Dactylorhiza Hatagirea (D.Don) Soo: Himalayan Marsh Orchid

Abstract

This chapter gives an account of Dactylorhiza hatagirea, a high-value Himalayan medicinal plant. Nearly endemic to the Hindu-Kush Himalayan region, the plant belongs to the family orchidaceae. It is found in alpine and sub-alpine regions of India and nearby countries at an elevation of 2800–4200 m asl. Its population density has been reported to be 0.60–2.19 individuals/m². Numerous factors responsible for critically endangered status of this species are over-extraction and illegal trading to meet high market demand, excessive weed proliferation, overgrazing leading to destruction of plant parts, low genetic diversity and poor seed germination rate. It has many vernacular names such as Salam panja, Hatajadi, Panch aunle, Himalayan marsh orchid and Angmo-lakpa and issued in several traditional systems of medicine. Indigenous people use it for treating numerous health problems such as diarrhoea, dysentery, bone fracture, wounds and sex stimulant. It contains many phytochemicals such as Dactylose A, B; Dactylorhin A-E, resveratrol and trans-stilbene. Its pharmacological importance has been established by several studies examining its anti-microbial, antioxidant, anti-cancerous, anti-inflammatory, antipyretic and neuropharmacological activities.

6.1 Introduction

The Himalayas are spread over 18% of the India subcontinent, contain 50% of India’s forest cover and harbour 30% of endemic species. Out of the 8000 higher plant species found in the Indian Himalayan region, 1748 species are known for their medicinal use (Semwal et al. 2010). Medicinal plants are those plants in which one or more parts of the plant produce certain substances capable of being used for therapeutic purposes or function as a precursor for drug synthesis. Plants containing secondary metabolites such as glycosides, alkaloids, tannins, volatile oils and nutrients possess medicinal properties. Since ancient times, medicinal plants are being used by people in healthcare for treating a wide range of health problems. Several traditional systems of medicine use medicinal plants, such as Ayurveda and Unani. The medications formulated from the medicinal plants are known to boost the natural recovery power of the body and have greater cultural acceptability and compatibility with human body and exhibit fewer side effects (Ranpal 2009).

India is a treasure trove of well-documented and well-practised traditional knowledge related to herbal medicine which has a great economic potential if capitalized by promoting its use in the developed world with an ever-increasing interest in herbal medicines. About 2000 miraculous medicinal plant mentioned in Ayurveda are widely renowned for being both safe and effective in curing many bodily ailments. D. hatagirea is also an extensively used medicinal orchid of high value in Ayurveda, Siddha and Unani system of medicine (Giri and Tamta 2010; Thakur 2019).

As per WHO estimates, traditional medicine is extensively used in developing countries by about 80% of the people in these countries. India’s rich cultural heritage is also evident from the extensive knowledge of traditional herbal medicine which is found in the Vedic literature, especially the Rigveda, Charaka Samhita and Sushruta Samhita. Many of the remotely located areas of Indian Himalayas have rich traditional knowledge regarding the use of these medicinal plants which are yet to be documented. Lack of modern healthcare facilities make the people living in these regions highly dependent on these plants for their health problems (Semwal et al. 2010).

Orchids, known for their beautiful and fragrant flowers, are the largest family of flowering plants after Asteraceae, with around 25,000–35,000 species worldwide. These are grown and traded for numerous reasons such as medicinal products and ornamental plants. Chinese people are credited to be pioneers in cultivation and usage of orchids in healthcare system. In India, orchids have been used in medicinal practices since Vedic times. Orchids have been used in the treatment of nerve disorders, debility, bone fractures, fever, skin problems, etc. (De 2020; Magar et al. 2020; Wani et al. 2020; Kumar et al. 2021).

Dactylorhiza, derived from Greek words dactylos meaning finger and rhiza meaning root, was named by Necker ex Nevski in 1937 is a member of Orchidaceae family. Dactylorhiza consists of almost 75 species, which are found in Northern Temperate Zone. D. hatagirea (D. Don) Soo is a perennial orchid native to the Himalayan region (Warghat and Sood 2013; Choukarya et al. 2019). Endemic to the Hindu-Kush region of the Himalayas, this species has numerous vernacular names such as Hath panja, Hatajadi and Salam panja, and it is used in many traditional systems of medicine such as Ayurveda, Unani, Amchi and Siddha. It is a critically endangered species as per Conservation Assessment and Management plan (CAMP). A number of reasons have been speculated to be the cause of its decreasing population out of which the prime cause is anthropogenic disturbance caused by habitat degradation, over-extraction and climate change which have resulted in small or fragmented patches of the plant population (Dhiman et al. 2019). The plant is very significant for both the indigenous and the scientific community owing to the numerous bioactive phytochemicals present in it such as resveratrol, Dactylorhins A-E, Dactylose A and B, militarin, loroglossin, flavonoids, alkaloids, glycosides, carotenoids and phylloquinone. Presence of these compounds gives the plant many of its medicinal properties such as antioxidant, anti-microbial, aphrodisiac, anti-tumour, anti-diabetic, anti-inflammatory, antipyretic and immunomodulatory properties (Dutta and Karn 2007). Indigenous people have been using this plant for treating numerous other health conditions such as wound healing, nerve tonic, cough and cold which are yet to be studied (Wani et al. 2020). The plant is in urgent need of conservation using both in situ and ex situ techniques to prevent the species from further deterioration and replacement of the already endangered population.

6.2 Botanical Description and Taxonomical Classification

Dactylorhiza hatagirea is a beautiful, food-deceptive orchid with medicinal properties which is endemic to the Hindu-Kush Himalayan region. Dactylorhiza is made up of two Greek words, “dactylos” which means finger and “rhiza” which means roots and was coined by Necker ex Nevski in 1937 (Warghat and Sood 2013; Thakur et al. 2018; Kumar et al. 2021).

