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Hibiscus anthocyanins-rich extract inhibited LDLoxidation andoxLDL-mediated macrophages apoptosis

Yun-Ching Chang a, Kai-Xun Huang a, An-Chung Huang a,

Yung-Chyuan Ho b, Chau-Jong Wang a,*a Institute of Biochemistry and Biotechnology, Chung Shan Medical University, No. 110, Sec. 1, Chien-KauoN. Road, Taichung 402, Taiwanb School of Applied Chemistry, Chung Shan Medical University, Taichung, TaiwanReceived 2 May 2005; accepted 21 December 2005

AbstractThe oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathogenesisof atherosclerosis. Anti-oxidativereagents, which can effectively inhibit LDL oxidation, may prevent atherosclerosis via reducingearly atherogenesis, and slowing downthe progression to advance stages. As shown in previous studies Hibiscus sabdariffa L. is anatural plant containing a lot of pigments thatwas found to possess anti-oxidative of activity. Therefore, in this study, we evaluated the anti-oxidative activity of Hibiscus anthocyanins(HAs) by measuring their effects on LDL oxidation (in cell-free system) and anti-apoptoticabilities (in RAW264.7 cells). HAs have beentested in vitro examining their relative electrophoretic mobility (REM), Apo B fragmentation,thiobarbituric acid relative substances(TBARS) and radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity assay. The anti-oxidative activity of HAs was definedby relative electrophoretic mobility of oxLDL (decrease of 50% at 2 mg/ml), fragmentation ofApo B (inhibition of 61% at 1 mg/ml),and TBARS assay (IC50: 0.46 mg/ml) in the Cu2+-mediated oxidize LDL. Furthermore, theaddition of >0.1 mg/ml of HAs could scavengeover 95% of free DPPH radicals, HAs showed strong potential in inhibiting LDL oxidationinduced by copper. In addition, todetermine whether oxLDL-induced apoptosis in macrophages is inhibited by HAs, we studiedthe viability, morphology and caspase-3 expression of RAW 264.7 cells. MTT assay, Leukostate staining analysis and Western blotting

reveals that HAs could inhibitoxLDL-induced apoptosis. According to these findings, we suggest that HAs may be used toinhibit LDL oxidation and oxLDL-mediatedmacrophage apoptosis, serving as a chemopreventive agent. However, further investigationsinto the specificity and mechanism(s)of HAs are needed._ 2006 Elsevier Ltd. All rights reserved.Keywords: Hibiscus Sabdariffa Linnaeus; Anthocyanins; Macrophage; LDL oxidation; Radical scavenging;Atherosclerosis

1. IntroductionAtherosclerosis is a complicated vascular disorder and aprincipal contributor to the pathogenesis of myocardialand cerebral infarction. The processes in atherosclerosisinvolves monocytes migration from the blood stream, then

their differentiation into macrophages, the uptake of lowdensitylipoprotein (LDL) by the macrophage scavengerreceptor, transformation of the lipid-laden macrophagesinto foam cells, smooth muscle cells proliferation andtransformation into foam cells, and thus the accumulationof foam cells leading to fatty streaks and subsequent plaqueformation (Glass and Witztum, 2001). LDL has beenfound to accumulate in atherosclerosis lesions and variousevidences indicate that an important part of the pathogenesisof atherosclerosis is the oxidative modification of LDL


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(Steinberg et al., 1989; Steinberg, 1995). Oxidative processesare likely to play a key role in determining the fate0278-6915/$ - see front matter _ 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.fct.2005.12.006Abbreviations: HAs, Hibiscus anthocyanins; PCA, protocatechuic acid;LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein;REM, relative electrophoretic mobility; TBARS, thiobarbituric acid relativesubstances; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ApoB, apoproteinB.* Corresponding author. Tel.: +886 4 24730022x11670; fax: +886 423248167.E-mail address: [email protected] (C.-J. Wang).www.elsevier.com/locate/foodchemtoxFood and Chemical Toxicology 44 (2006) 10151023

A Study of the Staining Effect of Roselle

(Hibiscus sabdariffa) on the HistologicSection of the Testis

Estudio de los Efectos de Tincin de la Rosa deJamaica (Hibiscus sabdariffa) en CortesHistolgicos de Testculo

Egbujo, E. C. ; Adisa, O. J. & Yahaya, A. B.

Histopathology Department, Jos University Teaching Hospital,Jos, Plateau State, Nigeria.

