1. Introduction

Plectranthus hadiensis (Forssk.) Schweinf. ex Sprenger. (Sin. Iriveriya) and Plectranthus amboinicus (Lour.) Spreng. (Sin. Kapparawalliya) are widely used medicinal plants in traditional and Ayurveda systems of medicine. Both species are belonging to genus Plectranthus in family Lamiaceae ^{[1, 2]}. Since both plants are belonging to the same genus and family, most of the morphological characters are similar. Due to this similar appearance, people use P. hadiensis instead of P. amboinicus and vice versa. Incorrect use of these plants in herbal formulations might adversely effect on therapeutic properties of herbal drugs. Moreover, there are more than 100 written recipes in Sri Lankan Ayurveda pharmacopoeia which contain P. hadiensis and P. amboinicus as major ingredients for the treatment of different ailments. ^{[3, 4]}. However, according to the existing literature P. hadiensis is mainly used for digestive system related diseases such as dysentery, vomiting, diarrhea, acute and chronic congestion of the liver while, P. amboinicus is mainly used for whooping cough, malaria ^[5], laryngitis, bronchitis, pneumonia, after measles, chicken pox, small pox, asthma and some other diseases related to the respiratory tract, burns, wounds, sores, insect bites and allergies, ringworms, skin ulceration. Although both plant species are widely used an array of traditional and Ayurveda recipes for the treatment of diverse range of ailments, comprehensive pharmacognostic studies are scattered and required for authentication and standardization of P. hediensis and P. amboinicus. ^{[6, 7, 8]} Therefore, in the present study, attempts have been made to compare agronomical, morphological and anatomical characters and preliminary phytochemical finger printing of P. hadiensis and P. amboinicus.

2. Materials and Methods

Plant materials of both P. hadiensis and P. amboinicus were collected from 3-4 months old, well grown healthy plants which have been maintained under similar soil and climatic conditions at institutional medicinal plant research plots. Herbarium specimens of both species were prepared and identified by systematic Botanist at National Herbarium and deposited at Institutional Herbarium (ITI/HTS 106 and 107).

Same aged well acclimatized potted plants of both P. hadiensis and P. amboinicus were established in 30x30 cm holes at 45 cm x 45 cm spacing at institutional research field (Lat: 6° 58’; Long: 79° 52', RF 2500 mm, sandy loam soil, Temperature 28±2°C). One kilogram of compost was added to each hole prior planting. Plants of each species were separately established in 100 cm x300 cm beds. Whole experiment contained more than 100 plants from each species. Irrigation, weeding and other aftercare operations were conducted as required. Number of leaves, number of branches, spreading, fresh weight, dry weight and fresh to dry weight ratio was obtained at two weeks interval up to 16 weeks. At least 20 plants were used to obtain growth data.

Specimens for both species were collected from the plants cultivated at same soil and environmental conditions as a measure to minimize the impact of environmental factors on the data collected. Distinguished morphological characters of both plants were collected and tabulated. At least twenty individuals per each species were used to record morphological data.

Free hand transverse sections of representative samples of stem, leaf and root were obtained from approximately same aged, healthy, plants from both P. hadiensis and P. amboinicus. Satisfactory sections were taken through an alcohol series and subsequently strained with 1% safranin in 50% ethanol. Stained material was mounted in glass slides using glycerin. Photomicrographs were taken by using Olympus, Model CX 31 digital microscope. For powder microscopical studies, crushed whole plant powder (3 g) of both plants were separately cleared by boiling with saturated chloral hydrate solution and mounted with drop of glycerin and examined under compound microscope. Line drawing was made for each character as described in literature ^[9].

Areal samples of both samples were cut into pieces and air dried for three days at room temperature (28±2°C). Samples were coarsely powdered and (5 g) was extracted with 50 mL of dichloromethane by using Soxhlet apparatus. Extract was filtered through Whatman No.1 filter papers and filtrate was concentrated under reduced pressure at 40°C using rotovapour.

