Mohammed Asad

Alkanna tinctoria, commonly called dyer’s alkanet (family-Boraginaceae), is used traditionally in Saudi Arabia to treat skin infections. A methanolic extract and a traditional formulation of the root used in folklore were prepared. LC-MS analysis was conducted to identify probable compounds present in the extract and the traditional hydrophobic formulation. The in vivo activity on excision wound was evaluated in diabetic mice while crystal violet assay was employed for in vitro evaluation. Human keratinocyte (HaCaT) cells were used to study in vitro cytotoxic effects. Several probable phytoconstituents were revealed by LC-MS analysis in the methanolic extract and the traditional formulation, and three of the constituents were the same. The extract ointment and traditional hydrophobic extract exhibited antibacterial and antibiofilm activity against both tested pathogens. The methanolic extract was relatively more cytotoxic on HaCaT cells compared to the hydrophobic formulation. The methanolic extract ointment did not significantly affect the wound healing, whereas the traditional formulation accelerated wound healing in diabetic mice. The results revealed that A. tinctoria in its traditional formulation is an effective wound healing agent but the methanolic extract of the plant does not affect the healing of wounds.

Phytochemicals are effective and are gaining attention in fighting against drug-resistant bacterial strains. In the present study, rutin and quercetin were tested for antibacterial, antibiofilm, and wound healing activities on excision wounds infected with MDR-P. aeruginosa in diabetic mice. Antibacterial and antibiofilm activities were studied in vitro using broth dilution assay and crystal violet assay, respectively. These phytochemicals were tested alone for wound-healing activities at different concentrations (0.5% and 1% in ointment base) and in combination with gentamicin to evaluate any additive effects. Rutin and quercetin demonstrated effectiveness against MDR-P. aeruginosa at higher concentrations. Both phytochemicals inhibited biofilm formation in vitro and contributed to the healing of diabetic wounds by eradicating biofilm in the wounded tissue. Rutin at a low concentration (0.5%) had a lesser effect on reducing the epithelization period and regeneration of the epithelial layer compared to quercetin. When combined with gentamicin, quercetin (1%) displayed the maximum effect on epithelium regeneration, followed by rutin (1%) in combination with gentamicin. Both phytochemicals were found to be more effective in controlling biofilm and wound-healing activities when used as an additive with gentamicin. The study supports the traditional use of phytochemicals with antibacterial, antibiofilm, and wound-healing activities in managing diabetic infections.

The investigation of novel phytochemicals for the prevention and treatment of infections caused by multidrug-resistant pathogens is gaining attention. The current study evaluated the in-vitro antimicrobial activity of kaempferol against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Its in-vivo efficacy in inhibiting these pathogens was determined using an excision wound model in nicotinamide-streptozocin- -induced diabetic rats. Kaempferol displayed an inhibitory effect against the tested bacteria both in vitro and in vivo. It also healed excision wounds at a 1% (w/w) concentration. An increase in antioxidant enzymes in wounded tissue was observed on kaempferol treatment. A reduction in the MRSA and P. aeruginosa counts in wounded tissue together with a reduced epithelization period was observed when compared to the infected control. A thicker epithelium, new capillaries, and a decrease in inflammatory cells were detected by hematoxylin and eosin staining. Furthermore, an increase in collagen fibers and their deposition was observed by Masson’s trichrome staining. Kaempferol at 40 µM did not display any toxicity for human keratinocytes grown in media containing high glucose and it did not affect the expression of the pro-healing cytokines genes vascular endothelial growth factor (VEGF) and transforming growth factor-b-1 (TGFβ1). Kaempferol displayed antibacterial and antioxidant actions but did not increase the expression of proliferative genes.

Chlorogenic acid is known to have antibacterial action against a number of pathogens. This study provides insights into the potentiality of chlorogenic acid in the treatment of chronic infection in wounds. The anti-infective and healing effect of chlorogenic acid in infected wounds was evaluated using diabetic rats. Antibacterial activity of chlorogenic acid against Methicillin-Resistant Staphylococcus Aureus (MRSA) and Multidrug-Resistant (MDR) Pseudomonas aeruginosa was evaluated in vitro. A decrease in the epithelization period (pin vitro. An increase in the expression of proliferative genes Vascular Endothelial Growth Factor (VEGF) and Transforming Growth Factor-b1 (TGF-b1) was observed in Human keratinocytes (HaCaT) grown in the presence of chlorogenic acid (p<0.001). The study confirms that chlorogenic acid supports wound healing with antibacterial, antioxidant, and proliferative effects of pro-healing cytokines genes. This agent may hold the potential for the management of infectious diabetic wounds.

