Plant Pigments ( Antioxidants ) of Medicinal Plants Malva Silvestris L . and Malva Moschata L . ( Malvaceae )

Qualitative-quantitative structure of plant pigments in wild plants Malva silvestrs L. and Malva moschata L. (Malvaceae), which were collected in  locations in Sarajevo area and surroundings, was tested during spring and summer in . Acetone extracts of both categories were made and rising paper-chromatography done for the purpose of qualitative analysis. Quantitative analysis was done by spectrophotometry. Chlorophyll a, chlorophyll b and xanthophylls presence was confi rmed by separation of pigments from acetone extract of these plant species. Spectrophotometric analysis of acetone extracts showed these results (given in mg/L): chlorophyll a ,, chlorophyll b , and carrotenoides ,. Data given in mg/g dry substance are: chlorophyll a ,x-, chlorophyll b ,x-, and carrotenoides ,x-. Pigments structure (in mg/L) in species Malva moschata is , for chlorophyll; , for chlorophyll b; and , for carrotenoides. Data given in mg/g are: chlorophyll a x-, chlorophyll b ,x-, and carrotenoides ,x-. Considering that species Malva moschata L. grows on ecologically clear soils as opposed to well-known medicinal species Malva sylvestris L., and considering the production of phytomass, phytochemical structure and pharmacological infl uence it can be considered very medical and be given advantage over this wider spread category.


Introduction
Plant pigments have a very important role in the protection of photosynthetic system, as well as protection of plant organism itself from the negative infl uences of sun radiation.Basic pigments chlorophylls have a special defense role, especially chlorophyll a and b.Accessory pigments carrotenoides are very important in that process ().Nowadays, chlorophylls and carrotenoides have a very important role in prevention and therapy of diff erent diseases of human beings, including immune system, diff erent forms of skin disease, and characteristic antioxidative infl uence is attributed to them.Chlorophylls show antimicrobial and deodorant activity, and they support vitamin B synthesis in organism.() Carrotenoides are protectors of chlorophyll.Th ey have an important role in strengthening of immune system, cardiovascular system, and act preventively against cancer ().Otherwise, plant pigments are water insolubile.Extremely lipofilic chlorophyll appears in most of these plants in two chemicaly related forms as bluish green chlorophyll a and yellow green chlorophyll b.Their ratio is most frequently :.Chlorophylls fluoridate lively red in short wave ultraviolet light ().Besides green chlorophylls, in less quantity, liposolubile orange red and yellow carrotenoides, are regular constituents of plastids in these plants.Th ose are unsaturated hydrocarbons, and in their chemical structure they are identical to tetraterpents.Because of numerous conjugated double bonds, they are orange or red colored lipofilic stains (lipochromes).β-carotene is the most frequent among them.Its chain ends form beta-ion rings, like in many others carrotenoides (α-karoten).Xanthophylls are derivatives of carotene that contain oxygen.Lutein, which corresponds to betacarotene, has one OH group on every ring.It does not act as vitamin, although it oxidizes even in darkness.Protective function is attributed to carrotenoides of photosynthetic apparatus in regard to chlorophyll ().For α and β-carotene, besides pro-vitamin activity, positive eff ects on immune system, cardiovascular disease was proven, and they reduce a risk from some forms of cancer, too ().Types of plant species Malva L. (family Malvaceae) have been used as origins of medical substances from ancient times.But, most of them, like M. sylvestris and M. moschata, are widely used as healthy wild vegetable in preparation of wide dietetics spectrum.Th is activity, in great part, can be attributed to participation of basic and accessory pigments, which appear in genus Malva species in larger quantity ().Both, flower and leaf of M. sylvestris and M. neglecta species are in offi cial usage, and they prescribe by numerous pharmacopoeia.Those are: German Pharmacopoeia (DAB  and DAB ), then Pharmacopoeia Helvetica edition guinta and edition sexta (Bern  and Bern ) and Ph Jug I and II.Th ey are used like infusions for respiratory organs treatment -Species pectorals and Species althaeae, and for skin disease treatment -Species emollients.Concentration of anthocyanins is sufficient for achieving therapeutic effect in making infusion for cough and tonsillitis.Apart from that, French pharmacopoeia (Pharmacopoeia Françoise IX edition, Paris, ) lists Malva alcea, Malva rotundifolia and Malva sylvestris as officinal.
It is important to mention that Malvae sylvestris fl oss is the offi cial drug in European pharmacopoeia (European Pharmacopoeia, fourth edition, Strasbourg, ), Czechoslovakian Pharmacopoeia Ph.Bs II (Pharmacopoeia Bohemoslovenica, edition secunda, Praha, ) and in Polish Pharmacopoeia III (Ph.Pol.)II -Pharmacopoeia Polska III, PZWL Warszawa, ).Basic metabolites in genus Malva specis are: mucus, pectin, starch, sucrose, irridoide glycosides, vitamins, some tannin, mineral substances, fats, anthocyanine, ferments and others (, , ).Since Malva silvestris, in the biggest part of its distribution, mainly grows in nitrifi ed soils, which are frequently loaded with heavy metals, pesticides and other residues, its usage is limited in humane medicine.Th erefore, it is important to fi nd and test species with similar pharmacological-physiological eff ects, and which grow on ecologically favorable soils.One of those species is Malva moschata L.. It fi nds its optimum in clear soils of mountain meadows, and it reaches high biomass production for the balanced collection and application in healthy phytopharmaceuticals production.Th e basic goal of this work is to test qualitative and quantitative contents of plant pigments in Malva silvestris species which grows on ecologically more burdened soils and M. moschata which grows on ecology more favorable soils, and after that estimate their potentials in modern dietetic and phytotherapy.

