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Identification, characterization, and palynology of high-valued medicinal plants.

1. Introduction

The use of herbal medicine for the treatment of diseases and infections is a safe and traditional therapy [1]. In developing countries, medicinal plants are attaining greater importance in the primary health care of individuals and communities. Medicinal plants form a large group of economically important plants that provide the basic raw materials for indigenous pharmaceuticals [2, 3]. The drugs are quite often adulterated or substituted by other low-quality plant material before harvesting and during handling and storage. Therefore, for attaining the quality assurance of herbal formulation in any system of medicine, emphasis should be given for good harvesting practices (GHP), good laboratory practices (GLP), and good manufacturing practices (GMP) [4]. One of the objectives of the authentication of the crude drug is to check its adulteration, which is useful in promoting the usage of genuine drugs contributing to the health care of human society. Herbal remedies used as medicine is often available in fragmentary form, and identification relies on microscopical rather than macroscopical evaluation. But it is quicker to find out the identity of a crude drug from its anatomy than from its chemistry. For proper identification and standardization of crude drugs, accurate anatomical, and morphological description is necessary, and this description must take into account all the diagnostic features. The authenticity of the crude drug is established by reference reported in the monograph and official pharmacopoeia. The evaluation of crude drug involves a number of methods such as organoleptic, microscopic, chemical, physiological and biological. Organoleptic evaluation includes shape, size, odor, taste, texture, and color of crude drug along with external marking. On the basis of gross morphology, drugs may be grouped as leaves, bark, root, rhizome, and so forth. Microscopic features such as spines, trichomes, spores, and epidermal structures may be examined which are used as diagnostic features in the identification of plant drugs. Taste and odor are extremely valuable tests when carried out carefully. Similarly for microexaminationl characteristics such as starch, calcium oxalate, epidermal cells, trichomes, fibers, and vessel stone cells are examined.

The selection of plant material would be based on availability of certain features which provide taxonomic evidence for their evaluation. The plant characters observed most commonly relate to gross morphology or at least to features readily by external examination with the naked eye. Morphological characters play significant roles for correct identification, characterization, and delimitation of taxa on the basis of available markers. The use of plants as therapeutic agents is paramount in virtually all systems of traditional medicine and especially in Unani medicine. It is necessary to develop a mechanism for quality assurance of plants used as drugs in these medical systems [5]. Quality assurance of herbal medicine seems to be little explored, and that is why standardization and authentication have taken a serious turn. It, therefore, seems obligatory to procure these indigenous drugs and certify their identity by taxonomic and chemical methods.

Pollen morphology is conducted as an aid to the morphological study and a significant tool for modern taxonomist for the delimitation of species. Pollen characters are useful in solving complicated problems of interrelationships between various taxa and assessment of their status in the classification, particularly with reference to the families, subfamilies, tribes, genera, species, and subspecies. Mature pollen grain size, exine sculpturing, and number of pores are the most distinctive features [6, 7]. Palynological data has been useful at generic and specific level [8]. This analysis also helps in qualitative analysis of drug powder and the correct identification of drug. It plays an important role in our daily life as well. Aerobiology has received much attention due to its wide application in allergology, forestry, agriculture, horticulture, archaeology, and plant geography [9, 10].

From the reported literature, it is evident that Achillea millifolium, Acorus calamus, Arnebia nobilis, Fumaria indica, Gymnema sylvestre, Origanum vulgare, Paeonia emodi, Peganum harmala, Psoralea corylifolia, Rauwolfia serpentine, and Vetiveria zizanioides are widely used in herbal medicines. They contain remarkable active constituents responsible for their biological activities, but these plants are not thoroughly investigated so far. Therefore, the main objective of the current study was to standardize these plants in line with the standards of quality detailed in the British Herbal Pharmacopoeia, World Health Organization (WHO) monographs, and other official publications.

