ANATOMICAL, BIOCHEMICAL AND PHYLOGENETIC STUDIES OF
STROBILANTHES BLUME SPECIES FROM NORTHERN WESTERN GHATS OF INDIA
A Thesis submitted to Goa University for the Award of the Degree of
DOCTOR OF PHILOSOPHY IN
BOTANY
By
MS. MARIA CINEOLA FERNANDES
Department of Botany Goa University
Goa 403206 INDIA
July 2020
CERTIFICATE DECLARATION
ACKNOWLEDGEMENTS LIST OF FIGURES
LIST OF TABLES
LIST OF PHOTO PLATES
1. INTRODUCTION ………. 1–5
2. REVIEW OF LITERATURE……… 6–18
3. MATERIALS AND METHODS……….. 19–34
4. RESULTS……….. 35–136
5. DISCUSSION……… 137–159
6. CONCLUSION……….. 160
7. SUMMARY………….…….………...…….…..………..……. 161–164
8. REFERENCES……….…….…. 165–195
ABBREVIATIONS PUBLICATIONS
CERTIFICATE
This is to certify that the thesis entitled “Anatomical, Biochemical and Phylogenetic studies of Strobilanthes Blume species from northern Western Ghats of India”
which is submitted herewith for the award of the Degree of Doctor of Philosophy in Botany of Goa University, Goa, India is the original research work carried out by Ms. Maria Cineola Fernandes under my guidance and to the best of my knowledge and trust that this work executed in this thesis has not formed part of award of any Degree or Diploma in any University or Institute.
Place: Goa Prof. S. Krishnan Date: (Research Guide)
I hereby declare that the thesis entitled “Anatomical, Biochemical and Phylogenetic studies of Strobilanthes Blume species from northern Western Ghats of India” is my original contribution and this work has not been submitted for any other degree or diploma of any other University or Institute. I solemnly declare that the present study is based on my own knowledge and idea. The literature related to the study has been cited. Due acknowledgements has been made wherever facilities and suggestions have been availed.
Place: Goa Maria Cineola Fernandes
Date: (Research Student)
To my Beloved Parents
from many people and I am extremely privileged to have got this all along the completion of my research. At the foremost I thank my Almighty God for giving me the strength to do my work and get through all the challenges during the course of this study.
I wish to express my sincere thanks and deepest respect to my research guide, Prof. S. Krishnan, Head, Department of Botany, for his valuable guidance, motivation and support during my entire work.
I am extremely thankful to former Heads of Department Prof. (Mrs.) Vijaya U.
Kerkar and Prof. B. F. Rodrigues, Department of Botany, Goa University for providing me all the facilities to carry out my work efficiently. My warm thanks to Prof. M. K. Janarthanam (Former Head and Former Dean) for his valuable suggestions during this study. I am extremely indebted to Prof. D. J. Bhat (Former Head and Former Dean), Prof. P. K. Sharma (Former Head and Dean), Dr.
Nandkumar Kamat and Dr. Rupali Bhandari for their guidance and non-teaching staff, Department of Botany, Goa University for their assistance during the course of my work.
My sincere thanks to VC’s nominee, Prof. B. F. Rodrigues and Prof. S. K.
Shyama for their useful suggestions during the course of the study and assessing my work with critical comments.
I owe my deep gratitude to Dr. J. R. I. Wood, Department of Plant Sciences, University of Oxford, for useful discussions, critical comments and guidance. I am very much grateful to Dr. Pieter Baas, Naturalis Biodiversity Centre, Department of Botany, Leiden, Netherlands for sharing his excellence in anatomy and helping me with valuable advice and clearing my doubts.
I am forever indebted to my teacher Dr. Emilia Mascarenhas for always accompanying me during the fieldtrips and providing me with her valuable discussions, encouragement, support and always pushing to achieve higher in life. I am also thankful to Dr. Jomy Augustine for his valuable discussions and explanations during the study.
I am thankful to the Director of Western Circle Herbarium, Pune (BSI) Dr. P. Lakshminarasimhan for allowing me to consult the herbarium specimens and their library. I extend my thanks to the authorities of Kew Botanical Gardens, London, for the photograph of the Type specimen. My thanks to Dr. Priyanka Ingle for help provided to deposit the herbarium specimens at BSI.
I am grateful to Prof. J. A. E. Desa, Department of Physics and Dr. Mahesh Majik, Department of Chemistry, Goa University for their help extended during the
study with their valuable subject knowledge. I am thankful to Department of Chemistry, Goa University for providing some of the chemicals required for the study. I am also thankful to Central Instrumentation Facility (USIC), Goa University for SEM studies.
I am thankful to the authorities of Gandhigram Rural Institute, Tamil Nadu;
Indian Institute of Technology (IIT), Madras; Anchrom, Mumbai; National Institute of Oceanography (NIO), Goa for providing the instrumentation facility. I am also thankful to Rajiv Gandhi Centre for Biotechnology (RGCB), Kerala for carrying out the gene sequencing.
I am grateful to the University Grants Commission (UGC), Maulana Azad National Fellowship, New Delhi, India for the fellowship during the tenure of my research.
I appreciate the kind help received from Dr. Siddharthan Surveswaran, Department of Life Sciences, CHRIST (Deemed to be University), Bangalore, Karnataka in phylogeny studies.
I am Indebted to my teachers, colleagues and friends who have helped me during my research: Dr. Maria Fonseca, Dr. Sharad Kambale, Dr. Mayur Nandikar, Dr. Gunadayalan Gnanasekaran, Mr. Shivaprakash Nedle, Mr. K. C. Kishor, Ms.
Shaila. I am thankful to Mrs. Savita, Ms. Ritika, Ms. Nitisha and Mr. Siddhant for giving me home stay at Satara and always accompanying me during my field trips at Maharashtra.
My special thanks to my dear friend Melvina D’souza for all her help, support and motivational discussions during my weak days.
My thanks to my colleagues from the Department of Botany, Goa University:
Dr. James D’Souza, Dr. Sidhesh Naik, Dr. Ravikiran Pagare, Dr. Anup Deshpande, Dr. Nisha Kevat, Dr. Rosy D’Souza, Ms. Nikita Verenkar, Ms. Rutuja Palkar, Ms.
Aditi Naik, Ms. Annie Nadar, Ms. Vaishali Gaonkar, Ms. Rutuja Kolte, Ms. Prabha Pillai, Mrs. Abhipsa Mahopatra, Mrs. Pallavi Konge-Randive, Ms. Akshatra Fernandes, Ms. Sankrita Gaonkar, Ms. Tanvi Prabhu, Ms. Apoorva Shet, Mr. Dhilan Velip, Ms. Sujata Dabolkar, Ms. Sheela Pal, Ms. Prabha Tiwari, Mrs. Smita Srivastava and Ms. Shravani Korgaonkar for help and support during my time at the University. My thanks to my colleagues from other Departments, Ms. Apurva Narvekar (Chemistry, GU), Mr. Jeyakanthan M (Physics, GU), Ms. Vijay laxmi J (Marine Science, GU) Ms. Shabnam Choudhary (Marine Science, GU) and Ms.
Ankeeta Amonkar (NIO) for their support during my research.
Lastly, I believe the real wealth is the good wishes, I am deeply Indebted to my family for their love, support, encouragement and appreciation at all times.
Maria Cineola Fernandes
No. No.
1. Map of northern Western Ghats of India (study area) and collection localities.
20
2. Syntype of S. reticulata Stapf [Cooke, T. K000883137] @ Royal Botanic Gardens, Kew.
42
3. EDS spectra of Strobilanthes spp. a. S. ciliata leaf; b. S. ciliata stem; c. S. callosa leaf; d. S. callosa stem; e. S. integrifolia leaf; f.
