Catechin, epicatechin, curcumin, garlic, pomegranate peel and neem extracts of Indian origin showed enhanced anti-inflammatory potential in human primary acute and chronic wound derived fibroblasts by decreasing TGF-ß and TNF-α expression.

BACKGROUND: Although chronic wounds are devastating and can cause burden at multiple levels, chronic wound research is still far behind. Chronic wound treatment is often less efficient due to delay in diagnosis and treatment, non-specific treatment mainly due to lack of knowledge of wound healing or healing resistance genes. It's known that chronic wounds do not progress towards healing, because it gets stalled in inflammatory phase of wound healing. OBJECTIVE: We aimed to use phytoextracts possessing excellent anti-inflammatory properties to regulate the unbalanced levels of cytokines responsible for increased inflammation. METHODS: Evaluation of anti-inflammatory activity of selected phytoextracts namely, Camellia sinensis (L.) Kuntze, Acacia catechu (L.f) Willd., Curcuma longa (L.), Allium sativum (L.), Punica granatum (L.) and Azadirachta indica A. hereafter, called as catechin, epicatechin, curcumin, garlic, pomegranate and neem extracts, respectively in Acute wound fibroblasts (AWFs) and Chronic wound fibroblasts (CWFs) using flow cytometry. RESULTS: The phytoextracts exhibited no cytotoxicity below 100 µg/ml on normal Human Dermal fibroblasts (HDFs), while garlic extract showed highest cell viability followed by catechin, epicatechin, curcumin, pomegranate peel and neem based on IC50 value. Garlic, catechin and epicatechin extracts showed highest anti-inflammatory activities for both TGF-ß and TNF-α in both AWFs and CWFs treated cells. After treatment of AWFs with catechin, epicatechin and garlic extracts, TGF-ß and TNF-α expression was significantly reduced compared to untreated AWFs and reached to almost normal HDFs level. Also, after treatment of CWFs with catechin, epicatechin and garlic extracts, TGF-ß and TNF-α expression was significantly reduced compared to untreated CWFs and was lesser than untreated AWFs. CONCLUSION: The present findings reveal the potential of catechin, epicatechin and garlic extracts for the treatment of acute and chronic wounds with excellent anti-inflammatory properties.

Standardized ready-to-use laboratory protocol to isolate primary human wound associated fibroblasts from infected wound samples for clinical applications.

Owing to the importance of fibroblasts in healing of wounds, it is necessary to isolate and culture them under in vitro conditions for the purpose of understanding the wound biology, drug discovery and development of personalized treatment. Although, several fibroblast cell lines are commercially available, they fail to represent the patient associated parameters. However, establishing a primary fibroblast culture, especially from infected wound samples, is challenging as the sample is more prone to contamination and number of live cells will be minimum in heterogeneous population. Also, it takes lot of efforts and resources for optimization of the protocol to get good quality cell lines from wound samples necessitating multiple trials, resulting in large number of clinical samples to be processed. To the best of our knowledge, for the first time we are reporting the standardized protocol to isolate primary human fibroblasts from acute and chronic wound samples. In this study, various parameters such as explant size (1-2 mm), explant drying time (2 min), transportation and growth culture media (antibiotics (working concentrations 1-3) and serum concentration (10%)) have been optimised. This can be altered for specific needs of cell in terms of both quality and quantity. Outcome of the work provides a ready-to-use protocol, which is very useful to those who want to initiate primary fibroblasts cell culture from infected wound samples either for clinical or research purpose. Further, these cultured primary wound associated fibroblasts have various clinical and biomedical applications in tissue grafting, treatment of burns and scars and wound regeneration especially in non-healing chronic wounds.

Human primary chronic wound derived fibroblasts demonstrate differential pattern in expression of fibroblast specific markers, cell cycle arrest and reduced proliferation.

INTRODUCTION: Although wound refers to simple cut in the skin, most wounds don't heal because of the various local and systemic factors that lead to its complexity and chronicity. Thus, prior understanding of the status of the wound is necessary and methods that can differentiate between the healing and non-healing wounds at a much earlier stage is crucial for a successful treatment. METHODS: The current study aims at differentiating Acute Wound Fibroblasts (AWFs) and Chronic Wound Fibroblasts (CWFs) based on differential expression of fibroblast specific markers such as Vimentin and Alpha Smooth Muscle Actin (α-SMA) and compare its cell cycle and proliferation. RESULTS: Immunostaining and western blotting analysis showed that, AWFs and CWFs differentially expressed vimentin and α-SMA, with AWFs and CWFs showing higher expression of vimentin and α-SMA respectively. AWFs showed higher distributions in G0/G1 (67.43% vs. 62.16%), S phase (22.61% vs. 8.51%) compared to CWFs. However, AWFs showed decreased distributions compared to CWFs in G2 + M phase (8.14% vs. 10.6%). Thus, it was observed that CWFs showed cell cycle arrest in the G1/G0 phase and inhibited DNA synthesis, which was further confirmed by reduced proliferation of CWFs. We suggest that, differential expression of the cell specific markers can be attributed to its pathophysiological status and chronicity of the wound and reduced proliferation rate of CWFs is due to lesser expression of vimentin, which is a key protein for in vitro cell proliferation. CONCLUSIONS: Outcome of the study serve as an immunological tool to guide the chronicity of the wound, which helps to understand the wound towards design of personalized care. The findings also represent a promising opportunity to gain insight into how cell cycle arrest can impact on wound healing and clinical outcomes.