2. Material and Methods

Fresh plants were collected from different localities of Khyber Pakhtunkhwa, Pakistan, for taxonomic studies both from wild and cultivated sources. Efforts were made to collect the plants when they were in flowering and fruiting periods for their correct botanical identification. Those plants which were not locally available, different herbaria of Pakistan were consulted for them like Herbarium PCSIR Laboratories Complex, Peshawar (PES), Quaid-i-Azam University herbarium, Pakistan, Museum of Natural History, Islamabad and Peshawar University herbarium.

The plants were identified by consulting Fascicles of Flora of Pakistan, Flora of British India [11], and other available literature. The detailed morphological characteristics of the species were established from fresh samples, herbarium specimen, or by consulting the literatures. In order to ensure a methodical study of the material obtained, herbarium samples were prepared according to the method of Fazal et al. [12] and stored at the Herbarium, PCSIR Laboratories Complex, Peshawar (PES), for future reference.

2.1. Microscopic Studies Crude Drugs. Macro- and microscopic characters of the crude drugs of Achillea millefolium, Acorus calamus, Arnebia nobilis, Fumaria indica, Gymnema sylvestre, Origanum vulgare, Paeonia emodi, Peganum harmala, Psoralea corylifolia, Rauwolfia serpentine, and Vetiveria zizanioides were studied as per Wallis [13] and Trease and Evans [14]. For microscopic characters, the plant material was finely powdered. Several samples were prepared in different mounting media, especially in chloral hydrate solution and water. For this purpose, one or several drops of the medium were placed in the center of a clean slide. A small amount of test material was sprinkled on this fluid. The precleaned cover slip, held with a tweezers, was placed on slide starting from oneedge. This edge should first make contact with themo unting medium; the glass is then lowered into place. This permits air bubble to escape. The slide was then observed under compound microscope, and the characters were recorded and compared with different pharmacopoeias and monographs for confirmation.

2.2. Organoleptic Evaluation/Macroscopic Studies. The organoleptic features of the plant were examined by using sensory organs or by using a magnifying glass. For organoleptic properties a panel of 9 members with different ages ranging from 23 to 30 years with 3 females and 6 males who were familiar with such characteristics was selected for analysis. The organoleptic properties of these medicinal plants were including color, odor, taste, external margins, apices, texture, external and internal marking, fracture, shape and size. Three groups of members were allowed to scale (1-9) these sensory properties. Descriptions for each score were as follows: 1 = good taste, 2 = bitter taste, 3 = moderate taste, 4 = color, 5 = good aroma, 6 = bad aroma, 7 = moderate aroma, 8 = smooth surface and 9 = rough surface. The Organoleptic test was performed in the Medicinal Botanic Centre (MBC) at the Pakistan Council for Scientific and Industrial Research (PCSIR) Labs Complex, Peshawar, Pakistan under suitable light conditions at temperature of 25 [+ or -] 2. The result from three independent groups was analyzed by Statistix 8.1 software for mean values and probability.

2.3. Plant Material for Palynological Study. Flowers and pollens of Achillea millefolium and Paeonia emodi were collected from Kaghan, Fumaria indica from Haripur, Peganum harmala from Lakki Marwat, Origanum vulgare, Psoralea corylifolia, and Rauwolfia serpentina from experimental farm PCSIR Laboratories Complex, Peshawar (Figure 1). The flowers were dried, and few of the plant pollens were freshly isolated and stored in vials containing acetic anhydride.

2.3.1. Methodology. Pollens samples were isolated from their anthers with the help of forceps and needle under a dissecting microscope. The pollens were prepared for scanning electron microscopy by standard method described by Erdtman [15]. The pollen grains, suspended in acetic anhydride, were placed on slide. They were crushed with the help of a glass rod, and the debris was removed with the help of a needle. The drop of acetic anhydride containing the suspended pollen grains was poured on the metal stub. The stub was placed for drying for approximately 30 minutes and then was coated with gold in a sputter chamber (ion-sputter JFC-1100) with coating restricted to 150[degrees]A 16]. Scanning electron microscopy (SEM) examination was performed on Jet microscope JSM-T200. The measurements were based on 10-15 readings for each specimen. Pollen characters like shape, class size aperture, and exine ornamentations were studied. Microphotographs were taken at the Centralized Resource Laboratories, University of Peshawar, Pakistan.