S. integrifolia stem; g. S. ixiocephala leaf; h. S. ixiocephala stem.
82
4. EDS spectra of Strobilanthes spp. a. S. heyneana leaf; b. S.
heyneana stem; c. S. reticulata leaf; d. S. reticulata stem; e. S.
sessilis var. ritchiei leaf; f. S. sessilis var. ritchiei stem.
83
5. EDS spectra of Strobilanthes spp. a. S. sp. ‘137262’ leaf; b. S. sp.
‘137262’ stem; c. S. barbata leaf; d. S. barbata stem; e. S. lupulina leaf; f. S. lupulina stem.
84
6. XRD spectra of leaf (a) and stem (b) Strobilanthes spp. A. S.
integrifolia; B. S. ixiocephala; C. S. ciliata; D. S. callosa; E. S.
heyneana; F. S. lupulina; G. S. barbata; H. S. sp. ‘137262’; I. S.
sessilis var. ritchiei; J. S. reticulata.
87
7. XRD spectra of S. heyneana showing prominent calcite reflection (104).
88
8. Methanolic extractive values of leaf and stem of Strobilanthes spp. 105 9. Quantification results. a. Lupeol in leaf samples; b. Lupeol in stem
samples; c. Lupeol comparison in both leaf and stem µg/200 mg of methanolic extracts of Strobilanthes species. Results presented as mean ± SD of two samples; *P<0.05.
112
10. GC-MS chromatogram of methanolic extract of Strobilanthes spp.
a. & b. S. callosa leaf and stem; c. & d. S. ciliata leaf and stem; e.
& f. S. integrifolia leaf and stem; g. & h. S. ixiocephala leaf and stem.
115
11. GC-MS chromatogram of methanolic extract of S. heyneana. a.
leaf; b. stem.
116
12. DPPH radical scavenging activity of L-ascorbic acid and Strobilanthes spp.
126
13. DPPH radical scavenging activity of L-ascorbic acid and 127
Strobilanthes spp.
14. ABTS radical scavenging activity of BHT and Strobilanthes spp. 129 15. ABTS radical scavenging activity of BHT and Strobilanthes spp. 130 16. Comparison of DPPH/ABTS assays to measure antioxidant
capacity in Strobilanthes spp.
130
17. The Randomized Axelerated Maximum Likelihood (RAxML) phylogeny based on ITS data. Bootstrap support values ≥50% are shown above the branches and Bayesian posterior probabilities (≥0.90) below the branches. Sequences from this study are indicated in blue. Country wise distributions of Strobilanthes species are provided.
134
18. The Maximum Likelihood (ML) phylogeny based on the combined cpDNA and mtDNA data. Bootstrap support values ≥50% are shown above the branches and Bayesian posterior probabilities (≥0.90) below the branches. Sequences from this study are indicated in blue. Country wise distributions of Strobilanthes species are provided.
135
19. Venn diagram for metabolites identified from leaf and stem of five Strobilanthes species.
152
20. Proportion of leaves and stem of five Strobilanthes species containing different groups of compounds.
153
Plate No.
Title Page
No.
1. Strobilanthes species from northern Western Ghats of India. a. S.
barbata Nees; b. S. callosa Nees; c. S. ciliata Nees; d. S. heyneana Nees; e. S. integrifolia Kuntze; f. S. ixiocephala Benth.; g. S.
lupulina Ness; h. S. reticulata Stapf var. reticulata; i. S. sessilis Nees var. ritchiei C. B. Clarke; j. S. sp. ‘137262’; k. S. reticulata Stapf var. nov.
37
2. Habitat and morphology. a. S. barbata Nees; b. Enlarged view of S.
barbata showing the unique habit (winged stem) and flower; c. S.
callosa Nees; d. & e. Flowers and fruits of S. callosa; f. & g. Dried old population of S. callosa and showing the new growth.
38
3. Habitat and morphology. a. & b. S. ciliata Nees; c. & d. S.
heyneana Nees; e. & f. S. integrifolia Kuntze.
40
4. Habitat and morphology. a. & b. S. ixiocephala Benth.; c. & d. S.
lupulina Nees; e. & f. S. reticulata Stapf var. reticulata.
43
5. Habitat and morphology. a. & b. S. sessilis Nees var. ritchiei C. B.
Clarke; c. & d. Dried population of S. Sessilis var. ritchiei; e. & f.
S. sp. ‘137262’.
44
6. Habitat and morphology of S. reticulata Stapf var. nov. a. Distinct clumps on rocky plateau; b. flower (flowering periodicity after 7 years); c. habit (sessile leaves).
45
7. Stem anatomy of Strobilanthes spp. a. S. ciliata (showing distinct brachysclereids in the centre of pith); b. S. integrifolia (few cystoliths in pith cells); c. S. callosa (sclereids distributed in pith cells); d. S. ixiocephala (raphides concentrated in centre of pith cells); e. S. reticulata (hexagonal shaped pith cells with few sclereids); f. S. sessilis var. ritchiei (hexagonal shaped pith cells with sclereids); BSc, brachysclereids; Cyt, cystoliths; Sc, sclereids (X100).
47
8. Stem anatomy of Strobilanthes spp. a. S. heyneana (sclereids and raphides in the hexagonal pith cells, cystoliths in the cortex); b. S.
sp. ‘137262’ (cystoliths in the cortex, sclereids in pith cells); c. S.
barbata (hexagonal pith cells with cystoliths and sclereids (X40);
d. & e. S. barbata (showing magnified view of winged stem) (X100); f. S. lupulina (hexagonal pith cells with sclereids) (X100);
Cyt, cystoliths; Sc, sclereids (X100).
48
9. Stem anatomy showing the distribution of cystoliths, raphides, sclereids in Strobilanthes spp. a. S. ixiocephala showing raphides in the pith cells; b. S. heyneana (hexagonal shaped pith cells with uniformly distributed sclereids); c. S. ciliata; d. S. barbata; e. S.
heyneana; f. S. sp. ‘137262’; g. S. lupulina showing cystoliths in hypodermal cells and outer cortex; h. Pith cells of S. integrifolia showing cystoliths; Cyt, cystoliths; R, raphides; Sc, sclereids (X400).
51
10. Leaf anatomy of Strobilanthes spp. a. S. ciliata leaf (broad U- shaped vascular bundle) (X40); b. S. integrifolia leaf (narrow U- shaped vascular bundle) (X40); c. S. callosa leaf (v-shaped vascular bundle) (X40); d. S. ixiocephala leaf (broad U-shaped vascular bundle) (X40); e. S. reticulata leaf (narrow U-shaped vascular bundle) (X100); f. S. sessilis var. ritchiei leaf (narrow U- shaped vascular bundle) (X40); g. S. heyneana leaf (broad U- shaped vascular bundle) (X40); h. S. sp. ‘137262’ leaf (V-shaped vascular bundle) (X40); i. S. barbata & j. S. lupulina leaf (broad U- shaped vascular bundle) (X40).
56
11. Leaf anatomy of Strobilanthes spp. a. S. callosa ground tissue of leaf midrib region showing cystoliths; b. S. ciliata collenchyma cells of lower epidermis of leaf showing sphaeraphides; c. S.
integrifolia showing the arrangement of cystoliths in collenchyma cells of upper epidermis of leaf; d. S. sessilis var. ritchiei collenchyma cells of lower epidermis of leaf showing fewer cystoliths; e. S. sp.‘137262’ ground tissue with cystolith; f. S.
sp.‘137262’ observed prominent sclereid in the mid-rib region of leaf; g. S. sessilis var. ritchiei collenchyma cells of upper epidermis and the epidermal cells of leaf showing cystoliths; h. S.
callosa leaf showing cystoliths in the collenchyma cells of upper epidermis. Cyt, cystoliths; Sc, sclereids; Sph, sphaeraphides (X400).
