1. Introduction

Herbal medicine is gradually getting popular among people in both developing and developed countries due to its less /no adverse side effects. Quality evaluation of herbal materials for safety and efficacy by using multi-component systems and acceptable analytical methodologies is fundamental as it strengthens their quality, safety and efficacy ^{[1, 2]}. With respect to quality control, correct identification of the species concerned from commonly available adulterants or substitutes, in fresh, dried or powdered state is of prime importance ^[3]. Since adulterants/ substitutes closely resemble the genuine material, macroscopic or microscopic evidences alone cannot always provide evidence for complete identification ^[4]. On the other hand misidentification of species and the subsequent substitution with unsuitable material could lead to a real danger in the preparation and administration of herbal medicine ^[5]. Serious adverse affects of substitution and misidentification of species. which are used for the Chinese Traditional Medicine have been reported ^{[6, 7]}. Therefore, implementation of rigorous standardization procedure for correct botanical identity, part of the plant, appropriate state of maturity, harvesting, processing and storage practices by using multi component analytical techniques is of primary importance in order to avoid harmful adulteration, substitution, contamination and degradation ^[8].

Munronia pinnata is an expensive, valuable, rear medicinal plant possessing many health claims and being used for the treatment of malaria, recurrent fever, dysentery and purification of blood in traditional systems of medicine in Sri Lanka ^{[9, 10]}. Andrographis paniculata is commonly available, less expensive medicinal plant used for the treatment of scabies, boils, skin eruptions, and chronic undetermined fevers ^[11]. Moreover, incorporation of cheaper, abundant substituent /adulterant Andrographis paniculata instead of authentic M. pinnata is becoming a very common practice in the open herbal markets in Sri Lanka (Personnel communication). Even though, this substitution has been practiced since long times, available information on comparative pharmacognostic, physicochemical, phytochemical and antioxidant capacity of this pair of plants are scares. In the present study attempts have been made to compare gross macroscopical and microscopical characters, physicochemical, physicochemical properties and antioxidant capacity of M. pinnata and A. paniculata as important pharmacognostic parameters in herbal drug standardization process.

2. Materials and Methods

Both M. pinnata and A. paniculata plants were collected from the institutional research plots where authenticated plants were maintained under similar soil and climatic conditions. Herbarium specimens of both plants were prepared and deposited (HTSMP 17 & HTSAP-18) in the institutional herbarium.

Free hand transverse sections were made using razor blades. Suitable sections were selected and taken through an alcohol series (as 30% and 50% alcohol for 5 minutes each) and subsequently strained with 1% safranin in 50% ethanol. Stained material was made into temporary mounts using glycerin. Photomicrographs were taken using a digital camera attached to Olympus, Model CX 31 microscope.

Powder microscopical studies were carried out following standard protocol with slight modifications ^[12]. Small amount of ground, whole plant material of both M. pinnata and A. paniculata were separately mounted on labeled glass slide containing one to two drops of water and glycerol/ ethanol by using moist tip of a needle. After placing the cover slip, it was warmed gently to remove air bubbles and observed under the microscope. Illustrations were made and photomicrographs were taken by using a digital camera attached to Olympus, Model CX 31 microscope.

About 10 g of coarsely powdered plant material of each species was separately extracted in 50mL of methanol by using Soxhlet apparatus. The extract was concentrated at 45C using rotovapour (Buchi Rotavapour, Type-R-114A29 B-480, Switzerland).

The phytochemical screening tests for alkaloids, flavonoids. saponins, steroid glycosides and tannins were performed according to the method described by Farnsworth ^[13].

The Thin Layer Chromatography (TLC) was performed according to the method described by Stahl ^[14] with some modifications. About 8 μL of the extract was spotted on TLC plates (Pre-coated silica gel 60 A 20 X 20cm; 0.2mm thickness) and developed using chloroform: dichloromethane: cyclohexane: methanol (5:4:1:0.4) as the mobile phase. They were observed under UV 366 nm and after spraying with Vanillin-Sulfuric acid. Spots were marked, specific colour of the spots was recorded and R_f value for each spot was calculated.

Total antioxidant capacity was determined using Ferric Reducing Antioxidant Power (FRAP) assay according to standard protocol ^[15]. Methanolic extract (100 μL) was mixed with 900 μL of freshly prepared FRAP reagent of pH 3.6 containing 2.5 mL of 10 mmol/L, 2,4,6-Tripyridyl-s-Triazine (TPTZ) solution in 40 mmol/L, HCl plus 2.5 mL of 20 mmol/L FeCl₃ and 25 mL of 300 mol/L acetate buffer. After incubating for 4 minutes, absorbance of the reaction mixture was measured at 593 nm using the spectrophotometer (Shimadzu, UV Mini 1240, Japan). Trolox was used as e standard solution.

