Aspects of pharmacognostic evaluation of the herbs for a traditional antimalarial (Maloff-HB) powdered formulation in Ogbomoso, Nigeria

Maloff-HB is a documented traditional oral powdered herbal drug in Ogbomoso, Nigeria whose botanical constituents, ascorbic acid and mineral elements composition have been quantiﬁed but there is inadequate information on the pharmacognostic properties of the nine herbal materials for its formulation. This study therefore sought to elucidate the bark and wood anatomy of eight of the nine herbs used in the formulation, and identify the diagnostic markers for their authentication. The conventional anatomical techniques of transverse sectioning (TS) and tissue maceration (TIM) were used to draw out 21 characteristics from the root barks of the eight woody species studied. In addition, 41 features of the wood in the roots of three of the species were drawn using TS, transverse longitudinal sectioning (TLS), radial longitudinal sectioning (RLS) and TIM. Following staining, mounting and microscopic examinations, the observed qualitative and quantitative features were taxonomically described in accordance with the provisions of International Association of Wood Anatomists, and their diagnostic values among the medicinal herbs were explored. Bark anatomical markers that are clearly diagnostic of the species studied included features of the secondary cortex, phloem rays, axial parenchyma, sclereids and resin ducts. In the wood, these included features of the vessels in the TS and variable ray characteristics in the TLS. The two artiﬁcial keys obtained from discontinuities in qualitative and quantitative features observed in the barks and the woods are useful tools for reliable identiﬁcation of the herbal materials studied.


Introduction
Nature has been a source of medicinal agents for thousands of years. Plants in particular have been identified and used for therapeutic purpose since the start of human history, and an impressive number of modern drugs have been isolated from them and other natural sources. 1,2 Plantbased medicines have continued to play an essential role in health care, with about 80% of the world's human population relying mainly on traditional medicine for their primary health care. 3 Extensive use of herbal medicine calls for accurate and efficient means of authenticating herbal materials which are the ingredients of the drugs. This is necessary for two main reasons. Firstly, the growing market for herbal drugs worldwide has endangered many international trading companies and generated an increase in herbs of questionable quality. Secondly, herbal drugs are often taken as combinations which generate unique problems of authentication such as determining if there is species confusion of different herbs sharing one name or one herb using different names, and if correct herbs have been included in a particular proprietary medicine. 4 Pharmacognosy is the science that deals with medicinal products of plant, animal, microbes or mineral origin in their crude or unprocessed state. 5 The subject matter of systematic pharmacognosy involves macroscopic and microscopic examination of herbal materials in order to recognize and identify unknown drugs. Microscopic evaluation of plant parts is particularly useful for assuring the identity of the material and as an initial screening test for impurities or adulterants. 6 These efforts which ensure botanical authentication are important aspects of herbal drug standardization. 7 Many poisoning incidents caused by misuse or confusion of herbal medicines have raised international concerns, and hence the necessity for authentication of the constituent herbs towards their safe and effective use. 4 Therefore, compilation of pharmacognostic parameters based on macroscopic observations, microscopic evaluation and physicochemical analyses of herbal materials is the first step towards establishing the identity and the degree of purity of crude drugs from wild sources. 8 Malaria fever is a major public health problem in Nigeria 9 and is responsible for over 70% of out-patient hospital visitation with a great toll on productivity. 10 The constant evolution of the malaria parasite has rendered the cheapest and most widely available antimalarial treatments ineffective, more so with the recent reports about the increasing resistance of Plasmodium falciparum to artemisin-based compounds. 11 Nowadays, anti-malarial drug resistance has become one of the most important challenges to malaria control efforts. 12,13 If we should rely on herbal drugs as saving grace in the management of this dreaded disease, then concerted efforts towards establishing an acceptable authentication of the herbal material are a must do. This is an area of focus in this study.
This study has laid emphasis on enumeration of bark and wood anatomical features for standardization and authentication of medicinal plant parts used for Maloff-HB, a documented oral powdered antimalarial herbal formulation in Ogbomoso, Nigeria. Although, nine herbal materials are used for Maloff-HB 13 , one of them i.e. the vines of Cassytha filiformis L. is non-woody; so the study focused on the other eight. The objectives of this study were to elucidate the bark and wood anatomical composition of the herbal materials used for producing Maloff-HB in order to come up with microscopic markers for diagnosing its constituent herbs; and to generate bark and wood anatomy-based diagnostic keys which can be useful as handy tools for authenticating the identities of the constituents of the herbal formulation. These are with a view to preventing misidentification and misrepresentation of the herbal materials 14 , and the attendant consequences. [15][16][17][18]

