Abstract

Introduction

Ajisamat, Haji Samat or Tongkat Haji Samat is a commonly used plant in Malaysian traditional plant medicines.The aqueous extract of the dried roots of the plant have been traditionally used by the aborigines and certain rural Malays for wellness, enhancing stamina and for its ergogenic effect. This plant has been used for generations to an aphrodisiac in Kelantan and Terengganu (Azmi et al., 2011). The names Ajisamat, Haji Samat or Tongkat Haji Samat were initially believed to correspond to Jackia ornata (syn. Jackiopsis ornata) of family rubiaceae (Department of Forestry Malaysia, 2005). Later it appeared that the name referred to other two plants of different species and family i.e. Prismatomeris glabra (Korth.) Valeton, rubiaceae, that the name referred to other two plants of different species and family i.e. Prismatomeris glabra(Korth.) Valeton, Rubiaceae, and Salacia macrophylla Blume, Celastraceae. The appearance of the vegetative parts including roots of both plants are nearly similar. Therefore, this study was carried out in an attempt to characterize the morphological, microscopic and chemical profiles of both types of Ajisamat plants.

Salacia macrophylla is a woody liana or climber found in swamps, lowland forest, on the mountain slopes as well as in old rubber estates. Usually it climbs up trees to several meters in height (Metcalfe and Chalk, 1965). Plants of this genus are known to possess pentacyclic triterpenes with highly oxidized A/B rings, as well as thiosugars, salacinol and kotalanol (Dhanabalasingham et al., 1996; Muraoka et al., 2008). These compounds have been reported to show a variety of biological activities such as cytotoxicity, antimicrobial, ntifertility, anti-inflammatory, antimalarial and a-glucosidase inhibitory properties (Jeller et al., 2004; Maregesi et al., 2010).

Prismatomeris glabra is a slender tree and usually found in keranga and undisturbed mixed dipterocarp forests up to 700 m altitude in Peninsular Malaysia, Sumatra and Borneo (Slik, 2006). Usually it is found on hillsides and ridges, along rivers and streams. It can grow on sandy to clay soils. The genus Prismatomeris usually contains anthraquinones and iridoids, compounds having cytotoxic (Hao et al., 2011) and antitumor activities (Krohn et al., 2007).

Materials and methods

Plant materials

Salacia macrophylla Blume, Celastraceae, was collected from a swamp forest near Taiping, Perak, Malaysia, while Prismatomerisglabra(Korth.) Valeton, Rubiaceae, was collected in Puchong, the dry lowland rain forest reserve, Selangor, Malaysia.

Life specimens of both plants were brought to Forest Research Institute Malaysia, Kepong, Selangor, Malaysia, and were identified by a botanist of specimens of both plants were deposited in the Herbarium of Faculty of Pharmacy, UiTM, Puncak Alam Campus under accession number UiTM191/HAB28 (P. glabra) and UiTM 278/TAS003 (S. macrophylla).

Macromorphological characterization

The macromorphological examination includes visual observation of the size, shape, color and was carried out following the method outlined by World Health Organization (WHO, 1998).

Micromorphological characterization

Micromorphological inspection was carried out on samples of the dried roots of the plants following the botanical microtechnique procedures described by teh (1996). The dried roots were boiled and sectioned (25 µm in thickness) with a reichert Jung sliding microtome slider. The obtained sections were bleached, stained with safranin, alcian green/blue and phloroglucinol, dehydrated, mounted with canada balsam and then studied under microscope. Microscopical examinations of the powdered roots of both plants were carried out by using the method modified from WHo (1998). The root powders of both plants were thoroughly mixed with two drops of glycerol on a glass slide and observed under the microscope.

Chemical characterization

Chemical characterization of both plant material included determination of solvent extractive values, ash values and color reaction tests.

Ethanol and water extraction amounts were determined following the methods described in WHO (1998) and Malaysian Herbal Monograph (2009). Briefly, the root powder S. macrophylla was used to calculate the values of the hot and cold extraction in water and ethanol, while the powder of root of P. glabra was extracted with only cold water and cold ethanol due to insufficient sample.

Measurements of total, acid-insoluble and water-soluble ashes were made according to the procedures discribed in Wiart and Kumar (2001) and in WHO (1998), while determination of sulfated ash was carried out following the method described in British Pharmacopoeia (2010).

