Hypericum perforatum L induced wound healing process in Freund's adjuvant arthritic rats.
Abstract: Hypericum perforatum L (Hp) beneficial properties in wound healing have been known since the era of Hippocrates. The aim of the study was to investigate the restoration quality of incised skin under locally oily Hp Rotoel[R] treatment in Freund's adjuvant arthritis Wistar rats. Histological skin specimens of rats were evaluated under light microscopy (x20 HE). Controls and adjuvant arthritis rats under Hp had a more rapid formation of granoulomatous tissue compared with non treated controls. Hp treatment ameliorated inflammatory laboratory findings. In conclusion Hp treatment seems to contribute to more rapid wound repair and its application may be considered helpful in surgical practice.

Key words: Hypericum perforatum L, wound, Freund's adjuvant
Article Type: Report
Subject: Arthritis (Care and treatment)
Wound healing (Methods)
Adjuvants, Pharmaceutic (Research)
St. John's wort (Health aspects)
Authors: Sapounakis, C.
Kotsiou, A.
Potamianou, A.
Tsamouri, M.
Vovou, J.
Tesseromatis, C.
Pub Date: 03/22/2011
Publication: Name: Australian Journal of Medical Herbalism Publisher: National Herbalists Association of Australia Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2011 National Herbalists Association of Australia ISSN: 1033-8330
Issue: Date: Spring, 2011 Source Volume: 23 Source Issue: 1
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Australia Geographic Code: 8AUST Australia
Accession Number: 254971770
Full Text: Introduction

A wound is defined as the tissue disruption of the cellular and anatomic continuity. The process of wound healing consists of integrated cellular and biochemical events leading to re-establishment of structural and functional integrity with regain of strength of injured tissue. The management of wound healing has been a primary care of people since the beginning of civilization. Nature offers an immense source of medicinal herbs in the service of humanity. The art of herbal healing is deep rooted in folk medicine.

Hypericum genus (St John's wort), originating in Europe, West Asia and North Africa, is one of the oldest and the best experimentally and clinically examined herbal remedies in various cultures and medical systems.

Different species of the genus Hypericum (Hypericaceae) have been used in traditional medicine to treat various diseases. Hypericum perforatum L has been shown to possess significant wound healing properties since Hippocrates era (Hippocrate 1839). Hypericum perforatum particularly has been administered for skin wounds treatment, eczema, gastrointestinal and psychological disorders (Bombardelli 1995, Barnes 2001, Sapounakis 2009). Hypericum perforatum and other species of the genus Hypericum possess anxiolytic, antiviral, antimicrobial, analgesic, anti-inflammatory and antioxidant properties, while recently interest has been directed towards the antidepressant activity of this plant extract (Axarlis 1998, Flausino 2002, Mattace Raso 2002, Brenner 2000, Rabanal 2005).

In Canary Islands folk medicine, flowers, leaves and fruit infusions of various Hypericum taxa, especially of the endemic Hypericum reflexum L, have been used as external wound ulcer treatment and also as sedative, diuretic, vermifuge, antihysteric and antidepressant agents. The methanolic extract as well as the chloroform and butanol fractions of Hypericum reflexum were reported to possess antibacterial properties against gram positive bacteria (Darias 1986, Rabanal 2002, Herrera 1996). Although Kumar and Singh (2001) have demonstrated the analgesic and anti-inflammatory activity of Hypericum extracts, these properties have not yet been fully elucidated (Kumar 2001).

Inflammatory conditions such as rheumatoid arthritis (RA) induce numerous pro-inflammatory mediators, such as interleukines and tumor necrosis factor (TNF) that activate several intracellular signal transduction mechanisms (Firestein 1999, 2003). It has been reported that patients with RA are prone to ulceration of soft tissues (Firth 2009). Freunds adjuvant arthritis (AA) of rats is a widely used experimental model that mimics human chronic arthritis immune disorders (Ruth 2001, Colpaert 1982, Hirschelmann 1986). In acute inflammations, such as carrageenan induced pleuritis model, Hypericum perforatum plant species have been shown to exert beneficial effect (Menegazzi 2006).