Collection and preparation of herbal materials
Eight of the nine herbal materials for the formulation of Maloff-HB powdered drug as listed in Table 1 were collected from medicinal herb sellers in Ogbomoso Nigeria, located around latitude 8.1333N and longitude 4.2567E. The materials were authenticated based on consultations with some experts in the Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso. Where applicable, further authentication was performed with the assistance of experienced traditional medicine practitioners from within and outside the town. The barks were prepared for sectioning 19 by cutting them into pieces of about 2cm × 2cm which were rehydrated by boiling in water for about 10 minutes. The re-hydrated barks were fixed in Formal-Acetic-Alcohol (FAA) prepared in the v/v ratio 5:5:50:40 of formaldehyde, glacial acetic acid, 95% ethanol and distilled water respectively. 20 Wood samples were collected as short segments of roots, each of which was cut as discs of about 1cm to 2cm thickness after debarking; they were similarly rehydrated and fixed in FAA, ready for sectioning. 21

Treatment of sections of barks and wood for microscopic observation
Each thin section of barks and woods were rinsed clean of FAA in several changes of water on a microscope slide. Sections of root and stem barks were stained in 1% ethanolic safranin for 10 minutes. Rinsing was done with water until no excess stain bled out of the sections. This was followed by counter-staining with fast green, and another round of destaining with water. The sections were then dehydrated in 30%, 50%, 70%, 90% and absolute ethanol for 2 minutes each. Staining of wood sections was also done with 1% ethanolic safranin for 10 minutes before de-staining, counter-staining and dehydration as earlier described. After dehydration, each tissue section, bark or wood was cleared in pure xylene for 20 minutes 22 and mounting was done in few drops of Canada balsam.

Tissue maceration
Wood tissue maceration was carried out using a modified form of Jeffrey's method i.e. by boiling a small block of re-hydrated wood for 5 to 10 minutes in about 5ml of concentrated nitric acid to which a few crystals of potassium chlorate had been added. 23 Maceration of the barks was done with cold treatments i.e addition of concentrated nitric acid and crystals of potassium chlorate without application of heat. In doing this, the scaly part or rhytidome was first peeled off by means of a knife, leaving only the secondary phloem part as the predominant tissue for subsequent treatments. The tissues were left to stand in this solvent overnight (i.e. between 10 and 12 hours) to soften. 24 For both the woods and the barks, softened tissue in concentrated nitric acid was rinsed in several changes of water and transferred onto a microscope slide in a few drops of water. With the bottom of a pair of forceps, the softened tissue was macerated by tapping it gently for some minutes and then teased out into its various components on the microscope slide by means of the pointed ends of the forceps. 25 Wood and bark macerates were stained with safranin for 10 minutes and temporary mounting was carried out in a few drops of glycerin.