2.4. Statistical Analysis. The data for statistical analysis was collected from three independent experiments. Mean values were obtained by using Statistix software (8.1). Mean values are significantly different when P < 0.05.

3. Results

In the present investigation, the detailed taxonomic, pharmacognostic, and biological studies of eleven medicinal plants were carried out. These plants are Achillea millefolium, Acorus calamus, Arnebia nobilis, Fumaria indica, Gymnema sylvestre, Origanum vulgare, Paeonia emodi, Peganum harmala, Psoralea corylifolia, Rauwolfia serpentine, and Vetiveria zizanioides (Figure 2).

3.1. Morphological, Palynological and Crude Drug Description. The detailed taxonomic and morphological studies of eleven medicinal plants along with their local names, family, distribution, part used, organoleptic characters, macro- and microscopical description of plant material of interest, and pollen grain morphology were carried out. The results have been arranged in alphabetical order by species name as shown in Table 1.

4. Discussion

The detailed taxonomical description is intended for quality assurance at different stages of processing of crude drug for product manufacturing. For this purpose, the morphological studies were carried out through identification of taxonomic markers. For confirmation, these characters were compared and confirmed by consulting Fascicles of Flora of Pakistan Nasir [17-19]; Nazimuddin and Qaiser [20]; Qaiser [21]; Ali [22, 23]; Ghafoor [24]; Hedge [25]; Jafri [26]; Li et al. [27]; and other available literatures.

In present study, pollen grain morphology of Origanum vulgare, Paeonia emodi, Psoralea corylifolia, and Rauwolfia serpentina is reported for the first time. The scanning revealed that pollen grains of these plants are scarbate, foveolate, and tectate with smooth surfaces. The pollen grains of A. millefolium were found to be compositous, that is, echinate and tectate with spiny exine and 3 pores. The pollens of F indica were in line with the results of Perveen and Qaiser [8] namely, they are triporate, oblate-spheroidal, pore more or less circular, operculate, annulate sexine thicker than nexine, fossulate-foveolate, and exine is 2.5 [micro]m thick. The pollens of P harmala are suboblate-subprolate and tricolporate, ornamentation is striate-rugulate and exine 1.2 [micro]m, sculpturing reticulate, consisting of lumina and muri, perforate, pit or hole diameter less than 1 [micro]m 28].

The pharmacognostic studies include the collection/procurement of various parts of these medicinal plants, their identification, standardization, and authentication through various taxonomic markers and macro- and microscopic characters. The evaluation of crude drug which eventually enters the commercial market is obviously of considerable importance. Companies involved in the crude drug sale generally avoid special recommendations on the use of a product. There are no applicable standards of quality for crude drugs, and they are not usually standardized with respect to the concentration of active constituents. For these reasons it is necessary to subject such crude drugs to various standards of quality, purity, and safety, if acceptable consumer usage is to be achieved.

In the present study, crude drug study of leaf of G. sylvestre, aerial parts of O. vulgare, tubers of P emodi, and seeds of P. harmala was reported for the first time. Previously Hyde [29] performed the crude drug study of aerial parts of A. millefolium indicating that fragments of leaves, small flower heads, and stem pieces are visible, while microscopic characters include trichomes long, uniseriate, with pointed terminal cell, glandular trichomes compositous, with about 4 pairs of cells. Leaf epidermal cells are elongated with sinuous anticlinal walls, while stomata are anomocytic. The unpeeled drug of A. calamus is covered with a thin brown cork. The peeled drug is creamy yellow in color with fewer root scars. The section has a large stele separated by a yellowish line from the cortex. Khatoon et al. [30] and Arora et al. [31] studied the root and root bark of A. nobilis, which indicated that they are furrowed and they are too deep that cylindrical form of root is lost and irregular segments are formed.