The aim of this study therefore was to investigate the wound healing effect of Hypericum perforatum flowers sojaoil extract (Rotoel[R]) in a model of experimental trauma in Freund's adjuvant arthritic Wistar rats.

Material and methods Animals

Four groups (I, II, III, IV) of 8 male Wistar rats each, aged 5-6 weeks and weighting 250 [+ or -] 15 g were used. Group I and II served as control. Animals were housed in a controlled environment with 12 h light/12 h darkness and had access to standard rodent chow and water ad libitum. The animals were treated in accordance with standard principles (Washington 1985).

Freund's adjuvant induced arthritis

The animals of group III, IV were intradermally injected with 0.1 mL complete Freund's adjuvant (CFA) (Sigma-Aldrich) into the right hindpaw as in previous studies (Anagnostou 2009, Tigka 2005). Each mL contained 1 mg of Mycobacterium tuberculosis (H37Ra, ATCC 25177 Sigma-Aldrich St. Louis USA), heat killed and dried, 0.85 mL paraffin oil and 0.15 mL mannide monooleate. Two days after the CFA injection the experimental arthritis manifestations were observed. The evaluation of the CFA induction was assessed via the clinical signs, e.g. changes in weight (organ weight/body weight) of adrenals, spleen, thymus and the increases in paw diameter.

Experimental wound induction

All animals were anesthetised with ether pro narcosi, depilated in dorsal area and skin trauma was performed with a punch out 6 mm instrument for dermal biopsy until the muscular aponevrose in order to have a similar trauma in length and deepness in all animals.

Treatment

The animals of groups II and IV were treated daily with a local application of 10 [micro]L Hypericum perforatum oily preparation (Rotoel[R] Jukunda) containing 10% Hypericum flos using a micropipette.

Specimens preparation

Central sections of skin specimens obtained 5 days after the local application of Rotoel[R] in the incised dorsal area of animals, were embedded in paraffin in sections of 5[micro]. Three sections of every sample were stained with Haematoxilin-Eosin and evaluated under the light microscopy (x20).

Blood collection

Blood was collected and laboratory parameters (rheumatoid factor, CRP, total proteins, albumin, globulin, interleukin-2, interleukin-6) were measured in order to assess the successful induction of experimental adjuvant arthritis. Commercial enzyme linked immunosorbent assay (ELISA) kits for the quantitation of IL- 2 and IL-6 were employed. All ELISA kits were used according to the instruction manuals.

Statistical analysis

The data from the experimental and control rats were statistically analyzed by t- test .

Results

Clinical manifestation of Freund's adjuvant arthritis

Adjuvant arthritis evaluation resulting from clinical manifestation showed an augmentation in right paw diameter in groups III and IV compared with the controls. Similarly body weight was decreased in the CFA arthritic group (p<0.05) and was non statistically significantly re-established with Hypericum perforatum treatment.

The relative organ weights of spleen and adrenals were increased in CFA arthritic animals (p<0.05) while thymus relative weight declined (p<0.05) (Table 1).

The inhibition of paw edema was calculated with reference to the control group. Hypericum perforatum treatment demonstrated a non statistical significant curative effect on the systemic changes which occur in adjuvant arthritic rats (mean value [+ or -] SD).

Laboratory findings of experimental v. control groups

The laboratory findings indicate an enhancement of rheumatoid factor (Rf), C-reactive protein (CRP), globulin, interleukin-2, interleukin-6, while albumin and total proteins were decreased in the adjuvant arthritis groups compared with the controls. The albumin/globulin ratio was reversed in CFA arthritic animals. Freund's adjuvant arthritis changed all the measured inflammatory parameters statistically significantly (p<0.05). In addition a tendency to the arthritis profile amelioration was observed under the influence of Hypericum perforatum (Table 2).