Correspondence to:

SUMMARY: This study describes the preparation and use ofRoselle (Hibiscus sbddariffa) for the differential staining oftesticular tissue sections to find out its staining effect on nuclear,cytoplasmic and other structures. Various treatments usingmodifications of the plant extract in water were carried out onsections of the rabbit testis. Various levis of differentiation of

nuclear and cytoplasmic structures as well as other structures ofthis organ was obtained especially when 1% eosin was applied asa counter stain. The best staining result was obtained when ironalum was used to mordant the extract and when the extract,mordanted with potassium alum was acidified using acetic acidand used to stain the sections. Modification of the aqueousextract to an alkaline pH using ammonia gave the pooreststaining effect. Roselle extract therefore shows reasonablepotential as a candidate nuclear stain especially when modanted
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with iron alum or mordanted with potassium alum and acidifiedwith acetic acid.

KEY WORDS: Hibiscus sabdariffa; Staining; Testis;Histologic section.

RESUMEN: Este estudio describe la preparacin y utilizacin deRoselle (Hibiscus sabddarifa) en la tincin diferencial de corteshistolgicos de tejido testicular, para conocer su efecto sobre latincin nuclear, citoplasmtica y otras estructuras. Diversostratamientos utilizando modificaciones de extracto de la planta enel agua, se llevaron a cabo en cortes de testculos de conejo.Distintos niveles de diferenciacin de las estructuras nucleares ycitoplasmticas, as como de otras estructuras de este rgano, sehan obtenido principalmente cuando se utiliza como tincineosina al 1%. El mejor resultado de tincin se obtuvo cuando seutiliz alumbre de hierro y cuando ste fue acidificado con cido

actico y se us para teir los cortes histolgicos. La modificacindel extracto acuoso a un pH alcalino utilizando amonaco, dio unacoloracin ms dbil. El extracto de rosa de Jamaica muestra unrazonable potencial para tincin nuclear, especialmente, cuandoes mezclado con alumbre de hierro o aluminio de potasio yacidificada con cido actico.

PALABRAS CLAVE: Hibiscus sabdariffa; Tincin; Testculo;Corte Histolgico.

INTRODUCTION

Staining techniques originated from the second half of the lastcentury (Kolliker, 1852). Stains have been used to enhanceaccurate descriptions of the microscopic structure of tissues,which is necessary forhistopathologic diagnosis. Plant and insectparts have found place in histological staining due to theircolouring and dying effect. Examples of plant and insect partsthat have found place in histological staining as natural dyes areHaematoxylon campechiaumn, from which haematoxylin isobtained (Bohmer, 1865) and Dactylopius cacti, from which

carmine stain is obtained (Goppert & Cohn, 1849). Although mostof the dyes in current use in histopathology laboratories are ofsynthetic origin, natural dyes still hold promise as a potentialsource cheaper dyes and consequently providing employmentopportunities in developing countries. Aqueous extract ofHibiscussabdariffa has been recently used to stain lymph node and kidneybiopsies. The trial was however without clearly definedtreatments.


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The staining was done at 56 C for lh. The results obtained weresaid to be similar to that of silver impregnation techniques andthe conventional haematoxylin and eosin method.

This study aims at demonstrating the dyeing effect ofHibiscussabdariffa on tissue sections and to nd out the effect of

mordants; pH and duration of staining and temperature on thestaining property oHibiscus sabdariffa.

MATERIAL AND METHOD

Preparation ofHibiscus sabdariffa extract and stainingsolution. The dried succulent red calyx of Roselle (Hibiscussbddariffa) was purchased in the market and ground to powderyform using pestle and mortar, sieved and stored in a drycontainer. A measured quantity of the ground powder of Rosellewas brought to boil in water, and mixed by shaking vigorously.This was allowed standing for 30 minutes, then filtered to obtain

the coloured extract.

Fixation, Preparation of sections and staining of sections.

The selected pieces of testicular biopsy obtained from a rabbitwere fixed in 10% formalin. These were processed throughascending grades of efhanol and two changes of absolute ethanol,cleared in three changes of xylene and then infilterated withmolten paraffin wax (60C). The testicular biopsies were thenembedded in paraffin wax, sectioned at 5 |jm and stainedappropriately with various modifications of the extract astabulated below. They were subsequently des hydrated, cleared

and mounted with DPX. Photomicrographs of the sections werethen taken.

