Pure Appl. Biol., 4(3): 407-417, September- 2015 http://dx.doi.org/10.19045/bspab.2015.43017

Review Article

Picrorhiza kurroa: An ethnopharmacologically important species of Himalayan region

Maria Masood*, Muhammad Arshad, Rahmatullah Qureshi, Sidra Sabir, Muhammad Shoaib Amjad, Huma Qureshi and Zainab Tahir Department of Botany, PMAS- Arid Agriculture University, Rawalpindi, Pakistan *Corresponding author email: [email protected] Citation Maria Masood, Muhammad Arshad, Rahmatullah Qureshi, Sidra Sabir, Muhammad Shoaib Amjad, Huma Qureshi and Zainab Tahir. kurroa: An ethnopharmacologically important plant species of Himalayan region. Pure and Applied Biology. Vol. 4, Issue 3, 2015, pp 407-417. http://dx.doi.org/10.19045/bspab.2015.43017 Received: 15/06/2015 Revised: 20/08/2015 Accepted: 25/08/2015 Abstract Picrorhiza kurroa Royle ex Benth. commonly known as Kutki, belongs to family Scrophulariaceae. It is found in the Himalayan regions of China, Pakistan, India, Bhutan and Nepal. It is considered as an important medicinal plant which is mostly used in the traditional medicinal system for asthama, jaundice, fever, malaria, snake bite and liver disorders Different pharmacological activities of P. kurroa include anti-microbial, anti-oxidant, anti-bacterial, anti- mutagenic, cardio-protective, hepato-protective, anti-malarial, anti-diabetic, anti-inflammatory, anti-cancer, anti-ulcer and nephro-protective activities were recorded from this plant. So far, Iridoids (Picroside I and II), Cucurbitacins and Phenolic components are the different phytochemicals which are extracted from P. kurroa. The authentification of P. kurroa raw material for commercially available herbal/botanical products is essential and it is done by the DNA fingerprinting of P. kurroa. Because of the over-exploitation of P. kurroa for medicinal purposes, the conservational status of P. kurroa in different regions became endangered. It is the need of the hour to utilize different conservational strategies and save this medicinal wealth from extinction as it is widely used by the local people for curing different diseases and thus it cause immense pressure on the plant population. Keyword: Ethnopharmacology; Conservation; Extinction. Introduction regions of Nepal where it grows on the rock’s Geographical distribution crevices on the north facing slopes, cliffs and Kutki is a perennial herb found in the the turf of glacial flats. Himalayan region (Garhwal to Bhutan), West P. kurroa Royle ex Benth.is present in wild China, South-East Tibet and North Burma. It form in the north-western Himalayan region grows in wild form in alpine regions on rock from Kashmir to Sikkim [1]. It is found in the crevices and also in organic soils. In Nepal, North-Western Himalayan region from Kutki is found in abundance in alpine Kashmir to Kumaun and Nepal and Garhwal Himalayan region between altitudes of regions in India [2]. It is found in Nepal, 3500m and 4800m and also in the western China, India, Pakistan and Bhutan [3].

