Himalayan Medicinal Plants: Advances in Botany, Production & Research

By Mohar Singh

The Himalayan Region is a mega hot spot for biological diversity. It supports over 1,748 plants species of known medicinal value. This title focuses on origin and distribution of Himalayan herbs, their medicinal potential, industrial significance, and research advancements pertaining to molecular breeding and omics-based approaches.

• Discusses evolved secondary biochemical pathways often in response to specific environmental stimuli
• Reviews conservation efforts
• Presents an in-depth analysis of 12 key species

• Language: English
• Publisher: Academic Press
• Release date: Jan 20, 2021
• ISBN: 9780128234303

Book preview

Himalayan Medicinal Plants

Advances in Botany, Production & Research

Editors

Nikhil Malhotra

ICAR-National Bureau of Plant Genetic Resources Regional Station, Shimla, India

Mohar Singh

ICAR-National Bureau of Plant Genetic Resources Regional Station, Shimla, India

Table of Contents

Cover image

Title page

Copyright

Contributors

Chapter 1. Introduction

Chapter 2. Aconitum heterophyllum

2.1. Introduction

2.2. Origin and distribution

2.3. Medicinal properties

2.4. Phytochemistry

2.5. Adulteration and substitution

2.6. Omics-based advancements

2.7. Plant tissue culture–assisted progress

2.8. Conclusion and future perspectives

Chapter 3. Arnebia euchroma

3.1. Introduction

3.2. Physiological structure

3.3. Medicinal properties of Arnebia euchroma

3.4. In vitro production strategies

3.5. Physiobiological factors affecting shikonin production

3.6. Omics advancement to unravel the biosynthetic machinery of shikonin

3.7. Conclusion and future perspectives

Chapter 4. Dactylorhiza hatagirea

4.1. Introduction

4.2. Origin and distribution

4.3. Medicinal uses

4.4. Dactylorhin

4.5. Biotechnological interventions in Dactylorhiza hatagirea

4.6. Conclusion and future anticipation

Chapter 5. Fritillaria roylei

5.1. Introduction

5.2. Origin and distribution

5.3. Phytochemistry

5.4. Therapeutics potential/biological significance

5.5. In vitro conservation/morphogenesis

5.6. Omics advancements

5.7. Conclusions and future perspectives

Chapter 6. Picrorhiza kurroa

6.1. Introduction

6.2. Origin and distribution

6.3. Phytochemistry of Picrorhiza kurroa

6.4. Medical significance

6.5. Tissue culture status of Picrorhiza kurroa

6.6. Omics advancements in picrosides biosynthesis

6.7. Conclusions and future perspective

Chapter 7. Podophyllum hexandrum

7.1. Introduction

7.2. Taxonomy

7.3. Adulterant

7.4. Geographical distribution and status

7.5. Morphology

7.6. Chemical constituents

7.7. Molecular advancements

7.8. Good agricultural and collection practices

7.9. Medicinal uses

7.10. Conclusion

Chapter 8. Rauwolfia serpentina

8.1. Introduction

8.2. Origin and distribution

8.3. Medical significance of Rauwolfia

8.4. Phytochemical constituents of Rauwolfia

8.5. Omics strategies and advancements

8.6. Conclusion

Chapter 9. Rhodiola imbricata

9.1. Introduction

9.2. Geographical distribution

9.3. Biochemical composition

9.4. Pharmacological properties

9.5. Cultivation and propagation of Rhodiola imbricata

9.6. Genetic diversity

9.7. Omics

9.8. Conclusion and future prospects

Chapter 10. Saussurea lappa

10.1. Introduction

10.2. Botanical identification and classification

10.3. Botany of Saussurea lappa

10.4. Origin and geographical distribution

10.5. Biochemical/analytical properties

10.6. Therapeutic attributes of Saussurea lappa

10.7. Conservation status

10.8. Trade

10.9. Omics advancements in Saussurea lappa

10.10. Conclusion and future perspectives

Chapter 11. Stevia rebaudiana

11.1. Introduction

11.2. Medical significance of Stevia

11.3. Breeding attempts for Stevia improvement

11.4. Biochemical profile analysis of Stevia

11.5. Details on steviol glycoside biosynthesis pathway and the associated genes

11.6. Approaches to improve steviol glycosides in Stevia rebaudiana

11.7. Conclusion and future prospects

Chapter 12. Swertia chirayita

12.1. Introduction

12.2. Cytological studies of genus Swertia

12.3. Genetic diversity studies

12.4. Bioactivity and medicinal uses

12.5. Tissue culture studies in Swertia chirayita

12.6. Ecological status

12.7. Conclusion

Chapter 13. Trillium govanianum

13.1. Introduction

13.2. Classification, origin, distribution, and cytotaxonomy

13.3. Biochemical analysis

13.4. Medical significance

13.5. Molecular breeding and genetic mapping

13.6. Molecular database studies

13.7. Threat

13.8. Conclusions and future perspectives

Chapter 14. Valeriana jatamansi

14.1. Introduction

14.2. Origin and distribution

14.3. Morphology

14.4. Botanical classification

14.5. Agronomy technique

14.6. Phytochemistry

14.7. Conservation approach

14.8. Molecular characterization

14.9. Omics approach

14.10. Formulated products

14.11. Conclusion and future perspectives

Chapter 15. Withania somnifera

15.1. Introduction

15.2. Botany

15.3. Origin and distribution

15.4. Medical significance

15.5. Major challenges in ashwagandha

15.6. Phytochemicals

15.7. Omics advancements

15.8. Crop improvement interventions

15.9. Conclusions and future perspectives

Chapter 16. Zanthoxylum armatum

16.1. Introduction

16.2. Distribution

16.3. Morphocytological studies

16.4. Biochemical analysis

16.5. Mineral elemental analysis

16.6. Genomics

16.7. Medical significance

16.8. In vitro regeneration

16.9. Conclusion

Author Index

Subject Index

Copyright

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Contributors

Ashrita

Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Academy of Scientific and Innovative Research, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Ashwani Bhardwaj,     Defence Institute of High Altitude Research, Defence R & D Organization, Leh, Ladakh, India

