A Review of Swertia chirayita (Gentianaceae) as a Traditional Medicinal Plant

Swertia chirayita (Gentianaceae), a popular medicinal herb indigenous to the temperate Himalayas is used in traditional medicine to treat numerous ailments such as liver disorders, malaria, and diabetes and are reported to have a wide spectrum of pharmacological properties. Its medicinal usage is well-documented in Indian pharmaceutical codex, the British, and the American pharmacopeias and in different traditional medicine such as the Ayurveda, Unani, Siddha, and other conventional medical systems. This ethnomedicinal herb is known mostly for its bitter taste caused by the presence of different bioactive compounds that are directly associated with human health welfare. The increasing high usage of Swertia chirayita, mostly the underground tissues, as well as the illegal overharvesting combined with habitat destruction resulted in a drastic reduction of its populations and has brought this plant to the verge of extinction. The increasing national and international demand for Swertia chirayita has led to unscrupulous collection from the wild and adulteration of supplies. The aim of this review is to provide a synthesis of the current state of scientific knowledge on the medicinal uses, phytochemistry, pharmacological activities, safety evaluation as well as the potential role of plant biotechnology in the conservation of Swertia chirayita and to highlight its future prospects. Pharmacological data reported in literature suggest that Swertia chirayita shows a beneficial effect in the treatment of several ailments. However, there is lack of adequate information on the safety evaluation of the plant. The pharmacological usefulness of Swertia chirayita requires the need for conservation-friendly approaches in its utilization. Providing high-quality genetically uniform clones for sustainable use and thereby saving the genetic diversity of this species in nature is important. In this regard, plant biotechnological applications such as micropropagation, synthetic seed production, and hairy root technology can play a significant role in a holistic conservation strategy. In addition to micropropagation, storage of these valuable genetic resources is equally important for germplasm preservation. However, more advanced research is warranted to determine the activities of bioactive compounds in vitro and in vivo, establish their underlying mechanisms of action and commence the process of clinical research.


INTRODUCTION
One of the prerequisites for the success of primary health care is the availability and use of suitable drugs. Traditional medicine is still the most affordable and easily accessible source of treatment in the primary healthcare system. Medicinal plants have always been a potential source to cure different diseases, either in the form of traditional preparations or as pure active principles, and they are frequently the only source of medicine for the majority of people in the developing world.
Swertia, a genus in the family Gentianaceae include a large group of annual and perennial herbs, representing approximately 135 species. Swertia species are common ingredients in a number of herbal remedies. In India, 40 species of Swertia are recorded (Clarke, 1885;Kirtikar and Basu, 1984), of which, Swertia chirayita is considered the most important for its medicinal properties. S. chirayita was first described by Roxburgh under the name of Gentiana chyrayta in 1814 (Scartezzini and Speroni, 2000). S. chirayita, common name: "Chiretta" (Figure 1) is a critically endangered medicinal herb that grows at high altitudes in the sub-temperate regions of the Himalayas between 1200 and 2100 m altitudes from Kashmir to Bhutan (Bentley and Trimen, 1880;Clarke, 1885) on the slopes of moist shady places (Gaur, 1999; Figure 2). Its widespread uses in traditional medicine have resulted in over-exploitation from the natural habitat and it is Abbreviations: ABTS, 2,2-azino-bis (3-ethylebenzothiazoline-6-sulphonic acid); ACE, Acetone; BA, 6-benzyl-adenine; BHA, Butylated hydroxy anisole; BHT, Butylated hydroxytoluene; 2,4-D, 2,4-Dicholorophenoxyacetic acid; DPPH, 2,2diphenyl-1-picrylhydrazyl; DW, Dry weight; EtOH, Ethanol; EA, Ethyl acetate; FRAP, Ferric Reducing Antioxidant Power; GA 3 , Gibberellic acid; HEX, Hexane; IAA, Indole-3-acetic acid; KN, Kinetin; MeOH, Methanol; NAA, Naphthalene Acetic Acid; PE, Petroleum ether. now on the verge of extinction in the wild. S. chirayita is also known by an array of names such as Anaryatikta, Bhunimba, Chiratitka, Kairata in Sanskrit, Qasabuzzarirah in Arab and Farsi, Chiaravata in Urdu, Sekhagi in Burma, and Chirrato or Chiraita in Nepal (Joshi and Dhawan, 2005). Some authors have described S. chirayita as an annual (Anon, 1982;Kirtikar and Basu, 1984) and others as a biennial or pluri-annual (Edwards, 1993). This ethnomedicinal herb is known mostly for its bitter taste caused by the presence of different chemical constituents such as amarogentin (most bitter compound isolated till date), swerchirin, swertiamarin, and other bioactive compounds that are directly associated with human health welfare (Joshi and Dhawan, 2005). Due to its excessive over-exploitation from the natural habitat, narrow geographic occurrence (Bhat et al., 2013) and unresolved inherent problems of seed viability and seed germination (Badola and Pal, 2002;Joshi and Dhawan, 2005), alternative approaches for propagation and conservation are urgently required to avoid the possible extinction of this important species. Consequently, S. chirayita has been receiving increasing attention from a wide range of researchers as evident from the number of publications appearing in the literature (Chen et al., 2011;Nagalekshmi et al., 2011;Ghosh et al., 2012;Kumar and Chandra, 2013Fan et al., 2014;Sharma et al., 2014Sharma et al., , 2015Padhan et al., 2015;Zhou et al., 2015). However, a comprehensive review detailing the documented ethnomedicinal uses, pharmacological properties and safety evaluation carried out on S. chirayita and identifying the existing knowledge gap is lacking. In this review, we document the medicinal uses and phytochemical properties of S. chirayita. Future prospects including the potential conservation approaches to ensure a continuous supply for both local and international expanding markets and safety FIGURE 2 | Natural distribution of Swertia chirayita. The shaded area represents the natural habitat of Swertia chirayita in the Himalayan Region. evaluation on uses of the species for medicinal purposes are highlighted.

