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Ethnopharmacological relevance: Ben-Cha-Lo-Ka-Wi-Chian is one of a thai traditional remedy that has been used as an antipyretic drug and comprising from five root species in an equal part by weights namely Capparis micracantha DC., Clerodendrum petasites S. Moore, Harrisonia perforata (Blanco) Merr. Ficus racemosa L., and Tiliacora triandra(Colebr.) Diels. In an exhaustive review and few data, it has been contamination and adulteration form upper ground of plant used also.
Aim of the study: in this study was aim to undertaken the pharmacognostic evaluation to provide the data base of each species in term of the roots of plant used.
Materials and methods: All five root species were collected from wild or non-cultivated from several places through Thailand. It was conducted in term of the morphological character including whole plant, microscopic of transverse section, histological of roots powdered, fragments of crude drugs, pharmacognostic character parameter and 3D-HPLC fingerprint profile.
Results and conclusions: Five root species were distinguished from morphological, 3D-HPLC chromatogram and established the useful key identification for identify each crude powdered species that can be resolve the problems from adulteration and contaminate in traditional medicine market. Moreover, all information will be used for ensure the medicinal plant for consumer protection.
Ben-Cha-Lo-Ka-Wi-Chian Remedy (BLW) is an important traditional medicine that have been practiced for long history and used as an antipyretic drug in Thailand. Fist revealed of these remedy has been in the national traditional medical textbook named "Paad sard song kro" which printed by Phra-ya Pis-sa-nu-pra-saad-vej.(ministry of education, 1999). Base on the characteristic of Thai traditional medicine, it has usually been prepared in form of the composition of the formulae or the remedies that means one remedy is composed from several kinds of plant species in a variety of ingredients. For that reason, BLW is also a remedy which is combines from five roots species in an equal part by weights including Capparis micracantha DC.(CAPPARIDACEAE), Clerodendrum petasites S. Moore(VERBANCEAE), Harrisonia perforata (Blanco) Merr. (SIMAROUBACEAE), Ficus racemosa L.(MORACEAE) and Tiliacora triandra(Colebr.) Diels (MENISPERMACEAE). Nowadays, BLW is also the first range in The Thai Traditional medicines which is notified in the List of Herbal Medicine Products of the National List of Essential Drugs A.D. 2006 and the government has also been recommended to be used in a Primary Health Care.
Whereas, BLW is well know and a lot of Thai traditional practitioners have been used in which increasing of commercial interested. Unfortunately, from the observation and the traditional medicine market survey it was revealed that some of BLW ingredients are being sold in mixed form or have adulterated and contaminated with another part of plants especially with upper ground parts of plant (not only roots) or substances with low quality of raw plant. Whether or not, from these problems it could be caused the consumer problems in the further.
Regarding to the report of World Health Organization: WHO (2003), whilst the tendency of traditional medicine has been widely increasing used and the efficacy has also been investigated by the researcher all around the world, the number of patients who experiencing negative health consequences caused by the use of poor quality of raw medicinal plant materials as a major cause a problems. Simultaneously with Yi zeng et al (2004) also concerned about the major problems for the standardization of the oriental traditional medicines are still difficult to improve and poor successful. Since the standardization for the formulae are more difficult than the western medicine.
A perusal of the literature reviewed demonstrated that few of knowledge studied for botanical identifications in each species in BLW remedy. Only T. triandra species was revealed the botanical identification data which provide by Department of Medical Sciences, in Thai Herbal Pharmacopoeia 1998 volume l as a whole plant and the pharmacognositc of F. racemosa bark was investigated by Koipillai BABU et. al. (2009), both were not specific in root. Therefore, this study was devoted investigated to find out the pharmacognostical studies including 3D-HPLC fingerprint profiles of all the five roots species for a formal scientific reference monograph on the identifying the characteristics of each plant species, particularly for the part of roots. Besides, an endeavor has also been made to find out the presence or absence of some histological characters of each dried powdered for established the useful key identification. Finally the consumer or stakeholders can be convenient used this key identification to detected the contamination or adulteration powdered by basic knowledge and low technology.
2. Material and methods
2.1 Plant materials
Each of five roots species were collected from 14 different places through Thailand including the North, North-East, Central and South prefecture during the winter time (October-December) 2008. All set of crude drugs were authenticated by one of the authors Ruangrungsi N. and identified by comparison with the herbarium, Department of National Parks, Wildlife and Plant Conservation, Ministry of Natural Resources and Environment, Bangkok. Each sample was air dried and ground to coarse powders. Voucher specimens were deposited at college of Public Health Sciences, Chulalongkorn University, Thailand.