It is a temperate and perennial ground dwelling herb. It grows in abundance in moist meadow wetland, with porous soil having rich humus and sloppy mountains, especially in the moist and shady regions. The hardy tuberous geophyte remains erect even during heavy snowfall (Maikhuri et al. 1998; De 2020; Magar et al. 2020; Kumar et al. 2021). The height of the plant has been reported to be different in various studies conducted in different regions and is thought to be affected by a number of factors such as age of the plant, topography of the area, soil and climate. The height of the plant ranges from 20 to 60 cm (Dutta and Karn 2007; Chamoli and Sharan 2019). The Trans Himalayan Ladakh Region of India has even taller plants; the height of the plant ranging from 70 to 90 cm. The mean diameter of the plant is 0.77 cm when measured at a height of 5 cm above the ground (Ranpal 2009; Warghat and Sood 2013).

The tuberoids of this plant are fleshy and flattened, with palmately divided 2–5 lobes (Fig. 6.1). In order to survive the arid conditions, the underground stem is thickened in which water can be stored in large quantities. The moisture content of the tubers is 79%. The leaves are palmately lobed and shaped lanceolate with sheathing leaf base. The mean length of leaves is 15 cm, speckled and arranged through the length of the stem. The stem is erect, hollow and obtuse (Maikhuri et al. 1998; Warghat and Sood 2013; Warghat et al. 2013; Chamoli and Sharan 2019; Magar et al. 2020). The flowers are 1.7–1.9 cm in length and possess curved spur with green bracts. The structure of the flower consists of 3 sepals forming the outer whorl and 3 petals forming the inner whorl out of which 2 are similar and one is modified (rounded and lobed) called as “lip”. The gynostemium is massive, located at the centre of the flower and consists of the female pistil to which the male stamens are attached. The colour of the flowers varies widely and can be lilac purple, rosy, purple, pink, red or white. When compared to the length of the plant, the inflorescence is short, with compact raceme which is 5–15 cm long, in which the axillary buds bear 25–50 flowers (Fig. 6.2) (Warghat and Sood 2013; Magar et al. 2020; Kumar et al. 2021). The phenological stage of initiation of growth begins in the spring season, i.e. May. Its vegetative phase is in the summer months of June–July during the time of melting of snow, the flowering period is from June to July while the fruiting period is from August to September. It is an early flowering species since its flowers emerge at the time when the other co-occurring species are still in their vegetative phase. During the winters, the plant survives below the ground as tubers (Dutta and Karn 2007; Thakur et al. 2018; Dhiman et al. 2019; Kumar et al. 2021). The seeds are monocotyledonous, minute (200–1700 μm) and dark-brown to black in colour. The seeds lack endosperm and therefore require symbiotic association with a fungi for germination in normal conditions (Aggarwal and Zettler 2010; Giri and Tamta 2012; Warghat et al. 2013; Warghat et al. 2014). Pollination takes place mainly by insects. Dispersal of seeds by wind enhances gene flow among the populations (Chamoli and Sharan 2019). Its mode of propagation is through seeds and also root cutting. For higher yield there should be a gap of 5 years between cultivation and harvesting of the plant; however, it is harvested after 2–3 years sometimes (Maikhuri et al. 1998; Giri and Tamta 2010).

Fig. 6.1

Tubers of Dactylorhiza hatagirea. (Source: https://commons.wikimedia.org/wiki/File:Paanch_aaule.jpg)

Fig. 6.2

Dactylorhiza hatagirea. (Source: https://commons.wikimedia.org/wiki/File:Dactylorhiza_hatagirea_(7832429742).jpg)

Synonyms: Orchis latifolia var. indica, Orchis hatagirea D. Don (Ranpal 2009; Chamoli and Sharan 2019).

Taxonomy:

• Kingdom: Plantae

• Division: Angiosperms

• Class: Monocots

• Order: Asparagales

• Family: Orchidaceae

• Subfamily: Orchidoideae

• Tribe: Orchideae

• Sub-tribe: Orchidinae

• Genus: Dactylorhiza

• Species: hatagirea (Ranpal 2009; Warghat and Sood 2013)

Vernacular names: Dactylorhiza hatagirea is known by different names in different regions and languages such as panja, Salam panja, Hath panja or Hatajadi in Uttarakhand; Salam panja in Kashmir, Wanglak or Angmo-lakpa in various parts of Ladakh and Hatajari (Uttaranchal), Panch aunle, Hatajadi (Nepali), Aralu, Salap (Sanskrit), Ongu lakpa (Sherpa), Lob (Gurung), Spotted Heart Orchid or marsh orchid in English, Buzidan and Salam Misri in Unani, Salam panja in Ayurveda, Hatajadi in Kumaon, Ambolakpa and Hath panja in Sowa Rigpa and Zhang Lie Lan in Chinese (Ranpal 2009; Giri and Tamta 2010; Pant and Rinchen 2012; Warghat and Sood 2013; Sirohi and Sagar 2019b; Kumar et al. 2021).

Parts used: Whole plant, rhizome, leaves, flowers.

6.3 Distribution: India and World

D. hatagirea occurs in the temperate zone and is nearly endemic to Hindu-Kush region, in the central and western Himalayas, the biodiversity hotspot (Badola and Aitken 2003; Bhatt et al. 2005; Pant and Rinchen 2012; Chamoli and Sharan 2019). The species is reported to occur in the sub-alpine and alpine regions of India (Jammu and Kashmir, Ladakh, Uttarakhand, Himachal Pradesh, Arunachal Pradesh, Sikkim), Nepal, Pakistan, Tibet, Bhutan and Afghanistan at an altitude of 2800–4200 m above sea level (Giri and Tamta 2010; Pant and Rinchen 2012; Choukarya et al. 2019; Magar et al. 2020; Wani et al. 2020).

The Conservation Assessment and Management Plan (CAMP) has identified and listed this plant as critically endangered, the Convention of International Trade in Endangered Species (CITES) has listed it under Appendix II and is critically rare as per IUCN (Bhatt et al. 2005; Murkute et al. 2011; Chamoli and Sharan 2019; Wani et al. 2020).