Color reaction tests were done following the method prescribed by the Malaysian Herbal Monograph (2009). One gram sample of powdered root was treated with each of the reagents from a specific list at room temperature and the observation on the colors produced was done under normal light. The reagents were concentrated sulfuric acid, concentrated hydrochloric acid, sodium hydroxide 5%, potassium hydroxide 5%, ammonium hydroxide 25% and ferric chloride 5%.

Phytochemical analysis

The powder of the roots of S. macrophylla (170 g) was successively extracted with n-hexane, chloroform and ethyl acetate, while the powder roots of P. glabra (400 g) using hexane, ethyl acetate and methanol.

TLC analyses were performed on silica gel 60 F₂₅₄ plates (Merck, Germany). Spots were detected at 254 and 366 nm; cerric sulfate was used as a spray reagent for the detection of triterpenes, while the characterization of anthraquinones was achieved with Bornträger reaction (5% KOH in ethanol). Column chromatography was carried out using silica gel (40-63 µm, Merck, Germany). The ¹ H-NMR, ¹³ C-NMR, ¹ H-¹ H-COSY, HMQC and HMBC spectra were recorded on Bruker Avance II 400 and Bruker Avance III 500 MHz NMR spectrometers; chemical shifts are in ppm (δ) relative to the SiMe₄ as internal standard and coupling constants are in Hz. MS were measured on Jeol HX 110 mass spectrometers (m/z) and Agilent 6220 LC/MS TOF.

Results and Discussion

Macromorphological characterization

Salacia macrophylla plant is slender with few branches (Fig. 1A). The petioles are grooved and the stipule is present. The leaves are simple, broadly elliptic in shape and glabrous. The leaf base is symmetrical (Fig. 1A) and arrangement of the petioles is subopposite (Fig. 1B). The roots of S. macrophylla are hard taproots, yellowish-brown in color and some with lateral roots. The root bark exfoliates easily. The outer layer is brown and the internal layer appears orange (Fig. 1C-D).

The second type of Ajisamat plant, Prismatomeris glabra, is a small plant, similar in appearance to the coffee plant. The leaves are simple, broadly elliptical in shape with an entire margin, and are oppositely arranged (Fig. 2A). Their scars or stipules are present and interpetiolar (Fig. 2B). Flowers are white with five petals, are black drupes when mature (Fig. 2C). The roots are hard woody taproots, yellowish-orange color, with lateral roots (Fig. 2D). The roots of this species also show the unusual feature of an orange inner bark.

Generally both plants show a similar physical appearance. The leaf shape is similar with acute to acuminate apices. They are truly opposite for P. glabra, while slightly subopposite for S. macrophylla.However the two species can be distinguished by the presence of stipules or stipule scars and the raised midvein in P. glabra leaves.

Micromorphological characterization

The micromorphological inspection of both types of Ajisamat plant revealed distinctive differences in the anatomical structures between the plants.

In P. glabra, the vessels elements are solitary, few are in pairs and lacking tyloses (Figure 2E). The vessel size for P. glabra can be categorized as extremely small (12-25 µm) and the number of vessels as very numerous (more than 40 vessels per mm²). The parenchyma of P. glabra appears as both paratracheal and apotracheal axial. The paratracheal type is mostly of 2-cells wide, forming short tangential lines, while the apotracheal type includes uniseriate short tangential lines extending from rays to rays (Fig. 2 G-I). No brachysclereids were observed in P. glabra root bark (Fig. 2H).

Tangential longitudinal section (TLS) of P. glabra root reveals tracheids, vessels with alternate wall pitting and simple perforation plates (Fig. 2J). The parenchyma is irregularly distributed among fibers, some with inclusion of calcium oxalate crystals in the form of bundles of raphides (Fig. 2F).

The anatomical structure of tangential sections of the roots of both types of Ajisamat plants are presented in drawings in Fig. 3 and Fig. 4. The summary of the distinctive anatomical features between the two plants is presented in Chart 1.

Microscopical studies of root powder of both plants were carried out. The diagnostic features of powdered roots of S. macrophylla and P. glabra are presented in Figs. 5 and 6 respectively. Their differential identification is therefore relatively easy. S. macrophylla is characterized by abundant brachysclereids and solitary calcium oxalate crystals, while P. glabra contains numerous raphides and no sclereids.