RESULTS AND DISCUSSION

The haematoxylin and eosin staining of the testis (Fig. 1) showeda deep red stained outer fibrous layer, the tnica albugenae,beneath which are seen a number of seminiferous tubules cut invarious plains. Between the tubules are connective tissuescontaining blood vessels and groups of interstitial cells. Eachseminiferous tubule is lined by several layers of cells, which whenviewed under high power magnification reveis an indistinct cell

boundary and prominent nuclei. The outer rows of nuclei belongto sustentacular cells and spermatogonia. Passing inward towardsthe center of the tubules are large nuclei of spermatocytes andmany smaller nuclei of spermatids. No matured spermatozoawere seen.
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In the test slides stained with Roselle extract mordanted withpotassium alum and counter stained with eosin (Fig. 2), thesection of the testis showed a better nucleacytoplasmicdifferentiation compared to that given by the haematoxylin andeosin method. The slight improvement in nuclear staining ascompared to when the extract was not mordanted could beattributed to the possible formation of a mordant and dyecomplex known as a 'dye-lake'. These are similar to thehaematoxylin-mordant complex, which are usually positivelymcharged, and therefore behave as cationic dye at low pH. Whenused to stain tissues, cationic dye-mordant complexes areattracted to negatively charged sites, displaying a particularaffinity for polyphosphates. This explains the affinity for thenuclei (Marshall & Horobin, 1973). The poor nuclear/ cytoplasmicdifferentiation seen in the use of unmordified Roselle extract andeosin as compared to haematoxylin and eosin may highlight theneed for an auxochrome which stabilizes a staining reaction asobtained in the haematoxylin-eosin method.


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Roselle extract mordanted with potassium alum, acidified withacetic acid and counterstained with eosin, (Fig. 3) showed that inthe testis, the nuclear staining is particularly enhanced. Thenuclear/cytoplasmic differentiation is markedly enhancedalthough affinity for eosin seemed to be reduced. This shows thatthe Roselle dye molecules are charged and that the ionic chargescould be varied using acids or alkalis and therefore, its applicationas a nuclear or cytoplasmic stain made possible. The reducedcytoplsmic staining when acetic acid was incorporated in theroselle extract also suggest that the presence of nucleic acids in

the cytoplasm, though not in the same quantity as in the nuclei,attracts and therefore reacts with the dye resulting in theblockage of reactive sites that would have been taken up by thecounterstain.


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This has been documented by Birkedal-Hasen (1973), where hereported a similar phenomenon in the heamatoxylin and eosinmethod. The implication of this is that, where cytoplasmicstructures or other structures are of interest, an appropriate pHthat would favour the uptake of the stain would have to beselected.

When roselle extract mordanted with potassium alum, alkalinizedwith ammonia was applied on the testicular section andcounterstained with eosin (Fig. 4) the nuclear/ cytoplasmicdifferentiation was markedly reduced and the counterstainseemed to stain all the structures uniformly resulting in the so-called "pink disease". This observation showed that an alkalineenvironment creates an anionic electrical environment, whichnaturally favored the acidic dye (eosin).


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The use of iron alum as mordant with roselle extract (Fig. 5) onthe section of the testis showed a good nuclear/ cytoplasmicdifferentiation as observed with the potassium alum counterpart,but in this case the connective tissues were more clearlydemonstrated. The increased nuclear/ cytoplasmic differentiationand demonstration of other connective tissue can be attributed tothe facts that iron alum tends to form a stronger dye- lake bondfhan potassium alum.


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In conclusin Roselle can be a good substitute to haematoxylin asa nuclear stain especially when mordanted with iron or potassiumalum. Acidifying with acetic acid has also been shown to enhancenuclear staining. The limitation of this study has been the inabilityto identify fhe iso-electric point (pH point) at which bofh nucleiand cytoplasm of the cells could be stained simultaneously. Otherfactors that could be investigated are optimal temperatures for itsuse and compatibility with other counter stains.

REFERENCES

Birkedal-Hansen, H. Eosin staining of gelatine. Histochemie,36:73-87, 1973. [ Links ]

Bohmer, F. Zur pathologischen, Anatomie der Meningitiscerebromedularis epidemia.Aerztl. Intelligenzb. (Manchen),12:539-50,1865. [ Links ]

Goppert H. R. & Cohn, F. Ueber die Rotation des Zellinhaltes vonNitella flexilis. Bot. Z., 7:681, 1849. [ Links ]

Kolliker, A. Manual of human Microscopio Anatomy. London,Hippolyte Bailliere, 1852. [ Links ]

Marshall P. N. & Horobin R.W. Measurements of the afnities ofbasic and 'mordant' dyes for various tissue substrates.Histochenue, 36:303-12, 1973. [ Links ]


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Correspondence to: Dr. James O. Adisa P. O. Box 1109, JosPlateau State NIGERIA.Email: [email protected]

Received: 12-02-2008, Accepted: 26-08-2008.
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