6.3.1 Population Density

According to previous studies, the population density of the species seems to differ in different regions of the Himalayas and often have patchy distribution. Uniyal et al. (2002) reported the population density of the plant to be 4756.1/ha in the Upper Gori Valley, Kala (2000) reported it to be 161,900/ha in the Indian-Trans Himalayan Region, 42,000/ha in Nanda Devi Biosphere Reserve Area and 10,000/ha in Valley of Flowers (Maikhuri et al. 1998; Bhatt et al. 2005). The density was also reported to be higher in the protected areas when compared to the unprotected areas, i.e. 0.60–1.89 individuals/m² in unprotected areas and 0.70–2.19 individuals/m² in protected areas (Uniyal et al. 2002; Bhatt et al. 2005; Chapagain et al. 2021). In the Manaslu Conservation Area in central Nepal, the population density has been recorded to be 2.18 individuals/m² with relative frequency being 7.48% (Bhattarai et al. 2014). In the Western Himalayan region of India, the areas with high frequency of plant extraction have been reported to have plant density of about 1–6.6 individuals/m² while regions with low frequency and intensity of plant extraction have13.8–46.29 individuals/m² (Thakur et al. 2018).

Orchid populations worldwide have been adversely affected due to habitat destruction and changes in the land use patterns resulting to changes in the natural habitat. The decline in population has been more drastic in the unprotected areas while the population has remained almost stagnant in protected areas (Swarts and Dixon 2009; Gonzalez et al. 2011; Kusum 2014; Chapagain et al. 2021). A large number of factors have together led to its dwindling population from its wild habitats, of which anthropogenic factor is the prime cause. Some of these factors have been discussed below.

Over-exploitation: The plant is collected by local inhabitants for domestic uses and by local herbal healers for treating various health conditions (Semwal et al. 2010). Owing to its high medicinal potential, the species is in increasingly high demand both in domestic and international markets with the market value of its dry tubers being Rs. 2700–3200 per kg. It is estimated that its demand is nearly 5000 tons every year, and collection of raw materials from the wild species has been the most common way to meet this demand. In India, the annual consumption of its salep is estimated to be around 7.38 tonnes which is valued at USD 83,333 (or Rs. 50 Lakhs) (Olsen and Helles 1997; Badola and Pal 2002; Kala 2004; Murkute et al. 2011; Warghat and Sood 2013; Warghat et al. 2013; Warghat et al. 2014; Wani et al. 2020).

Illegal trading: There is a wide gap between the demand and the supply of this plant which leads to traders collecting and selling the plant illegally in both national and international markets. Almost 90–100 mature plants are harvested to obtain 1 kg of dried roots which the local inhabitants could collect at a rate of Rs. 100–200 (Chaurasia et al. 2007; Warghat and Sood 2013).

Excessive weed proliferation: Most of the dominant associates of D. hatagirea are inedible such as Anemone tetrasepala, Anaphalis triplinervis, Morina longifolia and Polygonum polystachyum (an over-growing species) which increase the pressure on the plant, making it easier for the inedible species to proliferate (Bhatt et al. 2005).

Overgrazing: A study reported that the livestock were taken to higher regions of the valley by the locals, and the resultant trampling leads to disturbance in the plant life cycle due to the destruction of the parts of the plant above the ground and underground part of the plant getting exposed. Grazing also causes proliferation of weeds further suppressing the growth of the desired species (Ranpal 2009; Warghat and Sood 2013).

Low rate of propagation and poor seed germination: Vegetative propagation is very slow in the species, and it has only 0.2–0.3% seed germination rate. Due to the absence of metabolic machinery and endosperm, very few out of the millions of seeds present in the orchid capsule germinate (Vij 2002; Giri and Tamta 2012; Warghat et al. 2014). Also, the species has slow rate of growth, low capability of regeneration along with requirement of mycorrhizal association and high pollinator specificity. Since the seeds of this plant lack endosperm, they require fungal association to grow in their natural environment (Bhatt et al. 2005; Pant and Rinchen 2012; Bhattarai et al. 2014; Kumar et al. 2021).

Low genetic diversity: D. hatagirea has low genetic diversity which differs across different locations, with only 40% of the genetic diversity attributable to differences within populations and the rest 60% to among population (Ranpal 2009; Warghat et al. 2013). Inter Simple Sequence Repeats (ISSR) and random amplified polymorphic DNA markers when employed to study genetic diversity among populations of D. hatagirea revealed the occurrence of moderate genetic variations among populations (Warghat et al. 2012; Warghat et al. 2013). The causal factors for this are localized distribution of the species, fragmented habitats and rapidly deteriorating population of the species. This has led to reduction in the ability of the species to evolve in the absence of new allelic varieties immigrating into the population (Magar et al. 2020). The deleterious effects of genetic drift and inbreeding pose genetic risk to these species by affecting population fitness and genetic diversity (Thakur et al. 2018).

Other than these improper collection and cultivation practices mainly due to inadequate knowledge, poor conservation practices resulting from lack of awareness, destruction and degradation of forests, global climate change are some other crucial factors (Ranpal 2009; Pant and Rinchen 2012; Bhattarai et al. 2014; Warghat et al. 2014). There is a need to take serious efforts at the national level to prevent depletion, along with both ex situ and in situ conservation, protection and management strategies to reinsure rapid recovery of plant populations; otherwise, the species will likely vanish even before its complete medicinal importance has been established (Pradhan 1975; Kusum and Verma 2014; Thakur et al. 2018). One of the imperative ways of ex situ conservation is tissue culture. Protocorm development and mass multiplication of D. hatagirea under in vitro and in vivo conditions have been undertaken by Warghat et al. (2014). Asymbiotic seed germination was tested in 10 media. Highest seed germination (37.12%) and maximum protocorm formation (23.40%) was seen in Lindemann (LD) medium followed by BM-1 and Murashige and Skoog (MS) medium. Protocorms were further cultured in MS media containing different concentrations and combinations of growth regulators Indole Butyric Acid (IBA) (0–3 mg/L) and Kinetin (Kin) (0–3 mg/L). Maximum growth and development were seen in MS medium supplemented with IBA (3 mg/L) and kin (1 mg/L). After 28–30 days, number of shoots and roots were recorded to be 18.12 and 8.25, respectively. While the mean length of shoot was 17.80 cm and root length was 8.02 cm. In a different study, plant growth hormone 6-Benzylanimopurine (BAP) was used along with IBA in the MS medium, and maximum growth and development were recorded at the concentration 4 mg/L IBA and 3 mg/L BAP. Number of shoots and roots were recorded to be 43.50 and 15.00, respectively. While the mean length of shoot was 31.06 cm and root length was 14.20 cm after 28–32 days. Fully growth plantlets were placed in different combinations of potting mixtures of which 100% plantlet survival was seen in the mixture containing cocopeat, vermiculite and perlite in the ration of 1:1:1, along with highest number of plantlets, i.e. 25, shoots (75) and roots (23), longest shoot length (18.8 cm) and root length (44.7 cm) after transplantation in the greenhouse for a month (Warghat et al. 2014; Popli et al. 2016).