Results of physico-chemical parameters and antioxidant activity were analyzed by general linear model (GLM) ANOVA test followed by Duncan’s Multiple Range Test (DMRT) and presented as means ± SE.

3. Results and Discussion

Present study compared the prominent morphological, anatomical, powder microscopical, preliminary phytochemical, physico-chemical parameters and antioxidant capacity of M. pinnata and A. paniculata which could be used in herbal drug standardization process.

Figure 1. Comparison of morphological features of Munronia pinnata (Left) and Andrographis panicuala (Right) [1. Mature plant of M. pinnata, 2. Mature plant of A. paniculata, 3. Different stages of flowering and fruiting of M. pinnata, 4. Different stages of flowering and fruiting of A .paniculata; A. Flower, B. immature fruit, C. Mature fruit, D. Seeds].

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Basic morphological features of M. pinnata and A. paniculata are shown in Figure 1 and distinguished polymorphic vegetative and reproductive characters are given in Table 1.

As shown in Table 1, flower type, number of petals, number of sepals, flower size, fruit type could be considered as distinguish reproductive characters while leaf, stem and petiole characters exhibit prominent polymorphic vegetative characters. These characters could be used in differentiating Munronia pinnata from its adulterant in the raw material.

Table 1. Distinguished polymorphic vegetative and reproductive characters of Munronia pinnata and Andrographis paniculata

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Figure 2. Distinguished anatomical and powder microscopical features of Munronia pinnata and Andrographis paniculata (Epidermal peels and sections of the leaf midrib, petiole and stem of M. pinnata and A. paniculata [A-Transverse section of leaf petiole of M. pinnata, B- Transverse section of leaf midrib of M. pinnata, C- Transverse section of leaf petiole of A. paniculata, D- Transverse section of leaf midrib of A. paniculata, E- Transverse section of stem of M. pinnata F- leaf sections showing different types of crystals; G - Transverse section of stem of A. paniculata H- stem longitudinal section of A. paniculata showing crystals and pitted fibers I- Lower epidermis with stomata of M. pinnata, J - Upper epidermis of M. pinnata, K- Lower epidermis with stomata of A. paniculata, L- Upper epidermis of A. paniculata, M-Leaf midrib section of M. pinnata showing glandular and non glandular trichomes, N- Rosette crystals in phloem tissue, O- Glandular sessile trichome of M. pinnata, P- Leaf sections showing different types ofcrystals; R - different types of trichomes present in M. pinnata, S - Different types of trichome present inA. paniculata T-Trichomes; UE- upper epidermis; LE- lower epidermis; GT-Glandular trichomes; SGT-Sessile glandular trichome; CRY- Rosette crystals; PH-Phloem; P-Parenchyma cells; PL- Palisade; XYXylum;UE-Upper epidermis; UT- unicellular trichome, LE-Lower epidermis; TWF- Thick wall fiber,MGT- multicellular glandular trichome, MSGT- multicellular sessile glandular trichome, UTAT-unicellular two armed trichome].

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Important anatomical features of M. pinnata and A. paniculata are presented in Figure 2.

Table 2 and Figure 2 summarize the distinguished leaf stem and powder microscopic features which are useful for proper identification of M. pinnata and A. paniculata. Type of stomata, epidermal cell margins, present of different kinds of trichomes, shape of palisade layers, shape of the stem, collenchyma densely at the corners of the stem and availability of different types of crystals were key polymorphic features of leaf of M. pinnata and A. paniculata.

Table 2. Distinguished polymorphic anatomical and powder microscopic characters of Munronia pinnata and Andrographis paniculata

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Presence of leaf fragments with lower epidermis showing anomocytic stomata are prominent character in M. pinnata while leaf fragments with diacytic stomata, collumner palisade are characteristic in A. paniculata. Presence of solitary as well as clustered crystals, two armed hairs, fragments of single palisade layer, idoblasts attached to parenchyma tissue fragments and oil globules were observed in powder of M. pinnata.