Pushpender Bhardwaj,     Defence Institute of High Altitude Research, Defence R & D Organization, Leh, Ladakh, India

Kirti Chawla

Plant Tissue Culture and Genetic Engineering, National Agri-Food Biotechnology Institute (NABI), Mohali, Punjab, India

Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India

Aditya Dogra,     Department of Biotechnology, Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh, India

Varun Garla,     Department of Information Technology, Shoolini Institute of Life, Sciences and Business Management, Solan, Himachal Pradesh, India

Surendra Prakash Gupta,     Department of Life Science, Shri Vaishnav Institute of Science, Shri Vaishnav Vidhyapeeth Viswavidhalaya, Indore, Madhya Pradesh, India

Vikrant Jaryan,     Department of Botany, Sant Baba Bhag Singh University, Khiala, Jalandhar, Punjab, India

Anaida Kad,     University Institute of Engineering and Technology (UIET), Panjab University, Chandigarh, India

Mamta Kashyap,     Department of Biotechnology, Shoolini Institute of Life Sciences and Business Management, Solan, Himachal Pradesh, India

Anita Kumari,     Department of Ornamental Plants and Agricultural Biotechnology, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel

Pankaj Kumar,     Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Pawan Kumar

Institute of Plant Science, Agricultural Research Organization (ARO), The Volcani Center, Rishon LeZion, Israel

Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India

Rahul Kumar,     Faculty of Agricultural Sciences, DAV University, Jalandhar, Punjab, India

Varun Kumar,     Department of Ornamental Plants and Agricultural Biotechnology, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel

Vishal Kumar,     Govt. Senior Secondary School, Bhadwar, Kangra, Himachal Pradesh, India

Swaran Lata,     Himalayan Forest Research Institute (HFRI), Conifer Campus, Shimla, Himachal Pradesh, India

Nikhil Malhotra,     ICAR-National Bureau of Plant Genetic Resources Regional Station, Shimla, Himachal Pradesh, India

Ramgopal Mopuri,     Institute of Animal Science, Agricultural Research Organization (ARO), The Volcani Center, Rishon LeZion, Israel

Avilekh Naryal,     Defence Institute of High Altitude Research, Defence R & D Organization, Leh, Ladakh, India

Mahinder Partap

Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Academy of Scientific and Innovative Research, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Pratap Kumar Pati,     Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India

Archit Pundir,     University Institute of Engineering and Technology (UIET), Panjab University, Chandigarh, India

Mohammed Saba Rahim,     Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, Punjab, India

Shiv Rattan,     Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Joy Roy,     Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, Punjab, India

Anil K. Sharma,     Department of Biotechnology, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India

Ashutosh Sharma,     Faculty of Agricultural Sciences, DAV University, Jalandhar, Punjab, India

Himanshu Sharma,     Agri-Biotechnology Division, National Agri-Food Biotechnology Institute, Mohali, Punjab, India

Indu Sharma,     Department of Botany, Sant Baba Bhag Singh University, Khiala, Jalandhar, Punjab, India

Neha Sharma,     Division of Crop Improvement, ICAR-Central Potato Research Institute, Shimla, Himachal Pradesh, India

Shivani Sharma,     Himalayan Forest Research Institute (HFRI), Conifer Campus, Shimla, Himachal Pradesh, India

Vikas Sharma,     Department of Molecular Biology and Genetic Engineering, Lovely Professional University, Jalandhar, Punjab, India

Vikas Sharma,     Department of Botany, Sant Baba Bhag Singh University, Khiala, Jalandhar, Punjab, India

Kirti Shitiz,     Defence Laboratory, Defence Research & Development Organization, Jodhpur, Rajasthan, India

Baldev Singh,     Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India

Jagdish Singh,     Himalayan Forest Research Institute (HFRI), Conifer Campus, Shimla, Himachal Pradesh, India

Joginder Singh,     Himalayan Forest Research Institute (HFRI), Conifer Campus, Shimla, Himachal Pradesh, India

Mohar Singh,     ICAR-National Bureau of Plant Genetic Resources Regional Station, Shimla, Himachal Pradesh, India

Pradeep Singh,     Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India

Pramod Kumar Singh,     Department of Biosciences, Christian Eminent College, Indore, Madhya Pradesh, India

Vijay Singh,     Department of Botany, Mata Gujri College, Fatehgarh Sahib, Punjab, India

Archit Sood,     Institute of Plant Sciences, Volcani Center, Agricultural Research Organization, Rishon LeZion, Israel

Hemant Sood,     Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan, Himachal Pradesh, India

Ira Vashisht,     Crop Genetics & Informatics Group, School of Computational and Integrative Sciences (SCIS), Jawaharlal Nehru University, New Delhi, India

Ashish R. Warghat

Biotechnology Division, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Academy of Scientific and Innovative Research, CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, India

Chapter 1: Introduction

Nikhil Malhotra, and Mohar Singh     ICAR-National Bureau of Plant Genetic Resources Regional Station, Shimla, Himachal Pradesh, India

Abstract

The medicinal plants of the Himalayas are from ancient times for their multiple uses. The traditional harvesting practices of these valuable plants have altered over the time due to several reasons, including advent of market forces that have resulted in declining of many existing populations. The assessment of secondary metabolites obtained from these plants having wide therapeutic potential has garnered considerable interest to obtain understanding of their chemistry, analytical methodologies, biosynthetic mechanisms, and pharmacological activities. The use of conventional and contemporary approaches along with the formulation of sustainable harvesting practices and suitable regulations is required for the future conservation and management of medicinal plants in the Himalayas.

