Indian Journal of Traditional Knowledge Vol. 17 (1), January 2018, pp 106 - 112.

Mazus pumilus (Burm. f.) Steenis; Pharmacognosy

Saiqa Ishtiaq 1*, Uzma Hanif 2 & Muhammad Shaharyar Khan Afridi 1

1Punjab University College of Pharmacy, University of the Punjab, Allama Iqbal Campus-54000, Lahore, Pakistan; 2Department of Botany, Government College University, Lahore, Pakistan Email: [email protected] Received 07 February 2017; revised 13 April 2017

Mazus pumilus (Burm. f.) Steenis, is a well-known traditional medicinal plant belonging to family Mazaceae. The research work is about the pharmacognostic standardization of M. pumilus which includes; macroscopic features and microscopic evaluation of leaf, stem and roots. TS of leaf, stem and root showed the arrangement of the different cells. Histochemistry of TS of leaf, stem and root gave distinctive results with conc. HCl, phloroglucinol, ferric chloride, iodine solution and Sudan III which indicated the presence of Ca +2 oxalate crystals, lignin, tannins, starch and oil cells, respectively. Powder study of leaf depicted the presence of fibres, epidermal cells, resinous matter and vessels. The powdered study of stem showed collenchyma, vessels, fibers, cortex cells with tracheids, and helical vessels. While, root powder contained pithed vessels, cork cells, parenchyma and phelloderm. The quantitative analysis of TS of leaf was also performed for the establishment of leaf constants. In fluorescence analysis of herb, different colors were observed under ordinary light, short and high wavelength UV light. Phytochemical analysis of the methanolic extract of whole herb confirmed the presence of alkaloids, glycosides, flavonoids, saponins, sterols, triterpenoids, carbohydrates, proteins and tannins. All these results will help in identification, confirmation and quality characterization besides, laying down the pharmacopoeial standards for M. pumilus .

Keywords: Mazus pumilus , Mazus japonicas, Histochemistry, Pharmacognostic, Phytochemical, Pharmacognosy IPC Int. cl. 8: A61K 36/00, C07, C08, C12, A61K 38/00, A61K 39/00

Plant based medicines are now popularized species of the genus are edible 9. In an ethnobotanical worldwide and therefore there is a great demand of survey, it is found to be used as fodder for cattle10 . medicinal plants for herbal medicine system. To meet The herb also possesses antibacterial and antifungal the need for this purpose the standardization of these action 11 . Modern researches have proven that various plant materials is very important 1. Mazus is a genus of leaf extracts of M. pumilus has high potential of low growing perennial plants consisting of around 30 anticancer activity on human cancerous cell lines 12 . species. This genus is generally found in damp The juice of the herb is used in the cure of habitats in lowland or mountain regions of China, typhoid 13,14 . Due to its good antioxidant potential, the Japan, South East Asia, Australia and New Zealand 2. plant material has shown cardioprotective effect. Mazus pumilus (Burm. f.) Steens syn. Mazus japonius Recent phytochemical investigations of M. pumilus (Thunb.) Kuntze syn. Mazus rugosus Lour. is have revealed that it is enrich with saponins as a commonly occurring glabrous annual hairy herb in major class of secondary metabolites 15,16 . Punjab region of Pakistan 3,4 . M. pumilus is used as a In spite of a lot of attention to therapeutic effects; traditional medicine in different areas of its there isn’t any detailed pharmacognostic data geographical distribution by the local natives. The available on structural morphology and other plant has been used as a whole as well as by parts, physicochemical standards, as it is required for the specifically leaves 5. Ethnomedicinally, the whole identification and quality standardization of the plant. plant has been employed as a remedy for epilepsy 6. Therefore, an extensive anatomical, physicochemical The infusion of the whole plant is consumed as and phytochemical screening is required that will be tonic 2,3 . Similarly, whole herb is utilized as an helpful to avoid any ambiguity 17 . Morphological antifebrile, emmenagogue and aperitive 7,8 . Certain results are helpful in explanation of an exclusive drug with a major focus on quantitative and qualitative ______18 *Corresponding author microscopy . Therefore, the present research ISHTIAQ et al. : PHARMACOGNOSTIC STANDARDIZATION OF MAZUS PUMILUS (BURM. F.) STEENIS 107