Microscopic examination and data collection
Four prepared slides from each of the TS and macerated barks as well as the TS, TLS, RLS and macerated wood tissue were examined using an Olympus binocular microscope CH20i Model at 100X and 400X magnifications. 26 In the TS of the barks, observations were focused on cell types, morphology and arrangement of cork cells in the outer bark and the inner secondary phloem. In wood sections, attention was focused on such structures as type, morphology and cellular composition of rays in the TLS, shape, occurrence and distribution of vessels, and types of axial parenchyma in the TS; presence or absence of tylose in the vessels, and cellular composition of the rays in the RLS. 27 In the macerated bark and wood tissues, attention was focused mainly on fiber morphology, but observations were also recorded on types of vessel members and sclereids. These observations were recorded in accordance with the descriptions of the international association of wood anatomists 28 , and in drawings or photographs by means of Bresser microscope equipped with Photomizer SE Microcular camera attachment, 051012-VGA Model connected to a laptop computer. Some cell and tissue dimensions in wood sections such as height and width of rays in TLS, diameter and lumen width of vessels in TS, fiber length and width in macerated samples etc. were determined in micrometers using a calibrated eye piece micrometer accessory inserted in the ocular tube. 29 Making use of a calibrated ocular grid attached to the microscope, wood tissue composition in terms of density of fibers, vessels, and axial parenchyma per mm 2 area were calculated in the TS; and of wood rays in TLS. The relative abundance of these four tissue types were determined for each species by summing up the recorded mean values of their densities in a square millimeter area and computing the percentage of each in relation to the total.
Frequency of vessels with tylose was obtained from wood TS by randomly viewing 50 fields of microscope and noting the occurrence of vessel tylose in each with a tally. The frequency was then computed as a percentage of those fields of view in which tylose was present in relation to the total number of observed fields, i.e. 50. The frequency of vessels with varying shapes in TS (i.e. round or oval) was computed by first scoring each of the 50 microscope fields of view as present or absent with regards to each vessel shape. Thereafter, the number of views in which each shape was present was calculated as a percentage of the total of presence for the two.
The inner bark anatomical features that were quantified in relative percentage as appropriate included fibers, axial parenchyma, sieve tubes, rays and sclereids following the procedure earlier explained. 30 Twenty-one bark anatomical characters, consisting of nine qualitative and 12 quantitative (in replicates of 4 for percent tissue composition and of 10 for others), and forty-one wood anatomical characters, consisting of 19 qualitative and 22 quantitative (in replicates of 30) were compiled, making a total of 62.   these medicinal herbs were equally subjected to one way ANOVA, while the means in both cases were separated using multiple Duncan range test at α= 0.05. 31

Results
The results of the study are shown in Figure 1-8, and Tables  2-8 4. Discussion

Ethnopharmacological applications of the plant materials studied
Barks and wood of trees are very complex in structure and have the potential of storing many primary and secondary metabolites, some of which are useful in the drug industry. 32 For this reason, these items fall in the category of crude drugs, whose complex structure is also useful for their botanical identification to ensure their quality and purity. 33 Table 1 gives a highlight of the herbal materials (barks and/or woods) of the eight species studied as components of Maloff-HB, a traditional oral powdered drug for malaria fever in Nigeria. Researchers in other parts of the world have also listed some of these items as portions of herbal remedies for a wide range of health conditions: e.g. Mangifera indica. 34,35 , and Sarcocephalus latifolius. 36 While stem and root barks are frequently sold and used directly as medicinal herbs, wood is seldom so used unless as chewing sticks. 37,38 More commonly however, wood is medicinally used as whole roots or stem twigs i.e. in combination with its bark. 39 This account is not only confirmatory of wide application of the plant species studied as medicinal herbs, it is also a pointer to the necessity to ensure their identity and purity. The qualitative and quantitative bark and wood anatomical data obtained from this study are potentially useful to establish specific and higher taxonomic categories among these herbal materials. At the least, the barks of the species studied on the one hand, and their woods on the other hand can easily be distinguished from their adulterants.

