Comparative Evaluation of Liposome Encapsulated Hygrophila auriculata (Schumach.) Heine Root and Betulin on Wound Healing Activity through In silico, In vitro and In vivo approaches
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2023-12
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Abstract
The present study was conducted to evaluate the comparative wound healing activity
associated with Liposome-encapsulated Hygrophila auriculata (K. Schum.) Heine Root and
Betulin. The research was done in four phases. Initially, studies on phytochemicals and free
radical scavenging activities were carried out on various extracts from H. auriculata leaves
and roots. Subsequently, in silico studies were conducted to identify lead compounds against
wound targets from secondary metabolites of H. auriculata roots. Phase III involved the
synthesis and characterization of liposomes using EEHA (Ethanol Extract of H. auriculata)
and betulin. In the final phase, the in vitro and in vivo wound healing potential of the
synthesized liposomes was assessed using the human keratinocyte cell line, HACAT, and
swiss albino rats. The qualitative phytochemical analysis revealed the presence of eight major
phytoconstituents: alkaloids, phenols, tannins, flavonoids, proteins, carbohydrates, cardiac
glycosides, and terpenoids. The ethanol extract of H. auriculata demonstrated significant
scavenging effects on DPPH, FRAP, and ABTS, compared to other solvent extracts. Due to
its superior phytochemical content and free radical scavenging capability, the ethanol extract
was selected for further investigation. Enzymic antioxidants (catalase, superoxide dismutase,
peroxidase, polyphenol oxidase, and glutathione S transferase) and non-enzymic antioxidants
(α-tocopherol, polyphenols, and ascorbic acid) were evaluated in the ethanol extract. Notably,
the roots exhibited the highest concentration of antioxidants, prompting the selection of roots
for subsequent studies. LC-MS analysis of H. auriculata root identified 15 significant
compounds, and their physicochemical properties were examined. Ten ligands were analyzed
for docking characteristics with proteins crucial for wound healing prognosis. Among the
top-scoring complexes, since betulin exhibited the highest affinity, LB (Liposome
encapsulated betulin) was synthesized and further evaluated for in vitro and in vivo studies
along with the LHA (Liposome encapsulated H. auriculata). Liposome ointment
formulations (LHA and LB) were developed using both betulin and H. auriculata root extract
for a comparative analysis of their wound healing properties.
The synthesized liposomes demonstrated favourable characteristics in FTIR, XRD,
TEM, and ZETA potential measurements, indicating enhanced stability. In antibacterial
activity, liposomes of H. auriculata root exhibited superior inhibition against all bacterial
species, with zone of inhibition values ranging from 12.3 ± 0.57 mm to 22.2 ± 2.3 mm.
Cytotoxicity assessments on HACAT cells revealed an IC50 of 125.77 μg/ml and 97.65 μg/ml
For LHA and LB respectively revealing less cytotoxicity.
The Wound Healing assay results show both H. auriculata root and betulin liposomes
effectively promote wound healing in Human Skin Keratinocytes (HACAT), reducing wound
area in an incubation-dependent manner. Flow cytometry after 48 hours of H. auriculata
liposome treatment revealed the expression of Collagen 1 (78.35%) in HACAT cells, when
compared to the standard control human Epidermal Growth Factor (hEGF- 84.89%). In in
vivo studies, 10% LHA liposome ointment demonstrated the highest wound contraction on
day 16 and subsequent post-wounding days (p < 0.01) compared to the standard ointment
(Povidone). LB ointment also showed significant wound contraction on all post-wounding
days (p < 0.01). Histological examination indicated normal healing, and reduced IL-6, CRP,
and procalcitonin levels were observed in treated rats compared to untreated rats.
Haematological parameters showed a more favourable decline in macrophages,
T lymphocytes, platelets, and ESR in the LHA treated rats than in LB treated rats. This study
underscores the importance of synergistic effects (LHA) in enhancing efficiency, especially
when compared to the impact of a single bioactive compound (LB).
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Zoology