C C odorata Extract Upregulates Adhesion Complex Proteins and Fibronectin Production by Human Keratinocytes Phan et al 2000b

This study demonstrated that the C. odorata extract increased the expression of several components of the adhesion complex and of fibronectin by human keratinocytes. Using indirect immunofluorescence, an increased expression (dose-dependent) of laminin 1, laminin 5, collagen IV, and fibronectin was

Figure 4 Reepithelialization of in vitro scratch wounds by C. odorata extract. Phase-contrast micrographs of a single representative field immediately after wounding. (a) and (b) Control and C. odorata extract treatment at 0 hr. (c) and (d) Control and C. odorata extract treatment at 34 hr after wounding. Wound reepithelialization was faster in the group treated with 10 ng/mL extract (d). (With permission for reprint from Wound Repair and Regeneration.)

Figure 4 Reepithelialization of in vitro scratch wounds by C. odorata extract. Phase-contrast micrographs of a single representative field immediately after wounding. (a) and (b) Control and C. odorata extract treatment at 0 hr. (c) and (d) Control and C. odorata extract treatment at 34 hr after wounding. Wound reepithelialization was faster in the group treated with 10 ng/mL extract (d). (With permission for reprint from Wound Repair and Regeneration.)

found (Fig. 5). The expression of the p1 and p4 integrins was upregulated by the extract at low concentrations (0.1 and 1 Ag/mL), but the expression was decreased at higher doses of the extract (10-150 Ag/mL) (Fig. 5).

Extracellular matrix (ECM) and basement membrane zone (BMZ) components as ''adhesion'' molecules serve several critical functions for effective wound repair and are crucial to basal cell regeneration and wound reepithelialization. BMZ components serve as a scaffold for tissue organization and as a template for tissue repair. These components are produced and regulated by keratinocytes, fibroblasts, and cytokines (Uitto et al., 1996).

The adhesion complex proteins are essential to wound healing, especially to stabilized epithelium, and this effect of the C. odorata extract could contribute to the clinical efficacy of the extract used for the treatment of burns and wounds.

D. Investigation of Antioxidant Effects of C. odorata Extract on Cultured Skin Cells from Oxidative Damage: Identification of the Extract Active Compounds (Phan et al., 2001a,d)

Oxidants are involved in burn injury and tissue repair. Oxygen free radicals may contribute to further tissue damage in the events following skin injury and an overabundance is known to impair the healing process. Antioxidants, on the other hand, significantly prevent tissue damage and stimulate wound healing. H2O2 and HX/XO were applied to a skin cell culture to serve as an in vitro wound-healing model for oxidative damage and the protective antiox-idant effects of C. odorata were tested on this model. With the use of total crude extract, the concentrations at 400 Ag/mL and 800 Ag/mL showed maximum and consistent protective effects against the oxidant toxification

Figure 5 Enhanced expression and secretion of adhesion complex proteins and fibronectin by human epidermal keratinocytes treated with C. odorata extract. Magnification is at 400x. In vivo skin localization (indicated by arrows; e = epidermis, d = dermis, e-d j = epidermal-dermal junction) of laminin 1 (a), laminin 5 (d), collagen IV (g), fibronectin (j), ß1 integrin (m), and ß4 integrin (p). There was no expression of laminin 1 in keratinocytes maintained in basal medium (b). There was expression of laminin 1 in cells treated with 50 Ag/mL extract (c). A little extracellular secretion of laminin 5 was maintained in basal medium (e), this extracellular secretion increased and became more dense and extensive with 50Ag/mL of C. odorata extract in culture medium (f). Collagen IV, a structural protein of the epiderm-dermal junction, was only weakly expressed by keratinocytes maintained in basal medium (h), but there was increased expression and extracellular secretion by cells treated with 50 Ag/mL extract (i). Only a few keratinocytes expressed fibronectin when maintained in the basal medium (k), but there was a high increase in the expression and extracellular secretion of fibronectin by keratinocytes treated with 50 Ag/mL extract (l). There was very weak expression of the ß1 integrin in keratinocytes cultured in basal medium (m), but it was stimulated by C. odorata extract at 0.1 Ag/mL (p). ß4 integrin, an important component of epidemal-dermal junction, was expressed by keratinocytes in basal medium (q), but increased by cells treated with 0.1 Ag/ml C. odorata extract. (With permission for reprint from the European Journal of Dermatology.)

of cells in low or high doses of oxidants. The 50-Ag/mL concentration also had significant and slightly protective effects on fibroblasts against both H2O2-and HX/XO-induced damage. For keratinocytes, the dose-dependent relationship of oxidant toxicity was seen only in H2O2 but the protective strength of the extract correlated with the oxidant dosage. The extract at 400 Ag/mL and 800 Ag/mL showed dose-dependent effects on both low and high concentrations of oxidants. The concentration at 50 Ag/mL had no effect on keratinocytes (Phan et al., 2001a).