Botanical Description
S. chirayita is an annual/biennial herb 0.6-1.5 m tall. It has an erect, around 2-3 ft long stem, the middle portion is cylindrical, while the upper is quadrangular, with a prominent decurrent line at each angle. Its stem is orange brown or purplish in color with large continuous yellowish pith (Bentley and Trimen, 1880;Joshi and Dhawan, 2005). Leaves are lanceolate, in opposite pairs, no stalks, acuminate, cordate at the base, sessile, five to seven nerved and 4 cm long (Scartezzini and Speroni, 2000). The root is simple, yellowish, somewhat oblique, or geniculate, tapering and short, almost 7-8 cm long and usually half an inch thick (Bentley and Trimen, 1880;Scartezzini and Speroni, 2000). Flowers are small, numerous, tetramerous, large leafy panicles, green-yellow, and tinged with purple and green or white hairs (Scartezzini and Speroni, 2000;Joshi and Dhawan, 2005). The calyx is gamophyllous with four lobes, corolla-lobes four twisted and superimposed, united at the base where they have pairs of nectaries on each lobe covered with long hairs. Stamens 4, opposite the corolla lobe, at the base of the corolla. Ovary unilocular with ovules laminal placentation parietale; two stigmas. Capsules are egg-shaped, 2-valved with a transparent yellowish pericarp. Seeds are numerous, very small and dark brownish in color (Chandra et al., 2012). Multi-colored corolla and the presence of nectaries support cross-pollination in S. chirayita.

