MORPHOLOGICAL AND MICROSCOPIC ANALYSIS OF FIVE CURCUMA SPECIES GROWN IN SRI LANKA USING MULTIVARIATE TESTHTML Full Text
MORPHOLOGICAL AND MICROSCOPIC ANALYSIS OF FIVE CURCUMA SPECIES GROWN IN SRI LANKA USING MULTIVARIATE TEST
Herath Mudiyanselage Indika Chandralal Herath 1, Galabada Arachchige Sirimal Premakumara 2 and Thuppahige Don Chandima Manjula Kumara Wijayasiriwardene * 2
Faculty of Graduate Studies 1, University of Colombo, Colombo, Sri Lanka.
Industrial Technology Institute 2, Colombo, Sri Lanka.
ABSTRACT: Curcuma is an important genus used in Traditional Medicine in the world. Due to similar morphological characters and same Sinhalese vernacular names of Curcuma species, adulteration or substitution takes place. The current study was conducted to understand the similarities in morphological and microscopic characters by analyzing data using multivariate test on five species available in Sri Lanka; C. albiflora, C. aromatic, C. longa, C. oligantha, and C. zedoaria. Whole plants of Curcuma species were collected in 2016, from wet and dry zones of Sri Lanka. Voucher specimens of the plants were authenticated from National Herbarium, Peradeniya, Sri Lanka. Morphological characters of five Curcuma species and microscopic characters of five Curcuma species using 5 observations of each plant were analyzed. An experiment was conducted as per WHO guidelines and other published data. Statistical tests were performed using Minitab 17. Multivariate test was used to determine the complex relationship among variables by simple correspondence analysis. Analyses examined the relationships between the 25 observations and the associations between variables in two dimensions and similar morphological and microscopic observations were identified from their positions. Statistical analysis of the current study showed differentiation by morphological and microscopic characters of Curcuma species. Morphologically C. zedoaria and C. longa are similar. Microscopically, C. zedoaria and C. albiflora showed more similarity, In contrast, C. aromatica and C. longa clustered as another group. Therefore, this analysis can be used to identify commercial samples of Curcuma species more effectively.
Multivariate, Curcuma, Albiflora, Zedoaria, Oligantha, Aromatica, Longa
INTRODUCTION: Purpose and Rationale: Among plant geniuses, Curcuma claimed to have clinically valuable medicinal plants in indigenous and traditional medicine in the world 1. Genus Curcuma (family Zingiberaceae) comprises about 100 species all over the world, only 5 species are reported in Sri Lanka (C. albiflora, C. aromatica, C. longa, C. oligantha, and C. zedoaria) 2.
Curcuma is claimed as a potential source of raw material in herbal medicine to combat a variety of ailments such as arthritis, cancer, diabetes, cough, skin disorders, and oxidative stress-related pathogenesis, etc 3-7. Medicinally, most of the Curcuma species have medicinal importance with its action such as anti-inflammatory, hypocholesterolemic, choleretic, anti-microbial, anti-rheumatic, antiﬁbrotic, antivenomous, antidiabetic, antihepatotoxic anticancerous, anti-oxidant, toxicant, larvicidal, pheromone, insecticidal, anti-plasmodium, mutagenic, genotoxic, hyper-protective, platelet aggregation inhibitor, anti-arthritic, COX-1 inhibitor, antiviral, antiproliferative, cytotoxic, and apoptosis etc. properties 8.
In addition to its medicinal importance, Curcuma oil is used in aromatherapy and the perfume industry 9. Biologically active compounds have been isolated and detected in Curcuma species in the past decades.
Four species, namely C. longa, C. aromatica, C. oligantha, and C. zedoaria are used in Traditional Medicine in Sri Lanka. C. longa is one of the most valuable species of the Curcuma genus. C. aromatica, C. longa, and C. zedoaria have been used in various indigenous medical systems. Under the name Harankaha, regionally people use various plants for their herbal preparations; C. zedoaria, C. aromatica, C. albiflora, and Zingiber zerumbet 2. Due to its similar morphological characters and same Sinhalese vernacular names, these species are likely to be adulterated. Statistical analysis of the characters of plants concerning similarity can put into the same group. Therefore, the present study was undertaken to analyze the morphological and microscopic features of five Curcuma species statistically using multivariate test 10.