Ground samples of leaf, stem and root of P. hadiensis and P. amboinicus (200 g per each) were distilled using Clevenger light oil arm for 4 hours. Oil samples were collected and the volume of the oil was recorded. Results are presented as an average triplicates ±SD.

The chemical constituents of volatile oil were analyzed using a Gas Chromatograph ( Shimadzu GC 2010) connected with a 3m x 3mm stainless steel, flam Ionize Detector (FID). Injector temperature 230 °C and detector temperature was 240 °C. Argon was used as carrier gas. Column temperature was programmed from 60 °C to 225 °C and injected volume was 0.3µI. compounds were identified by retention data and boosting of authentic standard compounds.

The thin layer chromatography (TLC) was performed according to the method described in WHO guidelines with some modifications. Briefly concentrated filtrate was dissolved in minimum quantity of dichloromethane and 5 µL of extract was spotted in pre-coated florescent silica gel 60A 20 × 20 cm; 0.2 mm thickness, 20 cm x20 cm aluminum plates (Merck KGaA, Darmstadt, Germany). Plates were developed in chloroform: dichloromethane: cyclohexane (3:1:1) and spots were observed by spraying with vanillin- sulphuric acid. The Rf values for each spot were calculated as previously described ^[9].

All quantitative morphological and physical yield (both dry and fresh weight) data are presented as an average of 20 plants and ±SD. Results on essential oil content are presented as mean of triplicate ± SD.

3. Results and Discussion

Medicinal plants are an important source for drugs. However, selection of correct species, use of correct plant part, harvesting of plant material at optimum physical as well as chemical maturity are important to obtain proper quality materials. Moreover, good agricultural practices (GAP), quality control and standardization of raw materials by using multiple techniques, such as morphological, phycochemical, anatomical, microscopical, and chromatographic techniques have been suggested by World Health Organization.

Some of the important growth and yield parameters of the plant height, plant spreading and number of branches per plant were recorded in two weeks intervels up to 16 weeks. Plant fresh weight, plant dry weight, plant fresh to dry weight ratio of P. hadiensis and P amboinicus were recorded in monthly interval and presented in Figure 1. At least 20 plants were randomly selected for the collection of agronomical data.

Results clearly showed that all growth parameters of both plant species were increased with the time forming sigmoid shape growth curve. However, comparatively higher number of shoots per plant were observed in Plectranthus amboinicus while spreading was higher in Plectranthus hadiensis (Figure 1). In addition to the growth parameters, plant fresh weight, plant dry weight, dry to fresh weight ratio of both species were recorded as major yield components in monthly intervals commencing from eight weeks after planting.

Figure 1. Variation of growth and yield parameters of Plectranthus hadiensis and Plectranthus amboinicus [A & B = Growth parameters of Plectranthus hadiensis and Plectranthus amboinicus; C&D= Yield/growth parameters of Plectranthus hadiensis and Plectranthus amboinicus. Values are a mean of 20 plants]

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Plant fresh and dry weights of both species were increased with the maturity. However, fresh materials required for making one kilogramme of dry materials (fresh to dry weight ratio) was decreased with the maturity (Figure 1). Results clearly exhibited that if the plants are harvested at immature stage (before 12 weeks of age), it required higher amount of fresh materials to obtain 1 kg of dry materials (more than 8 kg of fresh materials to make 1 kg of dry materials). However, when plants are harvested after 12 weeks of maturity, it requires only 5.1 kg of fresh materials.

In the present study attempts were made to establish some important agronomical data, which are highly essential for establishment of commercial cultivation of Plectranthus hadiensis and Plectranthus amboinicus. Our study revealed that, major yield components, which are mainly responsible for the physical yield of the plant such as plant height, plant spreading and number of shoots per plants, are maximum at the fully matured stage (4 months after planting) and hence the higher physical yield could be obtained in fully matured stage for both plant species. Previous studies pointed out that Plectranthus hadiensis grown in both shade and full sunny conditions gave higher yield in 4 months age ^[10]. Therefore, our findings are in the agreement with previous studies. Further, increase of fresh weight and dry weight of materials are in agreement with previous workers, who investigated the increase of seed dry and fresh weight of Phyllanthus amarus with the maturity ^[11].