MEDICINAL USES
S. chirayita a traditional Ayurvedic herb is used by different indigenous population groups in multiple ways for several medicinal purposes ( Table 1). The whole plant is widely used by local people for the treatment of hepatitis, inflammation, and digestive diseases (Bhatt et al., 2006). The wide range of medicinal uses include the treatment of chronic fever, malaria, anemia, bronchial asthma, hepatotoxic disorders,  Mukherji, 1953;Kirtikar and Basu, 1984;Joshi and Dhawan, 2005; Whole plant Used in British and American pharmacopeias as tinctures and infusions Joshi and Dhawan, 2005 Root Serves as a drug and an effective tonic for general weakness, fever, cough, joint pain, asthma, and the common cold Kirtikar and Basu, 1984;Joshi and Dhawan, 2005 Whole plant For headaches and blood pressure, the leaves and chopped stems are soaked overnight in water. A paste is prepared and filtered with 1 glass of water. The preparation is consumed once a day for 2-3 days de Rus Jacquet et al., 2014;Malla et al., 2015 Whole plant For Tremor fever, whole S. chirayita plants are cut into small pieces and boiled in 1/2 L of water until the volume is reduced to less than half glass. The filtered water is stored in a glass bottle and half spoon is given to children once a day for 2 days. For adult, the posology is 1 spoon once in a day for 2 days and varies to three times a day until cured de Rus Jacquet et al., 2014 Whole plant Boiled in water and one cup of decoction is taken orally to cure malaria Shah et al., 2014 Whole plant Paste of the plant is applied to treat skin diseases such as eczema and pimples Joshi and Dhawan, 2005;Malla et al., 2015 Whole plant Liver disorders; stomach disorders like dyspepsia and diarrhea, intestinal worms Mukherji, 1953;Joshi and Dhawan, 2005 Whole plant Hiccups and vomiting, ulcers, gastrointestinal infections, and kidney diseases Kirtikar and Basu, 1984 Whole plant Used in combination with other drugs in cases of scorpion bite Nandkarni, 1976 Whole plant Used in excessive vaginal discharge Jadhav and Bhutani, 2005 liver disorders, hepatitis, gastritis, constipation, dyspepsia, skin diseases, worms, epilepsy, ulcers, scanty urine, hypertension, melancholia, and certain types of mental disorders, secretion of bile, blood purification, and diabetes (Karan et al., 1999;Banerjee et al., 2000;Rai et al., 2000;Saha et al., 2004;Chen et al., 2011). Recently, S. chirayita extracts showed anti-hepatitis B virus (anti-HBV) activities (Zhou et al., 2015). Traditionally, decoctions of this species are used for anthelmintic, hepatoprotective, hypoglycemic, antimalarial, antifungal, antibacterial, cardiostimulant, antifatigue, antiinflammatory, antiaging, antidiarrheal, as protectant of the heart and also help in lowering blood pressure and blood sugar (Schimmer and Mauthner, 1996). Herbal formulations such as Ayush-64, Diabecon, Mensturyl syrup, and Melicon V ointment (Edwin and Chungath, 1988;Mitra et al., 1996) contain S. chirayita extract in different concentrations for its antipyretic, hypoglycaemic, antifungal, and antibacterial properties. Furthermore, the curative value of this herb has also been recorded in ancient Ayurveda medicine systems and other conventional medical systems. The widespread uses of S. chirayita in traditional drugs have resulted in considerable chemical analysis of the plant, and active principles which attribute the plant its medicinal properties. S. chirayita is also used in British and American pharmacopeias as tinctures and infusions (Joshi and Dhawan, 2005). The whole plant is used in traditional remedies but the root is mentioned to be the most bioactive part (Kirtikar and Basu, 1984).

PHARMACOLOGICAL ACTIVITY
The varied ethnobotanical uses of S. chirayita have led to the initiation of various pharmacological investigations. Previous research demonstrates that the S. chirayita extracts exhibit a wide range of biological activities, such as antibacterial, antifungal, antiviral, anticancer, anti-inflammatory, and others like antidiabetic and antioxidant activities (Verma et al., 2008;Alam et al., 2009;Arya et al., 2011;Chen et al., 2011;Laxmi et al., 2011). Concurrently, a diverse range of in vitro and in vivo test systems has been used to evaluate the pharmacological properties of S. chirayita. Evidence-based laboratory investigations indicate that aqueous, alcoholic and methanolic extracts of S. chirayita possess a number of promising pharmacological properties. The whole plant of S. chirayita have been reported to be used for the treatment of antibacterial and antifungal activity (Alam et al., 2009;Laxmi et al., 2011;Rehman et al., 2011). Anti-hepatitis B virus activity of S. chirayita extracts was also studied on HepG 2.2.15 cells line (Zhou et al., 2015). The whole plant of S. chirayita has been reported for the anti-inflammatory and hypoglycemic activity (Banerjee et al., 2000;Kar et al., 2003;Alam et al., 2011;Das et al., 2012;Verma et al., 2013). Chen et al. (2011) investigated the 70% ethanolic extract of S. chirayita for antioxidant activities by using antioxidant tests including reducing power and beta-carotene assay. The results showed that 70% ethanolic extracts exhibited high DPPH scavenging activity (IC 50 = 267.80 µg/mL). Table 2 presents a summary focusing on the pharmacological evaluations using in vitro and in vivo systems whereas Table 3 provides antioxidant potential of S. chirayita.