Keywords

Conservation; Ecology; Himalayas; Medicinal plants; Phytochemicals

The Himalayan center of plant diversity is a narrow band of biodiversity lying on the southern margin of the Himalayas, the world’s highest mountain range with elevations exceeding 8000  m (Barthlott et al., 2005). The Himalayan region, likewise other biomes of the world, is known since centuries for harboring a rich wealth of extremely valuable medicinal plants (Kala, 2005, 2010). The Indian Himalayas are home to more than 8000 species of vascular plants of which 1748 possess medicinal properties (Singh and Hajra, 1996; Samant et al., 1998; Joshi et al., 2016). At present, the trade of these plants from the Himalayas to the other parts of the world is speeding up due to increase in their demand, which subsequently affect the traditional collection practices of medicinal plants (Olsen, 1998; Bhat et al., 2013). Earlier, traditional healers mainly practiced the harvesting of medicinal plants but with the high demand at regional to international markets, many of the untrained collectors begin to participate in collection (Sharma and Kala, 2016), which has also resulted in adulteration of plant material (Sagar, 2014). Numerous wild and cultivated medicinal plants possessing bioactive compounds known as secondary metabolites have been utilized as curative agents since ancient times and medicinal plants have gained importance recently, not only as herbal medicines but also as natural ingredients for the cosmetic industries. As a result, a significant number of Himalayan plants now figure in the Red Data Book of rare, endangered, and threatened medicinal plants and require urgent conservation efforts (Rana and Samant, 2010; Goraya, 2011). Some of the important medicinal plants species of the Indian Himalayas include Aconitum heterophyllum (Atis), Dactylorhiza hatagirea (Salampanja), Picrorhiza kurroa (Kutki), Podophyllum hexandrum (Bankakdi), Rauwolfia serpentina (Sarpagandha), etc.

Secondary metabolites are a unique group of compounds produced by plants to protect against various biotic and abiotic factors (Kennedy and Wightman, 2011). These compounds, however, do not influence the primary metabolic activities such as growth and reproduction of plants (Arbona et al., 2013). The major classes of secondary metabolites include phenolics, alkaloids, tannins, saponins, lignins, glycosides, and terpenoids. Some of these compounds have become an integral part of plant–microbe interactions toward adapting to environmental irregularities. They regulate symbiosis, induce seed germination, and show allelopathic effect, i.e., inhibit other competing plant species in their environment. Moreover, these compounds induce adverse physiological activities such as reduced digestive efficiency, reproductive failure, neurological problems, and gangrene and also possess high toxicity. The discovery of such unique compounds in the majority of Himalayan medicinal plants has inspired many scientific communities to explore their potential applications in various industries. The use of natural bioactive compounds and their products is thereby considered the most suitable source of alternative medicine. Thus, there is an unprecedented task to meet the increasing demand for plant secondary metabolites from flavor and fragrance, food, and pharmaceutical industries. However, their supply has become a major constraint since their large-scale cultivation is very limited. Moreover, it is difficult to obtain a constant quantity of compounds from cultivated plants as their yield fluctuates due to several factors including genotypic variations, geography, and edaphic conditions along with harvesting and processing methods.

Further, in situ and ex situ conservations are the most efficient methods to conserve genetic diversity of plants, including medicinal herbs. Traditionally, in situ conservation efforts have utilized the delineation of protected areas, whereas ex situ conservation efforts have included in vitro approaches and gene banks. However, conservation efforts have taken new dimensions with the advent of new technologies in recent years. As per the present scenario, these new approaches have integrated with traditional well-developed methods of conservation. The recent technological developments in high-throughput next-generation sequencing and other molecular biology techniques have provided greater opportunities to identify and characterize a large number of genes involved in important metabolite pathways. However, linking genotype to phenotype, predicting gene regulations, and ascertaining mutations require the utilization of vast genomic information and encompass the incorporation of intraspecific and environmental variability.

Unfortunately, there remains a paucity of information relating biological activities of essential metabolites with the ethnobotanical uses of the plants. In many cases, this may be due to the activity residing in nonvolatile components. Additionally, many researchers have neglected bioactivity screening related to ethnopharmacological uses. Thus, detailed work should be carried out to identify phytochemicals associated with biological activities, which support traditional uses of medicinal plants. Moreover, an integrated approach including conventional as well as emerging technologies should be utilized for the effective conservation of Himalayan herbs (Sharma and Kala, 2018). The latest ecological analysis methods coupled with whole-genome and transcriptome sequencing, metabolic engineering, and big data analytics should become an integral part of programs for the conservation and genetic improvement of the Himalayan plant wealth for future generations. Further, increased interdisciplinary collaboration and multiinstitutional focus for a resolute effort to this effect is urgently required. We encourage the preservation of traditional knowledge and uses of Himalayan medicinal plants and hope that additional steps should be undertaken to protect and maintain the Himalayan ecology.

References

1. Arbona V, Manzi M, de Ollas C, Gómez-Cadenas A. Metabolomics as a tool to investigate abiotic stress tolerance in plants. Int. J. Mol. Sci. 2013;14:4885–4911.

2. Barthlott W, Mutke J, Rafiqpoor D, Kier G, Kreft H. Global centers of vascular plant diversity. Nova Acta Leopold. 2005;92:61–83.

3. Bhat J, Kumar M, Bussmann R.W. Ecological status and traditional knowledge of medicinal plants in Kedarnath Wildlife Sanctuary of Garhwal Himalaya, India. J. Ethnobiol. Ethnomed. 2013;9:1–18.