Histological evaluation

The specimens of skin incision area demonstrated that the control animals treated with Hypericum perforatum had a much more rapid formation of granoulomatous tissue in comparison to the non treated groups.

The CFA arthritis rats demonstrated a delay in skin healing process, but Hypericum perforatum treatment induced a more rapid formation of granulomatous tissue, advanced wound healing and wound shrinkage compared with non treated animals.

Discussion

The healing properties of the Hp oil extract local administration are obvious in the controls and arthritic animals in accordance with the traditional local use of Hp as a wound healing agent. The local application of H. perforatum is already considered a safe medium for the facilitation of cesarean wound healing and the minimisation of scar formation, pain and pruritus (Samadi 2010).

The model of adjuvant arthritis was successfully induced as implied by the augmentation of paw diameter, one of the major factors assessing the degree of inflammation and the efficacy of various potential anti-inflammatory substances.

All parameters of inflammation were attenuated by H. perforatum extract. Parallel with Hp beneficial effect on the wound healing in control animals, Freund's adjuvant arthritis injuries tend to be repaired after Hp treatment as is verified by the biochemical findings. This is in accordance with similar data demonstrating analgesic and anti-inflammatory effects of various Hypericum species in mice models either with tetradecanoylphorbol acetate (TPA) induced ear inflammation (Rabanal 2005, Sanchez-Mateo 2006), zymosan induced organ dysfunction syndrome or of carrageenan induced pleuritis (Sanchez-Mateo 2006, Di Paola 2007).

The anti-inflammatory effect of Hp is attributed by Bezakova et al (1999) to its isolated constitutes anthrone, anthroquinone, emodin hypericin and pseudohypericin while its analgesic effect may be attributed to the presence of flavonoids, tannins and saponins (Sanchez-Mateo 2006, Bezakova 1999). Moreover the Hypericum anti-inflammatory effect is ascribed to the lipoxygenase activity inhibition and to the decrease of prostaglandine E (Bezakova 1999, Hammer 2007).

It must be mentioned that oil extracts of Hypericum perforatum (Oleum Hyperici) healing effect is independent of lesion origin since it is reported to exert gastroprotective, healing and palliative activity on gastric mucosa ulcers induced by NSAIDs such as indomethacin (Zduni 2009).

Furthermore in an evaluation study of Hypericum patulum leaf extract skin wound healing activity in rats compared with nitrofurazone ointment it was documented that leaf extract ointments lead to more effective tissue regeneration than the antibacterial agent (Mukherjee 2000).

It must be noted that although the present study as well as other reports confirm the healing effect of Hp after local application, they did not show considerable systemic Hp activity since it did not ameliorate the whole Freund's adjuvant arthritis multiple organ dysfunction syndrome. Probably the way of administration is responsible for the low systemic concentration of the active substances due to the local application.

It may be concluded that Hypericum perforatum treatment contributes to a more rapid tissue repair of the wound area probably via its constituents even in local application, showing a reduced tendency on the inflammation parameters and this may be useful in surgical practice.

The above results confirm the use of Hypericum perforatum as a potential wound healing agent, a property recognised in folklore medicine.

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Sapounakis Constatin is a Medical Doctor and General Practioner, Department of Pharmacology, Medical School, University of Athens, Greece

Kotsiou Antonia PHD is a pharmacist in the Department of Pharmacology, Medical School, University of Athens, Greece

Potamianou Aggella MD PHD is a Biopathologist in the Department of Microbiology, Asklipio General Hospital of Voula, Athens, Greece

Tsamouri Madalaine MD PHD is a pathologist in the Department of Pathology, Red Cross Hospital, Athens Greece

Vovou Joanna PHD is a biologist in the Department of Pharmacology, Medical School, University of Athens, Greece

Tesseromatis Christine MD PHD is an Associate Professor of Pharmacology Department of Pharmacology, Medical School, University of Athens, Greece

Sapounakis C (1), Kotsiou A (1), Potamianou A (2), Tsamouri M (3), Vovou J (1), Tesseromatis C (1) *