Chloroplast pigments separating by method of chromatography
One-dimensional rising paper-chromatography was used for determination of pigments.Chromatographic paper Whatman N° was used, and standard technique and reagents.Acetone extract of pigment was used as a sample.Eluent (medicinal gas:petroleum-ether:ac-etone=::) was used for separation.Time of separation was from  to  minutes.Th e distance from the start line corresponds molecular mass values in reverse order.Th us, the closest to the start line will be: -Chlorophyll b Mr =  (green-yellow fraction) -Chlorophyll a Mr =  (light green fraction) -Th en above chlorophyll a in wider area xanthophylls place and their molecule masses range from Mr= for cryptoxanthene to Mr= for violaxanthene.Colors of this fraction are diff erent nuances of yellow.-Carotene fraction Mr= yellow orange color is the closest to the front line ().

Spectrophotometric analysis of chlorophyll and carrotenoides from acetone solution
Quantitative analysis of chlorophyll a and b and carrotenoides is based on their extraction with acetone, followed by spectrophotometric analysis.Measure-ments are done on wavelengths from  nm, because it is absorbance maximum for chlorophyll a, and  nm, because it is absorbance maximum for carrotenoides.Measurements were done on spectrophotometer (Bansch & Lomb, General vertretong und Service -Stelle, Digitana AG , Zurich).Reading of absorbance is on wave length (λ) ,  and  nm, and after that calculations are done: -chlorophyll a = , x A -, x A -chlorophyll b = , x A -, x A -carotene = , x A -, x (a+b) where ,; ,; ,; ,; , are molar absorbance coeffi cients according to Wetsttein for absolute acetone and thickness of quvete of  cm, and A-light absorption.Calculated values are in mg/L, and then we calculate raw substance in mg/g according to the following formula: where: C = content of certain fraction in mg/g of raw substance C = concentration of pigments calculated according to formula (mg/L) V = extract volume R = extract dilution (if it exists) G = sample mass (dry or raw material)  = factor of transforming of g into mg ().

QUALITATIVE ANALYSIS
Chloroplasts pigments separation by one-dimensional paper-chromatography Paper chromatograms were obtained for every tested species.Identifi cation of pigments was done based on characteristic color: chlorophyll b is light green fraction, chlorophyll a is dark green, and xanthophylls -yellow; and also on the bases of their position on chromatogram.Presence of chlorophyll a; chlorophyll b and xanthophylls was proved in analyzed species of Malva genus.
Chromatogram of cultivated carrot -Daucus carota L. var sativa was used as reference (Figures . and .).