SAFETY EVALUATION
Concerns regarding safety of conventional drugs are vital issues of pharmaceutical industries. Studies have indicated that some commonly used medicinal plants may be mutagenic or cytotoxic especially over a long period of use (Verschaeve and Van Staden, 2008). There is increasing evidence on the toxicity of crude extracts and isolated compounds from different plant species (Koorbanally et al., 2006). However, despite its long history of use in traditional medicine, there is still a lack of scientific information concerning the safety evaluation of S. chirayita. It can be traced through the medicinal history as a nontoxic and safe ethnomedicinal herb and has been mentioned in medical papyri to expel fever, relieve headache, inflammation, and to stimulate the central nervous system. S. chirayita extracts, did not cause obvious toxic effects in mice as there were no significant differences in body weight and body temperature between the treated and control groups (Alam et al., 2011;Das et al., 2012). A clinical study by Medda et al. (1999) concluded that S. chirayita revealed no evidence of toxicity in both liposomal and niosomal forms. Furthermore, stringent efforts are required to further delineate the well-documented toxicological properties involving toxicity and mutagenic tests to evaluate the safety of this plant. Nevertheless, rigorous clinical studies involving different mechanisms are still needed to confirm the safety of S. chirayita in traditional medicine so that it can be used safely and effectively. Despite the fact that the benefits of medicinal plants is globally acknowledged, the need for better insight on the safety evaluation remains essential, so as to differentiate between toxic effects and pharmacological importance of plant extracts (Aremu and Van Staden, 2013).

SWERTIA CHIRAYITA CONSERVATION
Destruction of plant resources is a normal occurrence. The current speed of extinction through human interferences is estimated to be approximately 100-1000 times faster than the natural speed of extinction (Chapin et al., 2000). Due to developmental activities in the Himalayan region, wild populations of many medicinal plants, including S. chirayita are reduced to the verge of extinction. S. chirayita is traded and used mostly as a traditional drug. Due to its multiple uses the demand is on the rise by both national and international trading leading to increasing over harvesting of wild populations. This has resulted in drastic reductions of its populations. Lack of comprehensive data on annual harvested and traded plants of S. chirayita is also a major concern. According to the International Union of Conservation of Nature (IUCN) criteria, S. chirayita conservation status has been categorized as "critically endangered" (Joshi and Dhawan, 2005). S. chirayita is among the 32 most highly prioritized medicinal plants of India as identified by The National Medicinal Plant Board, Government of India (http://www.nmpb. nic.in).
The implication of losing this plant species due to extinction lies not only in the loss of genes useful for plant development or in the biosynthesis of new compounds but also the loss of potentially novel compounds of pharmaceutical or nutraceutical benefit. In order to meet the escalating demand in national and international trade markets of raw plants, cultivation must be escalated. There are limitations in the use of seed propagation, due to low viability, and low germination percentages (Badola and Pal, 2002;Chandra et al., 2012). Biotechnology offers new means of improving biodiversity and biotechnological approaches such as micropropagation techniques ( Figure 1E) has received more attention and may play a vital role in the establishment of genetically uniform plants for the Swertia industry. It is believed that the development of efficient micropropagation protocols, can guarantee an adequate supply of S. chirayita plants (devoid of environmental-imposed constraints) with subsequent reduction in uncontrolled harvesting pressure on wild populations. Several studies reported on micropropagation, somatic embryogenesis and acclimatization procedures with the capacity to produce many uniform S. chirayita clones throughout  the year Chandra, 2013, 2014;. As shown in Table 5, micropropagation protocols have successfully been established for S. chirayita using different explants. Synthetic seed technology is also an applied application of modern plant biotechnology which offers tremendous potential for easy handling, micropropagation and plant germplasm conservation through cryopreservation (Ara et al., 2000;Sharma et al., 2013a;Perveen and Anis, 2014;Gantait et al., 2015). Successful implementation of synthetic seed technology for mass propagation and short-term storage of genetically uniform clones require manipulation of in vitro tissue culture systems that are able to transform into complete plantlets (Ara et al., 2000). Recently,  reported on synthetic seed production and plant regeneration of S. chirayita from somatic embryos. However, further studies are required to improve technology so that it can be used on a commercial scale.
Many plant secondary metabolites accumulate in roots (Flores et al., 1999) but harvesting of these organs is destructive. Therefore, in the recent past Agrobacterium rhizogenes induced hairy root technology has received attention and engaged a new platform of applied research in generating pharmaceutical lead compounds. The large scaling-up of hairy root cultures is of importance for biotechnological applications (Guillon et al., 2006). Attempts have been made to standardize A. rhizogenes transformed root cultures for production of active secondary metabolites under in vitro conditions of S. chirayita (Keil et al., 2000). For commercialization of S. chirayita adventitious roots and to elucidate the feasibility