The diagnostic value of bark anatomy in the species studied
The eight plant species studied can be separated using their bark anatomy characteristics. Four of the species namely, Alstonia boonei, Enantia chlorantha, Parquetina nigrescens and Sarcocephalus latifolius have rays in their inner barks, while rays are not observable in the other four species. The first three of the four species mentioned here have secondary cortex /phelloderm in their inner barks but the fourth, i.e. S. latifolius lacks the tissue. The three species are however, distinguishable in that the number of layers and thickness of phelloderm in the three showed significant variation at p< 0.001 (Table 4) Moreover, E. chlorantha is the only species among those studied in which cork cambium was visible; P. nigrescens lacks sclereids/stone cells, while A. boonei and E. chlorantha, both of which have sclereids in them differ in that the former possesses the brachyand macro-sclereids, with significantly higher percentage of these strengthening cells, but the latter, only macrosclereids, with significantly lower percentage by volume at p <0.001 (Table 4)The other four species examined, which lack the rays, also do not possess phelloderm; the species are Calliandra haematocephala, Mangifera indica, Okoubaka aubrevellei and Pterocarpus osun. The last three of these species all have sclereids and resin ducts but the first, i.e. C. haematocephala does not. These three species can however be distinguished by the fact that only macrosclereids are observable in P. osun, while both brachy-and macro-sclereids are found in M. indica and O. aubrevellei. Furthermore, these two species can be separated on account of their significantly wide variation (at p <0.001) in relative percentage composition by volume of fibers, axial parenchyma, sieve tubes and sclereids (Table 4).
Ohemu et al. 40 conducted organoleptic, macroscopic and microscopic evaluation of stem bark of E. chlorantha. Their observations on powdered bark revealed the presence of brachy-sclereids, cork cells, bundles of phloem fibers, polygon-shaped parenchyma cells and numerous prismshaped calcium oxalate crystals. The results of the present study are in part, similar to those of Ohemu et al. 40 in terms of presence of sclereids in the bark of E. chlorantha. However, only macro-sclereids were those observed, and no crystals were found. Alam and Saqib 41 carried out a microscopic examination of powdered bark of Gaultheria trichophylla to reveal useful diagnostic features of the species. Additionally, these researchers examined the fluorescence properties of the powdered bark in different reagents, and conducted organoleptic evaluation of the whole and powdered barks and generated information usable for diagnosing the medicinal plant. In consonance with the observations made from the current study, Kotina et al. 42 examined the anatomy of the leaf and bark of Warburgia salutaris, and reported combinations of anatomical characters to be of diagnostic value for this important medicinal plant from South Africa. Such characters observed by these authors in the bark included scattered large druses and numerous small ones, secretory cells, thin-walled fiber-like sclereids, and sieve tubes having compound sieve plates on the lateral and oblique cross walls.
Babu et al. 33 examined the macroscopic, microscopic and HPTLC profiles of the barks of four species of Ficus sold as medicinal herbs in Indian markets. Similar to the exercise carried out in this study, these authors described and used the qualitative and quantitative anatomical features of the outer and inner barks to distinguish between the four species of Ficus studied namely, F. racemosa, F. virens, F. religiosa and F. benghalensis. Kumar et al. 43 also evaluated the pharmacognostic characters of the root bark of Holoptelea integrifolia, an important medicinal plant in India. They, not only found the qualitative and quantitative anatomical features of the bark distinctive enough to identify and decide the authenticity of this drug, but in addition, recommended the inclusion of these diagnostic features as microscopic standards in Indian herbal pharmacopeia.
The argument so far has pointed to two important facts. Firstly, that all the plant species examined for their bark anatomical characteristics in this study are well-known for their diverse medicinal value worldwide, and secondly, that the complex nature of stem/root bark anatomy lends itself to applications in pharmacognostic studies of herbal materials. While empirical data on bark anatomy have been successfully employed to diagnose a long list of medicinal plants (e.g. 8 ; 33 ; 42 ; and 44 ), available literature appears to be deficient in pharmacognostic studies of these medicinal plants from Ogbomoso Nigeria from the point of view of their bark anatomy. The dearth of information from this direction is yet another justification for this study. The results of bark anatomy obtained in this study are diagnostic enough to establish the species identities of the eight medicinal herbs studied. Qualitative and quantitative information, especially on the elements of the secondary phloem can be employed as suitable quality control measures to ensure purity, safety and efficacy of these drugs. Figure 7 is confirmatory of the diagnostic value of these features, being a key for unambiguous authentication of these important medicinal herbs.