Similar study was undertaken by Giri and Tamta (2012) with different basal media and several concentrations of plant growth regulators (PGRs) for in vitro propagation of green pod, shoot bud, tuber and leaf segment. Green pods culture resulted in poor, slow and difficult seed germination with 57% of treatments resulting in failure of germination. Good seed germination was seen in only 1 out of 28 treatments used which was in half strength MS medium supplemented with peptone (1 g/L), morphoinoethane sulphonic acid (1 g/L) and activated charcoal (0.1%). Average seed germination was also seen in only one, i.e. Knudson C (KC) medium with kinetin (1 mg/L) and activated charcoal (0.1%). Only four treatments resulted in the formation of protocorm-like bodies (PLBs); however, none of them multiplied in any culture. Only few plantlets were obtained of which none could survive when transferred to soil for hardening. In shoot bud culture, only MS medium supplemented with thidiazuron (TDZ) resulted in sprouting (11.10%). However, the shoots died in the shoot multiplication medium. Similarly, no rooting was observed in tuber and leaf segment culture. Vegetative propagation of the apical (consisting of dormant shoot bud), middle and basal tuber segments was also carried out with the help of PGRs, in which only the apical segments resulted in sprouting and rooting (Giri and Tamta 2012).

Use of robust molecular markers can be an efficient way of conservation and genetic improvement of the species. However, absence of genotypic linkage disequilibrium in populations and cross-specific amplification have been anticipated for use of efficient markers in conservation genetic studies. A study by Lin et al. (2014) for assessing intra- and inter- population genetic diversity of D. hatagirea in China resulted in identification of 14 simple sequence repeat (SSR) markers for different repeats. Study of variability on the basis of morphological, biochemical and isoenzyme patterns has indicated significant diversity among the populations (Chauhan et al. 2014). In the recent years, use of “omics” approaches has increased immensely in plant research to gain better understanding of mechanisms behind various pathways and processes. Advanced next-generation sequencing (MGS) techniques have commissioned the role of “omics” and have enabled genome and transcriptome sequencing for characterization of chloroplast genome, phylogenomic relationship and ecological divergence among the species of the genus dactylorhiza. Studies have reported use of ngs platforms nova-seq and genome analyzer IIx (both from Illumina Inc.) for elucidation the biosynthetic mechanisms of secondary metabolites dactylorhin, resveratrol and stilbenes from tissues of D. hatagirea. This transcriptomic characterization has been used to identify molecular cues linked with environmental factors such as freezing stress (Dhiman et al. 2019; sood 2021)

6.4 Phytochemical Composition

The mature tubers of the plant contain glucosides, starch, mucilage, loroglossin, phosphate, chloride and volatile oils. Chemically, the major constituents of the plant are dactylorhins A–E, dactyloses A, B and lipids (Table 6.1) (Maikhuri et al. 1998; Dutta and Karn 2007; Ranpal 2009; Warghat and Sood 2013).

Table 6.1 Phytochemicals present in Dactylorhiza hatagirea (Kizu et al. 1999; Wani et al. 2020)

Synthesis of Dactylose A and B takes place from l-ascorbic acid and 4-hydroxybenzyl alcohol via 2-c-(4-hydroxybenzyl)-α-l-xylo-3-ketohexulofuranosono-1, 4-lactone. Enzymatic emulsion of Dactylorhin A and Dactylorhin E using almond emulsion gives Dactylorhin A. Loroglossin is formed on hydrolysis of the compound (-2-3-dihydroxy-2-2-methylpropyl) butanedioic acid which is formed from enzymatic emulsion of Dactylorhin B and Dactylorhin D using cellulose (Dutta and Karn 2007; Warghat and Sood 2013). Qualitative phytochemical analysis of the hydroalcoholic extracts of the crude powder of the roots of D. hatagirea as shown the presence of flavonoids, saponins and carbohydrates, with total flavonoid compounds (TFC) content being 0.866 mg/100 gm of quercetin equivalent of dry extract sample (Choukarya et al. 2019). Its resveratrol content has been found to be 3.21 μg/100 mg of fresh weight (f. wt.) and trans-stilbene is 2.49 μg/100 mg f. wt. (Dhiman et al. 2019).

The phytochemicals have great medicinal potential and are therefore highly valued in both local and international markets. Alkaloids act as defensive elements which protect against predators, particularly mammals owing to their general toxicity and the ability to deter. They also have analgesic, anti-inflammatory and adaptogenic activities which play a role in alleviating pain, along with developing resistance to disease and stress (Hartmann 1991; Gupta 1994; Ali and Prasad 2016). Flavonoids have a range of beneficial biological activities and are generally nontoxic and an important component of diet. Glycosides and terpenoids are necessary for disease prevention and therapeutic effects in traditional medicine (Nakatani 2000). Saponins from plant sources are also responsible for some pharmacological effects like anti-inflammatory, molluscidal, anti-microbial, antispasmodic, anti-diabetic and anticancer, hypocholesteromic, antioxidant, anticonvulsant, analgesic, and cytotoxic activities (Ali et al. 2011). Generally saponins are toxic, but their consumption by human beings has been shown to have numerous beneficial effects on human health (Price et al. 1987).

The production and composition of secondary metabolites in plants is dependent on numerous factors such as physiological variations which includes the developmental stage of the plant organ, diurnal and seasonal variations which may be monthly or annual, the activity cycle of the pollinators, part of the plant being used, the type and location of the secretory structures which often have heterogeneous distribution within the plant body, presence of mechanical or chemical injuries, environmental conditions including climate, presence of diseases and pests, edaphic factors, geographic variations, genetic factors, evolution, etc. (Figueiredo et al. 2008; Magar et al. 2020). Plants growing at higher altitude, in natural environments (or the wild varieties) have higher amounts of bioactive compounds as compared to the ones growing at lower altitudes or the cultivated varieties (Bahuguna et al. 2000; Badola and Aitken 2003).