In this study, H2O2- and HX/XO- generated superoxides (O2—) were employed as an in vitro model to cause cultured skin cell injury, on which the protective effects of the C. odorata extract fractions were assessed.

The effects of column fractions from the extract on human dermal fibroblasts and epidermal keratinocytes, which are pivotal and crucial in cutaneous wound repair, were further evaluated. Hydrogen peroxide and superoxide radicals generated by the HX/XO reactions were employed to induce oxidative stress to in vitro cell cultures. The cytotoxicity of the oxidants and the protective effects of the extracts were indirectly assessed via cell viability. The most active antioxidant compounds were also identified using HPLC and LC-MS technology. Fractions B and C consistently showed the most protective effects on skin cell cultures injured by hydrogen peroxide

Figure 6 Protective effect of the column fractions of C. odorata extract on H2O2 and xanthine-oxidase-induced damage to human dermal fibroblasts. Cells were incubated for 3 hr at 37°C with 2 x 10~4 mol/L H2O2 (6A) or 2 x 10~2 units/mL xanthine oxidase (b) with or without fractions. Cells were then washed and assayed by the MTT assay. Bars represent mean + SEM of seven wells. Comparison with cells exposed to generated oxidants with or without fractions is indicated (p < .001). Fractions B and C show perfect protection on fibroblasts injured by oxidants.

Protective effect of the column fractions of C. odorata extract on H2O2 and xanthine-oxidase-induced damage to human epidermal keratinocytes. Cells were incubated for 3 hr at 37°C with 2 x 10~4 mol/L H2O2 (c) or 2 x 10~2 units/ml xanthine oxidase (d) with or without fractions. Cells were then washed and assayed by the MTT assay. Bars represent mean + SEM of seven wells. Comparison with cells exposed to generated oxidants with or without fractions is indicated. Fractions A, B, and C show good or almost complete protection on keratinocytes injured by oxidants (p < .001, .05, respectively).

Human keratinocytes exposed to hydrogen peroxide damage without (e) or with (f) C. odorata protection. (e) Keratinocytes were killed under toxicity of hydrogen peroxide. (f) Keratinocytes were protected by C. odorata extract fraction. (With permission for reprint from the Biological and Pharmacological Bulletin.)

and superoxide radicals in vitro (Fig. 6). Chemical analysis of these fractions (see Table 1) indicated that the major constituents in fraction B were p-coumaric acid ( p-CA = 4-hydroxycinnamic acid) and p-hydroxybenzoic acid (p-HBA = 4-hydroxybenzoic acid) and that the major compound in fraction C was protocatechuic acid (PCA = 3,4-dihydroxybenzoic acid). Minor compounds in fraction B were ferulic acid (FA = 4-hydroxy-3-methoxycin-namic acid) and vanillic acid (VA = 4-hydroxy-3-methoxybenzoic acid) and a tetrahydroxy-monomethoxyflavanone. The same flavanone, VA and p-CA, were minor constituents in fraction C. p-HBA is an important natural antioxidant. It is well established as an in vitro effective hydroxyl radical scavenger and has Trolox equivalent antioxidant activity (TEAC) (Rice-Evans et al., 1996; Ohsugi et al., 1999). Other published pharmacological characteristics of p-HBA are antibacterial and hypoglycemic properties (Orjala et al., 1993; Cho et al., 1998). The presence of p-HBA in the extract can account for its bacterial-growth inhibition in human wounds observed previously by clinical researchers.

Figure 6 Continued.