comprises of anatomical, structural and histochemical using microscope. Slides of powdered leaves, stem evaluations of the leaf, stem and root, along with the and roots were prepared according to the prescribed whole herb assessment of physicochemical parameters procedures. Photographs were captured through the (ash values, extractive values), fluorescence analysis and microscope with digital camera 23 . preliminary phytochemical properties. Quantitative evaluation Methodology Microscopic examination is employed for the quantitative evaluation and for this purpose some Plant material The plant was collected from botanical garden of specific histological features including, stomatal index, vein-islet and vein termination number and Government College University Lahore Pakistan, and 24,25 was authenticated by Dr. Uzma Hanif, Assistant palisade ratio were noted . Professor, Department of Botany, Government College University Lahore Pakistan. A specimen of Fluorescence analysis plant was deposited in herbarium of Government Ultraviolet fluorescence analysis of whole herb was College University, Lahore Pakistan, under voucher carried out by treating them with different reagents No: GC. Herb. Bot. 2270. All plant parts were dried and was observed in ordinary light and UV light (short wavelength of 254 nm and long wavelength of under shade, were powdered and preserved in brown 26 containers at dry place. 366 nm) .

Pharmacognostic evaluations Preliminary phytochemical analysis The preliminary phytochemical analysis of whole Macroscopic evaluation herb was carried out according to the standard All macroscopic evaluations were carried out on 5 procedures 27 . samples of each part. The taxonomical description was made according to the related articles and the Physicochemical analysis 19 data given in books . For the purpose of physicochemical analysis, whole herb was powdered. The powdered samples were Microscopic evaluation subjected to analysis for their extractive values in Fresh leaves, stem and root were immediately fixed chloroform, ethyl acetate and n-butanol, along with total in formalin: acetic acid: 70 % alcohol (5:5:90) for 24 ash, water soluble ash and acid insoluble ash values 28,29 . h. TS of leaf, stem and root were made by commonly used blade and razor method. Sections were stained Results 20 with safranin and fast green dye . Macroscopic evaluation Histochemistry Mazus pumilus is an annual herb growing to 20 cm. The sections of leaf, stem and roots were taken and It is a branched herb, average 7 cm tall; stems hispid. they were treated with various chemicals to note the Lower leaves are opposite and closely packed, to 2 x chemical reaction and color change. Inferences were 1.1 cm, obovate, obtuse, crenate to serrate, upper ones made to evaluate the particular reaction for alternate, smaller. histochemical analysis in the plant tissues used, accordingly. Phlorogucinol, concentrated HCl, Iodine Microscopic evaluation solution, Ferric chloride and Sudan III solution were TS of Leaf showing vascular bundles with xylem used to locate the presence of Ca +2 oxalate crystals, and phloem tissue in the central area, which are lignin, starch, tannins and oil globules, respectively. surrounded by the circular cells of collenchyma, with After treatment with particular reagent the sections an outer boundary of epidermal cells (labelled as a, were observed under light microscope and their b&d in Fig. 1A). Trichomes were observed on the photographs were taken using digital camera 21,22 . surface of epidermal cells (labelled as c and a in Figs 1A&B). Abundant stomata were seen in the lower Powder study epidermis (Fig. 1C). Chloral hydrate (75 %) solution was used as Vascular bundles were observed around the central clearing reagent prior to observation of powder drug pithy portion of the stem TS. Outer to the vascular 108 INDIAN J TRADIT KNOWLE, VOL. 17, NO.1, JANUARY 2018

Fig. 2 — T.S. of stem of M. pumilus m; a: Vascular bundle with xylem and phloem; b: Pith; c: Cortex; d: Epidermal cells

Histochemistry of stem demonstrated the Fig. 1 — Microscopy of the leaf of M. pumilus +2 presence of Ca oxalate crystals, tannins, lignins and oil globules, as an effervescent response was observed conc. HCl, megnata coloration when treated with phlorogucinol, greenish black coloration in case of ferric chloride solution. While, the presence of oil globules were confirmed by the treatment with Sudan III, when the globules turned pinkish red. The presence of aleurone and oil cells was also confirmed in powder microscopy of stem. However, it didn’t show any change when exposed to iodine solution which confirms the absence of starch granules in the stem.