58
12. Stomatal types of Strobilanthes spp. Diacytic or caryophyllaceous stomata. a. S. ciliata; b. S. integrifolia; c. S. callosa; d. S.
ixiocephala; e. S. reticulata; f. S. sessilis var. ritchiei; g. S.
heyneana; h. S. sp. ‘137262’; i. S. barbata; j. S. lupulina. Note: On close examination, each guard cell pair has one very small subsidiary cell and two successively greater ones (anisocytic pattern), clearly seen in S. ixiocephala (diacytic & anisocytic type of stomata).
61
13. Scanning Electron Micrographs of Strobilanthes spp. showing stomata on the abaxial surface. a. S. ciliata (X500); b. S. ciliata (X2000); c. S. integrifolia (X500); d. S. integrifolia (X2000); e. S.
callosa (X500); f. S. callosa (X2000); g. S. ixiocephala (X500); S.
ixiocephala (X2000).
62
stomata on the abaxial surface. a. S. reticulata (X500); b. S.
reticulata (X2000); c. S. sessilis var. ritchiei (X500); d. S. sessilis var. ritchiei (X2000); e. S. heyneana (X500); f. S. heyneana (X2000); g. S. sp. ‘137262’ (X500); S. sp. ‘137262’ (X2000).
15. Scanning Electron Micrographs of Strobilanthes spp. showing stomata on the abaxial surface. a. S. barbata (X500); b. S. barbata (X2000); c. S. lupulina (X500); d. S. lupulina (X2000).
64
16. Scanning Electron Micrographs of Strobilanthes spp. showing trichomes on the abaxial and adaxial surface. a. S. barbata adaxial surface showing epidermal cells with a peltate glandular trichome (X2000); b. S. sessilis var. ritchiei adaxial surface with a 4-celled non-glandular trichome (X500); c. S. heyneana adaxial surface with 9 basal cells surrounding the non-glandular trichomes (X150);
d. S. sp. ‘137262’ adaxial surface with a 4-celled non-glandular trichome (X500); e. S. sp. ‘137262’ adaxial surface showing non- glandular trichomes (X150); f. S. callosa abaxial surface with peltate glandular trichome (X2000); g. S. callosa adaxial surface with peltate glandular trichomes and non-glandular trichomes (X150); h. S. integrifolia adaxial surface showing epidermal cells with a wrinkled head of a peltate glandular trichome (X1000).
66
17. Scanning Electron Micrographs of Strobilanthes spp. showing trichomes on the abaxial and adaxial surface. a. S. ixiocephala adaxial surface showing epidermal cells with 4-celled non- glandular trichomes and peltate glandular trichomes (X150); b. S.
ixiocephala adaxial surface with high density of papillae on the cell wall surface of non-glandular trichome (X2000); c. S. ixiocephala abaxial surface showing peltate glandular and non-glandular trichome (X500); d. S. lupulina adaxial surface profuse distribution of non-glandular trichomes and peltate glandular trichomes (X100); e. S. lupulina adaxial surface showing epidermal cells with peltate glandular trichome and non-glandular trichome showing papillae on the trichome cell wall (X1000); f. S. lupulina abaxial surface showing the profuse distribution of peltate glandular trichomes and sparse non-glandular trichomes (X500).
67
18. Scanning Electron Micrographs of Strobilanthes spp. showing trichomes on the abaxial and adaxial surface. a. S. ciliata abaxial surface with peltate glandular trichome (X500); b. S. reticulata abaxial surface showing profusely distributed peltate glandular trichomes and non-glandular trichomes (X100); c. S. reticulata abaxial surface showing papillae on the cell wall of the wing region of non-glandular trichomes (X1000); d. S. reticulata adaxial surface showing non-glandular trichomes and peltate glandular trichomes (X300).
68
19. Petiole anatomy. a. S. barbata; b. S. callosa; c. S. ciliata; d. S.
heyneana; e. S. integrifolia; f. S. ixiocephala; g. S. lupulina; h. S.
reticulata; i. S. sessilis var. ritchiei; j. S. sp. ‘137262’ (X40).
71
20. Petiole anatomy of Strobilanthes spp. showing structure of main vascular bundle a. S. barbata (circular); b. S. callosa (U-shaped).
Col, collenchymas; Cyt, cystoliths; Sph, sphaeraphides; Tc, tannin cell (Illustrations).
72
21. Petiole anatomy of Strobilanthes spp. showing structure of main vascular bundle a. S. ciliata and b. S. heyneana (deeply crescent- shaped). Col, collenchymas; Cyt, cystoliths; Sc, sclereids; Tc, tannin cell (Illustrations).
73
22. Petiole anatomy of Strobilanthes spp. showing structure of main vascular bundle a. S. integrifolia and b. S. ixiocephala (deeply crescent-shaped). Col, collenchymas; Sc, sclereids; Tc, tannin cell (Illustrations).
74
23. Petiole anatomy of Strobilanthes spp. showing structure of main vascular bundle a. S. lupulina (deeply crescent-shaped); b. S.
reticulata (crescent shaped). Col, collenchymas; Tc, tannin cell (Illustrations).
75
24. Petiole anatomy of Strobilanthes spp. showing structure of main vascular bundle a. S. sessilis var. ritchiei (crescent-shaped); b. S.
sp. ‘137262’ (deeply crescent-shaped). Col, collenchymas; Cyt, cystoliths; Sph, sphaeraphides; Tc, tannin cell (Illustrations).
76
25. Petiole anatomy of Strobilanthes spp. showing cystoliths, raphides, sclereids, sphaeraphides, styloids. a. S. ciliata showing sclereids in the ground tissue; b. S. ciliata showing raphides in the cells of ground tissue; c. S. ciliata showing cystoliths in the collenchyma cells of abaxial surface; d. S. callosa showing sphaeraphides in the cells of ground tissue on the adaxial surface; e. S. callosa showing cystoliths in the collenchyma cells and in the cells of ground tissue on the abaxial surface; f. S. ixiocephala showing raphides in the cells of ground tissue; g. S. ixiocephala showing styloids in the collenchyma cells of abaxial surface; h. S. sessilis var. ritchiei showing the arrangement of cystoliths in the collenchyma cells of abaxial surface (X400).
78
26. Petiole anatomy of Strobilanthes spp. showing cystoliths, raphides, styloids. a. S. heyneana showing cystoliths, styloids in the collenchyma cells of adaxial surface; b. S. barbata showing deposition of styloids and cystoliths in ground tissue; c. S. lupulina showing styloids, cystoliths, raphides in the cells of ground tissue;
d. S. lupulina showing styloids, cystoliths, raphides in the collenchyma cells of the adaxial surface (X400).
79
28. TLC studies of selected Strobilanthes spp. a. TLC chromatogram of leaf extract (before spray reagent); b. TLC chromatogram leaf extract (after spray reagent); c. TLC chromatogram of stem extract (before spray reagent); b. TLC chromatogram of stem extract (after spray reagent). S1, S. callosa; S2, S. ciliata; S3, S. integrifolia; S4, S. ixiocephala; S5, S. heyneana.
107
29. HPTLC profile showing the identification of lupeol in methanolic leaf (a) and stem (b) extracts. a. MS1 to MS5 (S. callosa, S. ciliata, S. integrifolia, S. ixiocephala, S. heyneana); b. MS6 to MS10 (S.
callosa, S. ciliata, S. integrifolia, S. ixiocephala, S. heyneana) using toluene:ethylacetate:glacial acetic acid:formic acid (16:2:1:1 v/v/v/v) solvent system scanned at 540nm (white light).
110
30. HPTLC profile during the quantitative analysis of Lupeol in methanolic leaf and stem extracts of selected Strobilanthes spp. a.