The diagnostic value of wood anatomy in the medicinal herbs studied
The results of this study have clearly resolved the three medicinal plant species studied as follows: In Sarcocephalus latifolius, the vessels occur as only solitary units; rays in TLS are all heterocellular with bi-convex and constricted (i.e. dumb-bell) shapes. Additionally, no linear rays, while the wood fibers are significantly (P<0.001) longer (>1000µm) than in the other two species i.e. Calliandra haematocephala and Parquentina nigrescens ( Table 7). In these two species however, vessels, apart from occurring in solitary units in TS, are also found in groups/radial chains; both homocellular and heterocellular rays occur in the TLS, whose general shapes include, but not limited to monoconvex and linear; and mean fiber length is significantly (P <0.001) shorter (<900µm). These two species can however, be diagnosed in that C. haematocephala has significantly narrower vessels (mean diameter of about 60µm), and less predominant (below 40%) in linear ray composition. These are against the mean vessel diameter of 200µm or more (Table 7)and more predominant (>60%) linear rays (Table 8) in P. nigrescens. In addition, there are bi-convex shaped rays, but no vessel tylose in C. haematocephala, while the reverse is the case in P. nigrescens. Pandey 45 stated that among others, qualitative and quantitative features of wood vessels, parenchyma, rays and fibers are reliable diagnostic and phylogenetic indices. The findings from the present study are in consonance with Pandey's position and have further confirmed that these features have the potential for diagnosing the three herbal materials studied, which may otherwise be impossible using only morphological characterization. 46 In the eight woody species of Hypericum studied by Peronne et al. 47 , the number and mean diameter of vessels showed interspecific differences. So also the difficulty posed by the identification of Cola acuminata and C. nitida when not in fruit was reported by Jensen et al. 48 to be resolvable using wood anatomical features. In particular, wood fiber composition, types and amount of crystals have proved very strongly of diagnostic value in the classification and delimitation in the Cola species studied. Some of the features reported by these authors to be useful diagnostic markers were also found to be so useful in the current study.
Marques et al. 49 reported homogeneous wood anatomical features in the genus Psychotria but the statistical analyses based on qualitative and quantitative features allowed the separation of the nine species they studied as distinct taxa. According to these authors, the woods of Psychotria were characterized by slightly distinct growth rings, diffuse porosity, solitary vessels or in radial multiples of 2-6 or clusters of 3-5 vessel elements with terminal and lateral simple perforation plates, septate fiber-tracheids and rare axial parenchyma. In the present study, similar observations were made on the woods of the medicinal plants examined, with a number of the features found to be diagnostic after showing significant statistical differences.
So far, two main facts can be pointed out from the three medicinal plant species examined for their wood anatomical charactersistics in this study. The first is that these species are well-known for their diverse medicinal value, and secondly, that the complex nature of wood anatomy can be reliably used in pharmacognostic studies of these herbal materials. While empirical data on wood anatomy have been successfully used to diagnose many medicinal and non-medicinal plants (e.g. [47][48][49][50], available literature seems to be poor in pharmacognostic studies of these three medicinal plants from Ogbomoso Nigeria from the point of view of their wood anatomy. By and large, the results of this study are confirmatory of the view that qualitative and quantitative characteristics of wood anatomy are reliable in pharmacognostic evaluation of medicinal plants. In particular, they are diagnostic of the three plant species examined as revealed by the entries in Figure 8,

Applicability of the diagnostic keys for authentication of the medicinal herbs studied
In order to identify any of the medicinal herbs studied , the user enters each of the keys in Figure 7 and 8 at the center and proceeds centrifugally (toward the circumference) by selecting the characters applicable to the unknown specimen at the successive rings of compartments. This process progressively narrows down on the choice of the possible identities (i.e. sectors) of the unknown specimen until only one choice is achieved, which represents its identity. These keys, which follow the single-access format 51 can also be used to authenticate any of the medicinal herbs suspected or supplied under a given name. As an illustration, if a user in applying the key in Figure 7 suspects the identity of a plant to be Enantia chlorantha, confirmation is done by evaluating the specimen based on characters 1, 2, and 5, and if desirable, 8 and 14 in addition. Thus, if given a key, and the assurance that a suspected taxon is included in that key, the first step is to locate the position of the taxon in the key and then work on that key along the established route of identifying the taxon, paying particular attention to only those characters leading to the taxon name, and ensuring that all such, rather than most of the statements are in agreement with the observable features of the specimen in the hand. Going by the easy mode of navigation, and the possibility for confirmation of suspected identity, it is clear that the functionality attributes of the keys generated from this study outweigh those of the dichotomous key, the most widely used tool for plant identification. 52

Conclusion
The salient bark anatomical characteristics which were diagnostic of the eight plant species studied included occurrence of secondary cortex (or phelloderm); presence/absence, types, distribution and relative abundance (%) of sclereids; presence/absence, cellular composition and shape of phloem rays; arrangement, types and abundance (%) of axial parenchyma; and presence/absence of resin ducts. On the other hand, the notable wood anatomical features that are clearly diagnostic of the three species studied were in the TS (namely: occurrence and diameter of vessels, and abundance of wood parenchyma); and the TLS (namely: cellular composition, width, height, thickness and morphology of wood rays). Discontinuities in qualitative and quantitative features of the bark and wood anatomical observations yielded two taxonomic keys whose clarity, simplicity and diagnostic value are evident in avoiding misidentification of these medicinal herbs.

Source of Funding
None.

Conflict of Interest
None.