Another hydroxybenzoic acid, protocatechuic acid (PCA), was detected in fraction C of the extract as a major compound. PCA also is a strongly antioxidant phenolic acid. A number of reports described PCA as a potential chemopreventive agent against carcinogenesis and tumor promotions (Tanaka et al., 1995). PCA isolated from traditional herbal medicines showed strong inhibitory activity against superoxide anion radical (O2~) (Ohsugi et al., 1999). PCA was also found to have similar protective effects as caffeic acid in terms of exhibition of potent protection on cultured endothelial cells against oxidized low-density lipoproteins (Vieira et al., 1998). In the studies of a polyphenolic extract from Cudrania cochinchinesis, another medicinal plant

Figure 6 Continued.

for wound healing, PCA was the major constituent and was speculated to be responsible for protection of fibroblasts and endothelial cells against hydrogen peroxide- and HX/XO-induced damage and to be a stimulator of fibroblast proliferation (Tran et al., 1997). A third hydroxybenzoic acid, vanillic acid (VA), was found in both fractions B and C. This compound has anti-inflammatory activity in vitro (Harborne et al., 1999).

The C. odorata extract also contains two hydroxycinnamic acid derivatives, p-coumaric acid (p-CA) and ferulic acid (FA). p-CA is a major constituent in fraction B and a minor one in fraction C. Hydroxycinnamic acids such as p-CA, FA, and caffeic and chlorogenic acids are among the

TABLE 1 Major and Minor Constituents of Fractions A, B, and C

Purified Compound fraction Constituents class

A Tamarixetin3

Trihydroxy-monomethoxy-

flavanone3 Pentamethoxy-flavanone3 Dihydroxy-trimethoxy-

chalcone3 Eupatilin3

5,6,7,4'-Tetramethoxy-

tetramethoxyflavone Kaempferide Protocatechuic acid

B p-Coumaric acid3

Flavonol Flavanone

Flavanone Chalcone

Flavone Flavone

Flavone

Flavonol Hydroxybenzoic acid

Hydroxycinnamic acid

Retention UV spectrum Molecular time (min) (nm) mass (m/z)

19.8 254,369 316

19.1 290, 330sh 302

19.3 290, 330sh 374

23.1 237,373 330

21.4 269,342 344 22.4 265, 322 342

22.2 275, 341 358

23.2 264, 362 300

4.4 259,294 154

11.7 290sh, 309 164

p-Hydroxybenzoic acid3

Ferulic acid

Vanillic acid

Tetrahydroxy-monomethoxy flavanone3 C Protocatechuic acid3

Vanillic acid p-Coumaric acid

Tetrahydroxy-monomethoxy flavanone

Sinensetin

Rhamnetin

Hydroxybenzoic acid Hydroxycinnamic acid Hydroxybenzoic acid Flavanone

Hydroxybenzoic acid

Hydroxybenzoic acid

Hydroxycinnamic acid

Flavanone

Flavone

Flavonol sh, shoulder or inflection. a Major constituents.

8.1 259,292 168 3

o 5T

4.4 259,294 154 I

11.7 290sh, 309 164 3

17.3 290, 340sh 318

20.7 240, 266sh, 372

21.4 255,371 316

oo w most widely distributed phenylpropanoids in plant tissues (Rice-Evans et al., 1996) and their biological activities have been widely investigated. They are potential chemopreventive agents against carcinogenesis and tumor growth (Rice-Evans et al., 1996) and they are also recognized to be strong free-radical scavengers to hydroxyl radicals (Rice-Evans et al., 1996; Zang et al., 2000).

Hydroxycinnamic acid derivatives were also found to be effective inhibitors of xanthine oxidase. The phenolic OH group, present in their molecules, displays an important contribution to the XO-inhibitory activities. The absence of this group in the molecule induces the reduction of the inhibition on XO (Chang et al., 1994; Chan et al., 1995). Other antioxidant activities of hydroxycinnamic acid derivatives have been reported. They were able to scavenge reactive species of oxygen and nitrogen (Nakayama, 1994; Harborne et al., 1999). The presence of representatives of two groups of antioxidant phenolic acids (hydroxybenzoic and hydroxycinnamic acids) in fractions B and C can account for their strong inhibition of the cytotoxicity of superoxide radicals and hydrogen peroxide (H2O2).

To investigate the synergistic effect of the C. odorata lipophilic fractions on skin cells, solutions of pure p-HBA, PCA, p-CA, and FA (Sigma) in similar concentrations to those of C. odorata fractions were applied on fibroblasts and keratinocytes injured by H2O2 or HX/XO. No protection was observed in these experiments, suggesting that the single compounds provided less effect than the mixture of compounds in the C. odorata fractions. On the other hand, phenolic compounds in the extract worked in a synergistic manner to protect skin cells from oxidative damage, leading to enhanced healing.