MATERIALS AND METHODS: Whole plants of Curcuma species were collected in the year 2016 from wet and dry zones of Sri Lanka in the flowering season; C. albiflora (Kitulgala, Kegalle and Erathna, Ratnapura district), C. aromatica (Erathna, Ratnapura), C. longa (Kahathuduwa, Colombo district), C. oligantha (Hebarawa, Badulla district), C. zedoaria (Gonapola, Colombo district). Voucher specimens of the plants (herbariums) were authenticated and deposited in the National Herbarium, Peradeniya, Sri Lanka, and Herbal Technology Section, ITI for future reference. Morphological and microscopical characters of five Curcuma species using 5 observations of each plant were used. To study Pharmacognostical parameters, preparation, preservation, and storage of plants and experiments were done according to WHO guidelines and other published data 11-14. Morphological studies, leaf, flower, and rhizome, were observed through Leica MS 5 microscope separately, and thin hand-cut specimens were observed under Labomed (Sigma, Labo America, Inc. U.S.A.) microscope with 100x and 400x magnification for the microscopic studies.
Statistical Analysis: Statistical tests were performed using Minitab 17. Multivariate test was used to determine the complex relationship among variables. Ten morphological features and eight microscopic features were analyzed by simple correspondence analysis and by cluster variable method. Analyses examined the relationships between the 25 observations and the associations between variables in two dimensions and similar morphological and microscopic observations were identified from their positions 10, 15-19.
RESULTS AND DISCUSSION:
Morphological Characters of Five Curcuma Species: Some of the morphological characters are reported in Table 1.
TABLE 1: MORPHOLOGICAL COMPARISON OF FIVE CURCUMA SPECIES 19
|Morphological features||C. albiflora||C. aromatica||C. longa||C. oligantha||C. zedoaria|
|Height||35 ± 15 cm||170 ± 25 cm||120 ± 15 cm||50 ± 15 cm||160 ± 20 cm|
|Length of petiole||9 cm||90 cm||18 cm||1.5 cm||15 cm|
|Size of lamina||15±3×7±1cm||50±20×12±2 cm||40±10×7±1 cm||23±5×6±1 cm||45±20×10±2 cm|
|Number of leaves||5-6||5-7||3-5||5-12||4-6|
|Peduncle height||12 cm||5-8 cm||6-7 cm||5-7 cm||5-8 cm|
|Size of inflorescence||7 × 6 cm||15-30 × 9 cm||10-15 × 5-7 cm||3-7 cm||10-18 × 6-8 cm|
|Calyx||1.3-1.8 cm||2 cm||1.5 cm||1.5 cm||8 mm|
|Leaf lower surface||Glabrous (0)||Pubescent (2)||Glabrous(0)||Glabrous(0)||Lightly pubescent(1)|
By cluster variable analysis of morphological characters of five species, C. longa and C. zedoaria have similar morphological characters Fig. 1. In the row profile, petiole length, and lower surface of C. aromatica has shown a higher percentage (12 - 13% and 13.3 - 13.7%) respectively Table 2. Lower surface was more pubescent in C. aromatica.
Table 3 gives a summary of the decomposition of the 10 × 25 contingency table into 9 components. The column labeled. Inertia contains the χ squared / n value accounted for by each component. Of the total inertia, 0.1368 %, 58.2% was accounted for by the first component, 32.38% by the second component, and so on.