The authentication of the crude drug material is necessary for many reasons. Firstly, a new source of drug material must be specified if it is to be used commercially. Secondly, people using plants for medicine must know the source of their material; otherwise they may not be able to gain their objectives, with failure to duplicate pharmacological results obtained with extracts from subsequent lots of material. This is a real problem in cases where the source of the material is not authenticated. Thirdly, when a drug has become an article of commerce, it becomes adulterated with unsuitable ingredients; here, microscopy can be used to give an indication of purity.

Authors' Contribution

Hina Fazal: study design, collection, identification and crude drug studies. Nisar Ahmad: Palynological studies. Bilal Haider Abbasi: Revised the whole manuscript.

http://dx.doi.org/10.1155/2013/283484

Acknowledgment

The authors are thankful to the Higher Education Commission (HEC) of Pakistan for the support to complete this research work.

References

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Hina Fazal, (1) Nisar Ahmad, (2) and Bilal Haider Abbasi (2)

(1) Pakistan Council of Scientific and Industrial Research (PCSIR) Laboratories Complex, Peshawar 25120, Pakistan

(2) Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan

Correspondence should be addressed to Nisar Ahmad; nisarbiotech@gmail.com

Received 10 April 2013; Accepted 14 May 2013

Academic Editors: U. Feller, A. Ferrante, and H. Verhoeven

TABLE 1

(1) Botanical name            Achillea millefolium L.

Local name                    Baranjasaf

Family                        Asteraceae

Distribution in Pakistan      Swat, Hazara, Kaghan, and Peshawar

Plant material of interest    Aerial parts

Organoleptic                  Fragrant odour, taste is bitter

Macroscopic characters        Green fragments of the pinnate leaves;
                              the downy segments are linear and finely
                              pointed. Many small flower heads are
                              surrounded by long felted hairs. Stem
                              pieces are ribbed, rounded, pithy,
                              downy, and green to violet red.

Microscopical characters      Trichomes are long, uniseriate, with
                              pointed terminal cell. Glandular
                              trichomes are compositous, with about 4
                              pairs of cells. Leaf epidermal cells are
                              elongated with sinuous anticlinal walls.
                              Stomata anomocytic. Calcium oxalate is
                              absent.

Pollen grain morphology       Pollen grains are compositous, that is,
                              echinate and tectate about 30 [micro]m
                              in diameter, with a coarsely spiny exine
                              and 3 pores; spines are more than 1
                              [micro]m (Figure 3).

Traditional uses              Used to relieve fever, delivery pain,
                              and diarrhea and is hepatoprotective.

(2) Botanical name            Acorus calamus L.

Local name                    Bach

Family                        Araceae

Distribution in Pakistan      Chitral, Peshawar, Kashmir, Rawalpindi,
                              and Poonch

Plant material of interest    Rhizome.

Organoleptic                  Pungent, taste is bitter

Macroscopic characters        Rhizome is covered by brown cork and
                              deeply wrinkled longitudinally. It bears
                              triangular leaf scars and hair-like
                              fibers on the upper surface. The lower
                              surface has small root scars. The peeled
                              drug is cream yellow in color. Fracture
                              is sharp producing a granular, white,
                              and spongy surface.

Microscopical characters      Powder is yellowish white, consisting of
                              oval-shaped parenchymatous cells.
                              Yellow-brown oleoresin, and starch
                              grains are present. A few xylems are
                              present.

Traditional uses              Used to cure diabetes and high blood
                              pressure.

(3) Botanical name            Arnebia nobilis Reichb. f

Local name                    Ratanjot

Family                        Boraginaceae

Distribution                  Endemic species of Afghanistan

Plant material of interest    Root/root bark

Organoleptic                  Smell aromatic, tasteless

Macroscopic characters        Root color is purple brown, twisted,
                              deeply furrowed, and irregular. Length
                              of segments is 5-10 cm and 3-6 cm in
                              diameter. It is covered with papery
                              layers of the same color.

Microscopical characters      Outermost xylem with broad vessels and
                              innermost with groups of radially
                              arranged narrow vessels while the middle
                              region is occupied by alternate rings of
                              clusters of broad and narrow vessels and
                              presence of pith.