MS1 to MS2; b. MS3 to MS4; c. MS5 to MS6; d. MS7 to MS8; e.
MS9 to MS10 scanned at 540nm (white light).
111
31. Antioxidant studies in selected Strobilanthes spp. a. DPPH radicals are scavenged by antioxidants through the donation of protons forming the reduced DPPH; b. ABTS radicals are scavenged by antioxidants through the donation of protons forming the reduced ABTS.
125
LIST OF TABLES Table
No.
Title Page
No.
1. GenBank accession numbers of DNA sequences acquired from NCBI database.
31
2. Primers used for sequencing of ITS, matK, rbcL, trnL and matR genes.
33
3. List of Strobilanthes species studied from Northern Western Ghats of India.
35
4. Comparative stem anatomical characteristics of Strobilanthes species.
52
5. Comparative stem anatomical characteristics of Strobilanthes species.
53
6. Comparative leaf anatomical characteristics of Strobilanthes species.
59
7. Comparative leaf anatomical characteristics of Strobilanthes species.
59
8. Stomatal characteristics of abaxial leaf surface of Strobilanthes species.
69
9. Comparative anatomical characteristics of petiole of Strobilanthes species.
80
10. Comparative anatomical characteristics of petiole of Strobilanthes species.
80
11. Atom percentage of elements of S. ciliata and S. callosa (leaf &
stem).
85
12. Atom percentage of elements of S. integrifolia and S. ixiocephala (leaf & stem).
85
13. Atom percentage of elements of S. heyneana and S. reticulata (leaf
& stem).
85
14. Atom percentage of elements of S. sessilis var. ritchiei and S. sp.
‘137262’ (leaf & stem).
86
15. Atom percentage of elements of S. barbata and S. lupulina (leaf &
stem).
86
18. Fluorescence analysis of leaf and stem powder of Strobilanthes ciliata.
93
19. Fluorescence analysis of leaf and stem powder of Strobilanthes integrifolia.
94
20. Fluorescence analysis of leaf and stem powder of Strobilanthes ixiocephala.
95
21. Fluorescence analysis of leaf and stem powder of Strobilanthes callosa.
96
22. Fluorescence analysis of leaf and stem powder of Strobilanthes reticulata var. reticulata.
97
23. Fluorescence analysis of leaf and stem powder of Strobilanthes sessilis var. ritchiei.
98
24. Fluorescence analysis of leaf and stem powder of Strobilanthes heyneana.
99
25. Fluorescence analysis of leaf and stem powder of Strobilanthes sp.
‘137262’.
100
26. Fluorescence analysis of leaf and stem powder of Strobilanthes lupulina.
101
27. Fluorescence analysis of leaf and stem powder of Strobilanthes barbata.
102
28. pH values of leaf and stem of Strobilanthes species. 103 29. Loss of moisture on drying of leaf and stem powder of Strobilanthes
species.
103
30. Ash values of Strobilanthes species of leaf and stem powder of Strobilanthes species.
104
31. Methanolic extractive values of leaf and stem of Strobilanthes species.
105
32. Rf values of Thin Layer Chromatography of leaf and stem of selected Strobilanthes species.
108
33. Quantity of Phytochemical Lupeol in leaf and stem of Strobilanthes Species
109
34. Compounds detected in methanolic extracts of Strobilanthes species (leaf and stem) using Gas Chromatography Mass Spectrometry (GC-MS).
117
35. IC50 value of L-ascorbic acid and Strobilanthes species (leaf and stem) extract.
127
36. IC50 value of BHT and Strobilanthes species (leaf and stem) extract.
128
37. Strobilanthes species, sample location, voucher and GenBank accession numbers of ITS, matK, rbcL, trnL, and matR sequences.
133
38. Datasets in this study: Number of taxa, aligned characters, constant characters, Parsimony informative characters, Parsimony uninformative characters and best fit nucleotide model.
132
1. INTRODUCTION
Introduction
1
1. INTRODUCTION
The Genus Strobilanthes Blume (Acanthaceae) consists of ca. 450 species (Mabberley, 2017) distributed in the tropical regions of Asia, and found growing naturally in the wet tropical evergreen and moist forests (Wood, 1994, 1995, 1998;
Wood et al., 2003, 2009). The Indian subcontinent has nearly 150 species (Karthikeyan et al., 2009), of these 59 species are noted in peninsular India (Venu, 2006) and 64 species in the Western Ghats (Augustine, 2018). It is the second largest genus of the family Acanthaceae which comprises of perennial flowering herbs and shrubs popularly known as ‘karvi’, in the Sahyadri range of Western Ghats, India. In Strobilanthes, some species bloom annually, others are plietesials i.e. they grow without blooming for several years and then produce huge quantity of flowers, release seeds and die.
Blume (1826) instituted the genus Strobilanthes based on Strobilanthes cernua Blume and other species from Java. Nees (1832, 1847) described various additional species from India. Anderson (1867) described 103 species in Strobilanthes under 7 sections-Endopogon, Eustrobilanthes, Amentianthes, Goldfussia, Secundiflori, Paniculati and Leptacanthus. On the basis of seed number, the morphologically similar species, were referred to separate genera. This approach to generic delimitation of recognizing a broad Strobilanthes has been followed to a great extent by most authors, for example C.B. Clarke (1884), followed Anderson treatment.
Bremekamp (1944) proposed completely new classification. He divided Strobilanthes sensu Anderson (1867) into almost 50 smaller genera principally with distinguishing characters such as structure of the pollen grain and characters of the seed testa. He split Strobilanthes s.l. which occurs in some of the recent Indian Floras, recognised by Bremekamp as Carvia, Didyplosandra, Diflugossa, Goldfussia, Leptacanthus, Mackenziea, Nilgirianthus, Phlebophyllum, Pleocaulus, Supushpa, Thelepaepale and Xenacanthus. However, the majority of the newly described genera were small (less than 15 species or monotypic like Carvia callosa, Supushpa scrobiculata, Thalepaepale ixiocephala), and around 176 species were left unassigned to genera. Bremekamp’s classification was never completed to include all species of
2
Strobilanthes and hence was never accepted by all others. Moreover, many generic characters were inaccurately described and unsatisfactory in defining the group hence, Terao (1983), Wood (1994), Carine and Scotland (1998) rejected Bremekamp’s system pointing out that it was riddled with inconsistences, separating closely related species into separate genera and using characters that were not as clear-cut as he had claimed.
More recent molecular studies, especially Moylan et al. (2004) reported the phylogenetic relationship among the members of sub-tribe Strobilanthinae using the sequence data of Chloroplast trnL-F, nuclear ribosomal internal transcribed spacer (ITS) and morphology. Parsimony and maximum likelihood analysis trnL-F indicated that the Strobilanthinae sensu Bremekamp formed a single monophyletic group, which could not be easily split into smaller component genera. Consequently, a single expanded genus Strobilanthes is generally accepted and the present study follows this approach treating Strobilanthes a broad, well-supported genus.
The pioneering work on the species diversity of Strobilanthes in India was done by a well-known German botanist and natural philosopher Christian Gottfried Daniel Nees von Esenbeck (1832, 1847). Nees described 45 species of Strobilanthes from India and 11 species of them from Western Ghats. Wight (1838–1853) described 4 species of Strobilanthes and several new species of flowering plants. Beddome (1868–1883) added 8 species to Strobilanthes and other flowering plants. Anderson (1867) described 55 species from India. Clarke (1884, 1907) added 53 species from India and other south-east Asian countries Bentham (1851, 1861, 1876) added two species from India. Gamble (1888, 1923, 1924) described two species from Western Ghats. John R.I. Wood the world authority of Strobilanthes presently as a Senior Research Associate in the Department of Plant Sciences, University of Oxford. He has described more than 50 species of Strobilanthes and his considerable work to the Genus has added three species from Western Ghats (1994, 1995, 1998, 2003, 2009).