TABLE 2: ROW PROFILE OBTAINED FROM MORPHOLOGICAL CHARACTERS
|HT - Plant Height||0.012||0.013||0.014||0.015||0.017||0.059||0.061||0.064||0.067|
|LL - Leaf Length||0.015||0.016||0.017||0.018||0.015||0.050||0.069||0.055||0.059|
|LW - Leaf Width||0.031||0.036||0.031||0.036||0.031||0.054||0.058||0.054||0.058|
|PL - Petiole Length||0.013||0.014||0.013||0.015||0.011||0.126||0.133||0.140||0.129|
|NL - Number of Leaves||0.034||0.041||0.048||0.034||0.041||0.034||0.048||0.048||0.041|
|FPH - Flower Peduncle Height||0.065||0.070||0.062||0.075||0.081||0.027||0.032||0.038||0.043|
|IL - Inflorescence Length||0.026||0.026||0.023||0.023||0.026||0.056||0.113||0.075||0.094|
|IW - Inflorescence Width||0.035||0.035||0.029||0.023||0.035||0.052||0.058||0.047||0.064|
|CX - Calyx||0.034||0.037||0.048||0.045||0.048||0.050||0.053||0.053||0.053|
|LLS - Leaf Lower Surface||0.000||0.000||0.000||0.000||0.000||0.133||0.133||0.133||0.133|
|HT - Plant Height||0.065||0.042||0.047||0.043||0.046||0.045||0.017||0.022||0.019|
|LL - Leaf Length||0.064||0.040||0.050||0.043||0.048||0.045||0.023||0.028||0.024|
|LW - Leaf Width||0.054||0.031||0.036||0.031||0.036||0.031||0.027||0.031||0.027|
|PL - Petiole Length||0.137||0.025||0.028||0.035||0.027||0.032||0.002||0.002||0.002|
|NL - Number of Leaves||0.034||0.020||0.027||0.034||0.041||0.027||0.034||0.054||0.082|
|FPH - Flower Peduncle Height||0.043||0.032||0.032||0.038||0.038||0.032||0.027||0.027||0.032|
|IL - Inflorescence Length||0.105||0.038||0.056||0.049||0.053||0.045||0.011||0.015||0.011|
|IW - Inflorescence Width||0.070||0.029||0.029||0.035||0.041||0.041||0.023||0.029||0.035|
|CX - Calyx||0.056||0.040||0.042||0.045||0.037||0.040||0.040||0.037||0.042|
|LLS - Leaf Lower Surface||0.133||0.000||0.000||0.000||0.000||0.000||0.000||0.000||0.000|
|HT - Plant Height||0.021||0.020||0.055||0.062||0.059||0.057||0.058||0.510|
|LL - Leaf Length||0.025||0.027||0.045||0.064||0.050||0.055||0.059||0.178|
|LW - Leaf Width||0.031||0.027||0.045||0.049||0.045||0.054||0.054||0.039|
|PL - Petiole Length||0.002||0.002||0.021||0.025||0.022||0.017||0.028||0.126|
|NL - Number of Leaves||0.061||0.048||0.027||0.034||0.041||0.027||0.041||0.026|
|FPH - Flower Peduncle Height||0.032||0.038||0.022||0.022||0.027||0.032||0.032||0.033|
|IL - Inflorescence Length||0.019||0.026||0.019||0.023||0.023||0.026||0.019||0.047|
|IW - Inflorescence Width||0.041||0.041||0.035||0.035||0.041||0.047||0.052||0.030|
|CX - Calyx||0.045||0.048||0.021||0.019||0.024||0.021||0.021||0.007|
|LLS - Leaf Lower Surface||0.000||0.000||0.067||0.067||0.067||0.067||0.067||0.003|
TABLE 3: ANALYSIS OF CONTINGENCY TABLE OBTAINED FROM MORPHOLOGICAL CHARACTERS
Since the number of components was not specified, Minitab calculates 2 components. The column labeled Qual (quality) in Table 4 is the proportion of the row inertia represented by the two components. The rows plant height, petiole length, and peduncle height, with the quality of 0.950, 0.996 and 0.931 respectively were observed, which are best represented among the rows by the two-component breakdown.
The column labeled Inert is the proportion of the total inertia contributed by each row. Thus, petiole length contributes 42.1% to the total χ squared statistic. The column labeled Corr represents the contribution of the component to the inertia of the row. Thus, Component 1 accounted for most of the inertia of petiole length (Coor = 0.906) but explains little of the inertia of plant height (Coor = 0.073). Contr, the contribution of each row to the axis inertia, shows that petiole length and peduncle height contribute the most, with plant height and calyx contributing to a smaller degree, to Component 1.
TABLE 4: ROW CONTRIBUTIONS
|Name||Qual||Mass||Inert||Component 1||Component 2|
|Number Of Leaves||0.748||0.026||0.111||-0.544||0.507||0.096||0.374||0.241||0.082|
|Leaf Lower Surface||0.645||0.003||0.014||0.663||0.612||0.015||-0.153||0.033||0.001|
Microscopic Characters of Five Curcuma Species: Some of microscopic characters of five Curcuma species were reported Table 5.