2.2 Morphological identification
The unique characteristic of each root species was carried out by macroscopic and microscopic. For the macroscopic, the characteristic of whole plants, shape, size, color, odour, surface or fracture appearance and other visual inspection of crude drug were investigated. For microscopic, two sessions including transverse section and dried root powdered(ground and sifted through a 250 micron sieve) were studied under microscope(Zeiss Axioskop, Germany) with and with-out salfranin staining solution namely anatomical and histological.
2.3 Physio-chemical identification
The pharmacognostic character including moisture content, loss on drying, total and acid insoluble ashes content and extractive values (ethanol and water) were investigated as followed World Health Organization(WHO) guideline standard methods (WHO, 1998) In brief, the moisture content was investigated by Azeotropic method with saturated toluene. Loss on drying was conducted by gradually heating to 105°C until constant weight, kept in desiccator to cool and weight. Total ash was carried out by accurately weighed 4 g of coarse root powdered into tared crucible, ignite it by increasing the heat until 600°C to afford its white (carbonless) then cool in desiccator and weight. Continually used the crucible containing the total ash for acid insoluble by adding 25 ml of 2M hydrochloric acid and boiling for 5 minutes then filtrates on an ashless filter-paper for collected the insolulble matter, transfer the neutral filter-paper containing the insoluble matter to the original crucible, ignited at 600°C to constant weight, allow residue to cool in desiccators then weigh without delay. Extractive values were determined by ethanol and distill water separately. Four gram of each coarse root powdered in closed conical flask was macerated with 100 ml of each solvent, and allow standing for 18 hrs. at room temperature after shaking well for 6 hrs., filtrated and adjusted to 100 ml by marc rinsing solvent. Transferred 20 ml of aliquot into evaporating dish, evaporated to dryness and further dry at 105°C for 6 hrs. until the constant weight. All the experiments of each sample were conducted in triplicates.
2.4 Three- Dimensional (3-D) HPLC analysis
3D-HPLC was performed on a Agilent 1100 3D-HPLC system(Agilent Technologies, Tokyo, Japan) equipped with a photodiode-array detector using a column(4.6 x 250 mm) TSK gel ODS-80Ts(Tosoh Corp., Tokyo, Japan) and kept at 40°C. Each compound was successively separated at a flow rate 0.8 ml/min. by using elution of the mobile phase 10 mM phosphoric acid-acetonitrile linear gradient (95:5-5:95, 60 min). All the measurements were done in triplicate.
3.1 Morphological identification
The comparative macroscopical characters of five species in BLW remedy were studied and evaluated as revealed in Table 1. Additionally, for more details of each species in term of picture of whole plant, anatomy of transverse section, histology of dried roots powdered and the fragment of dried root crude drugs were showed in the Figure 1 to 5.
3.1.1 Capparis micracantha DC. (Fig. 1)
Whole plant is half erect shrub, oblong of leaves, armed with very short conical straight thorns and white flower with petals oblong, long filament and style as show in the figure 1.1. The fragment of dried root crude drug is harden, grayish yellow, coarse surface and quite big sized (Fig. 1.3).
Transverse section illustrated clearly two part layer of cortex and xylem phoem layer. In the layer of epidermis has abundance of sclereid next to 4-5 layer of periderm and a group of sclereids are distributed in the part of cortex layer in the several size of parenchyma of cortex. It has a lot of primary phloem tissue all around the endodermis layers. In the cortex and xylem phoem, several of xylem ray with starch granules, big of xylem vessel and has no pith are appeared as shown in Fig. 1.2.
Powdered of dried root crude drug is grayish yellow, several sizes of sclereids and starch granules and fragment of pitted vessels. The part of xylem in radial longitudinal section showing wood fiber with medullary ray and fragment of fibers, those are containing starch granules (Fig 1.4).
3.1.2 Clerodendrum petasites S. Moore. (Fig. 2)
Whole plant is erect shrubs or small trees, generally unarmed, opposite leaves, inflorescence axillary cyme, large flowers and showy, mostly white or sometime red, calyx campanulate, truncate and 5-dentate as in figure 2.1. The root is conical small size and hardens, less branch root and root hairs. The fragment of crude drug is small, harden, the color of surface cutting is soft white, smooth, glisten and normally empty of pitted (Fig 2.3).
Transverse section shown wider xylem vessel, which are consists of several size of xylem ray with starch granules and variety of xylem vessel size. On the other hand, the narrow size of parenchyma of cortex is composed of 5-7 layers of parenchyma containing prism crystal. It is clear endodermis layer between cortex and xylem layer. Five layers of periderms and the outer side is easy to lose. The parenchyma of pith is containing with starch granules (Fig. 2.4).