Holarrhena pubescens seeds are used in the treatment of various diseases, especially diabetes and associated complications, in different parts of the world. The present study was undertaken to determine the hypolipidemic and antihyperlipidemic effects of methanolic extract of H. pubescens seeds in rats. The extract was subjected to LC-MS analysis to determine the chemical constituents. The hypolipidemic action was studied by determining the effect of 28-day oral administration of seed extract on serum cholesterol, serum triglycerides, and serum HDL-cholesterol levels. The antihyperlipidemic action was studied in rats fed with a high-fat diet containing cholesterol and saturated fat, and the same lipid parameters were estimated during 28-day treatment. To elucidate its probable mechanism of action, in vitro studies on the inhibition of lipid accumulation in preadipocytes, DPP-IV inhibitory effect, and lipase enzyme inhibition were studied. The seed extract reduced serum levels of cholesterol and triglycerides in both normal rats and animals fed with a high-fat diet without a significant effect on HDL-cholesterol levels. The seed extract was highly effective in inhibiting lipase enzyme activity but showed a modest effect on the inhibition of lipid accumulation and DPP-IV. The results demonstrated that H. pubescens seed extract has hypolipidemic and antihyperlipidemic effects mediated probably through inhibition of lipase enzyme activity.

Achillea fragrantissima, a desert plant commonly known as yarrow, is traditionally used as an antimicrobial agent in folklore medicine in Saudi Arabia. The current study was undertaken to determine its antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) and multi-drug-resistant Pseudomonas aeruginosa (MDR-P. aeruginosa) using in vitro and in vivo studies. A biofilm model induced through an excision wound in diabetic mice was used to evaluate its effect in vivo. The skin irritation and cytotoxic effects of the extract were determined using mice and HaCaT cell lines, respectively. The Achillea fragrantissima methanolic extract was analyzed with LC-MS to detect different phytoconstituents, which revealed the presence of 47 different phytoconstituents. The extract inhibited the growth of both tested pathogens in vitro. It also increased the healing of biofilm-formed excision wounds, demonstrating its antibiofilm, antimicrobial, and wound-healing action in vivo. The effect of the extract was concentration-dependent, and its activity was stronger against MRSA than MDR-P. aeruginosa. The extract formulation was devoid of a skin irritation effect in vivo and cytotoxic effect on HaCaT cell lines in vitro.

The study developed a simple and inexpensive method to induce biofilm formation in-vivo for the evaluation of the antibiofilm activity of pharmacological agents using Swiss albino mice. Animals were made diabetic using streptozocin and nicotinamide. A cover slip containing preformed biofilm along with MRSA culture was introduced into the excision wound in these animals. The method was effective in developing biofilm on the coverslip after 24 h incubation in MRSA broth which was confirmed by microscopic examination and a crystal violet assay. Application of preformed biofilm along with microbial culture induced a profound infection with biofilm formation on excision wounds in 72 h. This was confirmed by macroscopic, histological, and bacterial load determination. Mupirocin, a known antibacterial agent effective against MRSA was used to demonstrate antibiofilm activity. Mupirocin was able to completely heal the excised wounds in 19 to 21 days while in the base-treated group, healing took place between 30 and 35 days. The method described is robust and can be reproduced easily without the use of transgenic animals and sophisticated methods such as confocal microscopy.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infection worldwide. Clove oil’s ability to inhibit the growth of MRSA was studied through in vitro and in vivo studies. The phytochemical components of clove oil were determined through gas chromatography-mass spectrometry (GC-MS) analysis. The antibacterial effects of clove oil and its interaction with imipenem were determined by studying MIC, MBC, and FIC indices in vitro. The in vivo wound-healing effect of the clove oil and infection control were determined using excision wound model rats. The GC-MS analysis of clove oil revealed the presence of 16 volatile compounds. Clove oil showed a good antibacterial effect in vitro but no interaction was observed with imipenem. Clove bud oil alone or in combination with imipenem healed wounds faster and reduced the microbial load in wounds. The findings of this study confirmed the antibacterial activity of clove oil in vitro and in vivo and demonstrated its interaction with imipenem.

Frankincense (Boswellia sacra oleo gum resin) is reported to possess antimicrobial activity against several pathogens in-vitro. The antimicrobial effects of frankincense oil and its interaction with imipenem and gentamicin against methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant P. aeruginosa were determined through in-vitro methods and an in-vivo study using a rat pneumonia model. Frankincense oil was subjected to GC-MS analysis to determine the different volatile components. Antibacterial effects against MRSA and MDR-P. aeruginosa was evaluated and its MIC and MBC were determined. For the rat pneumonia model (in-vivo), oil was administered at a dose of 500 mg/kg and 1000 mg/kg followed by determination of CFU in lung tissue and histological studies. Frankincense oil did not show a very potent inhibitory effect against MRSA or MDR-P. aeruginosa; the oil did not affect the zone of inhibition or FIC when combined with imipenem or gentamicin indicating a lack of interaction between the oil and the antibiotics. Furthermore, there was no interaction between the antibiotics and the frankincense oil in the in-vivo model. The result of the study revealed that frankincense oil has a weak inhibitory effect against MRSA and MDR-P. aeruginosa, and it did not show any interaction with imipenem or gentamicin.