Other than these, numerous other compounds have been found from different parts of the plant such as indole alkaloids (1H-Indole by strictosidine synthase), Naphthoquinone (Napthalane-1,4-dione), ascorbic acid ((R)-5-((S)-1,2-dihydroxyethyl)-3.4 dihydroxy furan-2(5H)-one), phylloquinone (2-methyl-3((7R, 11R,E),3,7,11,15-tetra methylhexadec 2-en-1-yl) naphthalene-1,4-dione), militarine, albumin, butanedioic acid, hydroquinone, loroglossin (bis(4-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl) tetrahydro-2H-pyran-2-yl)oxy)benzyl) (2R,3S)-2,3-dihydroxy-2-isobutylsuccinate; C₃₄H₄₆O₁₈), pyrocatechol, glucomannan, glycoside, saponin, tannin, carotenoids (synthesized from zeaxanthin-dioxygenase and prolycopene isomerase), terpenoids, steroids, phylloquinone (vitamin K) (synthesized from 2-carboxy-1,4-naphthoquinone phytyltransferase) (Kizu et al. 1999; Ali and Prasad 2016; Dhiman et al. 2019; Kawra et al. 2020; Wani et al. 2020). The metabolites dactylorhin A, B and E have also been isolated from the orchids Gymnadenia conopsea R. Br. and Coeloglossum viride (Li et al. 2009; Sood 2021). The structures of phytochemical present in D. hatagirea have been shown in Fig. 6.3.

Fig. 6.3

Structures of secondary metabolites isolated from Dactylorhiza hatagirea

6.5 Ethnomedicinal Uses

As per the estimates of WHO, almost 80% of the world’s population in developing countries depends on traditional medicine for their healthcare needs. The Himalayas lying in the Northern region of India and covering around 18% subcontinent, harbours about 30% of India’s endemic species. It is a biodiversity hotspot of not just the wide variety of endemic plant species found in the region but also of the rich traditional knowledge of their medicinal uses, much of which remains largely undocumented (Semwal et al. 2010). Since the outreach of modern medicinal services is inadequate in our country, people in many remote areas are still dependent on locally available plants for medicinal properties. D. hatagirea, is one such plant which is extensively used in traditional Indian medicinal systems such as Ayurveda, Unani, and Amchi since time immemorial for treating myriad health conditions, and in numerous different ways, which tend to differ across regions (Pant and Rinchen 2012).

Orchids are widely used as “salep” which is a flour prepared by grinding dried tubers of D. hatagirea, with glucomannan being its major constituent it is known to have high nutritional value and is considered a nervine tonic and immunomodulator. Salep is also a high quality astringent which possesses aphrodisiac properties (Baral and Kurmi 2006; Warghat and Sood 2013; Verma 2014; Dhiman et al. 2019). It is widely used in many systems of medicine such as Ayurveda, Amchi, Unani and Siddha. It is estimated that the annual consumption of the salep is about 7.38 tonnes (Dhiman et al. 2019). In Ladakh, milk-containing salep is boiled and consumed as a rejuvenating tonic. According to local people, in adverse conditions, consumption of 25 gm powdered tubers is sufficient for a day’s diet. Decoction prepared using salep, sugar and flavoured spices is used as a nutraceutical (Warghat and Sood 2013).

In the local households of Ghasa, Nepal, D. hatagirea is commonly used. Paste of the rhizome is prepared and applied on cuts, burns, skin problems and infectious wounds for swift recovery. After cleaning and drying, power of its rhizome is prepared, which is also used as a spice or consumed with milk for health benefits (Ranpal 2009).

The indigenous people in Rudraprayag, Uttarakhand, consider the plant to be useful for its therapeutic potential and use it along with other ingredients to cure numerous common health problems like diarrhoea, cough, dysentery, weakness, burns, etc. For treating normal weakness, roots of D. hatagirea along with other ingredients such as kunja (wild rose) pulp, Shilajeet are mashed together forming paste and consumed in the form of pills. Extracts of fresh tubers is consumed as a remedy for cough. Boiled tubers extract along with milk is known to be beneficial in healing bone fracture. A mixture of refined tubers, misri and milk is consumed to control spermatorrhoea. Juice obtained from the fresh roots of the plant is taken for curing stomachache and for treating wounds due to burns, the tubers are rubbed on stones and the paste, thus obtained is applied on the affected part. Tuber paste is also consumed orally to cure diarrhoea. It is also used to cure bleeding and wounds (Pant and Rinchen 2012; Chamoli and Sharan 2019; Ojha et al. 2020).

The ethnomedicinal uses of orchids found in Kashmir Himalayas by the inhabitants of the region has been studied by Shapoo et al. (2013). The study results revealed that the plant D. hatagirea was being used in different forms and dosages along with certain dietary restrictions in order to cure numerous health conditions. Almost 100–200 mL of dried tubers in powdered form along with sugar mixed in water is consumed daily and food items containing excessive fat is avoided for stomachic. For treating headache, poultice is prepared by crushing fresh tubers and applied 1–2 times a day along with avoiding consumption of cold water. A paste prepared by adding crushed fresh tubers and turmeric powder is applied once daily during the bedtime and cold water is avoided for treating fracture. For treating cough and cold, decoction is prepared by boiling dried flowers and tubers of the plant in water for 5 min followed by adding honey and is consumed 2–3 times. A mixture of crushed tubers and milk along with honey/sugar is consumed once or twice daily as vermifuge and also for curing nervous system weakness. Decoction of dried flowers and tubers boiled in water for 5 min is consumed twice a day by the person suffering from diarrhoea. Poultice prepared from crushed leaves and tubers once or twice per day. Powdered and dried plant along with ghee is consumed twice or thrice daily for general weakness after delivery. A mixture of fresh tubers, milk, sugar and almonds is believed to have aphrodisiac activity. Cold water, sugary, fatty foods and pickles are avoided with most of these preparations (Shapoo et al. 2013).

Several ethnomedicinal plants are used in the postpartum period to reduce childbirth-related complications among the Marwari community in Jodhpur, Rajasthan, and one of them is D. hatagirea. The plant along with numerous other medicinal plants such as Acacia Senegal, Coriandrum sativum, and Withania somnifera are powdered and a ladoo is prepared out of it with the help of ghee and is consumed by pregnant women as both prophylactic measure and for management of postpartum complications. D. hatagirea is believed to function as a uterine tonic and diuretic (Goyal 2017). Himalayan marsh orchid is also in many other areas for curing debilitated condition after childbirth and for increasing regenerative fluids (Pant and Rinchen 2012).