Powdered are composed of several sizes of boreded pitted vessels fragment, parenchyma in a section view, fragment of fibers, starch granules and prism crystal of calcium oxalate. And also has part of xylem in radial longitudinal section showing wood fiber and medullary ray. The powdered is strong smell, soft white, glisten, delicate and the test is bitter (Fig 2.4).
3.1.3 Harrisonia perforata (Blanco) Merr. (Fig. 3)
Whole plant is a climbing to erect prickly shrub. The branches are armed with short, sharp spines, the leaves pinnate with unpaired terminal leaflet and the leaflets are ovate-lance-shaped. Flowers are a pedicel with 4-5 merous, triangular lobes,petals are lance-shaped which are red outside and pale red to white inside. The fruit is globular, exocarp of leathery texture and freshly (Fig. 3.1). The fragment of crud drug is harden and plain surface. The outermost is grayish in color and leathery texture (Fig. 3.3)
Transverse section shown big size of xylem vessel, which are galore distributed in phloem layer and enclosed with xylem parenchyma and some of them are containing with the starch granules. In the parenchyma pitted vessel part, there are attached with abundant of xylem fibers. The parenchyma is containing rosette aggregate crystals, which are distributed in the parenchyma of cortex. The primary phoem tissue and the fiber are arranged by conical shape in the cortex layer. Epidermis is thickness and small cell (Fig 3.2)
Powdered are appeared with several type of histological character. There are part of xylem in tangential longitudinal section showing wood fiber and modullary ray, fragment of pitted vessels, varieties size of starch granules, the parenchyma in sectional and longitudinal view, fragment of fiber and prism crystal of calcium oxalate and also port of xylem in radial longitudinal section showing wood fiber and modullary ray (Fig 3.4). The powdered are soft, grayish-yellow, mind smell and the test is bitter.
3.1.4 Ficus racemosa L. (Fig. 4)
Whole plant is tree or banyans, evergreen or occasionally deciduous, the crown often irregular and shabby, woody epiphytic climbers or stranglers, creepers. The trees whose branches send down aerial roots that thicken (pillar roots). Leaves arranged spirally or alternate, simple to palmately lobed. Fruits are in large clusters on the main branches and trunk, ripening rose-ripe, subglobose to pyriform and soft (Fig. 4.1). The fragment of crude drug is dark red-brown, coarse surface. The outermost is thin and leathery texture (Fig 4.3).
Transverse section discloses the big conical shape of primary phoem tissue and phloem fiber in the cortex regions that also containing with variety size of parenchyma of cortex. The outermost layer is consisted of 4-6 layer of periderm. The thin vascular cambiums are along the outermost boundary of phloem region, which are consisted with variety size of xylem vessels and also distributed by xylem fibers. The pitted zoned is 0.05 mm wide in the central of TS (Fig 4.2).
The powdered of F. racemosa is dark red-brown in color, soft, distend, mind bitter of taste and smell. The histological character of the powdered is containing several kind of fragment of pitted vessels and fiber. The variety shapes of calcium oxalate and variety form of starch granules are also found. The part of xylem in radial longitudinal section are often showing wood fiber and medullary ray and are often found the xylem parenchyma in longitudinal view (Fig 4.4).
3.1.5 Tiliacora triandra Diels. (Fig. 5)
Whole plant is climbing shrub, dioecious. The leaves are ovate, alternate, glabrous and dark-green. Flower is small and yellowish. The fruits are yellowish obovate druplets (Fig. 5.1). The fragment of crude drug is grayish-yellow, soft, leathery texture of epidermis and clear brown patches radiating from pitted (fig 5.3).
Transverse section showed abundant of several forms and sizes of starch granules in most of root cell, especially in parenchyma and sclereids. The epidermis is consisting of several layers of tabular cells with brownish contents. The cortex layer is composed of thin layer of parenchyma and the several sizes of sclereids are along with this region. In the phloem layer, abundant of xylem fibers are composed with broader xylem vessel and upper layer is phloem tissue. The abundant of starch granules are contained in reserved parenchyma and also in pitted vessels (Fig. 5.2).
The powdered is grayish-brown in color with a faint odor and an intensely bitter taste. In the histological character of the powdered, there are composed of several from and size of starch granule and fragment of pitted vessels. It is also found fragment of fibers with moderately thick-walled, cork in surface view and part of xylem parenchyma. The several kinds and size of sclereids and parenchyma in sectional view are found, whose cells contain a lot of starch granules (Fig 5.4).