Chemical characterization

For S. macrophylla, both ethanol and water extractives were of similar value when produced by the cold method. With the hot method, the extractives are about four times greater than those produced by cold method (Table 1). In contrast, by cold method P. glabra produces a water extractive almost six times more abundant than the ethanol extractive.

The ash values obtained for S. macrophylla and P. glabra are presented in Table 2. Regardless of the type of the ashes, the values for P. glabra appear to be consistently higher than that of ash, especially for the sulfated ash values. Similarly, Shinkar (2007) reported that high values of acid-insoluble ash are related to the high content of calcium oxalate in plants. For P. glabra, calcium oxalate crystals were found to be abundantly present in the form of raphide needles. Therefore, the high ash values for this plant are likely due to the high content of calcium oxalate.

Chart 2 summarizes the color changes resulting from the reactions of root powders with reagents prescribed by the Malaysian Herbal Monograph (2009) for color reaction tests. Some distinctive differences in colors were produced were observed between the two types of Ajisamat plants that indeed could be used for fast discrimination between them.

Phytochemical analysis

Repeated column chromatography (silica gel) of the n-hexane extract afforded S. macrophylla afforded β-sitosterol Patra et al., 2010 and four pentacyclic triterpenes namely, pristimerin (1) (Khalid et al., 2007), netzahualocoyene (2) (Dhanabalasingham et al., 1996) and netzahualocoyonol (3) (Jeller et al., 2004). The chloroform extract, when subjected to a similar fractionation process, produceded 2,3,7-trihydroxy-6-oxo-1,3,5(10), 7-tetraene-24-nor-friedelane-29-oic acid methyl ester (4) (Ankli et al., 2000). Structure elucidations of compounds (1-4) were carried out by comparison of spectral data with literature. Triterpenes 1-4 belong to the D:A-friedo-nor-oleanane group and show the rare quinone-methide moiety. Such derivatives are significant chemotaxonomic characteristic of the family Celastraceae (Gamlath et al., 1990).

Chemical studies on the methanolic Prismatomeris glabra extract led to the isolation of four anthraquinones. These compounds were isolated by MPLC (Medium Pressure Liquid Chromatography) and preparative thin layer chromatographic techniques. They were identified as 1,3-dihydroxy-2 methoxymethyl-9,10-anthraquinone (5) (Reanmongkol et al., 2003), 1-hydroxy-3-methoxy-2-(methoxymethyl)-9,10-anthraquinone (6) (Joshi et al., 1955), 2-methyl-3-methoxy9,10-anthraquinone (7) (Burnett and Thomson, 1968; Sartori et al., 1995) and 1,3-dimethoxy-2-methyl-9,10-anthraquione (8) (Fraga et al., 2009).

This study further illustrates another case in which a plant vernacular name corresponds to two or more different species. Only the eyes of an expert botanist can differentiate the plants in their original habitat. It is not surprising that traditional healers are unable to differentiate them fully. The implementation of simple tests recommended notably by WHO would enable the differentiation of the two species clearly. It is proposed that the information gathered above be incorporated into future monographs for Salacia macrophylla and Prismatomeris glabra in the forthcoming volume of Malaysian Herbal Monograph. The chemical compositions of these plants are very different. The traditional claims can not be supported by any information from literature so far. Azmi et al. (2011) have earlier reported that P. glabra aqueous extract negatively affected the cognitive functions in mice. This combined with the known cytotoxicity of anthraquinones warrant caution on the use of this plant. To the best of our knowledge, no pharmacological/toxicological study was carried out on S. macrophylla. The above mentioned triterpenes are also known for significant cytotoxicity (Ankli et al., 2000; Setzer et al., 1998). Yet, due to their low water solubility, it cannot be ascertained that a water extract of this plant is toxic without further tests. S. macrophylla therefore needs to be fully investigated. This is an endeavour undertaken by our group and results will be reported in due time.

Acknowledgment

The authors wish to express their gratitude to Mr. Richard Chung and Mr. Kamarudin Salleh of Forest Research Institute Malaysia (FRIM) to identifying the plant species. This work was supported by Dana Kecemerlangan UiTM 600-RMI/ST/DANA 5/3/DST (225/200).

References

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