Intake of dried and powdered tubers along with milk is believed to enhance vigour in Ladakh. Its tubers are considered to have emollient, astringent, demulcent properties (Kumar et al. 2021). Juice from tuber is given for curing pyorrhoea while poultice prepared from root paste is applied on cuts and wounds (Vishwakarma and Karole 2021). A study on the use of medicinal plants for treating cold, cough and fever, by the Amchis (herbal practitioners of Amchi system of medicine) in Ladakh region of Himalayas, documented use of D. hatagirea (called Ambolakpa) for treating fever. The tubers of the plant are collected in the month of October, dried in shade and powdered along with Aconitum heterophyllum roots, Punica granatum seeds, fruits of Emblica officinalis, Terminalia chebula, leaves of Ficus religiosa, Azadirachta indica and mineral salt. It is consumed twice daily in the form of tablets (2–3) with warm water until recovery (Ballabh and Chaurasia 2007).

In the Kullu District of Himachal Pradesh investigation of the indigenous uses of the locally available medicinal plants on Parvati Valley found that the Himalayan Marsh Orchid was used as an expectorant, which enhances sputum secretion in the respiratory tract. It is also used for purifying blood, treating rheumatism, sexual disability, bone fractures, cuts, wounds and as an antibiotic (Sharma and Samant 2014).

The roots in the form of decoction are believed to be energy boosting and therefore, recommended for weak people especially by Balti and Brokpa ethnic groups in Ladakh (Haq et al. 2021). According to a study on ethnomedicinal plants in Devikund, Sunderdhunga Valley, Bageshwar District, Uttarakhand, the tuber extract of the plant is used in the treatment of whooping cough and fever (Sekar and Rawat 2011).

In the Sowa Rigpa system of medicine, the plant is known as d.bang-lag and is known to have spermatogenesis boosting, aphrodisiac and nourishing properties (Yeshi et al. 2017). A study in the Garam Chashma Valley, of the Chitral valley in Pakistan reported the use of D. hatagirea plant (indigenously called Juwari Joshu) for treating anaemia and as an aphrodisiac. The roots of the plant are dried and consumed orally in the form of powder while in the Hindu-Kush area it is mainly used as a nerve tonic and as sex stimulant. Ethnic groups residing in hilly districts of Nepal, such as Magar and Tangbeton, use D. hatagirea plant for treating head ache, stomachache, piles and typhoid (Miya et al. 2020; Birjees et al. 2021; Hassan et al. 2021).

Immunomodulatory potential of Dactylorhiza hatagirea: Certain bioactive substances derived from plant sources have also been reported to enhance immunocompetence. Although several chemical and synthetic immunomodulators are available in the market they suffer from major drawbacks such as nephrotoxicity, hepatotoxicity, gastrointestinal toxicity, neurotoxicity, cardiovascular toxicity, metabolic toxicity, among many others. This has led to an upsurge of interest in usage of plant-based immunomodulators. In plants, several compounds like glycosides, alkaloid, volatile oils, tannins, polypeptides, etc. also influence various physiological processes along with essential nutrients. Secondary metabolites from plants such as flavonoids, isoflavonoids, alkaloids, polysaccharides, glucans, have immunomodulatory potential (Shukla et al. 2012).

There are many medicinal plants which have been proven to be immunomodulators (Agarwal and Singh 1999; Kumar et al. 2012; Shukla et al. 2012; Sharma et al. 2017). Salep prepared from the roots of the plant D. hatagirea has been traditionally used as an immunomodulator (Choukarya et al. 2019). Several compounds from plant have potential to modulate the immune response. They can do so by virtue of their antioxidant and anti-inflammatory potential.

In vitro and human cells studies have shown that polyphenols like resveratrol have pro-inflammatory cytokine (such as TNF-α and IL-6) inhibitory properties. Resveratrol has both innate and adaptive regulation potential. It has shown NADPH oxidase inhibitory property in cell culture studies. Studies have also demonstrated that it inhibits spleen cell proliferation which is induced by concanavalin A, interleukin-2 or alloantigens. It also hinders IL-2, IFNγ, TNF-α and IL-12 production by lymphocytes and macrophages, respectively. It participates in T cell, natural killer cell and macrophage activation (Malaguarnera 2019; de Arruda et al. 2020).

6.6 Pharmacological Importance

6.6.1 Anti-microbial Activity

Indiscriminate use of commercial anti-bacterial drug use leads to multiple drug resistance. Hence, the anti-microbial potential of medicinal plants is being widely evaluated since these have fewer side effects and also prevent resistance build up against pathogens (Ranpal 2009).

Dactylorhiza hatagirea has been found to possess significant anti-microbial activity. In a study, Staphylococcus aureus, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa and Bacillus subtilis were used to test anti-microbial potential of the rhizome and aerial extract of plant. The plant extracts were prepared using petroleum ether, chloroform, methanol and water as solvents. Results showed that the chloroform extract of the aerial part formed the most active Zone of Inhibition (ZOI), i.e. 14 mm, for the bacteria E. coli. The solution was found to inhibit bacterial growth at concentration above 125 mg/mL. While the aqueous extract of the rhizome formed most active ZOI, i.e. 13 mm for the bacteria S. flexneri. The solution was found to inhibit bacterial growth above the concentration of 250 mg/mL. Both aerial and rhizome extracts were found to form ZOI in the range of 7–11 mm for the bacteria S. aureus for most of the solvents. Comparing ZOI of the plant extract with the standard antibiotics, namely Azithromycin, Amikacin, Ciprofloxacin, Norfloxacin and Nitrofurantoin, it was found that the ZOI of D. hatagirea was larger than Ciprofloxacin and similar to Norfloxacin for S. aureus, thereby indicating similar efficacy in inhibiting bacterial growth. For E. coli, the effectiveness of the aerial part of D. hatagirea was close to that of Ciprofloxacin which is surprising since this bacteria is highly resistant to many of the synthetic drugs. The ZOI value of Ciprofloxacin found to be nearly the same as that of aerial part of D. hatagirea, and similar results were obtained in case of rhizome part of bacteria Sh. flexneri. The results also concluded that D. hatagirea rhizome extract showed resistance against all Gram-positive and Gram-negative bacteria, with better effectiveness (except in case of E. coli), and hence had better anti-bacterial properties than the aerial part which exhibited resistance only against some bacteria (Ranpal 2009).