Spectrophotometric pigments analysis
Spectrophotometric analysis was done for quantitative analysis of pigments.Water was used as reference.Following results were obtained on the basis of measurements: (Table .).By entering these results in formula for pigments quantity calculation we got the values given in Table .Calculated values are in mg/L of acetone extract, and after that values are calculated according to given formula and they are in mg/g of dry mass for both species (Table  ().Leaves and fl owers of Malva silvestris were used for the treatment of diffi cult diseases of respiratory tract, including malignant diseases of this system.
Decoct of this plant is eff ective agent against middle ear infl ammation (, ,  ).Th is species has proven to be specially healthy food in soldiers feeding in nature, and in the cases of mass catastrophe as well (, , ).
In Malva moschata species, till now, no systematic studies of nutritive and pharmacological active substances was done.But, it is supposed that this species contains similar substances like the wider spread species and that it can be used in the same purpose.Malva silvestris usually grows on nitrifi ed ground, by roads, near traffi c arteries, on soils, which can be loaded with heavy metals, pesticides and other residuum with unfavorable infl uence on human health.Because of that this species from natural soils has or may have limited usage.Plant species Malva moschata, grows far from human settlements, in mountain or sub-mountain zone, by edges of meadows in the zone of coniferous forests and mixed deciduous and conifer forests, on the grounds which are natural and unburden with organic and inorganic pollutants.Th erefore, this species has excellent possibilities in preparation of diff erent nutritive and phytotopharmacs in relation to majority if species of Malva genus and it has big comparative advantage in this respect.

Conclusion
Wild medicinal plant species Malva silvestris and Malva moschata contain diff erent plant pigments or antioxidants.Chlorophyll a, chlorophyll b and xanthophylls presence was verifi ed by separation of pigments from acetone extract of these plant species.Spectrophotometric analysis of acetone extract showed these results (given in mg/L): chlorophyll a ,, chlorophyll b , and carrotenoides ,.Data given in mg/g dry substance are: chlorophyll a ,x - , chlorophyll b ,x - , and carrotenoides ,x - .Pigments content (in mg/L) in category Malva moschata is , for chlorophyll; , for chlorophyll b; and , for carrotenoides.Data given in mg/g are: chlorophyll a x - , chlorophyll b ,x -  , and carrotenoides ,x - .Th ere is high potential for application of plant pigments in the prevention and modern therapy therefore main and accessory plant pigments play important role as antioxidants and as anticancer means.Species Malva moschata has more advantages as an eff ective antioxidant means than other plant species Malva silvestris.
SULEJMAN REDŽIĆ ET AL.: PLANT PIGMENTS (ANTIOXIDANTS) OF MEDICINAL PLANTS MALVA SILVESTRIS L. AND MALVA MOSCHATA L. (MALVACEAE) Testing was done on plant material of wild plants Malva silvestris and Malva moschata.Th ese plants belong to a wider spread family Malvaceae, order Malvales, subclass Dillenidae, class Magnoliopsida and phylum Magnoliophytina().
SULEJMAN REDŽIĆ ET AL.: PLANT PIGMENTS (ANTIOXIDANTS) OF MEDICINAL PLANTS MALVA SILVESTRIS L. AND MALVA MOSCHATA L. (MALVACEAE) METHODS Methods of fi eld work Plant material was collected by hand in dry weather.Healthy specimens were collected in ecologically favorable soils.Surface parts of plant Malva sylvestris were collected in twenty locations in Sarajevo region.Malva moschata samples were collected from twenty locations on Igman mountain at altitude of - m in the summer and they were regularly conserved.Th is region of sampling is marked by coordinates: ° and °:' of east longitude and ° ' and ° of north latitude.Methods of laboratory work Determination of qualitative-quantitative pigments characteristics was done, after extraction, by one-dimensional rising paper-chromatography and by spectrophotometry (, , ).
SULEJMAN REDŽIĆ ET AL.: PLANT PIGMENTS (ANTIOXIDANTS) OF MEDICINAL PLANTS MALVA SILVESTRIS L. AND MALVA MOSCHATA L. (MALVACEAE) .) For most of C plants the ratio of chlorophylls a and b is , to :, while in C plants that ratio is bigger and usually is -:.Chlorophyll a and b ratio is very high and amounts to ,: in Malva silvestris, and that can be attributed to the specifi c structure of chloroplasts and to the fact that analyzed chlorophyll originates from leaf mesophylle.Chlorophyll a and b ratio in mountain plant Malva moschata is ,: and which is a result of high intensity of sun lights.It is a known fact that this ratio is lower in plants that grow in high intensity of light and they contain less chlorophyll a. Analysis of BOSNIAN JOURNAL OF BASIC MEDICALSCIENCES 2005; 5 (2): 53-58