Efficient Regeneration
In vivo shoot tip 0.5 mg/l BA + 1.0 mg/l GA 3 An efficient shoot proliferation Kumar and Chandra, 2013 In vitro flower production Axillary bud 1.0 mg/l BA + 70 mg/l Adenine sulfate In vitro flowering and effective protocol for regeneration Sharma et al., 2014 Somatic embryogenesis In vivo leaves 0.5 mg/l 2,4-D + 0.5 mg/l KN An efficient protocol for plant regeneration through somatic embryogenesis  Direct and Indirect regeneration for commercial application, hairy root technology is required along with various factors affecting the production of root biomass and bioactive compounds. Overall, micropropagation which is conducted under a controlled environment will help to prevent the current plant biodiversity conservation problems arising from over harvesting practices of wild populations and can profoundly improve the quality of bioactive secondary metabolites of this age old medicinal plant S. chirayita.

CONCLUSIONS AND FUTURE PERSPECTIVES
S. chirayita offers many promising prospects for both traditional and modern medicine. S. chirayita is apparently a potential herbal therapy for many ailments. This review summarized the existing ethnobotanical uses, phytochemistry, pharmacological activities, safety evaluation, and conservation status on S. chirayita. So far no serious side effects or toxicity of S. chirayita have been reported, but further toxicological studies are still needed to confirm the safety of S. chirayita in humans. Efforts are required for further studies, especially evaluating its biological activities in vivo and toxicological and mutagenic properties in order to better validate the safety of these different plant-derives compounds. In all probability there is a need for clinical trials to establish the efficacy of using S. chirayita in medicine. Due to its multiple uses the demand in both national and international markets is constantly on the rise. Overexploitation combined with habitat destruction has resulted in the drastic reduction of its population. For the successful commercialization of this critically endangered medicinal plant any proposed research must be viewed in a wider context that includes conservation practices and sustainable supply of raw plants. This will require innovative tools, which utilize biotechnological interventions, including micropropagation, cryopreservation, and bioreactors for the conservation, as well as for raising commercial production. In synthetic seed technology more detailed research is required mainly for improvement in germination frequency of synthetic seeds and subsequent plantlet growth in soil so that it can be used on a commercial scale. Additionally, in the near future, hairy root technology can be used as a model system and will also provide plant biotechnologists with powerful tools to improve the valuable phytochemicals of S. chirayita. Although efficient micropropagation protocols have been established, further studies focusing on seed biology and ways of improving bioactive secondary metabolites in cultivated S. chirayita would be beneficial for their commercialization. Quality control protocols to prevent misidentification and possible adulteration of S. chirayita are also needed. In summary, S. chirayita have been studied extensively in terms of taxonomy, ethnobotany, phytochemistry, biological activities, and conservation. However, new findings may increase the present therapeutic importance of S. chirayita and promote their future use in modern medicine, while novel biotechnological approaches are required for further conservation.

AUTHOR CONTRIBUTIONS
VK conducted the research and wrote the paper. JVS supervised the work and proof read the paper.

ACKNOWLEDGMENTS
VK is grateful to the National Research Foundation and the University of KwaZulu-Natal, South Africa for the financial support. Dr. M. Moyo and Dr. A. O. Aremu are thanked for reading the manuscript.