4. Goraya G. Conservation concerns for medicinal plants of Himachal Pradesh. ENVIS News Lett. Med. Plants. 2011;3:15.

5. Joshi R.K, Satyal P, Setzer W.N. Himalayan aromatic medicinal plants: a review of their ethnopharmacology, volatile phytochemistry, and biological activities. Medicines. 2016;3:6.

6. Kala C.P. Indigenous uses, population density, and conservation of threatened medicinal plants in protected areas of the Indian Himalayas. Conserv. Biol. 2005;19:368–378.

7. Kala C.P. Medicinal Plants of Uttarakhand: Diversity Livelihood and Conservation. Delhi, India: Biotech Books; 2010:188.

8. Kennedy D.O, Wightman E.L. Herbal extracts and phytochemicals: plant secondary metabolites and the enhancement of human brain function. Adv. Nutr. 2011;2:32–50.

9. Olsen C.S. The trade in medicinal and aromatic plants from central Nepal to Northern India. Econ. Bot. 1998;52:279–292.

10. Rana M.S, Samant S.S. Threat categorisation and conservation prioritisation of floristic diversity in the Indian Himalayan region: a state of art approach from Manali wildlife sanctuary. J. Nat. Conserv. 2010;18:159–168.

11. Sagar P.K. Adulteration and substitution in endangered, ASU herbal medicinal plants of India, their legal status, scientific screening of active phytochemical constituents. Int. J. Pharmaceut. Sci. Res. 2014;5:4023–4039.

12. Samant S.S, Dhar U, Palni L.M.S. Medicinal Plants of Indian Himalaya: Diversity Distribution Potential Values. Almora, India: G.B. Pant Institute of Himalayan Environment and Development; 1998.

13. Sharma N, Kala C.P. Utilization pattern, population density and supply chain of Rhododendron arboreum and Rhododendron campanulatum in Dhauladhar mountain range of Himachal Pradesh, India. Appl. Ecol. Environ. Sci. 2016;4:102–107.

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Chapter 2: Aconitum heterophyllum

Nikhil Malhotra¹, and Shivani Sharma²     ¹ICAR-National Bureau of Plant Genetic Resources Regional Station, Shimla, Himachal Pradesh, India     ²Himalayan Forest Research Institute (HFRI), Conifer Campus, Shimla, Himachal Pradesh, India

Abstract

Aconitum heterophyllum Wall (Ranunculaceae), also called Atis, is a high-value biennial herb native to northwest and east Himalayan regions of Indian subcontinent. Its nontoxic tuberous roots are commonly used as therapeutic ingredient in the Traditional Indian and Chinese Medicinal System for curing dyspepsia, abdominal pain, diabetes, and diarrhea. The aconites, including atisine, represent major constituents as well as marker compounds of A. heterophyllum. This chapter presents a rationalized summary and critical evaluation of progress made in this medicinal herb pertaining to past and present research along with future prospects. This will act as a baseline data as well as valuable source for different stakeholders and researchers working on various aspects of A. heterophyllum and/or genus Aconitum in times to come.

Keywords

Aconites; Aconitum heterophyllum; Atisine; Herbal drug; Metabolic pathway; Transcriptome; Tuberous roots

2.1. Introduction

Out of many important medicinal plants cultivated in present times, Aconitum species finds a key position for their conservation and cultivation. The genus Aconitum belongs to the family Ranunculaceae. There are ∼400 species of Aconitum occurring worldwide (Lane, 2004; Yin et al., 2019). In the northwest Himalayas, it is represented by 10 species and 2 varieties. Some of the important species of Aconitum are Aconitum balfourii, Aconitum bisma, Aconitum carmichaeli, Aconitum chasmanthum, Aconitum deinorrhizum, Aconitum ferox, Aconitum japonicum, Aconitum napellus, and Aconitum violaceum along with A. heterophyllum—the only nontoxic species of this genus (Chauhan, 2006; Buddhadev and Buddhadev, 2017). These herbaceous biennial plants are primarily natives of the mountainous parts of the Northern Hemisphere, growing in moisture retentive but well-drained soils on the mountain meadows (Tamura, 1995). These plants are tall, with erect stem being crowned by racemes of large and eye-catching blue, purple, white, yellow, or pink zygomorphic flowers with numerous stamens. The root is best harvested in the autumn as soon as the plant dies down and is dried for later use. In recent years, the demand for medicinal and aromatic plants has grown rapidly because of accelerated local, national, and international interest. Aconitum genus is the center of attraction in the field of herbal medicines because of its property of curing a wide range of diseases and, hence, the pressure on its natural habitat has increased. This is one of the most prized plant genuses which has been enlisted in the Red Data Book and is widely considered as a mystifying group due to fatal as well as therapeutic behavior (Tai et al., 2015). The pharmacological analysis of Aconitum species and their compounds have shown various therapeutic effects pertaining to cardiovascular and central nervous system (Dzhakhangirov et al., 1997; Friese et al., 1997; Ameri 1998; Polyakov et al., 2005) alongside anticancer (Solyanik et al., 2004), antimicrobial, and cytotoxic activities (Gavín et al., 2004; González et al., 2005). In recent years, a large number of studies have investigated the toxicological characteristics of Aconitum, its main alkaloids, and their derivatives (Xie et al., 2005; Fujita et al., 2007; Jaiswal et al., 2013, 2014). It has been observed that the whole plant of Aconitum is highly toxic with the concentration of toxic compounds higher in roots and flowers than in leaves and stems (Ding et al., 1993). The symptoms of toxicity affect mainly the central nervous system and the heart, with concomitant gastrointestinal signs. The cause of death is the development of ventricular tachyarrhythmia and heart arrest. No specific therapy exists for Aconitum poisoning, although cardiovascular supportive treatment is usually applied (Lin et al., 2004). The toxicity of Aconitum is mainly due to the diester diterpene alkaloids and monoester diterpene alkaloids such as deoxyaconitine, benzoylmesaconitine, jesaconitine, benzoylhypaconine, and benzoylaconine (Chinese Pharmacopoeia Commission, 2005; Srivastava et al., 2010; Nyirimigabo et al., 2015). Through various physical and chemical methods of treatment, highly toxic Aconitum alkaloids could be transformed into less toxic derivatives.

