methanol extract showed the highest level of anti-inflammatory activity than other solvent extracts as well as the positive control, Calendula.Denaturation of protein has been identified as one of the major causes of inflammation (Pillai and Nair 2014).During chronic inflammation, drugs play a crucial role in protecting tissue damage by preventing protein denaturation.When these proteins get denatured, they lose their structure and resulting in tissue damage.In this study, the anti-inflammatory potential of the C. guianensis extract was examined terms of inhibition of heat-induced denaturation of bovine serum albumin (BSA).When the BSA is subjected to heating, it gets denatured and becomes turbid and results in high absorbance at 660 nm.If a plant extract possess anti-inflammatory activity, it prevents protein denaturation and exhibit less turbidity so that absorbance will be low compared to negative control.Similarly, the methanolic extract of Senna auriculata leaves demonstrated 23.38% inhibition of protein denaturation (Prasathkumar et al. 2021).In the present work, high level of inhibition of BSA denaturation exhibited by the methanol extract of C. guianensis gives hope for its protective ability towards structural proteins.
The effect of C. guianensis on the migration of monocyte cells in the injured area is shown in Supplementary Figure S6.Photograph of the injured area at 0 th h revealed the removal of countable number of monocytes at the bottom of the 24-well plate.In untreated control, monocyte cell population was increased in the injured site after 24 h of incubation, which indicates migration of cells.But methanolic extract of C. guianensis treated monocytes exhibited migration of lesser number of cells compared to the control.In the case of Calendula extract (10 mg/mL), migration of large number of monocytes was noticed compared to C. guianensis extract treated cells.These results are suggestive that the C. guianensis can act as anti-inflammatory agent by inhibiting monocytes migration.Due to wrong or inappropriate signals, huge number of monocytes arrive at the wound site and cause damage to healthy cells and thereby resulting in chronic inflammation and delayed wound healing.So, the inhibition of monocyte migration could reduce the prolonged inflammation in chronic wounds, which in turn fasten the wound healing.Results of monocyte migration assay strongly suggested that C. guianensis has the potential to inhibit monocyte migration and reduce the chances of prolonged inflammation in chronic wounds.C. guianensis may block the MMP-9 activity in the monocytes and also inhibit the chemotaxis by suppressing signals such as IL-6 and TNF-alpha and thereby caused inhibition of cell migration.Similarly, Alternanthera tenella leaf extract inhibited the monocyte migration (Sathishkumar et al. 2016).

Conclusions
In the present study, in vitro assays were used to evaluate the wound healing mechanisms of an Indian traditional drug C. guianensis fruit.Active phytochemicals such as 2-furoic acid, 2,4-heptadienal, pyrazole and 6-hydroxyquinoline were deciphered first time in the methanol extract by GC-MS analysis.The methanol extract of C. guianensis has shown potent antioxidant, anti-inflammatory, anti-bacterial and angiogenic activities than other solvents.Marked anti-inflammatory activity of C. guianensis fruit extract was explored in this project in terms of moderate inhibition of protein denaturation and proteinase inhibition and also by maximum inhibition RBC membrane denaturation, which justified its usage in Indian traditional system of medicine to treat chronic wounds.Further, it was found that C. guianensis is safe to use as it has no cytotoxic effects.This study has given preliminary data to develop potent drug from C. guianensis extract for the treatment of chronic wounds.However, detailed in vivo investigations are needed to ascertain the healing mechanisms of phytochemical constituents of C. guianensis.