(1) Department of Pharmacology, Medical School, University of Athens, 75 Mikras Asias St, Goudi Athens 11527 Greece

(2) Department of Microbiology, Asklipio General Hospital of Voula, Athens Greece

(3) Department of Pathology, Red Cross Hospital, Athens Greece

* Corresponding author email: ctesser@med.uoa.gr
Table 1: Clinical manifestations of CFA arthritis with and
without Hypericum perforatum treatment in comparison with the
controls

           Groups                  Controls I

Body weight (g)                  250 [+ or -] 15
Thymus weight (g/g x 10-3)     1. 02 [+ or -] 0.04
Spleen weight (g/g x 10-3)     1.09 [+ or -] 0.13
Adrenals weight (g/g x 10-3)   0.05 [+ or -] 0.04
Paw diameter (mm)                5 [+ or -] 0.5

           Groups               Controls + Hp II

Body weight (g)                  248 [+ or -] 10
Thymus weight (g/g x 10-3)     1. 12 [+ or -] 0.07
Spleen weight (g/g x 10-3)     1.12 [+ or -] 0.09
Adrenals weight (g/g x 10-3)   0.05 [+ or -] 0.27
Paw diameter (mm)                5 [+ or -] 0.5

           Groups                     CFA III

Body weight (g)                  205 [+ or -] 12 *
Thymus weight (g/g x 10-3)     0.72 [+ or -] 0.11 *
Spleen weight (g/g x 10-3)     1.43 [+ or -] 0.12 *
Adrenals weight (g/g x 10-3)   0.08 [+ or -] 0.01 *
Paw diameter (mm)               8.5 [+ or -] 0.2 *

           Groups                    CFA + Hp IV

Body weight (g)                  215 [+ or -] 8 (#)
Thymus weight (g/g x 10-3)     0,98 [+ or -] 0.03 (#)
Spleen weight (g/g x 10-3)      1.35 [+ or -] 0.1 (#)
Adrenals weight (g/g x 10-3)   0.068 [+ or -] 0.03 (#)
Paw diameter (mm)               7.8 [+ or -] 0.3 (#)

Groups * I/III p <0.05 (#) II/IV p<0.5 (#) III/IV p<0.5

Table 2: Changes in laboratory findings in CFA arthritic animals
compared with controls with and without Hypericum perforatum
treatment

Groups M [+ or -] SD        Control          Controls + Hp II

Rf IU/ml                      >9.5                 >9.5
CRPmg/L                       >3.5                 >3.5
Total proteins         1.09 [+ or -] 0.13   1.12 [+ or -] 0.09
(g/dl)                  6.9 [+ or -] 0.1      7 [+ or -] 0.5
Albumin                  5 [+ or -] 0.5       5 [+ or -] 0.5
(g/dl)                 4.1 [+ or -] 0.01    4.25 [+ or -] 0.01
Globulin (g/dl)        2.41 [+ or -] 0.21   2.56 [+ or -] 0.04

Groups M [+ or -] SD     CFA arthritis III     CFA arthritis + Hp IV

Rf IU/ml               15.65 [+ or -] 0.01 *    10.5 [+ or -] 0.2 *
CRPmg/L                 5.3 [+ or -] 0.3 *      4.1 [+ or -] 0.02 *
Total proteins         1.43 [+ or -] 0.12 *    1.35 [+ or -] 0.1 (#)
(g/dl)                 6.15 [+ or -] 0.02 *     6.35 [+ or -] 0.2 *
Albumin                 8.5 [+ or -] 0.2 *     7.8 [+ or -] 0.3 (#)
(g/dl)                    3 [+ or -] 0.02       3.7 [+ or -] 0.02 *
Globulin (g/dl)          4.4 [+ or -] 0.2        4.01 [+ or -] 0.3

Groups * I/III p <0.05 (#) II/IV p<0.5 (#) III/IV p<0.5
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