A. heterophyllum Wall, commonly known as atis, is a rare diploid (2n  =  16) Himalayan plant species found between 2400 and 3600  m amsl (Fig. 2.1). Ayurveda classical texts of 15th–16th century introduced Abhava-Pratinidhi Dravya concept, wherein it was categorized as an abhava dravya (unavailable drug). Its roots are ovoid-conical, tapering downward to a print, 2.0–7.5  cm long, 0.4–1.6  cm or more thick at its upper extremity, gradually decreasing in thickness toward tapering end, externally light ash-gray, white or gray-brown, while internally starch white, external surface wrinkled marked with scars of fallen rootlet, and with a rosette of scaly rudimentary leaves on top. It is a cross-pollinated plant which flowers in the second year. The flowers are helmet shaped, bright blue or greenish blue in color and have a purple vein. For medicinal use, the roots from plants bearing fully developed tubers are collected (Kumar et al., 2016; Rajakrishnan et al., 2016). The tubers sometimes occur as a pair of mother and daughter tubers. Tuberization in A. heterophyllum is a distinctive process from young rootlet to fully mature storage roots which are committed to the storage of primary as well as secondary metabolites (Pal et al., 2015). A. heterophyllum has been listed as critically endangered medicinal herb by the International Union for Conservation of Nature and Natural Resources (IUCN, 1993; Nautiyal et al., 2002; CAMP, 2003; Srivastava et al., 2011), which has thereby prohibited the export of its plants, plant portions and their derivatives, and extracts obtained from the wild (Shah, 2005; Chinese Pharmacopoeia Commission, 2015). Owing to the huge cost for dried tuberous roots of A. heterophyllum (∼₹10,000 per kg), and an ever-rising demand of raw material (>20 tons per year) (Aneesh et al., 2009; NMPB, 2015), overharvesting of its tubers has been facilitated over the years. This reckless collection has led to reduction in its population in natural habitat. Although efforts have been done to maintain its population in farm fields by conventional breeding and propagation methods (Fig. 2.2), nothing has been significantly achieved in R&D programs globally (Rawat et al., 2016). Nontoxic active components like atisine, hetisine, and heteratisine, collectively termed as aconites, accumulating in tuberous roots of A. heterophyllum have wide pharmacological effects on immune, digestive, and nervous systems (Murti and Khorana, 1968; Pelletier et al., 1968; Mori et al., 1989; Rastogi and Mehrotra, 1991; Zhaohong et al., 2006; Nisar et al., 2009; Malhotra et al., 2014; Malhotra, 2017).

Figure 2.1  Mature Aconitum heterophyllum plant.

Figure 2.2  Field plantation of Aconitum heterophyllum at HFRI Farm, Shillaru, Himachal Pradesh, India.

Biotechnological interventions have substantially contributed in terms of higher aconites production and conservation in various Aconitum species, but the contemporary breakthroughs are still lacking in A. heterophyllum, besides a few research interventions made in the recent past. Although this plant has been circumspectly studied for its cultivation (Nautiyal et al., 2006; Srivastava et al., 2011), conservation and sustainable utilization (Pandey et al., 2005; Seethapathy et al., 2014; Kumar et al., 2016), cytology (Siddique et al., 1998; Rani et al., 2011; Jeelani et al., 2015), ecology (Nautiyal et al., 2002; Bhat et al., 2014; Jeelani et al., 2015), medicinal uses (Ukani et al., 1996; Nyirimigabo et al., 2015), phytochemical constituents (Gajalakshmi et al., 2011; Jaiswal et al., 2013; Jaiswal et al., 2014; Malhotra et al., 2014; Nagarajan et al., 2015a,b; Nyirimigabo et al., 2015; Kumar et al., 2016), reproductive biology (Siddique et al., 1998) along with reports on plant tissue culture (Giri et al., 1993, 1997; Jabeen et al., 2006; Solanki and Siwach, 2012), and OMICS-assisted approaches (Malhotra et al., 2014, 2016; Pal et al., 2015; Kumar et al., 2016), the comprehensive coverage of botany, production, and research advancements in A. heterophyllum have not been attempted till date. Thus, this chapter becomes very unique and important for the researchers and readers across the globe working on this high-value plant species.

2.2. Origin and distribution

Classification of the genus Aconitum has been extremely difficult because aconites are morphologically highly variable (Yang, 1990; Tamura, 1995; Luo, 2003). Numerous categorizations in this genus have been proposed (de Candolle, 1824; Nakai, 1953; Wang, 1965; Tamura, 1995), but due to the difference in explanation of features considered, these are still in great dispute. The major centers of Aconitum diversity are northwest and east Himalayas, southwest China, and Japan. Although the chloroplast DNA, nuclear ribosomal DNA (nrDNA), and nuclear internal transcribed spacer (ITS) sequence data have been used to study the phylogenetic relationships within Aconitum subgenus Aconitum (Kita et al., 1995; Kita and Ito, 2000; Luo et al., 2005) along with a study on chromosomal and molecular patterns (Mitika et al., 2007), significant information on adequate understanding of its phylogeny is still lacking. Moreover, separate studies for tracing the evolutionary history of each Aconitum species have not been done; therefore, no records are available for justifying the origin of A. heterophyllum also.

In India, A. heterophyllum is found and cultivated in the Himalayan states of Jammu and Kashmir, Ladakh, Himachal Pradesh, and Uttarakhand in the northwest along with Sikkim and Arunachal Pradesh in the east. It also occurs in Nepal, Bhutan, and parts of southwest China.

