Identification, Characterization, and Palynology of High-Valued Medicinal Plants

High-valued medicinal plants Achillea millefolium, Acorus calamus, Arnebia nobilis, Fumaria indica, Gymnema sylvestre, Origanum vulgare, Paeonia emodi, Peganum harmala, Psoralea corylifolia, Rauwolfia serpentina, and Vetiveria zizanioides were identified with the help of taxonomical markers and investigated for characterization and palynological studies. These parameters are used to analyze their quality, safety, and standardization for their safe use. Botanical description and crude drug description is intended for their quality assurance at the time of collection, commerce stages, manufacturing, and production. For this purpose the detailed morphology was studied and compared with the Flora of Pakistan and other available literatures. Here we reported the pollen grain morphology of Origanum vulgare, Paeonia emodi, Psoralea corylifolia, and Rauwolfia serpentina for the first time. Similarly the crude drug study of Gymnema sylvestre (leaf), Origanum vulgare (aerial parts), Paeonia emodi (tubers), and Peganum harmala (seeds) was also carried out for the first time.


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 2 The Scientific World Journal 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.

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.

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 one edge. This edge should first make contact with the mounting 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.

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±2. The result from three independent groups was analyzed by Statistix 8.1 software for mean values and probability.

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 ∘ 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.

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 < 0.05.

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.

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][18][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 m thick. The pollens of P. harmala are suboblate-subprolate and tricolporate, ornamentation is striate-rugulate and exine 1.2 m, sculpturing reticulate, consisting of lumina and muri, perforate, pit or hole diameter less than 1 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 Pollen grain morphology Pollen grains are compositous, that is, echinate and tectate about 30 m in diameter, with a coarsely spiny exine and 3 pores; spines are more than 1 m (Figure 3).

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

Local name Bach
Family Araceae 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.  The Scientific World Journal Microscopical characters Leaf epidermal cells with anomocytic stomata. Calcium oxalate is absent. Pollen grains spherical with a pitted exine.

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.

Local name Gurmar
Family Asclepiadaceae

Plant material of interest Leaf
Organoleptic Odourless, taste is slightly bitter

Macroscopic characters
Stems and leaves hairy stem are hollow, ridged, golden brown from the outer side thin bark and 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.  Traditional uses Used to cure fever, inflammation, and irritability of stomach and for aromatic properties.
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  The Scientific World Journal 9 become an article of commerce, it becomes adulterated with unsuitable ingredients; here, microscopy can be used to give an indication of purity.