Carine and Scotland (1998) worked on pollen morphology of Strobilanthes and its implication in the species delimitations is significant. Carine and Scotland (2002) noted sixty-five species distributed in south India and Sri Lanka where most of them
Introduction
3
are endemics. Venu (2006) described two species from south India and several other field taxonomists added new species to the genus Strobilanthes (Augustine, 2018).
Strobilanthes is derived from two Greek words – "strobilos" which means cone and "anthos" which means flower, and refers to the plant's inflorescence in which the floral characteristics namely; combination of filaments united to form a membranous sheath, a bifid stigma with a reduced posterior lobe, and two bundles or rows of hair on the inner posterior corolla wall that retains the style, which differs from other members of Acanthaceae (Bremekamp, 1944; Carine & Scotland, 1998, 2002); Manktelow, 2000; Wang & Blackmore, 2003; Carine et al., 2004). There is a complex structure ‘Stapetal curtain’ which divides the flower due to the close synorganisation between the filaments and corolla tube and this structure is not only present in the tribe Ruellieae but also elsewhere in the family Acanthaceae (Moylan et al., 2004).
The blooming cycle varies from four years (S. integrifolia Kuntze), seven years (S. barbata Nees, S. ixiocephala Benth., S. sessilis Nees var. ritchiei C.B.
Clarke, S. reticulata Stapf, S. callosa Nees, S. sp.), to twelve years (S. kunthiana T.
Anderson ex Benth.) or even sixteen years (S. scrobiculata Dalzell ex C.B. Clarke), after which the seeds disperse and the plants die (Venu, 2006; Mascarenhas &
Janarthanam, 2013; Augustine, 2018). The pliestesial flowering and complex reproductive characters makes the genus one of the taxonomically most difficult groups (Wood & Scotland, 2009). Hence, the taxonomy of the genus is still under improvement.
A number of species are cultivated for their attractive flowers and foliage as ornamental plants (S. auriculata var. dyeriana (Mast.) J.R.I. Wood, S. anisophylla var. isophylla (Nees) J.R.I. Wood) while, some are used for their medicinal value.
Strobilanthes ciliata Nees is a potential medicinal plant, endemic to Western Ghats of India and is widely used in ayurveda as a source of the drug ‘Sahacharya’ (Venu, 2006).
4
The indigenous people from Moreh area of Manipur uses the inflorescence of S. auriculata as a rare indigenous food plant as there is a taboo that it increases stamina and immunity to protect from several ailments and diseases (Ningombam et al., 2014). The indigenous and traditional food becomes an intrinsic part of the people of North-east and Manipuris (Singh, 2011).
OBJECTIVES OF THE PRESENT INVESTIGATION
In the present study, the ten Strobilanthes species are all endemic to India. The northern Western Ghats (NWG) has not been a well explored area of the Western Ghats, India with respect to Strobilanthes for more than a decade. Therefore, documenting the species of Strobilanthes from NWG became important. Several of the genera recognised by Bremekamp that occur in the present study are Carvia, Mackenziea, Nilgirianthus, Pleocaulus and Thelepaepale. The species in the present study under Bremekamp’s grouping are as Carvia callosa, Mackenziea integrifolia, Nilgirianthus barbata, N. ciliata, N. heyneana, N. lupulina, N. reticulata, N. sp., Pleocaulus ritchiei and Thelepaepale ixiocephala. All these segregated genera are grouped under a single genus Strobilanthes (Wood, 1994; Carine & Scotland, 1998).
The Identification of species of Strobilanthes in their vegetative stage is often found to be challenging due to the prolonged intervals between blooming. Distinct anatomical variations can form taxonomic characters to distinguish the species at vegetative stage. In the present study, Strobilanthes species render difficulties to identify during vegetative stage i.e. without flowers, since blooming period of this genus varies. Also, no comprehensive anatomical study of vegetative structures of Strobilanthes species exists which can form a key character. Although the vegetative anatomy of the family has been reviewed by Metcalf and Chalk (1950); Remadevi et al. (2006); Patil and Patil (2011, 2012); Sarpate and Tupkari (2012a); Tripp and Fekadu (2014), none of the works include the complete anatomy of Strobilanthes.
Taking this into consideration, this study presents a comparative anatomical account of stem, leaf and petiole of 10 species of Strobilanthes from Northern Western Ghats of India. The anatomical characterization was carried out to help in identification of different species at vegetative stage (Fernandes & Krishnan, 2019a).
Introduction
5
The Genus Strobilanthes contains several hidden chemical constituents which are medicinally important and not completely explored. Local species of Strobilanthes are used by the traditional people to cure different types of diseases. ‘Lupeol’ is one such triterpenoid found in Strobilanthes which have great therapeutic value. Do all species of Strobilanthes possess this compound? Are there any other bioactive compound or marker compounds other than lupeol in Strobilanthes?
Moylan et al. (2004) reported the phylogenetic relationships among the members of Strobilanthinae using the sequence data of Chloroplast trnL-F, nuclear ribosomal internal transcribed spacer (ITS) and morphology. Parsimony and maximum likelihood analysis trnL-F indicated that Strobilanthinae sensu Bremekamp as a single monophyletic group and which could not be easily divided into smaller component genera. Since, Strobilanthes species are with high potential medicinal value and differential flowering periods it is important to understand the phylogenetic relationship among the species using molecular data. Molecular data obtained during this study will help in understanding the phylogenetic relationship among the species.
Considering the above, the following specific objectives are proposed:
Objectives
1. Collection and documentation of Strobilanthes species from Northern Western Ghats of India.
2. Anatomical characterization (stem, leaf and petiole) of collected Strobilanthes species to distinguish them at vegetative stage.
3. Biochemical analysis of selected Strobilanthes species for identification and quantification of bio-active compounds using GC-MS / HPTLC.
4. Molecular sequencing of Strobilanthes species from Northern Western Ghats to understand phylogenetic relationship among the species.
2. REVIEW OF
LITERATURE
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2. REVIEW OF LITERATURE
2.1. Strobilanthes and Anatomy
Strobilanthes is a widely known genus taxonomically with ca. 450 species as reported (Mabberley, 2017). The anatomical studies in the Genus Strobilanthes especially with regards to vegetative characters are relatively scarce. In many taxa, ecological adaptation may cause extreme morphological variations, leading to false species concepts. Also, in Strobilanthes some taxa show their blooming/flowering cycle varying from 4 to after a period of 16 years, so proper identification with morphology becomes very difficult. In this case, anatomical studies can provide characters for identification of species at vegetative growth.
The comparative floral anatomy on sub-tribe Strobilanthinae sensu Bremekamp (1944) with a special mention of structural variation in inflorescence was carried out. Certain characters were identified which would provide evidences for phylogentic relationships within the group such as the presence or absence of an accessory bud, the presence or absence of a hollow style, the presence or absence of an abscission layer, the number of stamens comprising the androecium and the pattern of filament detachment. ‘Stapetal curtain’ is a complex structure which partitions the flower due to the close synorganisation between the filaments and corolla tube and this structure seems not only related to tribe Ruellieae but also have a wide distribution throughout the family Acanthaceae (Moylan et al., 2004).
Tripp et al. (2013) revised the classification of tribe Ruellieae and described subtribe Strobilanthinae. It emphasises to place all the segregate genera under an expanded genus Strobilanthes s.l. which will resolve the entire taxonomic problem.
The anatomical knowledge of genera within the tribe Ruellieae was analyzed to study the comparative leaf and stem anatomy to understand the similarities and differences between the taxa (Tripp & Fekadu, 2014). The leaf anatomical study was mostly uniform and conservative across the subtribes of tribe Ruellieae, and same as for stem and petiole anatomy with focus on the major anatomical feature in Acanthaceae i.e.