Two well-known antioxidant compounds, a-tocopherol and curcumin (Sigma), were also tested in comparison with C. odorata fractions. The a-tocopherol solution, at the same concentrations as the extract fractions, showed about 50% less protective effects on skin cells than fractions B and C. Curcumin has strong protective effect on fibroblasts and keratinocytes against H2O2 damage. But there was no effect on HX/XO-induced damage (Phan et al., 2001c).

Fraction A contained mostly methoxylated flavonoids (flavonols, fla-vanones, flavones, and a chalcone, see Table 1). Although a variety of different flavonoids had already been reported from C. odorata (Barua et al., 1978; Triaratana et al., 1991), our present investigations revealed that many more are present in the plant, especially flavanones. Some of the compounds have been demonstrated earlier to be responsible for the hemo-static effect of the C. odorata extract (Triaratana et al., 1991). The presence in fraction A of tamarixetin and kaempferide, which are the 4'-methyl ethers of the well-known antioxidant and anti-inflammatory flavonols quercetin and kaempferol, and of small amounts of protocatechuic acid can explain some of the protective effects on keratinocytes injured by superoxide radicals and H2O2. These mixtures of phenolic compounds in the lipophilic fractions of C. odorata are likely to be the major contributors to the antioxidant properties of this plant species. In addition, kaempferide has been reported to inhibit inflammation induced by tumor-promoting phorbol esters, and the flavone eupatilin, which is also present in fraction A, selectively inhibits 5-lipoxygen-ase of cultured mastocytoma cells (Harborne et al., 1999).

It has been scientifically demonstrated that the extract of C. odorata has therapeutic properties in some aspects of wound healing. The results of this study suggest that one of the possible mechanisms by which C. odorata contributes to wound healing could be the antioxidant effect of the phenolics present. PCA, p-HBA, also the p-CA, FA, and VA, the major constituents of fractions B and C, are also the main active compounds of many medicinal plants including plants for wound healing, and many biological activities and potential therapeutic applications of these compounds have been reported. Although these compounds are not new, this is the first time, to our knowledge, that these acids were identified in an extract of C. odorata and demonstrated to be responsible for the protection of cultured skin cells against oxidative damage. The flavonoids identified in the various fractions are likely to have a synergistic effect.

IV. CONCLUSION

Crude extract of C. odorata has been used successfully for treating wounds. In this study, the therapeutic properties of the C. odorata extract were demonstrated scientifically with:

enhanced proliferation of fibroblasts, endothelial cell, and keratino-cytes;

stimulation of keratinocyte migration;

upregulation of keratinocyte production of extracellular matrix proteins and basement membrane components;

protection of skin cells against oxidative damage;

antioxidant effects of a mixture of phenolic compounds found in the extract

The activities shown in this study provide an important scientific basis for understanding how the C. odorata extract could have benefited wound healing in vivo and leads to further speculation that these properties could explain the observed enhancement of wound healing. These findings also provide a rationale for the wide use of C. odorata preparations in tropical countries to treat burns and wounds, regardless of income and status, with the potential to improve the quality of life of patients at low cost.

The clinical efficacy of this plant extract or its preparations should be further investigated by prospective, randomized, controlled trials. Perhaps a new potent agent for wound healing could be developed from this plant extract.

ABBREVIATIONS

APCI-MS

Atmospheric pressure chemical ionization mass spectrom-

etry

ChA

Chlorogenic acid

CID

Collision induced dissociation

DAD

Diode array detection

DMF

N,N-Dimethyformamide

DMEM

Dulbecco's modified eagle medium

FA

Ferulic acid

FCS

Fetal calf serum

HBSS

Hanks' balanced salt solution

HPLC

High-performance liquid chromatography

h2o2

Hydrogen peroxide

HX-XO

Hypoxanthine-xanthine oxidase

KBM

Keratinocyte basal medium

KGM

Keratinocyte growth medium

MTT

[3-(4,5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bro

mide]

PBS

Phosphate-buffered saline

PCA

Protocatechuic acid

p-CA

para-Coumaric acid

p-HBA

para-Hydroxybenzoic acid

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