Traditional uses              Used to cure jaundice, kidney pain,
                              wound healing, and diarrhea.

(4) Botanical name            Fumaria indica (Hausskn.)

Local name                    Shahtra

Family                        Fumariaceae

Distribution in Pakistan      Swat, Hazara, Peshawar, Rawalpindi,
                              Lahore, Ziarat, Karachi, and Hyderabad

Plant material of interest    Aerial parts

Organoleptic                  Taste is slightly bitter

Macroscopic characters        Fragments of green glabrous leaves.
                              Flower petals are shrunken and red-
                              violet. Fruits are flattened about 2 mm
                              in diameter and green containing one
                              seed. Stem pieces are light green or
                              brown in color, ribbed, and hollow.

Microscopical characters      Leaf epidermal cells with anomocytic
                              stomata. Calcium oxalate is absent.
                              Pollen grains spherical with a pitted
                              exine.

Pollen grain morphology       Triporate, Oblate spheroidal, pore more
                              or less circular, operculate, fossulate-
                              foveolate, and exine 2.5 [micro]m thick
                              (Figure 3).

Traditional uses              Juice of the plant is given in fever,
                              removing worms from abdomen, diabetes,
                              bladder infection, piles, and allergy.
                              Also used in the treatment of goiter, as
                              diuretic and antihelmintic.

(5) Botanical name            Gymnema sylvestre Retzius Spreng.

Local name                    Gurmar

Family                        Asclepiadaceae

Plant material of interest    Leaf

Organoleptic                  Odourless, taste is slightly bitter

                              Stems and leaves hairy stem are hollow,
                              ridged, golden brown from the outer side
                              thin bark and

Macroscopic characters        green and from the inside. Petiole 3-12
                              mm; leaf obovate to ovate, 3-5.5 cm,
                              thick papery, adaxially pubescent to
                              glabrous, abaxially, and glabrous.

Traditional uses              Leaf juice is used in eye diseases and
                              snakebites. It is also used to remove
                              the effect of wine and other narcotic
                              drugs.

(6) Botanical name            Origanum vulgare L.

Local name                    Satar

Family                        Lamiaceae

Distribution in Pakistan      Chitral, Swat, Hazara, Malakand,
                              Kashmir, and Rawalpindi

Plant material of interest    Aerial parts

Organoleptic                  Aromatic, taste is like trachyspermum.

                              Stems are branched, thinly to densely
                              pilose with spreading hairs, glabrous,
                              purplish, or green.

Macroscopic characters        Leaves are simple, entire, ovate, 5-30
                              mm, gland-dotted, apex acute, or obtuse,
                              with scattered hairs, petiolate.

Microscopical characters      Trichomes, green oval cells, epidermal
                              and parenchymatous cell, and pollen
                              grains are present.

Pollen grain morphology       Profusely reticulate, lumina, and muri
                              are present.

Traditional uses              Used as fresh fodder and for washing
                              utensils, as diuretic, and is also used
                              in toothache and earache.

(7) Botanical name            Paeonia emodi Wall. ex Royle

Local name                    Mamaikh

Family                        Paeoniaceae

Distribution in Pakistan      Chitral, Swat, Hazara, and Ziarat

Plant material of interest    Tubers

Organoleptic                  Aromatic, taste is bitter.

Macroscopic characters        Tubers are cylindrical, straight, or
                              curved, 5-9 cm in length, 1.2-2.2 mm in
                              diameter, externally dark brown with
                              thick bark, internally light brown, bark
                              1-2 mm in diameter; ridges and furrows
                              are present; few furrows are too deep.

Microscopical characters      Powder is yellowish to pink in color,
                              parenchymatous cells are visible, starch
                              granules are abundant, and calcium
                              oxalate crystals, oil globules, and
                              reticulate vessels are present.

Pollen grain morphology       Sculpturing scarbate with smooth
                              surface.

Traditional uses              Its powder is used in dysentery and
                              chronic diarrhea, applied externally for
                              rheumatism. Rhizome is given to children
                              to bite during teething, used in
                              backbone ache, as tonic, emetic, and
                              cathartic.