3.2 Physiochemical identification
The percentage of moisture content, loss on drying, total ash content and acid insoluble ash are tabulated in pharmaconostic character table(Table 2) as well as extractive value, which including ethanol and water extract. The percentage by dry weight of each species was investigated from n=42 from 14 places throughout Thailand by grand mean and polled standard division. The average amounts of moisture content and loss on drying of all species were not difference. There were around 6-8 % of dry weight. In case of total ash content, F.racemosa and C. micracantha were shown the high values to be 5.94±0.34 and 4.91±0.34 % of dry weight respectively. Whereas, the investigated in acid insoluble ash content studies C. micracantha was demonstrate the highest percentage to be 2.09±0.59 % by dry weight as well. Whilst, the extractive values including ethanol and water, T.triandra was performed the first one whose given a highest value to be 1.22 ±0.13 and 2.17±0.25 % by weight respectively. By the water extract C.micracantha has also exposed not different values of T. triandra (2.16±0.46 % by dry weight).
3.3 Three- Dimensional (3-D) HPLC analysis
The chromatograms and spectrum that representative constituents of each species extract in BLW remedy extracts were illustrated in Figure 6. All of the major peaks in each species chromatogram were measurement by maximum absorption wavelength (UV-spectrum) and Identified by comparing with the data base in The Dictionary of natural products Program (DNP 19.1) by Tayle & Francis Group. As results of 3D-HPLC, all chromatograms from each species were clear identifications and can be distinguished between species by the quantity of their constituents and different of retention times.
4. Discussion and conclusion
The pharmacongnostic specification is plays an important role in term of the medicinal plant. Underlining the specification is emphasize the requirement to ensure the quality or standard of medicinal plants by using the standard methods that have been provided by the WHO in the manual of Quality control method for medicinal plant materials.
In present study, Ben Cha Lo Ka Wi Chin remedy ingredients, which including form five root plant species was selected to examination since the increasing interested in this remedy and the government has been encouraged to use this remedy as a antipyretic drug in primary health care. Even though all the five species are easily to characterize when in form of whole plant but it could be difficult when all the root of five species on the basis of powdered or fragment of crude drug. Additionally, only root part should be used for these remedy since the national traditional medical textbook as mention before. Therefore, all five root species was consider for investigated and provide the evidence data base in order to differentiate all the root species. Additionally, this is a great interest to give a clear standard for identification or reliable characters for specified plant materials from adulteration or contamination in the traditional medicine market also.
Therefore in this study, the series of pharmacognostic parameters, which is a useful tool for setting standards for crude drugs, was selected for undertaken and future information can be set like a key identification for each root species. Hence various physiochemical parameter, morphology and 3D-HPLC fingerprints were established to substantiate the standardization of five root species in BLW remedy.
The morphological result was conducted; all plant material was collected from wild or non-cultivated trees that mean no any contaminants in each species, by the macro- and microscopic character. It can be quick characterizing each plant species by the results of whole pant and transverse section part. By the way, from the histological characters studies herein established the useful key for identify the powdered as shown in Figure 7. It is a utilized key for distinguish or segregate all the root plant powdered that may be cause by adulteration or contaminate in the market. In the key identification figure, it can be used as an easy way to distinguish or ensure the root powdered by the present and absent of some histological characters.
Regarding to the results of physio-chemical identification that has been provided valuable information for its quality or standard of the medicinal plants material, the parameter of moisture content and loss on drying was demonstrated not difference results of all root species. F.racemosa was exhibited the high value in total ash content and C. micracantha was also shown high % weight in acid insoluble ash content. In term of extractive value, T. triandra was demonstrated high amount in both ethanol and water extractive value. Even though plant materials are obtained from non-cultivated tree populations, the collecting of medicinal plant, the process of preparing the crude drug, and the time that they keep the crude drug, most of the factor can be caused the quality of the crud drugs in the market. Therefore, the parameter standardization of the medicinal plant should be done and carried out to avoid unauthentic medicinal plants.
Likewise, the 3D-HPLC was displayed clear 3D-HPLC fingerprint chromatogram that can be use to verify each species. From the chromatogram, all plant species were representative differences constituents and difference retention times. Namely, C. micracantra was exposed major peak in early of retention time as same as T. triandra but they still shown clear different chromatogram. All of major peaks in each species were also exhibited the different constituents due to the difference of their UV spectrum. Thus, the entire constituent from all majors' peaks in each species is much difference from all extract crude drug.
From the investigation on five root species of Ben Cha Lo Ka Wi Chian remedy, the results can be used as a useful information for all the people who would like to use this remedy or only single herb from the ingredient in which the propose of consumer protection. It also utilized as an evidence base to confirm the quality or standard of the material drug for biological or clinical investigation in the future.
Conflict of interest statement