6.6.2 Antioxidant Activity

Free radicals have been speculated to be a critical factor in the development of many diseases of the humans such as ischaemia, atherosclerosis, cancer and central nervous system-related problems. Antioxidants typically function by quenching ROS and/or forming chelates with catalytic metal ions. Synthetic antioxidants such as butylated hydroxyl anisole (BHA) and butylated hydroxyl toluene (BHT) are considered quite unsafe and toxic. And so, sources of natural antioxidants which are both safe for human consumption and bioactive, capable of neutralizing free radicals, are constantly being researched (Ranpal 2009; Kumar et al. 2012). The antioxidant activity of D. hatagirea based on inhibitory concentration value (IC₅₀) using DPPH assay has been estimated to be 0.065–0.21 mg/mL (Sirohi et al. 2019; Kawra et al. 2020). The maximum antioxidant activity in D. hatagirea using DPPH assay has been found to be 40.13% and the maximum ferric reducing antioxidant power has been found to be 0.33% (Ali and Prasad 2016). The inhibitory concentration value using hydrogen peroxide and nitric oxide radicals has been found to be 53.01 μg/mL and 62.50 μg/mL for D. hatagirea as compared to 17.92 μg/mL and 24.17 μg/mL for ascorbic acid, respectively (Sirohi et al. 2019).

6.6.3 Aphrodisiac Activity

Dactylorhiza hatagirea is frequently used for its aphrodisiac properties by traditional medicine practitioners. A study on Wistar strain albino rats showed beneficial effects of the plant extract on the sexual organ and sexual behaviour. The study results showed that the resulting anabolic effect was similar to that of testosterone treatment. There was an increase in the weight of the sexual organs which indicates increased production of steroidal hormones. Rats which were treated with lyophilized extract experienced 2.5 times more attraction compared to the non-treated ones while the testosterone treated animals exhibited 2 times more attraction. Significant increase in copulation and number of bouts in the treated animals was observed. There was also an increase in number of ejaculating animals. Mount latency time decreased by 36% in animals treated with D. hatagirea extract as compared to 34% in testosterone-treated animals. Also there was 36% reduction in intromission as post-ejaculatory latency in the animals treated with D. hatagirea, and 17% reduction in group treated with testosterone as compared to the control group (Thakur and Dixit 2007a). Lyophilized aqueous extract of D. hatagirea exhibits aphrodisiac activity both in vitro and in vivo. An increase in the sperm count (141 × 10⁶) was observed in Wistar strain albino rats administered with 100 mg/kg body weight D. hatagirea extract which was significantly higher than the sperm count of the rats in both control group (110 × .10⁶) as well as in testosterone-treated group (121 × 10⁶). The Penile Erection Index (PEI) was higher in treated group (i.e. 49.8) as compared to the control group (24.6). Relative increase in inducible nitric oxide release was also observed, which was 12.9 μM for D. hatagirea and 4.93 μM for the control group (Thakur et al. 2011). D. hatagirea also increases the pendiculatory activity in male rats which reflects an enhancement of sexual behaviour. Administration of 200 mg/kg body weight of extract increased mean number of yawning to 1.33 on 14th day from 0.31 (on 0 day) and stretching increased to 1.36 on 14th day from 0.44 (on 0 day) which was also higher than the control group, i.e. 0.33 and 0.44, respectively. Average number of sperms/chamber of WBC counting chamber was also higher, i.e. 86.4 as compared to 61.4 in case of control group after 30 min incubation (Thakur and Dixit 2007b).

6.6.4 Anti-cancerous Activity

The anti-cancerous activity of the D. hatagirea extract was evaluated using MCF-7 and MDA-MB-231 cell lines, for which HEK-293 was used for normal cell line. In this study, MCF-7 and HEK-293 were grown in Dulbecco’s Modified Eagle Medium (DMEM) and MDA-MB-231 in Leibovitz (L-15). The results of the cytotoxicity study showed that the root and shoot extracts had difference in the percentage viability of the cells being tested, which was also dose dependent. The reduction in the number of cells was not significant, even with increasing concentration of plant extract. Percentage viability of root extract treated HEK-293 cells, increased to merely 94.44%, compared to 98.55% for the concentrations 1000 μg/mL and 250 μg/mL, respectively. While the percentage viability of shoot extract treated HEK-293 cells, decreased from 99.56 to 92.41% for concentrations 250 μg/mL and 1000 μg/mL, respectively. When MDA-MB-231 cell line was treated with root extract, there was significant reduction in viable cell population from 96.86% at 250 μg/mL to 82.38% at the concentration 1000 μg/mL. In case of shoot extract, the percentage viability of root extract decreased from 99.65% at 250 μg/mL concentration to 83.81% at the concentration 1000 μg/mL. The percentage viability of root extract treated MCF-7 cells declined from 99.17% at 250 μg/mL concentration to 84.24% at 1000 μg/mL concentration. Similarly, the percentage viability of shoot extract treated MCF-7 cells declined from 96.53% at 250 μg/mL concentration to 87.09% at 1000 μg/mL concentration, respectively. For HEK-293, the IC₅₀ value was 9900 μg/mL for root extract and 6362.5 μg/mL for shoot extract. Hence based on the study, it can be concluded that the extracts of the plant D. hatagirea can be used to treat cancer which functions by killing the cancerous cells without causing significant harm to normal cells (Popli and Sood 2017).