On the other hand, transverse sections of root +2 Fig. 3 — T.S. of root of M. pumilus showing a: Epidermis; b: unveiled the presence of Ca oxalate crystals, Cortex; c: Pericycle; d: Pith. tannins, lignins and starch granules. Production of bundles, cortex cells were observed with outer layer of tiny bubbles which were seen on the slide under the epidermal cells of stem (labelled as a, b, c & d in Fig.2). microscope, megnata coloration of the section and dark coloration of the of the cells were clear signs The TS of root was almost elliptical in shape and +2 showed marginal epidermal cells, inner cortex, of the presence of Ca oxalate crystals, tannins and pericycle and rounded pith cells (labelled as a, b, c & lignins, respectively. In addition to that, on d in Fig. 3). exposure to iodine solution, blue shade appeared within the slide in certain cells. This proved the Histochemical analysis of TS presence of starch granules in the root. It was Histochemical analysis of the leaf, stem and root further verified in the powder microscopy of root. was carried out. The transverse sections were exposed Unlike stem, the TS of root is devoid of oil cells. to conc. HCl, phlorogucinol, ferric chloride, iodine Powder microscopy solution and Sudan III. The histochemistry of leaf showed the presence of Ca +2 oxalate crystals, tannins, The powder microscopy of leaf shows; reticulate and lignins as it gave effervescence with conc. HCl, vessels with inter costal region in which Ca +2 oxalate megnata coloration with phlorogucinol and black crystals were distributed, thin asymmetric elongated coloration with ferric chloride solution. However, it fibers with pointed edges, boarder pitted grouped didn’t show any change when exposed to iodine vessels, vein islets with brownish resinous matter solution and Sudan III, which confirms the absence of associated with it and brick shaped rectangular starch granules and oil globules. epidermis cells (Fig. 4). ISHTIAQ et al. : PHARMACOGNOSTIC STANDARDIZATION OF MAZUS PUMILUS (BURM. F.) STEENIS 109

Fig. 6 — Powder microscopy of the root of M. pumilus Fig. 4 — Powder microscopy of the leaf of M. pumilus A: Group of bordered pithed vessels; B: Parenchyma with starch A: a. reticulate vessels, b. inter costal region, c. Ca +2 oxalate granules; C: Pith cells; D: Phelloderm; E: Secondary phloem with crystals; B: group of vessels; C: epidermal cells; D: Vein islet +2 Ca oxalate crystals; F: Thick walled cork cells with resinous matter; E: Fiber secondary phloem tissues with Ca +2 oxalate crystals, dense walled either penta or hexagonal cork cells (phellem), small irregular shaped parenchyma cells with starch granules in small aggregates, and thick walled polygonal pith cells (Fig. 6).

Quantitative evaluation The quantitative evaluation helped in establishing leaf constants including; palisade ratio (7.0 ± 0.31), stomatal index (11.35 ± 0.41), vein-islet number (11.0 ± 0.55) and vein termination number (15.5 ± 0.76) (Table 1).

Fluorescence analysis The fluorescence analysis of whole herb revealed various colors of the extracts under ordinary light, Short wavelength (254 nm) UV light, and Long wavelength (366 nm) UV light indicating the presence of fluorescent compounds in the methanolic extract (Table 2).

Fig. 5 — Powder microscopy of the stem of M. pumilus Preliminary phytochemical analysis A: Cortex; B: Aleurone and oil cells; C: Helical vessels of stem; The phytochemical screening of whole herb mainly D: Collenchyma tissues in longitudinal view; E: Ca +2 oxalate revealed the presence of terpenoids, sterols, crystals; F: Single vessel with piths; G: Tracheid; H: Fiber glycosides, flavonoids, alkaloids, carbohydrates, tannins, saponins, and proteins (Table 3). The powder of stem highlighted the thin polygonal cortex cells, group of pithed vessels, +2 Physicochemical constants collenchyma, irregular shaped Ca oxalate crystals, The extractive value of whole herb in ethyl acetate collenchyma, elongated fibers of xylem, i.e. was high, followed by n-butanol. The chloroform tracheids, aleurone and oil cells with round to extractive value was the lowest as compare to other circular dispersed oil globules, flexible and twisted solvents (Table 4). The ash values of whole herb helical vessels and thin fibers with visible lumen showed high content of total ash and water-soluble and pointed edges (Fig. 5). ash followed by acid insoluble ash (Table 5). The powder microscopy of root revealed the presence boarded pitted vessels that were fused into Discussion each other, cells of phelloderm–a part of periderm Herbal drug’s purity, safety, identity, and quality with crisscross network, brick shaped quadrilateral can be maintained by means of standardization which 110 INDIAN J TRADIT KNOWLE, VOL. 17, NO.1, JANUARY 2018