2.3. Medicinal properties

From ancient times, A. heterophyllum has been used in different formulations in the Indian Ayurvedic System for curing various diseases. Balachaturbhadra Churna, Caspa Drops, Chandraprabha Vati, Chaturbhadraka Vati, Chitrakadi vati, Kutajghan Vati, Livex, Panchatikta Guggulu Ghrita, Rasnerandadi Kwatha, Satyadi Yoga, Shaddharana churna, and Sudarshan Churna are some of the popular multidrug herbal formulations in which A. heterophyllum is used as one of the main ingredients (Lather et al., 2010; Nariya et al., 2011; Ajanal et al., 2012; Sojitra et al., 2013; Joshi et al. 2014, 2016; Kumar et al., 2014; Selvaraj et al., 2014; Chaudhary et al., 2015; Gupta et al., 2015; Dhamankar and Jadhav, 2016; Baishya et al., 2020). These drugs find common use in the treatment of diarrhea, fever, indigestion, inflammation, helminthiasis, hyperlipidemia, and other ailments. Some of the important medicinal properties of A. heterophyllum are listed in Table 2.1.

2.3.1. Antibacterial activity

Ahmad et al. (2008) isolated the new aconitine type norditerpenoid alkaloids, 6-dehydroacetylsepaconitine, and 13-hydroxylappaconitine from the tubers of A. heterophyllum along with the known alkaloids lycoctonine, delphatine, and lappaconitine, which were screened for antibacterial activity against different bacterial strains. They showed antibacterial activity against diarrhea causing gram-negative bacteria Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, and Salmonella typhi. This report strengthens the use of A. heterophyllum as an antimicrobial and/or antihelminthic agent. In another study by Sinam et al. (2014), the root alkaloid extract of A. heterophyllum showed antibacterial activity against Bacillus subtilis, Bordetella bronchiseptica, Pseudomonas putida, Staphylococcus aureus, and Xanthomonas campestris.

Table 2.1

2.3.2. Antidiarrheal activity

The antidiarrheal activity of roots of A. heterophyllum may be attributed to an antisecretory and antienteropooling type effect as a result of reactivation of Na+ and K+ ATPase activity mediated through nitric oxide pathway. They cause either a decrease in mucosal secretion or increase in mucosal absorption, which allows the feces to become desiccated, thus retarding its movement through the colon (Prasad et al., 2014).

2.3.3. Antihelminthic activity

Aqueous and alcoholic extracts of tubers of A. heterophyllum gave encouraging results when evaluated against Pheretima posthuma, using piperazine citrate as standard. Time required for initial three paralytic attacks and deaths was used as parameters to evaluate the drug (Pattewar et al., 2012). It was revealed that a dose of 100% aqueous root extract was responsible for anthelmintic activity.

2.3.4. Antihyperlipidemic activity

The methanolic extract of tubers of A. heterophyllum had a hypolipidemic effect on diet-induced obese rats. It was observed that the pharmacological effect was due to the inhibition of hydroxymethylglutarate-Coenzyme A reductase and activation of lecithin-cholesterol acyltransferase. This resulted in lowering apolipoprotein B, total cholesterol, low-density lipoprotein cholesterol, and triglycerides in the blood serum along with the decrease in intestinal fat absorption and increase in apolipoprotein A with high-density lipoprotein cholesterol. These results supported the use of A. heterophyllum as an antihyperlipidemic agent (Subash and Augustine, 2012).

2.3.5. Antiinflammatory and antipyretic activity

For the assessment of antiinflammatory activity of A. heterophyllum, cotton-pellet–induced granuloma method was used. It was found that ethanolic extract of A. heterophyllum tuber had significant antiinflammatory activity, thereby providing scientific evidence for a traditional use as an antiinflammatory agent. Further, the antipyretic effects of roots of A. heterophyllum in the form of aqueous, chloroform, and hexane extracts were examined using the method of yeast-induced pyrexia, with aspirin as a standard antipyretic agent for comparison. These studies showed that the extracts were nontoxic with nonsignificant antipyretic activity (Verma et al., 2010).

2.3.6. Antioxidant activity

Prasad et al. (2012) demonstrated in vitro antioxidant activity of A. heterophyllum in different models which was attributed to low flavonoid and phenolic contents in its roots. Further, the root extracts of A. heterophyllum were tested for glycerol-induced acute renal failure in Wistar albino rats (Konda et al., 2016) and streptozotocin-induced diabetic rats (Rah et al., 2016), respectively. These studies revealed significant antioxidant property without any toxic effects.

2.3.7. Immunomodulatory activity

The immunomodulatory activity of ethanolic extract of A. heterophyllum tubers along with other medicines of the Ayurveda and Unani systems of medicine was investigated on delayed-type hypersensitivity, humoral responses to sheep red blood cells, skin allograft rejection, and phagocytic activity of the reticuloendothelial system in mice. It was found that the extract appeared to enhance the phagocytic function and inhibiting humoral component of the immune system. The results obtained from these preliminary studies showed that A. heterophyllum has immunomodulatory activity, which could possibly lead to synthesis of new immunomodulating agents of herbal origin (Atal et al., 1986; Gulati et al., 2002).

2.3.8. Nervous system stimulation

A. heterophyllum has the ability to make the sympathetic nervous system highly sensitive to physiological stimuli. It was found that while atisine had a hypotensive effect at every tested dose, the plant extract showed hypertensive properties. Hypertension produced by high doses of aqueous extract was attributed to the excitement of the sympathetic nervous system (Raymond-Hamet, 1954). Nisar et al. (2009) isolated two new diterpenoid alkaloids viz. heterophyllinine A and heterophyllinine B from the roots of A. heterophyllum, which were almost 13 times more selective in inhibiting the enzyme butyrylcholinesterase than acetylcholinesterase. These enzymes are involved in the transmission of nerve impulses.