‘Cystoliths’ (Tripp & Fekadu, 2014).
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Carine and Scotland (1998) studied the pollen morphology of Strobilanthes from southern India and Sri Lanka. Basically, two classes of shapes of pollen are recognised as spheroidal and ellipsoid (prolate/subprolate). In ellipsoid pollen grains, all were tricolporate and had pseudocolpi and distinguished primarily on the basis of tectal ornamentation. Spheroidal pollen grains recognised primarily on variation in number, distribution and form of apertures, and on sexine structure. It was observed that the variation in pollen morphology was much higher as compared to the previously studied Bremekamp (1944). Pollen morphology of Strobilanthes in China was studied for the taxonomic implications and recognised two pollen shapes as observed (Carine & Scotland, 1998). Three new pollen types were described, hence expanding the palynological diversity of the genus (Wang & Blackmore, 2003).
2.2. Anatomy and its significance in morphology
In Genus Satanocrater anatomical and morphological features reflects its adaptation to xeric environment. The anatomical features adapted to xeric conditions support the morphological and molecular data and helps to place Satanocrater in the tribe Ruellieae (Tripp & Fatimah, 2012). The Occurrence of cystoliths, acicular fibres, raphides is the significance of family Acanthaceae (Metcalf & Chalk, 1950; Inamdar et al., 1990; Kuo-Huang & Yen, 1996; Patil & Patil, 2011; Tripp & Fekadu, 2014).
The morphological adaptations are reflected in the anatomy of the plant. According to Fahn (1982) brachysclereids were produced in plants as responses towards physiological disturbances. Sclereids were reported to provide mechanical safety by giving supplementary vigour and firmness to the plants (Rao, 1957). Members of Acanthaceae, the two genera Ruellia and Dianthera put forth the distinct anatomical features within them and were quite interesting to know the similar combination of characters which are peculiar to the family (Holm, 1907).
Studies on anatomy prove that, it provides evidence for assessment in relation to systematics. The comprehensive study on nodal and leaf anatomy of Bonnetiaceae provided important characters such as foliar vascular bundles enveloped by a sheath composed of two concentric regions as in, an inner region consisted of multiple layers of fibres and an outer specialized endodermis composed of thin-walled cells with Casparian strips, which helped in segregation of Bonnetiaceae from Theaceae
Review of Literature
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(Dickison & Weitzman, 1996). Comparative stem anatomical study in Eriosyce sensu lato based on a combination of characters could be used as a basis for systematic of infrageneric taxa (Nyffeler & Eggli, 1997). On view of exploiting the systematic and taxonomy of seven west African species of Momordica of the family Cucurbitaceae, petiole and stem anatomy was carried out (Aguoru & Okoli, 2012). Some of the important evidences reported were the occurrence of grit cells below the epidermis of petiole, brachysclerides interspersed in cells of epidermal layer of petiole and also various other features were used to delineate the taxa in tribe Cucurbitoideae.
Anatomical characters can be used in defining the genus level placement as described (Noor-Syaheera et al., 2015). Certain characters studied in Acanthaceae members for the leaf anatomy viz. the presence and absence of brachysclereids, hypodermal layers, cystoliths, types of stomata and trichomes, shape of vascular bundles in midrib are unique and could not match with genus Avicennia with the characters which was placed into Acanthaceae, suggesting a revision on its placement.
Similarly, leaf anatomical studies of Polygonum s.l. in Iran were used to distinguish the different taxa having great morphological similarities with some of the species (Keshavarzi et al., 2012). Stem anatomy have solved problems in identification of taxa and the relevance of anatomical characters are been used in taxonomy of genus Rhipsalis (Calvente et al., 2008).
The importance of petiole anatomy with distinct anatomical structures shows either the close similarity or the distinctiveness of species to species (Olowokudejo, 1987). Howard (1962, 1979) had put forth the terminology and ontogeny of petiolar vascular systems. Hare (1943) reported the importance of petiole vasculature basically in response to mechanical needs; it holds the weight of the leaf as well as the wind action. However, certain factors of adaptation to the habitat and other factors must have brought diversity in petioles with immense variety of anatomical structures of taxonomic value (Hare, 1943).
2.3. Stomata and Trichomes
Studies have shown that the Stomatal Index (SI) of leaves differs with the light intensity encountered by the plant; the lower light, the lower the SI (Schoch et al.,
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1980). Glandular trichomes are also known as glands, which are concerned in the secretion of a variety of substances, e.g. salt solution, lipids (Elleman & Entwistle, 1982). They are also known to function in protection of the plants (Levin, 1973).
Trichomes also control the water cutback of the plants through temperature regulation, by either minimizing the intake of sunlight or by maximizing its dissipation once it has been absorbed (Johnson, 1975). Thus, the distribution of trichomes must be regulated by the habitat of the species (Agrawal et al., 2009; Hoof et al., 2008; Kenzo et al., 2008; Perez-Estrada et al., 2000; Ehleringer, 1984;
Coley, 1983; Sobrado & Medina, 1980).
2.4. Traditional Uses of Strobilanthes species
Various traditional uses have been recorded in the Genus Strobilanthes which contains several hidden chemical constituents which and are medicinally important.
Local species of Strobilanthes are used by the traditional people and the Adivasi to cure different types of diseases.
2.4.1. Strobilanthes auriculata Nees
S. auriculata is a pleiestial plant found on the hillocks of Moreh area of Manipur. The indigenous people collect the inflorescence of S. auriculata and prepare it in the form of condiments cooked or steam with indigenous small fish curry (Gnatokpo thonoba).
Other way, the inflorescence is steam ‘Poknom’ and taken orally to increase stamina and immunity against diseases. They do so since, there is a taboo of protecting them from various ailments. About 150g of inflorescence and equal quantity of
‘Phlogoconthus thyrsiformisi’ leaves are boiled together and the obtained decoction is then mixed with honey, taken to increase immunity against the cardiovascular diseases. Hence, S. auriculata is a medicinally important plant for the people of Manipuris (Ningombam et al., 2014).
2.4.2. Strobilanthes asperrima Nees
S. asperrima is native of India, Japan, Malaysia and rest of Asia. It is used as an anti- diabetic agent by the traditional healers of Chhattisgarh. Traditional knowledge put forth’s that the leaves are used as hypoglycaemic (Samal, 2013).
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2.4.3. Strobilanthes callosa Nees
S. callosa is a synonym of Carvia callosus commonly known as ‘Karvi’ found in the low hills of Western Ghats all along the west coast of India. The local adivasi and villagers uses S. callosa leaves for the treatment of inflammatory disorders, known as
‘Karvi’ in Mararthi (Sarpate & Tupkari, 2012). The juice obtained from the crushed leaves is believed to cure of stomach ailments (Agarwal & Rangari, 2003). Also, the leaves are rubbed on the body during cold and fever. It is used as a diuretic to treat arthritis (Sarpate & Tupkari, 2012). The stem bark is used as an emollient in formulations to cure the painful and ineffectual attempts to urinate or defecate and externally, the paste is applied for mumps and the flowers are also used as vulnerary (Singh et al., 2002).
2.4.4. Strobilanthes ciliata Nees
S. ciliata is one of the endemic species of Western Ghats, India that have got several medicinal properties. The plant has strong aroma and widely used in Ayurveda as the source of drug ‘Sahacharya’ (Venu, 2006). All plant parts serve as medicine. Roots are useful in the treatment of rheumatalgia, lumbago, sciatica, limping, chest congension, strangury, fever, leucoderma, skin diseases, inflammations, cough, bronchitis, odontalgia and general debility (Venkatachalapathi & Ravi, 2013). Stem is widely used for whooping cough, bronchitis, dropsy, leprosy and pruritus (Venkatachalapathi & Ravi, 2012).