The plants used for the morphological study were obtained from the well matured, cultivated plants under similar soil and climatic conditions. Therefore, the morphological characters observed could be considered as a true reflection of genetically determined morphology of both species. Despite the fact there were several monomorphic and polymorphic characters were observed, for the present study we have listed prominent polymorphic characters which are highly important for differentiation of both species. Out of listed characters, shape of the stem, pinkish purple stem and petiole, presence of a grove on petiole, coriaceous leaf texture could be highly valued for identification of P. hadiensis (Figure 2 and Table 1). Acclimatization or growing of plants in a same soil and climatic conditions prior morphological, anatomical and chemical parameter has been studied ^[12].

Figure 2. Morphological variations of Plectranthus hadiensis and Plectranthus amboinicus [A & B- well grown research plot of Plectranthus hadiensis; and Plectranthus amboinicus; C&D -Flowering branches of Plectranthus hadiensis; and Plectranthus amboinicus; E &F- Leaves of Plectranthus hadiensis and Plectranthus amboinicus; G&H- Branches of Plectranthus hadiensis; and Plectranthus amboinicus showing vegetative characters]

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Table 1. Prominent morphological characters of Plectranthus hadiensis (Forssk.) Schweinf. ex Sprenger.and Plectranthus amboinicus (Lour.) Spreng

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Results of the anatomical study revealed that the presence of different types of trichomes as main diagnosis anatomical features for both species. Among the trichomes, both simple and glandular trichomes were presence in abaxial and adaxial surface of leaves, stem and petiole cross sections. Some of the observed trichomes included short-stalked and long-stalked capitate trichomes, digitiform trichome, and large spherical headed trichomes. Moreover, with different shapes and sizes, subsidiary cells with wavier outer surfaces and glandular sessile and glandular stalk trichomes with multicellular heads were observed in the stem cross sections. The distinguished features of powder microscopy of P. hadiensis and P. amboinicus included are presence of simple and glandular trichomes scattered as broken fragments of alone or attached to epidermal cells of stem, petiole or leaf blade particles, fragments of lower and upper epidermal particles with stomata were observed in both species. Study of anatomical features of genus Plectranthus is well famous due to presence of an array of trichomes with special shapes and sizes. Results of the presence study on trichomes are in agreement with previous workers, who observed short-stalked and long-stalked capitate trichomes, long-stalked capitate trichome with a large spherical head of Plectranthus species as taxonomically prominent anatomical characters ^[13].

Figure 3. Main anatomical characters of Plectranthus hadiensis (Forssk.) Spreng. and Schweinf. Plectranthus amboinicus (Lour.) and ex Sprenger. [A- Plectranthus hadiensis; B- Plectranthus amboinicus; 1a & b –stomata, 2a & b- Multicellular trichomes, 3 a & b- cork tissues, 4 a &b Oil glands, 5a & b- glandular multicellular trichomes (x 40)]

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Essential oil content of different parts of the plants (roots, stems and leaves) of both Plectranthus hadiensis and Plectranthus amboinicus at different maturity stages with commercial samples are presented in Table 2.

Essential oil content and composition of medicinal plants play an important role in therapeutic properties. As demonstrated in Table 2, essential oil content of different parts (leaf, stem and roots) of Plectranthus hadiensis and Plectranthus amboinicus increased with the maturity. The higher essential oil contents of both species were reported in roots. Essential oil content different parts of both species harvested after 5 months of maturity approximately similar to essential oil content obtained from market samples. Further, essential oil content was increased as leaf< stem<roots. Results of the present study on essential oil content of both P. hadiensis and P amboinicus are in agreement with previous studies conducted for both essential oil content of P. hadiensis and P. amboinicus elsewhere ^{[14, 15]}. Presence of higher content of essential oil at 5 months aged maturity clearly validates the traditional claims of harvesting of both species at fully maturity stage. Moreover, root essential oil content was comparatively higher in P. amboinicus. It was varied from 0.75-1.34% in roots. Meanwhile previous workers pointed out that Plectranthus species are essential-oil-rich and it was more than 0.5% in dry matter basis ^[16].