(8) Botanical name            Peganum harmala L.

Local name                    Harmal

Family                        Zygophyllaceae

Distribution in Pakistan      Skardu, D.I.Khan, Peshawar, Hassan
                              Abdal, Quetta, Sibi, Zhob, Kalat,
                              Nakran, and Lakki Marwat

Plant material of interest    Seeds

Organoleptic                  Smell characteristic, taste is bitter

Macroscopic characters        Seeds are blackish brown, triangular,
                              2-3 mm long, and 1-1.5 mm in diameter.

Microscopical characters      Seed powder is brown, sclerenchymatous
                              cells of testa parenchyma cells, and oil
                              globules are present.

Pollen grains morphology      Pollens are suboblate-subprolate and
                              tricolporate; sculpturing is striate-
                              rugulate, reticulate, consisting of
                              lumina and muri, perforate, pit diameter
                              less than 1 [micro]m.

Traditional uses              Leaves are smoked to repel evil sight
                              and mosquitoes and remove bad smells. It
                              is a brain tonic, blood purifier, and
                              remedy for tapeworm and is used in ear
                              diseases.

(9) Botanical name            Psoralea corylifolia L.

Local name                    Babchi

Family                        Papilionaceae

Distribution in Pakistan      Peshawar, Rawalpindi

Plant material of interest    Seeds

Organoleptic                  Odour aromatic similar to elemi, taste
                              is bitter

Macroscopic characters        Seeds are greyish black, flattened,
                              dotted oblong, dotted, reniform, and
                              rough. 3/5 mm in length and 2/3 mm in
                              diameter. Black seed coat/testa.

Microscopical characters      Seed powder is greyish black.
                              Sclerenchymatous cells of testa,
                              parenchymatous cells of calyx, and
                              cotyledon are present. Epidermal cells
                              and oil cells are also visible.

Pollen grain morphology       Scarbate and tectate with smooth
                              surface.

Traditional uses              Used as blood purifier, antihelmintic,
                              and expulsion of gases and piles.

(10) Botanical name           Rauwolfia serpentina L.

Local name                    Choti Chandan, Chota Chand

Family                        Apocynaceae

Distribution in Pakistan      Peshawar, Karachi

Plant material of interest    Root

Organoleptic                  Odour is indistinct, earthy, reminiscent
                              of stored white potatoes; taste is
                              bitter.

Macroscopic characters        The root segments are 3-20 mm in
                              diameter, subcylindrical to tapering.
                              Externally light brown to greyish
                              yellow, rough or wrinkled
                              longitudinally, and smooth to the touch.
                              Fracture is short but irregular.

Microscopical characters      Phloem parenchyma and calcium oxalate
                              crystals are present, xylem parenchyma,
                              wood fibres, and tracheids are present.
                              Powder is brownish/reddish grey, and
                              starch grains are present.

Pollen grain morphology       Foveolate with pits on the surface and
                              scabrate.

Traditional uses              Used in high blood pressure,
                              sleeplessness, and in scorpion bite.

(11) Botanical name           Vetiveria zizanioides L.

Local name                    Khas

Family                        Poaceae

Distribution in Pakistan      Rawalpindi

Plant material of interest    Whole plant

Organoleptic                  Aromatic, taste characteristics

Macroscopic characters        Culms are present, leaf-blades, panicle
                              contracted, raceme 5-75 cm long. Sessile
                              spikelet, callus glabrous, glume
                              spinulose, and awnless.

Microscopical characters      Trichomes and oil cells are present, and
                              thin fibres, irregular cell structures,
                              and yellow-brown cells are present.

Traditional uses              Used to cure fever, inflammation, and
                              irritability of stomach and for aromatic
                              properties.
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Article Details
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Title Annotation:Research Article
Author:Fazal, Hina; Ahmad, Nisar; Abbasi, Bilal Haider
Publication:The Scientific World Journal
Article Type:Report
Date:Jan 1, 2013
Words:4314
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