2.4.5. Strobilanthes crispa Blume
S. crispa originated from Madagascar to Indonesia. It is commonly known as ‘Pokok pecah kaca’ in Malaysia. This plant is traditionally used to cure various ailments like cancers and diabetes (Menant, 1980; Fadzelly et al., 2006).
2.4.6. Strobilanthes cusia (Nees) Kuntze
S. cusia an herbaceous plant, is native of northeast India, Myanmar, Thailand, and southern China (Gu et al., 2014). This plant is known as Assam indigo, ‘Kum’ in Manipuri. It is an important medicinal plant species used traditionally in the state of Manipur, India. It is a shrub, glabrous, growing upto 5–6 ft tall in wild (Shahni &
Handique, 2013). This plant is often cultivated for the dye in Manipur valley. Leaves
11
are used to treat influenza, epidemic cerebrospinal meningitis, encephalitis B, viral pneumonia, and mumps (Tomonori et al., 2004). The root of S. cusia known as ‘Da- Ching-Yeh’ is widely used in the Chinese medicine to treat mumps, influenza epidemic, viral pneumonia, cerebrospinal meningitis, encephalitis B (Tomonori et al., 2004). It is also used for sore throat, inflammatory diseases with redness of skin, etc.
The entire fresh plant of S. cusia has anti-fungal activity and used to treat athlete’s foot.
2.4.7. Strobilanthes heyneana Nees
S. heyneana is a medicinal plant used in ayurvedic medicinal preparations in southern India. The plant possesses aspirin type of analgesic, anti-inflammatory and immunosuppressant activities (Anvitha & Monnanda, 2015).
2.4.8. Strobilanthes ixiocephala Benth.
S. ixiocephala is commonly known as ‘Waiti’ in Marathi and grows gregariously on the hills of Sahayadri (Agarwal & Rangari, 2003). Traditionally over the ages, the tribals have used the plant for the treatment of inflammatory disorders. This confirms its use in folk medicine as a valid anti-inflammatory and antimicrobial herbal drug with anti-rheumatic activity (Sarpate & Tupkari, 2012a).
2.4.9. Strobilanthes kunthiana T. Anderson ex Benth. and Strobilanthes cuspidata T. Anderson
S. kunthiana shrubs are found in Shola forest of Western Ghats. It is the species which flowers once in 12 years period. It is commonly known as ‘Neelakurinji’. The tribal people of Nilgiri hill uses S. kunthianus and S. cuspidatus to treat joint pains and inflammations (Desu et al., 2011, 2012).
2.5. Phytochemical studies in Strobilanthes species
The species of Strobilanthes are studied with regards to its medicinal properties and have been investigated for their therapeutically important compounds. One of the important phytochemical constituent of Genus Strobilanthes is ‘Lupeol’. This compound is a triterpenoid, which has been used as anti-protozoal, anti-inflammatory, anti-tumour and chemo-preventive agents (Gallo & Sarachine, 2009). It is one of the
Review of Literature
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components in anti-allergic formulations (Kovalenko et al., 2008). Also serves as a function in anti-aging creams, lotions, gels, and lip balms at levels of 0.2–3% w/w due to its ability to maintain the skin texture and integrity by promoting epidermal regeneration and replenishing cutaneous anti-oxidant enzymes depleted by environmental toxins. Some of the species investigated for their therapeutic properties are listed below.
2.5.1. Strobilanthes asperrima Nees
Phytochemical test reveals that there is presence of alkaloids, glycoside, flavonoids, phenolic and tannin compounds. It is used in the treatment of goitre, antitumor’s, tuberculosis, bactericidal, fungicidal and also possesses anti-oxidant activity (Samal, 2013).
2.5.2. Strobilanthes callosa Nees
Phytochemical investigation showed ‘Lupeol’ as a major ingredient by using 50%
petroleum ether as solvent (Agarwal & Rangari, 2003). The anti-inflammatory and antimicrobial activities of the benzene and ethanol extract of S. callosa, isolated triterpenoids were investigated in rat system. These results confirm the use of this plant in folk medicine as an anti-inflammatory and antimicrobial herbal drug (Singh et al., 2002). Also, the phytochemical constituents such as alkaloids, tannins, flavanoids, carbohydrates, saponins, steroids, terpenoids, phenolics, coumarins and fixed oil were present in the S. callosa extract. HPTLC method was used and validated for the quantification of lupeol (Sarpate & Tupkari, 2012). A triterpenic alcohol 19α-H- lupeol has been isolated from S. ixiocephala as the major component (Agarwal &
Rangari, 2003).
2.5.3. Strobilanthes ciliata Nees
The phytocompound lupeol isolation and quantification was done using HPTLC method and found to be the major constituent of S. ciliata (Venkatachalapathi &
Subban, 2012). Evaluation of antimicrobial activity using petroleum ether and methanolic extracts of S. ciliata was studied (Venkatachalapathi & Subban, 2013).
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2.5.4. Strobilanthes crispa Blume
S. crispa is proven scientifically that it contains high antioxidant properties. The presence of alkaloids, catechins, caffeine, flavonoids and tannins in the leaves of S.
crispa is contributing to the medicinal properties. This further can help to enhance the defense system especially towards the degenerative diseases (Ismail et al., 2000).
Various chemical constituents have been reported from S. crispa using different biochemical methods. Chemical compounds such as verbascoside (Soediro et al., 1987; Ahmed, 1999), p-hydroxy benzoic acid, ferulic acid, syringic acid, gentisic acid (Soediro et al., 1987), tritriacontane, stigmasterol (Afrisal, 2008) were identified.
Hyperglycaemic activity of S. crispa in the rat model system was reported (Fadzelly et al., 2006).
Anti-bacterial activity of methanolic extracts of S. crispa against Bacillus cereus was studied (Muskhasli et al., 2009). Jaksa et al. (2005) evaluated the effect of administration of S. crispus extract on the histology and tumour marker enzymes, glutathione S-transferase and uranyl diphosphate glucoronyl transferase in rat liver induced with hepatocarcinogen diethylnitrosamine and 2-acetylaminflourene. Even though there are various types of bioactive compounds isolated and identified from S.
crispa their contribution towards medicinal uses or pharmacological activities were not fully studied (Nurraihana & Norfarizan-Hanoon, 2013).
2.5.5. Strobilanthes cusia (Nees) Kuntze
Li et al. (1993) isolated seven compounds from whole plant of S. cusia: three triterpenes – lupeol, betulin and lupenone, two indole alkaloids – indigo and idirubin and two quinazolinone alkaloids – 4(3H)-quinazolinone and 2,4(1H,3H)- quinazolinedione. It also demonstrated that indirubin has anticancer activity and 4(3H)-quinazolinone has hypotensive action. Antibacterial activity of methanolic leaf extract was carried out to study the control of microbial pathogens (Shahni &
Handique, 2013). Three indole alkaloid glycosides, Strobilanthosides A–C, two known indole alkaloid glucosides and five phenylethanoid glycosides were isolated from aerial parts of S. cusia (Gu et al., 2014).
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2.5.6. Strobilanthes ixiocephala Benth.
The compound 19 α-H-lupeol was isolated as a major component (Agarwal &
Rangari, 2001). Essential oils from the inflorescence were reported as caryophyllene, fenchyl acetate, cardinal and new sesquiterpene ixiocephol which was studied from NMR and MS data (Agarwal & Rangari, 2003a).