As demonstrated in Table 3, p -Cymene, Gerenyl acetate and geraniol were identified in both species harvested after 3 and 4 months maturity. Since both plants are belonging to the same genus, presence of common compounds is unavoidable.

Table 2. Essential oil content of different parts of Plectranthus hadiensis (Forssk.) Schweinf. ex Sprenger. and Plectranthus amboinicus (Lour.) Spreng with maturity

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Table 3. GC analysis of essential oil

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Thin Layer chromatography is the widely used analytical technique in herbal materials or drugs standardization process due to its reliability, cost effectiveness and simplicity.

In the present study, we attempt to compare the TLC fingerprinting pattern of essential oil of areal parts of P. hadiensis and P. amboinicus at different maturity stages (2, 3, and 4 months after planting) with commercially available samples (market samples) in order to study the proper maturity stage with higher secondary metabolite (essential oil) content. As demonstrated in Figure 4, TLC finger print profiles observed after spraying with vanillin sulphuric acid exhibited nine spots for essential oil of areal parts of Plectranthus hadiensis while five spots for Plectranthus amboinicus at all maturity stages. Further, all these spots were also present in essential oil distilled from samples purchased from open market. Out of these spots, a prominent, yellow green (Rf 0.73) spot was characteristic to P. hadiensis, while violet-blue (Rf 0.61) and brown- red (R_f 0.55) were characteristic spots for essential oil of P. amboinicus. Moreover, intensity of spot colour of TLC profiles was increased with the maturity. Therefore, TLC fingerprinting technique could be easily incorporated for authentication of market raw materials of both species in the process of quality control and standardization of raw materials as well as finished products containing P. hadiensis and P. amboinicus as ingredients. Use of TLC fingerprint profiles for standardization of Plumbago indica ^[17], Acmella oleraceae ^[18], Gyrinops walla ^[19], Munronia pinnata and Andrographis paniculata ^[20] have been extensively used. Further increase of the colour intensity might be due to increasing of secondary metabolites with the maturity stage ^[21].

Figure 4. Thin Layer Chromatogram of essential oil of Plectranthus hadiensis and Plectranthus amboinicus at different maturity stages. [Ph₂ &Pa₂ = leaf essential oil of Plectranthus hadiensis and Plectranthus amboinicus after two months of transplanting; Ph₃ &Pa₃ = leaf essential oil of Plectranthus hadiensis and Plectranthus amboinicus after three months of transplanting; Ph₄ &Pa₄ = leaf essential oil of Plectranthus hadiensis and Plectranthus amboinicus after four months of transplanting; Phc &Pac = leaf essential oil of Plectranthus hadiensis and Plectranthus amboinicus collected from open market solvent system Methanol 1: cyclohexane 1:dicloromethane 4]

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According to the WHO guidelines good Agricultural and Collection Practices (GAP), proper identification of a plant species, quality control and standardization of herbal materials are considered key issues in the herbal drug standardization. In the present study we investigate the variation of major yield and growth parameters with the maturity, key taxonomically important morphological and anatomical features, powder microscopic analysis, essential oil content with maturity, TLC profile of essential oil with the maturity of Plectranthus hadiensis and Plectranthus amboinicus by using recommended protocols. Our results on morphological variations, anatomical and powder microscopic analysis and chemical analysis are in agreement with previous studies ^{[1, 22]}. Therefore, results of the present study either singularly or as a whole could be incoporated for standardization and quality control of P. hadiensis and P. amboinicus.

4. Conclusions

Results of the present study on morphological, anatomical, powder microscopical, TLC and essential oil contents are key parameters of quality control and stadadization of P.hadiensis and P.amboinicus. Therefore, results of the present study either singularly or as a whole could be incorporated for quality control and standardization of either raw materials finished products containing P. hadiensis and P. amboinicus as raw materials. Further, presence of higher physical growth of the plant, higher content of essential oil and TLC spots with higher colour intensity scientifically validate the traditional claims of harvesting of both plants at fully maturity stage ( after 12 weeks of age.

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