2.4. Phytochemistry

A. heterophyllum is a rich source of alkaloids, flavonoids, free fatty acids, and polysaccharides (Rajakrishnan et al., 2016; Paramanick et al., 2017). The main alkaloid reported in Aconitum is aconitine that is highly toxic (O’Neil et al., 2001). However, among the reported species, A. heterophyllum is the only nontoxic species with therapeutic potential (Chauhan, 2006; Jaiswal et al., 2014; Malhotra et al., 2014). The pharmacological properties of A. heterophyllum are attributed to the nontoxic active constituents, i.e., aconites, including atisine which comprises the major alkaloid constituents of this plant species (Chatterjee and Prakash, 1994; Srivastava et al.,2011). These constituents make it a safer herb to use when compared with other Aconitum species, since no purification process is mandatory for its purification or detoxification. Some of the important phytochemicals of A. heterophyllum are listed in Table 2.2.

The early investigations on the tubers of A. heterophyllum, beginning with 19th century works by Broughton, Wasowicz, and Wright, have been documented by Jowett (1896). Broughton was first to isolate atisine. Different salts (sulfate, hydrochloride, and platinichloride) were prepared from the alkaloid, and the molecular formula was deduced. Wasowicz showed that the aconitic acid is also present along with atisine besides suggesting slight modifications in the molecular formula of atisine. Wright proposed a new formula for atisine based on analysis of its aurichloride salt. Subsequently, investigations on the properties and composition of atisine and its salts were studied in great detail. No alkaloid other than atisine was found in such studies (Jowett, 1896). The structure of atisine and three other alkaloids hetisine, heteratisine, and benzoylheteratisine was confirmed by Jacobs and Craig (1942a,b).

Detailed studies on hetisine, atisine, and heteratisine helped in their structure elucidation (Solo and Pelletier, 1962; Aneja and Pelletier 1964; Pelletier and Parthasarathy 1965; Aneja et al., 1973). Further investigations on A. heterophyllum led to the isolation and structure elucidation of additional new diterpene alkaloids; atidine, F-dihydroatisine, hetidine, and hetisine as well as lactone alkaloids heterophyllisine, heterophylline, and heterophyllidine (Pelletier and Aneja, 1967; Pelletier et al., 1968). In 1982, a new entatisene diterpenoid lactone, atisenol, was isolated from the tubers of A. heterophyllum (Pelletier et al., 1982). Moreover, the structure and, most importantly, the stereochemistry of atisine and related alkaloids were established by Dvornik and Edwards (1964).

Table 2.2

From the reported literature, it is evident that alkaloids were the main focus of study in A. heterophyllum. Several pharmacological actions of A. heterophyllum have been attributed to their alkaloids (O’Neil et al., 2001). Later on, work on Aconitum alkaloids led to the isolation of two new aconitine-type norditerpenoid alkaloids 6-dehydroacetylsepaconitine and 13-hydroxylappaconitine along with known norditerpenoid alkaloids lycoctonine, delphatine, and lappaconitine (Ahmad et al., 2008). Although aconitine, which is the major alkaloid of other Aconitum species, is not a major constituent of A. heterophyllum, high-performance liquid chromatography (HPLC) studies carried out on the tubers from Kumaon and Garhwal regions of the Himalayas showed that aconitine is present in different populations and varies from 0.13% to 0.75% (dry weight basis) (Bahuguna et al., 2000; Pandey et al., 2008). HPLC studies on quantification of aconitine from tubers of A. heterophyllum from the Kashmir valley have reported 0.0014%–0.0018% aconitine (Jabeen et al., 2011). Similarly, a study by Bahuguna et al. (2013) reported higher content of atisine (0.35%) and aconitine (0.27%) in greenhouse-grown A. heterophyllum when compared with the naturally grown plants (0.19% and 0.16%, respectively). Further, a study by Malhotra et al. (2014) led to estimation of atisine in roots of plants of different age groups (1–3 years) which showed variation in atisine content. It increased from 0.14% in 1-year-old plants to 0.22% in 2-year-old plants and then decreased to 0.08% in the roots of 3-year-old plants. Atisine was not detected in shoots of A. heterophyllum. They also analyzed atisine/aconites content through HPLC and bromocresol green extraction method in 14 accessions of A. heterophyllum collected from different locations of Himachal Pradesh. The significant variation was observed among 14 accessions of A. heterophyllum as atisine content in roots ranged from 0.14% to 0.37% and total alkaloids (aconites) from 0.20% to 2.49%. Two accessions, namely AHCR and AHSR, showed the highest atisine content of 0.30% and 0.37%, as well as the highest total alkaloids content of 2.22% and 2.49%, respectively. Thereafter, steviol was quantified in roots of A. heterophyllum. It was found that high-content accession had 0.06% steviol which was sixfold greater as compared to roots of low-content accession (0.01%) (Kumar et al., 2016). Moreover, Kumar and Chauhan (2016) quantified steviol in leaves of A. heterophyllum, thereby providing a novel source for extraction of steviol which could benefit the harvesters to get additional economic returns on leaf biomass.

Soon after, extensive chromatographic separations along with mass and nuclear magnetic resonance spectroscopy analysis resulted in the isolation of three new diterpenoid alkaloids, 6β-methoxy, 9β-dihydroxylheteratisine, 1α,11,13β-trihydroxylhetisine, 6,15β-dihydroxylhetisine, and the known compounds namely atidine, heteratisine, hetisinone, iso-atisine, and 19-epi-isoatisine (Ahmad et al., 2017). Then, Obaidullah et al. (2018) isolated heterophylline-A and heterophylline-B, along with condelphine from the roots of the A. heterophyllum.