Presence of closed or arch shaped vascular rings in leaves and stems, different types of trichomes, anomocytic stomata in lower epidermis have been identified as common features of family Meliaceae. Further, stone cells in cortex as well as pericycle of the cork, various types of solitary or clustered crystals have been observed in the tissues of all organs ^[16]. They further highlighted the availability of secretary cells, distribution of solitary and clustered crystals as special diagnostic feature of family Meliaceae. Similarly, presence of quadrangular stem with dense collenchyma strands at angles, diacytic stomata and cystoliths in both upper and lower epidermis have been reported as distinguished diagnostic features of A. paniculata ^[17]. Moreover, morphological and anatomical features of leaves have been successfully used for the identification of leaves of Olivae folium and its counterparts ^[18].

Thin Layer chromatography (TLC) is the widely used analytical method in herbal drug standardization process due to its simplicity, rapidness and cost effectiveness ^[19]. In the present study, TLC fingerprints of leaf stem and root extracts of M. pinnata and A. paniculata were compared. The highest number of common spots both under UV 366 nm (R_f 0.35, 0.50, 0.71, 0.85) and after spraying with vanillin sulfuric (R_f 0.50, 0.78, and 0.88) in both M. pinnata and A. paniculata was observed in leaves followed by stem and root extracts. Moreover, two spots (R_f 0.16 and 0.70) were common for leaf, stem and root extracts of both plants after spraying with vanillin sulfuric. TLC fingerprints visualized under UV 366 nm exhibited some species specific spots for leaf (R_f 0.56, 0.59), stem (0.57) extracts of M. pinnata. Similarly, spots with R_f 0.32 and 0.72 were distinguished for leaf extracts of A. paniculata. The presence of similar spots in all three extracts as well as individual extracts indirectly validates the traditional claim of using A. paniculata as a substitute for M. pinnata since ancient times in traditional systems of medicine in Sri Lanka. In contrast presence of spots with different R_f values may be due to species specific chemical compounds present in different parts of these two species.

As presented in Table 2, leaf, stem and root extracts of M. pinnata and A. paniculata positively reacted on saponin, alkaloids, tannins, flavonoids and steroid glycosides. Present results are in agreement with previous studies ^[20] which reported the presence of saponin, alkaloids, tannins, flavonoids and steroid glycosides in A. paniculata.

As shown in Table 3, significantly higher values for all tested physicochemical parameters were observed for leaf extracts. In this study, it reveals that Total ash, Water soluble ash, Acid insoluble ash and moisture contents vary in the order as leaves > stem > root in both plants. In the mean while all these are higher in A. paniculata rather than M. pinnata except water soluble ash. Comparatively higher extractable matter content was exhibited in hot extraction over the cold extraction. The order of extractable matter content varied as leaf>stem> roots in both hot and cold extraction methods. This may be due to enhancement of extraction in hot extraction procedure. Observed higher extractable matter content of leaf are in agreement with previous studies ^[21], which reported the presence of comparatively higher extractable matter content in leaf extracts of A. pannuculata.

Table 3. Phytochemical analysis of different parts of Munronia pinnata and its’ adulterant Andrographis paniculata

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The antioxidant capacity (TAC) of plants is mainly contributed by the varying amount of active molecules/ ingredients present in different parts of the plant. Results of (TAC) showed the presence of antioxidant capacity to a considerable extent in all three plant parts tested. However, higher TAC was exhibited in A. paniculata compared to M. pinnata.

Table 4. Physico-chemical analysis of leaves, stems and roots of Munronia pinnata and its’ adulterant Andrographis paniculata

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Order of increase of TAC was leaf > stem > root for M. pinnata and stem > leaf > root for A. paniculata (Table 4). Presence of prominent antioxidant activity in leaf and stem extracts for A. paniculata has been reported by previous workers ^{[21, 22]}. In the present study pharmacognostical evaluation of distinguished morphological, anatomical, powder microscopical characters, physicochemical and phytochemical properties and in vitro antioxidant capacity have been investigated in order to differentiate authentic M. pinnata from A. paniculata. Procedures of morphological, anatomical powder microscopical, Thin Layer chromatography and antioxidant capacity were adopted from the standard methods described in literature ^{[12, 14, 15, 16, 23]}. In conclusion, it is clear that M. pinnata could be differentiated from A. paniculata by comparing above characters.

Table 5. Total Antioxidant Capasity of Munronia pinnata and Andrographis panniculata, TE- trolox equivalent

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4. Conclusion

Moreover, presence of certain phytochemical groups, and antioxidant capacity of M. pinnata and A. paniculata justifies the use of A. paniculata as a substitute for M. pinnata in traditional systems of medicine in Sri Lanka, which needs to be confirmed after further clinical trials. Information gathered through the present study could be directly used for the upgrading of Sri Lankan pharmacopeia.

Statement of Competing Interests

the authors have no competing interests.

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