Effect of the carotenoid-producing alga, Dunaliella bardawil, on CCl4-induced toxicity in rats.

Dunaliella bardawil is a carotenoid-producing alga that is being considered for use in nutraceuticals. To evaluate potential protective effects of consumption of this alga, rats were treated with two different doses of D. bardawil (2.5 and 5.0 g kg(-1) body weight [bw]) as a biomass suspension daily for 14 days. Animals were tested against Carbon tetrachloride (CCl4; 2 ml kg(-1))-induced liver toxicity as measured by various biochemical marker enzymes in liver and blood. All measurements were taken 6 h following the single dose of CCl4. The results of this study show that there was a slight, but statistically significant mean serum enzyme values, with D. bardawil treatment, compared to higher mean values in animals receiving CCl4 alone. Lipid peroxidation is measured by thiobarbituric acid-reactive substance (TBARS) activity was likewise slightly less elevated with algae treatment. The results also demonstrated protection against DNA strand breaks in hepatocytes, as measured by single cell gel electrophoresis. Liver histopathology was less severe with D. bardawil treatment, supporting the apparent protective action of 14-day treatment on hepatic oxidative injury.

Antioxidant property of Decalepis hamiltonii Wight & Arn.

Aromatic edible root of D. hamiltonii was subjected to the extraction of the antioxidant rich fraction. Different parts of root namely whole tuber, peel, tuber without peel and medullary portion were extracted with dichloromethane (European Patent No. W02005063272). The extract was found to contain flavor compound 2-hydroxy-4-methoxybenzaldehyde (2H4MB), which was identified by TLC and GC. Medullary portion was found to be rich in 2H4MB, (73.73 mg g(-1) dry tissue) followed by peel, containing 68.34 mg g(-1) 2H4MB. Different concentration of dichloromethane extracts were subjected for antioxidant assay by DPPH (1,1 dihydroxy 2-picryl hydrazyl) method, this has shown 44, 46.7% radical scavenging activity in case of medullary, peel extracts and 67.3% in case of pure 2-hydroxy-4-methoxybenzaldehyde at 100 ppm concentration, whereas ascorbic acid used as standard showed 94.3% activity. In beta-carotene linoleate model system (b-CLAMS) 43.46 and 45.7% antioxidant activity was observed in medullary and peel extracts at 100 ppm concentrations respectively, whereas standard 2-hydroxy-4-methoxybenzaldehyde exhibited 69.64% at 100 ppm and BHA (butylated hydroxyl anisole) 90.1% activity also at 100-ppm level. Similarly hydroxyl radical scavenging activity was found to be 48.36, 46.86, 48.26 and 73.60% in whole tuber, medullary, peel and standard 2-hydroxy-4-methoxy benzaldehyde respectively at 100 ppm levels. This is the first report on the antioxidant activity of D. hamiltonii. Results have shown that 2H4MB is one of the major constituents responsible for antioxidant activity. Hence the extract of D. hamiltonii can be utilized for the production of antioxidant rich fractions required for various health benefits.

Comparative evaluation of hepatoprotective activity of carotenoids of microalgae.

The present study deals with evaluation of the hepatotoprotective activity of carotenoids from two well-known microalgae, Spirulina platensis and Dunaliella salina. Carotenoids were extracted in hexane:isopropyl alcohol (1:1 vol/vol) and fed orally in olive oil to Wistar albino rats at a dose of 100 microg/kg of body weight/day (in terms of carotenoids). The degree of hepatoprotection was measured by estimation of biochemical parameters like serum transaminases [serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT)], serum alkaline phosphatase, total albumin, and total protein. The results were compared with those for a control group, a CCl4-induced hepatic damage group, and a group treated with synthetic beta-carotene (all-trans) at the same dose. The protein content of the CCl4-treated group, which received normal diet and a dose of toxin, showed a significant decrease, i.e., 3.92 mg/mL, whereas the protein levels were higher, i.e., 6.96 and 6.32 mg/mL, in the case of the Dunaliella and Spirulina, respectively, carotenoid-treated groups. The CCl4-treated group shown higher activity of transaminases (128.68 units/mL SGPT and 171.52 units/mL SGOT). However, the activity of SGPT was 62.83 units/mL for Dunaliella and 76.83 units/mL for Spirulina, i.e., carotenoids of Dunaliella showed a higher degree of protection. For serum alkaline phosphatase, the standard beta-carotene value was 81.52 units/mL, compared with 84.46 units/mL for the CCl4-treated group; however, natural algal carotenoids yielded 38.45 units/mL (D. salina) and 44.73 units/mL (Spirulina). The total albumin value diminished with CCl4 treatment (2.46 mg/mL); the effect was highest for Dunaliella, followed by the Spirulina carotenoid-treated group. The results clearly indicate that carotenoids from Dunaliella possess better hepatoprotection compared with those from Spirulina. High-performance liquid chromatography of the carotenoids indicated that Spirulina contains only beta-carotene and Dunaliella contains other carotenoids and xanthophyll. The increase in protection with Dunaliella indicates that mixed carotenoids exhibit better biological activity than beta-carotene alone. The results of this study indicate that carotenoids obtained from an algal source have a higher antihepatotoxic effect, compared with synthetic beta-carotene and with beta-carotene alone from a natural source.