2.5. Adulteration and substitution

Natural sources of medicinal plants are unable to meet demand for popular herbal products. Populations of many species have limited distribution in their natural habitats, requiring conservation strategies for protection. Unavailability of such medicinal plants has led to arbitrary substitution and adulteration in raw drug market (Kumar 2014a,b; Mishra et al., 2015; Shanmughanandhan et al., 2016; Ichim, 2019). Adulteration is a practice of substituting the original crude drug partially or fully with other substances which are either free from or inferior in therapeutic and chemical properties or addition of low grade or entirely different drug similar to that of original drug substituted with an intention of enhancement of profits.

A. heterophyllum has been substituted by Cyperus rotundus, commonly called Musta, in herbal-processing methods, thus affecting the quality of the herbal drug formulations (Venkatasubramanian et al., 2010; Adams et al., 2013; Kumar 2014b; Seethapathy et al. 2014, 2015). Being a low-cost substitute (∼₹30–50 per kg), it shares similar biological functions like antidiabetic, antidiarrheal, antipyretic, and treatment of urinary tract infections (Mitra et al., 2003; Uddin et al., 2006; Venkatasubramanian et al., 2010; Nagarajan et al., 2015b). Seethapathy et al. (2014) differentiated A. heterophyllum and C. rotundus by using nrDNA ITS sequence–based sequence characterized amplified region (SCAR) markers for validating adulteration in herbal drugs. The former was not detected through SCAR markers, while the latter was identified in complex mixtures of DNA extracted from commercial formulations.

2.6. Omics-based advancements

Understanding the biology of aconites biosynthesis has provided insights about the sites of biosynthesis and accumulation of aconites in A. heterophyllum. Comparative genomics was utilized for cloning the 15 genes of aconites biosynthetic pathway along with expression analysis of those genes in relation to atisine/aconites content in A. heterophyllum. Multiple genes of mevalonic acid/methylerythritol 4-phosphate (MVA/MEP) pathways showed elevated expression in high atisine/aconites content accession as compared to low-atisine/aconites content accession. This was the first attempt toward molecular understanding of atisine/aconites biosynthesis in A. heterophyllum. Eight genes viz. 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), phosphomevalonate kinase, isopentenyl pyrophosphate isomerase, 1-deoxy-D-xylulose 5-phosphate synthase, 2-C-methylerythritol 4-phosphate cytidyltransferase, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), and gerenyl diphosphate synthase with elevated expression in relation to aconites content could be used as suitable targets for developing gene markers for genetic improvement of A. heterophyllum (Malhotra et al., 2014).

With progress in modern technologies, transcriptomics has emerged as a powerful tool to capture traits of economic importance. The availability of whole transcriptome data could be used not only to discover candidate genes involved in tuberous root development and secondary metabolites production but also for understanding molecular basis of various biological processes in A. heterophyllum (Pal et al., 2015; Malhotra et al., 2016). Comparative next-generation sequencing transcriptome analysis between root and shoot tissues of A. heterophyllum predicted the candidate genes involved in the production of secondary metabolites. The in silico expression profiling for 15 genes identified 4 transcripts namely HDS, HMGR, mevalonate kinase, and mevalonate diphosphate decarboxylase with higher expression in root as compared to shoot transcriptomes. The pathway analysis performed for both the tissues suggested 341 and 329 mapped Kegg Orthologs (KOs) responsible for secondary metabolism in root and shoot transcriptomes, respectively, thereby attributing medicinal value to this plant species. In total, 77 interacting pathways associated with isoquinoline alkaloids biosynthesis were identified in root transcriptome of A. heterophyllum indicating how important primary and secondary metabolic pathways are connected with each other (Pal et al., 2015). Later, a study by Rai et al. (2017) has also revealed enrichment of essential biological processes and secondary metabolism in transcriptomes of Aconitum carmichaelii and A. heterophyllum.

A complete atisine biosynthetic pathway was also constructed connecting glycolysis, MVA/MEP, serine biosynthesis, and diterpene biosynthetic pathways in A. heterophyllum (Fig. 2.3). The study revealed phosphorylated pathway as a major contributor toward serine production in addition to repertoire of genes in glycolysis (glucose-6-phosphate isomerase, phosphofructokinase, aldolase, and enolase), serine biosynthesis (3-phosphoglycerate dehydrogenase and 3-phosphoserine aminotransferase), and diterpene biosynthesis (kaurene oxidase and kaurene hydroxylase) sharing a similar pattern of expression (2- to 4-folds) in roots compared to shoots vis-à-vis atisine content (0%–0.37%), thus suggesting their vital role in atisine biosynthesis (Kumar et al., 2016). Further, the biosynthetic machinery of tuberous roots was discerned to identify plausible key genes toward root biomass development by utilizing transcriptome datasets of A. heterophyllum. Four genes viz. ADP-glucose pyrophosphorylase, β-amylase, SRF receptor kinase (SRF), and expansin showed maximum contribution toward tuberous root development. There is possibility of altering the expression levels of these genes for improving tuberous root (biomass) yield for herbal drug industries. These results can be further explored to dissect the molecular regulation of tuberous root formation and growth in A. heterophyllum (Malhotra et al., 2016). Besides this, the role of ATP binding cassette transporters was also evaluated in tuberous roots (Malhotra, 2017).

2.7. Plant tissue culture–assisted progress

The use of plant tissue culture techniques has been employed for conservation of this medicinally important plant species. Plants of A. heterophyllum were obtained via somatic embryogenesis in callus derived from in vitro–raised leaf and petiole explants (Giri et al., 1993). Then, a method for the production of hairy roots of A. heterophyllum was developed by Giri et al. (1997). Embryogenic callus cultures were successfully transformed using Agrobacterium rhizogenes strains viz. LBA 9402, LBA 9360, and A4 for the induction