TABLE 5: COMPARATIVE MICROSCOPIC CHARACTERS OF FIVE CURCUMA SPECIES 20, 21
|Description||C. albiflora||C. aromatica||C. longa||C. oligantha||C. zedoaria|
|Number of layers of palisade||2||1||1||1||1|
|Periderm||15-16 layers||8-10 layers||2-4 layers||5-6 layers||14-15 layers|
|Primary vascular bundles||15-20 vascular bundles in the inner core||50-60 vascular bundles in the inner core of the outer zone||70-80 vascular bundles evenly distributed||15-20 vascular bundles in the inner core||90-120 bundles distributed evenly in the outer zone|
|Cambium||3-4 layers||2 layers||2 layers||2-4 layers||2-3 layers|
|Starch grains||Eccentric and concentric, Striations and hilum of few starch grains, Globular, a circular, elongated, oval and semicircular shape, 5-20/cell
small: 5-10 μm, medium: 15-25 μm, and large: 30 μm
|Numerous in the inner and outer core, spindle-shaped, eccentric, 5-20/cell, two sizes; 18 ± 1 μm and 24 ± 1 μm||Numerous in the inner and outer core, triangular shaped, eccentric, 12-20/cell
contain two sizes of starch grains; 45 ± 15 µm and 30 ±5 µm
|Less, triangular and dumbbell shape, 1-10/cell
small: 5-7 μm, medium: 12-16 μm, and large: 20-27 μm
|Numerous in inner and outer core, large and rod-shaped, eccentric, 5-20/cell
25-40 μm circular, 85-95 μm polygonal or sector-shaped, and 45-75 μm polygonal.
|Crystals||5 ± 2 µm and 10 ± 2 μm||5 ± 2 μm 12 ± 2 μm, 20 ± 1 μm, and rosette crystals||cuboidal (20 ± 5 μm), hexagonal (45 ± 5 μm), and diamond-shaped (35 ± 5 μm) prismatic crystals||prismatic crystals; 20 ± 2µm, 30 ± 2 μm, and 45 ± 2 μm||5 ± 2 μm 10 ± 2 μm, 20 ± 2 μm, and rosette crystals|
Morphological and microscopic pictures were displayed in Plate 1.
PLATE 1: COMPARATIVE MORPHOLOGICAL AND MICROSCOPIC STUDIES OF FIVE CURCUMA SPECIES
As per cluster variable analysis, C. albiflora and C. zedoaria shows similarity, C. aromatica and C. longa shows similarity by microscopical features Fig. 3. Further by column plot, C. aromatica and C. longa showed similar microscopical characters Fig. 4.
TABLE 6: ROW PROFILES OBTAINED FROM MICROSCOPICAL CHARACTERS
|Starch grains 2||0.016||0.018||0.020||0.022||0.025||0.026||0.026||0.027||0.026|
|Starch grains 3||0.067||0.065||0.067||0.067||0.067||0.000||0.000||0.000||0.000|
|Stomatal index U||0.040||0.040||0.053||0.026||0.040||0.040||0.040||0.042||0.046|
|Stomatal index L||0.045||0.049||0.042||0.045||0.049||0.048||0.047||0.050||0.046|
|Number of palisade||0.067||0.067||0.067||0.067||0.067||0.033||0.033||0.033||0.033|
|Primary vascular bundles||0.011||0.015||0.014||0.015||0.013||0.037||0.039||0.041||0.043|
|Starch grains per cell||0.018||0.036||0.043||0.054||0.072||0.018||0.072||0.036||0.054|
|Starch grains 2||0.027||0.032||0.033||0.034||0.035||0.037||0.021||0.029||0.027|
|Starch grains 3||0.000||0.000||0.000||0.000||0.000||0.000||0.000||0.000||0.000|
|Stomatal index U||0.033||0.040||0.040||0.040||0.053||0.040||0.026||0.040||0.026|
|Stomatal index L||0.045||0.031||0.031||0.028||0.035||0.035||0.024||0.024||0.028|
|Number of palisade||0.033||0.033||0.033||0.033||0.033||0.033||0.033||0.033||0.033|
|Primary vascular bundles||0.045||0.052||0.054||0.055||0.057||0.058||0.011||0.013||0.013|
|Starch grains per cell||0.018||0.043||0.051||0.058||0.065||0.072||0.007||0.018||0.036|
|Starch grains 2||0.023||0.026||0.091||0.101||0.087||0.094||0.098||0.168|
|Starch grains 3||0.000||0.000||0.100||0.167||0.134||0.122||0.145||0.081|
|Stomatal index U||0.040||0.026||0.045||0.046||0.042||0.052||0.046||0.000|
|Stomatal index L||0.024||0.028||0.049||0.052||0.045||0.049||0.052||0.001|
|Number of palisade||0.033||0.033||0.033||0.033||0.033||0.033||0.033||0.005|
|Primary vascular bundles||0.014||0.015||0.067||0.071||0.075||0.082||0.090||0.240|
|Starch grains per cell||0.011||0.014||0.018||0.025||0.032||0.054||0.072||0.050|
The table gives a summary of the decomposition of the 14 × 25 contingency table into 13 components. The column labeled. Inertia contains the χ squared / n value accounted for by each component. Of the total inertia, 0.64.03% is accounted for by the first component, 18.53% by the second component, and so on Table 7. The column labeled Qual (quality) in Table 8 is the proportion of the row inertia represented by the two components.