involve both qualitative and quantitative parameters. phlorogucinol demonstrated presence of lignins. Among qualitative parameters microscopy is simple Occurrence of oil globules in the pith cells of stem and economical measure to identify source material. was confirmed, as they turned pinkish red in color On the other hand, the quantitative parameters involve on exposure of Sudan III dye, though results were determination of stomatal index, vein islet, vein negative with leaf and root indicating they are termination number and palisade ratio. By these devoid of oil cells. Similarly, the iodine solution values the purity of drug can be illustrated. Literature gave positive result for starch granules in root, revealed that both qualitative and quantitative studies when its exposure turned them blue. While, it gave are important for identification of raw material during negative results for leaf and stem. drug manufacturing 30,31 . The powdered microscopy of the leaf of the herb The transverse section of leaf showed the basic depicted the reticulate vessels, with inter costal region profile of botanical anatomy showing the arrangement among them, along with that thick walled vessels, of vascular bundles, collenchyma tissues, trichome epidermal cells, thin irregular shaped fibers (Fig. 4). and epidermis. The abundant stomata were also The powdered micrographs of stem showed the observed in the lower epidermis (Fig. 1). The TS of helical vessels, crystals of Ca +2 oxalate, aleurone with stem vascular bundles arranged in circular form along oil forming cells. The compound and single vessels with pith cells, cortex and epidermis (Fig. 2). The TS with piths were also visible. Besides that, the basic of root also disclosed the presence of cortex, pith, structures including cortex, collenchyma tissues, thin epidermis and pericycle in the middle (Fig. 3). fibers pointing at the edges, and tracheids (Fig. 5). Histochemical assessment of transverse sections The powder study of root revealed bordered pithed were carried out to acquire a realistic image on vessels, pith cells, secondary phloem tissues with Ca +2 preparatory scale at cellular level utilizing conc. crystals. In addition to them, thick and thin cork cells HCl, phloroglucinol, ferric chloride solution, iodine were also observed. Phelloderm (modified cork cells) solution, and Sudan III, which demonstrated the was also seen (Fig. 6). existence of tannins, lignin and Ca +2 oxalate The quantitative evaluation of TS of leaf helped in crystals in the leaf, stem and root. Effervescent the establishment of leaf constants that proved to be a response of all parts to conc. HCl indicted the helpful tool to aid in the authentication and presence of Ca +2 oxalate crystals. Ferric chloride confirmation process of this therapeutically important solution determined the existence of tannins in all drug (Table 1). The fluorescence analysis is an parts by showing blackish coloration on exposure, appreciated, easy and direct method for the and megnata coloration due to the application of identification of fluorescent compounds. Different Table 1 — Leaf constants of leaves of M. pumilus compounds give fluorescence when they are exposed to short and long wavelength UV light 32 . The powder Leaf constants Range Mean ± SEM of whole herb gave various colors when observed Palisade ratio 6 – 8 7.0 ± 0.31 Stomatal index of lower epidermis 10.4 – 12.3 11.35 ± 0.41 under ordinary daylight, in short and long wavelength Vein-islet number 9.5 – 12.5 11.0 ± 0.55 of UV light Table 2. The preliminary phytochemical Vein termination number 13.2 – 17.8 15.5 ± 0.76 screening of methanolic extract of M. pumilus whole

Table 2 — Fluorescence analysis of whole herb, M. pumalis Observations S. No. Chemicals Ordinary light Short wave length Long wave length 245nm 366nm 1 Water Light green Purplish blue Dark green 2 Chloroform Dark green Reddish Yellowish green 3 Aniline Dark red Brick red Dark green 4 Powder Light green Bluish green Light green 5 66% H 2SO 4 Brownish green Light green Dark green 6 50% H 2SO 4 Pale yellow Brownish black Dark green 7 50% HNO 3 Lemon yellow Brownish green Dark green 8 5% NaOH Light green Brownish black Dark green 9 5% FeCl 3 Brown Dark grey Dark green 10 10% KOH Yellowish green Reddish green Dark green ISHTIAQ et al. : PHARMACOGNOSTIC STANDARDIZATION OF MAZUS PUMILUS (BURM. F.) STEENIS 111