Nutraceutical applications of garlic and the intervention of biotechnology.

Garlic (Allium sativum L.) is an important and widely cultivated plant with both culinary and medicinal uses stemming from its biological activities, which include antibiotic, anticancer, anti-thrombotic, and lipid-lowering cardiovascular effects. Though such medicinal use of garlic existed for centuries, there was little scientific support for its therapeutic and pharmacological properties. However, there has been a recent upsurge of research on garlic aiming to understand its exact mechanism of action in each case so that garlic and its products may have more judicious future applications. Since garlic is vegetatively propagated, its improvement for desired traits through conventional means is difficult. The intervention of biotechnological methods such as tissue culture and gene transfer protocols developed recently hold great promise for improving this crop. Due to new innovations in instrumentation and processing technologies coupled with more judicious experimental models, better products are foreseen in the market. The objective of this article was to review the recent developments made towards understanding the mechanism by which garlic imparts different therapeutic effects as well as to review what biotechnology can offer to improve this crop and its products.

Chemical composition, iron bioavailability, and antioxidant activity of Kappaphycus alvarezzi (Doty).

Kappaphycus alvarezzi, an edible seaweed from the west coast of India, was analyzed for its chemical composition. It was found that K. alvarezzi is rich in protein (16.24% w/w) and contains a high amount of fiber (29.40% w/w) and carbohydrates (27.4% w/w). K. alvarezzi showed vitamin A activity of 865 mug retinal equivalents/100 g of sample. It contained a higher quantity of unsaturated fatty acids (44.50% of the total), in which relative percentage of oleic acid was 11%, cis-heptadecanoic acid 13.50%, and linoleic acid 2.3% and 37.0% of saturated fatty acids (mainly heptadecanoic acid). K. alvarezziwas also found to be good source of minerals, viz 0.16% of calcium, 0.033% of iron, and 0.016% of zinc, which are essential for various vital biological activities. Bioavailability of iron by in vitro methods showed a higher efficiency in intestinal conditions than in stomach conditions. Ascorbic acid influenced higher bioavailability of iron. Successive extracts of n-hexane, acetone, ethyl acetate, ethanol, and direct extractables of chloroform/methanol (1:1 and 2:1) were screened for antioxidant activity using a beta-carotene linoleic acid model system (B-CLAMS), DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) model system and hydroxyl radical scavenging activity. The chloroform/methanol (2:1) extract has shown 82.5% scavenging activity at 1000 ppm. Acetone fraction extracts at the 1000 ppm level showed 63.31% antioxidant activity in beta-carotene linoleic acid system. The acetone extract showed 46.04% scavenging activity at 1000 ppm concentration. In the case of hydroxyl radical scavenging activity, all the extracts showed better activity at the concentrations of 25 and 50 ppm, where at the 50 ppm level ethyl acetate extract showed 76.0%, acetone 75.12%, and hexane 71.15% activity, respectively. Results of this study suggest the utility of K. alvarezzi (Eucheuma) for various nutritional products, including antioxidant for use as health food or nutraceutical supplement.

Study on wound healing activity of Punica granatum peel.

The methanolic extract of dried pomegranate (Punica granatum) peels showed the presence of a high content of phenolic compounds (44.0%) along with other constituents. This extract was formulated as a 10% (wt/wt) water-soluble gel and was studied for its wound healing property against an excision wound on the skin of Wistar rats. The activity was compared with that of a commercial topical antibacterial applicant. The wound healing activity was assessed by measuring the percent contraction in skin and estimation of collagen content in terms of hydroxyproline content. Healed skin was also subjected to histopathological studies to examine the microscopic changes. The animals treated with 2.5% gel showed moderate healing (55.8% and 40.8% healing compared with negative and positive controls, respectively), whereas the group treated with 5.0% gel showed good healing (59.5% and 44.5% healing compared with negative and positive controls, respectively). The amount of hydroxyproline increased by twofold in the group treated with 5.0% gel. Histopathological studies also supported the wound healing on application of the gels. The group of rats that received 5.0% gel showed complete healing after 10 days, whereas in rats treated with 2.5% gel, healing was observed on day 12, in contrast to the positive control animals receiving the blank gel, which took 16-18 days for complete healing. The results of this study may be extended to different types of wounds so that the formulation could be exploited to develop it as a topical dermatological formulation. High-performance liquid chromatography analysis of the extract showed the presence of gallic acid and catechin as major components.