2.5.7. Strobilanthes kunthiana T. Anderson ex Benth.
Phytochemicals extracted from S. kunthiana shrubs are phyto-constituents like carbohydrates, triterpenoids, phytosterols, flavonoids and tannins. The presence of these multiple phytoconstituents together makes them potential anti-inflammatory and anti-arthritic (Desu et al., 2011, 2012). Studies were confirmed through in-vitro methods done on wistar rats. Similar properties are known to occur in S. cuspidatus (Desu et al., 2012). Singh et al. (2014) evaluated antioxidant property by H2O2 and DPPH method in S. kunthiana flowers. The ethyl acetate, n-Butanol extract exhibited promising significant antioxidant activity on the other hand, n-hexane extract was devoid of any activity.
2.6. Molecular Studies
Traditional classification of plants was based on the morphological characters to their respective class, orders, families, genera and species and until recently it is followed in systematics. The development of the molecular techniques like molecular hybridization, cloning, restriction endonuclease digestions, protein and nucleic acid sequencing have provided new tools in phylogenetic relationships (Hamby &
Zimmer, 1992). Ning et al. (2012) carried out genetic diversity studies in the populations of S. cusia in the Fujian Province of China using RAPD markers. High level of genetic diversity was noticed among the different populations of Fujian.
2.6.1 Molecular Phylogeny
Molecular phylogenies are an accepted integral part of systematics and the results of such studies is a gene tree, hypothesizing relationships among genes or genomes (Doyle, 1992). It is now a routine, to provide the insights into the systematic relationship at all levels of plant phylogeny. The phylogenetic trees constructed are available at all levels of taxonomic hierarchy which plays an essential role in
15
comparative studies of various fields like ecology, molecular evolution and comparative genetics (Soltis & Soltis, 2000).
Phylogeny is essential to provide a better understanding of both patterns and processes of evolution, it also supplies the comparison of taxa in a phylogenetic context and provides the most meaningful insights into biology (Harvey & Pagel, 1991; Avise, 1994). The phylogenetic relationship is most accurate when aware of the past, present distribution patterns and knowing the biogeography of plants is useful tool of phylogenist (Thorne, 1989). Soltis et al. (1997) provided nuclear based phylogenetic hypothesis for angiosperms for comparison with the rbcL tree (Chase et al., 1993).
Internal transcribed spacers (ITS) of the ribosomal DNA are used to understand the phylogenetic relationship among plant species (Chase et al., 1993;
Clegg et al., 1994; Clark et al., 1995; Alvarez and Wendel, 2003). ITS region of 18S–
26S nuclear ribosomal DNA has established as a useful source of characters for phylogenetic studies in many angiosperm families. It can be easily amplified by PCR and also sequencing is possible using universal primers. It may be noted that ITS have a relatively minute site affected by insertions and deletions among sequences that can be used in phylogenetic analysis. ITS have enhanced the understanding of angiosperm phylogeny in several groups as: (a) substantiated the earlier unexpected findings; (b) determined the conflicts between other data sets (c) improved the resolution of species relationships and (d) provided direct evidence of reticulate evolution (Baldwin et al., 1995).
The maturase K (matK) gene is a gene with potential contributions to plant molecular systematic and evolution (Johnson & Soltis, 1994, 1995; Steele & Vilgalys, 1994; Liang & Hilu, 1996). It is located within the intron of the chloroplast gene trnK, on large single-copy section adjacent to the inverted repeat (Hilu & Liang, 1997). It has been used efficiently in understanding the systematics in the family’s Saxifragacaee (Johnson & Soltis, 1994, 1995), Poaceae (Liang & Hilu, 1996) and Leguminosae (Wojciechowski et al., 2004). The matK gene stands out among genes used in angiosperm systematics in its (a) nucleotide substitutions; (b) non-
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synonymous mutations; (c) insertion/deletion (Soltis & Soltis, 1998). The sequence information received from matK gene constructs an angiosperm tree more robust and congruence is high between matK tree and those based on multiple genes representing one, two or all three genomes (Soltis et al., 2003; Zanis et al., 2002; Kuo et al., 2011).
The rbcL gene encoding the large subunit of ribulose 1,5-bisphosphate carboxylase has provided the most extensive molecular data set for plant systematics (Chase et al., 1993). It provided opportunity to study plant evolutionary history (Clegg, 1993). It has helped to place a number of intricate angiosperm families including Podostemaceae, Aphloiceae and Ixerbaceae, and have strongly supported the topology for angiosperms (Soltis et al., 2000).
The chloroplast DNA regions have been widely used in phylogenetic studies and trnL gene is been used from intra and interspecific levels of subfamily and tribe levels (Mes et al., 2000). The sequence analysis of the trnL-trnF and trnL intron was used to distinguish the Cinnammomum species among C. cassia (Nees & T. Nees) J.Presl, C. zeylanicum Breyn., C. burmannii (Nees & T. Nees) Blume and C. sieboldii Meisn. (Kojoma et al., 2002). The trnL-trnF intergenic spacer was used for phylogenetic reconstruction in the Juncaceae and defined the major clade within Luzula and Juncus (Juncaceae) (Drabkova et al., 2004). The use of trnL gene has been also used in defining the relationships of Morus (Tribe Moreae, Moraceae) in 13 species (Nepal & Ferguson, 2012).
2.7. Molecular Phylogeny in Acanthaceae
McDade and Moody (1999) underlined the use of molecular data in the systematic studies of Acanthaceae to mark out the phylogenetic relationships and determine the lineages among the members of the family using the sequence data from the intron and spacer trnL-trnF chloroplast region. The results suggest strong support for monophyly of four major lineages within Acanthaceae as defined; the Acanthus, Barleria, Ruellia and Justicia.
The sequence data from the (nr-ITS) region and intron and spacer of the trnL- trnF cp region was used to study phylogenetic relationships within the vast family
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Acanthaceae (McDade et al., 2000). With respect to the phylogenetic relationships, the ITS hypothesis was completely congruent with the trnL-trnF results with some exceptions like Crossandra pungens Lindau. and two Acanthus species, which are strangely placed by nuclear ribosomal internal transcribed spacer data. The analysis does provide considerable resolution of relationships as monophyletic lineages and are arranged with respect to the previous classifications.
McDade et al. (2008) used sequence data from nr-ITS and four chloroplast noncoding regions, and Parsimony and Bayesian methods of analysis for comprehensive understanding of phylogenetic relationships among lineages of Acanthaceae s.l. which included all the known lineages of the family and Andrographideae. This also included 13 genera whose relationships remained puzzled. The results strongly supported most of the aspects including the inclusion of Avicennia in Acanthaceae. The use of molecular markers can provide an effective tool to assess the genetic polymorphism in assessing the intraspecific genetic variability in the mangrove species Acanthus ilicifolius Linn. to design the conservation strategy (Lakshmi et al., 1997).
Phylogenetic relationships within tribe Acantheae were studied from DNA sequence data of four regions (a) nr ITS; the chloroplast (b) rps 16 intron (c) trnG-S spacer and (d) trnL-F intron and spacer for 18 of 20 recognised genera and 82 of ca.
500 species. The results based on parsimony and Bayesian analyses were completely congruent and thus provided strong support for monophyly of ‘one-lipped corolla’
and ‘two-lipped corolla’ (two sub-lineages of Acantheae), imitating acceptable differences in corolla morphology (McDade et al., 2005).
The relationships of tribe Whitfieldieae was studied using molecular sequence data for two chloroplast loci (ndhF gene, trnL-trnF spacer and intron). Manktelow et al. (2001) studied morphological data which included the pollen structure (SEM) collected for enigmatic genera Whitfieldia, Chlamydacanthus and Lankesteria assessed in phylogenetic perspective. The results substantiate that Chlamydacanthus and Whitfieldia are closely related, ultimately Lankesteria is sister to these two genera together and the three genera included an expanded tribe Whitfieldieae.