Table 3 — Preliminary phytochemical analysis of whole herb, chloroform, ethyl acetate and n-butanol and the M. pumalis results are tabulated in Table 4. The ash values for the S. No. Group Name of tests Inferences powdered plant are calculated to figure out the 1 Proteins Millon’s test + + amount of siliceous material left over in the residue Ninhydrin test – and to determine the extraneous material adhered to 34 Biuret test + + the plant while its collection . The ash values of Xanthoproteic test – whole herb were carried out and the results are 2 Carbohydrates Barfoed’s test – presented in Table 5. Molisch’s test + + + Benedict’s test – Traditional significance of study 3 Saponins Foam test + The herb has been used as a traditional medicine. Bromine water test + This standardization approach will invoke the Legal’s test + scientists to work further for the assessment of the 4 Lipids Soap formation test – distinctive chemical constituents present in its 5 Tannins Ferric chloride test + + extract that are rendered responsible for its 6 Terpenoids Salkowaski test + biological activities and traditional uses. Their Liebermann’s test – chemistry and structural elucidation will assist in 7 Sterols Salkowaski test + Liebermann’s test + describing the mechanism of their actions. Besides that, Sulphur test + these constraints will help in the identification of the 8 Glycosides Bromine water test + + + correct herb while collection and minimize the chances Keller-killani test + + of adulteration, substitution or sophistication. Legal’s test + 9 Flavonoids Ferric Chloride test + + Conclusion Alkaline reagent test + The study of pharmacognostical features had shown Lead acetate test + the standards, which will be useful for the recognition of 10 Alkaloids Mayer’s Test – its distinctiveness and genuineness. Physicochemical Wagner’s test + parameters like ash values, and extractive values are all Hager’s test + indicators of the quality of material. These anatomical Dragendroff’s test + + + perspectives, pharmacognostical evaluations and + present, ++ moderately present, +++ strongly present, – absent physicochemical characterization, carried out in this Table 4 — Extractive values of whole herb, M. pumilus research work can be utilized as a reference for S. No. Parameters Percentage yield ± SEM authenticity of this plant material. 1. Chloroform 1.65 ± 0.131 2. Ethyl Acetate 8.87 ± 0.191 Declaration of interest 3. n-butanol 5.40 ± 0.263 We declare that we have no conflict of interest.

Table 5 — Ash values of whole herb, M. pumilus Acknowledgement S. No. Parameters Values % (w/w) The research work was funded by the Punjab 1 Total ash 15.15 ± 0.028 2 Acid insoluble ash 2.326 ± 0.116 University College of Pharmacy, University of the 3 Water soluble ash 15.233 ± 0.044 Punjab, Lahore Pakistan. herb, revealed the presence of various important References active constituents; carbohydrates, proteins, saponins, 1 Suresh PV, Shraddha SN & Gopalkrishnan B, alkaloids, glycosides, flavonoids, lipids, and Pharmacognostical studies of Nyctanthes arbor-tristis L. terpinoids (Table 3). stem bark-A common but less known folklore herb, Indian J Tradit Knowle , 12 (2) (2013) 284-287. The extractive values are an integral part of 2 United States: California Academy of science; Available physicochemical analysis and these quantitative from: http://www.inaturalist.org/taxa/126377-Mazus. values are determined for further standardization of 3 Vardhana R, Direct uses of medicinal plants and their identification , (Sarup & Sons. New Delhi, India), 2008, 223. the plant powder. These values give a clear idea about 33 4 Nasir E, Ali SI & Stewart RR, Flora of West Pakistan: an the yields of the extract in different solvents . The annotated catalogue of the vascular plants of West Pakistan evaluation was performed on whole herb with and Kashmir , Fakhri, 1972. 112 INDIAN J TRADIT KNOWLE, VOL. 17, NO.1, JANUARY 2018

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