6.6.5 Anti-diabetic Activity

Anti-diabetic potential of D. hatagirea has been studied by administering D. hatagirea root extract in diabetic rats. For in vitro analysis, hydroalcoholic root extract of the plant was used and the percentage inhibition of α-amylase was calculated, with acarbose serving as positive control. α-amylase was found to be dose dependent on D. hatagirea extract, with IC₅₀ value being 35.33 for acarbose and 224.45 μg/mL plant extract. For in vivo analysis, alloxan monohydrate was used to induce diabetes in Wistar rats, which functions by causing β-cell necrosis, ultimately resulting in insulin deficiency. This resulted in variation in the biochemical parameters such as an increase in blood glucose, increased cholesterol and triglyceride, decreased protein content and body weight. The effect of root extract was compared with reference drug “glibenclamide”. Gradual significant reduction in blood glucose level was observed in all treatment group from 250 mg/dL to 125.20 mg/dL for D. hatagirea (100 mg/kg), 245 mg/dL to 117.8 mg/dL for 200 mg/kg plant extract and 240–112.7 mg/dL for glibenclamide treated group after 15 days. Similarly, significant reduction was also observed for other biochemical parameters such as total cholesterol which reduced to 109.6 mg/ dL for 100 mg/kg plant extract and 105.1 mg/ dL for 200 mg/kg plant extract and 101.1 mg/ dL for glibenclamide treated group while for untreated diabetic group it was 180 mg/dL. For triglycerides, it was 95.5 mg/dL for plant extract (100 mg/kg), 92.5 mg/dL for plant extract (200 mg/kg) and 89.2 mg/dL for glibenclamide (600 μg/kg) treated. Total protein content also increased in the treated group, with its values being 7.90gm/dL for plant extract (100 mg/kg), 8.35 mg/dL for plant extract (200 mg/kg) and 8.75 mg/dL for glibenclamide (600 μg/kg) treated compared to 5gm/dL in the untreated diabetic group. A significant increase in the body weight was also observed in the weight of treated groups. In the plant extract treated group, it increased by 30 g (for 100 mg/kg extract) and 35 g (for 200 mg/kg extract) while for glibenclamide treated group it increased by 40 g. In case of untreated diabetic group, a reduction of 10 g was noted. This study supports the use of D. hatagirea for treatment of diabetes (Choukarya et al. 2019; Magar et al. 2020). The methanolic leaf extract of D. hatagirea has been shown to possess anti-diabetic property without any cytotoxic effect on the cells. D. hatagirea leaf extract (500 μg/mL) can inhibit the activity of α-amylase to 75% as compared to 85% by standard drug acarbose, with IC₅₀ values of 51 μg/mL and 210 μg/mL for acarbose and leaf extract, respectively. Similarly, it can inhibit α-glucosidase activity to 72% as compared to 94% using acarbose with IC₅₀ values of 39 μg/mL and 200 μg/mL for acarbose and leaf extract, respectively. Bioactives in the leaf extract can also reduce post-prandial blood sugar levels by enhancing cellular uptake of glucose by inducing GLUT-4 translocation inhibition (Alsawalha et al. 2019).

6.6.6 Anti-inflammatory Activity

A number of phytochemical compounds found in D. hatagirea have potential anti-inflammatory activity. The hydroalcoholic extract exhibits dose-dependent anti-inflammatory response in Carrageenan-induced paw oedema with results similar to that of standard drug Diclofenac, with maximum effect being at fourth hour, i.e. 39% and 56.34% inhibition for doses 100 mg/kg and 200 mg/kg, respectively, thereby supporting the traditional use of the plant in inflammation management. (Sharma et al. 2020).

6.6.7 Antipyretic Activity

Rise in body temperature results from increased prostaglandin E2 concentration in the brain. Antipyretic activity of D. hatagirea was assessed by administering increasing doses of D. hatagirea extract to Wistar rats with Brewer’s yeast-induced pyrexia. A dose-dependent relationship was found to exist between the amount of extract and decrease in body temperature. There was a significant reduction in the rectal temperature which started 1 h after administration of its extract and continued till 4 h (Sirohi and Sagar 2019a).

6.6.8 Neuropharmacological Activity

Soporific drugs or hypnotic drugs, which are psychoactive drugs used to treat insomnia and are also used as surgical anaesthesia. Hydroalcoholic extract of D. hatagirea has been found to be safe and dose-dependent in prolonging duration of sleep in Swiss albino male mice which increased from 35.30 min for the group receiving 100 mg/kg, to 57 min for the group receiving 300 mg/kg. It is being speculated that the sedative effect of the plant extracts could be the result of facilitation of GABAergic transmission. (Sirohi and Sagar 2019b).

6.6.9 Other Applications

D. hatagirea tubers are used in silk industries as sizing material. Due to their beautiful flowers, they have been widely used for decoration and ornamental purposes and in perfume industries. The plant stem and leaves are also used as fodder for the livestock, vegetables and as insect repellent (Wani et al. 2020). Further studies are however required to elucidate the mechanism behind these effects and their active compounds responsible for the same.

6.7 Conclusion

Himalayas are the biodiversity hotspot which harbour many endemic species of high value medicinal plants such as Dactylorhiza hatagirea. Also known as Himalayan marsh orchid, D. hatagirea has several vernacular names such as Salam panja and Hatajadi. It grows mainly in the moist and shady regions and belongs to the family “orchidaceae”. It is a critically endangered with less population density and patchy distribution. A number of factors such as over-exploitation, illegal trading, excessive weed proliferation, overgrazing, low rates of propagation, poor seed germination, low genetic diversity have resulted in decline in their population. Its major chemical constituents are responsible for its medicinal properties are dactylorhins A–E, dactylose A and B, starch, mucilage, loroglossin, glucomannan, saponin, etc. D. hatagirea has been a part of many traditional systems of medicine such as Ayurveda, Unani, Amchi and Siddha. Locally, it has been used to treat a number of health conditions such as cough, cold, cuts, burns, skin problems, diarrhoea, fractures, general debility, head ache, sexual disability and weakened immunity. Several studies have been undertaken to establish pharmacological efficacy of the plant. In vitro and in vivo studies have shown that the plant has significant anti-microbial, antioxidant, aphrodisiac, anti-cancerous, anti-diabetic, anti-inflammatory neuropharmacological activity. However, its use in numerous other health conditions, and the effects on human consumption remains largely unverified. Other than improving health, the plant is also used in other industries as sizing material, decoration and ornamental purposes, insect repellent, etc. There is an urgent need to take suitable measures to prevent this plant population from further deterioration and studies need to be undertaken to elucidate the mechanisms behind their health promotive effects and role of their bioactive components.