The rows large starch grains, small crystals, and medium size crystals, with the quality of 0.960, 0.965 and 0.902 respectively were observed, which are best represented among the rows by the two-component breakdown. Large starch grains contribute 32.4 % to the total χ squared statistic. Component 1 accounted for most of the inertia of large storage grains (Coor = 0.909) but explains little of the inertia of the number of starch grains per cell (Coor = 0.014). Contr showed that large starch grains contribute the most, with stomatal index contributing to a smaller degree, to Component 1.
TABLE 7: ANALYSIS OF CONTINGENCY TABLE OBTAINED FROM MICROSCOPICAL CHARACTERS
TABLE 8: ROW CONTRIBUTIONS OBTAINED FROM MICROSCOPICAL CHARACTERS
|Component 1||Component 2|
|2||Starch grains 2||0.643||0.168||0.059||-0.258||0.549||0.051||-0.107||0.094||0.030|
|3||Starch grains 3||0.960||0.081||0.324||-1.122||0.909||0.459||0.266||0.051||0.089|
|7||Stomatal index U||0.321||0.000||0.000||-0.048||0.016||0.000||0.209||0.305||0.000|
|8||Stomatal index L||0.349||0.001||0.000||-0.178||0.137||0.000||0.221||0.212||0.000|
|9||Number of palisade||0.713||0.005||0.008||-0.199||0.076||0.001||0.577||0.637||0.028|
|12||Primary vascular bundles||0.913||0.240||0.077||-0.090||0.074||0.009||-0.304||0.839||0.347|
|13||Starch grains per cell||0.072||0.050||0.049||-0.070||0.014||0.001||0.140||0.057||0.015|
CONCLUSION: Statistical analysis of the current study showed differentiation by morphological and microscopic characters of Curcuma species. Similar groups were identified; morphologically C. zedoaria and C. longa are similar according to the cluster variable method. Further C. albiflora and C. oligantha showed closer similarity than other species. Microscopically, C. zedoaria and C. albiflora showed more similarity. Moreover, C. aromatica and C. longa showed similarity.
But microscopically C. oligantha grouped into the separate group by cluster variable method. By column plot, C. aromatica and C. longa showed more similarity in terms of microscopic characters. As per morphological and microscopic characters, Curcuma species grown in Sri Lanka grouped to five different groups by simple corresponding methods. Therefore, this analysis can be used to identify more effectively.
CONFLICT OF INTEREST: Nil
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How to cite this article:
Herath HMIC, Premakumara GAS and Wijayasiriwardene TDCMK: Morphological and microscopic analysis of five Curcuma species grown in Sri Lanka using multivariate test. Int J Pharmacognosy 2017; 4(7): 224-31. doi link: http://dx.doi.org/10.13040/IJPSR.0975-8232.IJP.4(7).224-31.
This Journal licensed under a Creative Commons Attribution-Non-commercial-Share Alike 3.0 Unported License.
H. M. I. C. Herath, G. A. S. Premakumara and T. D. C. M. K. Wijayasiriwardene *
Faculty of Graduate Studies, University of Colombo, Colombo, Sri Lanka.
21 March 2017
19 May 2017
30 May 2017
01 July 2017