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Taxonomy and Morphology Pollen grains round, tri-colpate, with The complete botanical name is Picrorhiza incomplete or perforate tectum, the partial kurroa Royle ex Bentham. It has got two tectum micro-reticulate, colpus membrane synonyms i.e. Picrorhiza lindleyana (Wall.) smooth or occasionally coarse [10, 11]. Steud. and Veronica lindleyanaWall. Rhizome is 2.5–12.0 cm long and 0.3–1.0 cm In Greek, “picros” means bitter, while thick, sub-cylindrical, straight or to some “rhiza” means root.The specific epithet of extent curved, externally greyish-brown, plant is taken from the Punjabi name of the external surface is coarse due to longitudinal plant “Karu”, which means bitter [2]. furrows and spherical scars of roots, tip ends Vernacular names being used for P. kurroa in a growing bud enclosed by a crown of include Balakadulen, Hellebore, leaves (Figure 2). Root is elongated, tubular, hohwangryun, honglen, hunglen, honkadu, 5–10 cm in length and 0.5–1.0 mm in huhuanglian, kadu, kadukrohini, diameter, straight or marginally curved with kadugurohini, kalikutki, karupicrorhiza, a few longitudinal and dotted scars, mostly karru, katki, katu, katuka, Karukarohini, associated with rhizomes [12]. katukaa, Kaurohini, Kavi, Kauka, katuka, The anatomy of rhizome shows 20–25 layers katukarohini, katukarogani, katukarohini, of cork consisting of tangentially extended, katuki, katuko, katukurohini, katurohini, suberized cells; 1–2-layered cork cambium; katvi, kaur, khanekhaswael, kharbaqe-hindi, cortex single layered or not present, main kotkaanphraao, koouren, kotkaanphraao, cortex continues in some cases, 1 or 2 small kour, kurri, kuru, kutaki, kutki, kutta, rohini, sized vascular bundles (xylem and phloem) sutiktaka, tiktarohini, Tikti, xi present in the cortex. Vascular bundles are zanghuhuanglian [4-8]. surrounded by fibrous bundle sheath. Morphological and Taxonomic Features Secondary phloem is made up of parenchyma Picrorhiza kurroa Royle ex Benth. belongs to cells and a few dispersed fibers. 2–4 layered Scrophulariaceae family. This is a large plant thick cambium is present. Secondary xylem family, with around 200 genera and 3000 consists of tracheids, vessels, fibers and species, mainly found in the northern parenchyma cells. Vessels vary in size and temperate regions of the world. shape, tracheids long, thick walled,lignified, Scrophulariaceae includes the such as more or less cylindrical with blunt pointed popular garden plants (including tiny ends. Starch grains are abundantly present, alpines); and also some other plants grown 25–105 μm in diameter. Anatomy of root for their aesthetic value (include Penstemon, shows when root is young, it shows single Mimulus and Calceolaria) [9]. layered epidermis, some epidermal cells Kutki is a perennial herb with an elongated stretch forming unicellular hairs. rhizome. The leaves are basal and alternate, Hypodermis is single-layered. Cortex 8–14 approximately 5–10 cm long (Figure 1). layered, consisting of ovoid to polygonal, Terminal Spikes are present. Calyx divide thick-walled parenchymatous cells. Primary equally in 5 parts. The corolla has 4 or 5 stele, tetrarch to heptarch, enclosed by a lobes, 4–5 mm long, bilobate with lobes more single-layered pericycle and single layered or less spreading or nearly actinomorphic. thick-walled cells of endodermis. Mature Stamens 4, inserted on corolla tube, slightly roots show 4–15 layers of cork, 1–2 layers of didynamous. Stigma capitate. Fruit an acute cork cambium. Vessels vary in size and capsule, tapered at top, dehiscing into 4 shape, some tubular with tail-like, tapered valves, 12 mm long. Seeds numerous, ends; some barrel shaped with perforation on ellipsoid: seed coat very thick, transparent.

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Pure Appl. Biol., 4(3): 407-417, September- 2015 end walls or adjacent walls. Tracheids are extracts of rhizome of P. kurroa was checked cylindrical with tapered sharp ends [1]. against bacterial strains which belonged to The flowering period of Kutki is from June to gram positive and gram negative bacteria. August. It is found far away from the Ethanolic extract of rhizome of P. kurroa community and takes from hours to days to exhibited high antibacterial activity against walk to its growing habitat. It has been B. cereus, E. coli, K. pneumoniae, S. aureus, reported that plant has been harvested to near S .pyogens and S. typhi. The methanolic extinction. extract of rhizome showed greater Pharmacological Activities antibacterial activity against S. aureus and P. The pharmacological properties of aeruginosa, while hexane and acetone extract Picrorhiza kurroa Royle ex Benth. include showed intermediate activity against B. anti-microbial, hepatoprotective, anti- cereus, E. coli, K. pneumonia, P. aeruginosa, oxidant, anti-bacterial, anti-mutagenic and S. aureus, S. pyogens and S. typhi. Aqueous anti-cancer activities. extract of rhizome did not show antibacterial Rhizomes of Picrorhiza kurroa Royle ex activity against the tested bacterial strains Benth., when phytochemically screened [14]. showed the presence of bioactive To evaluate mutagenic potential of P. kurroa components, which have been linked to root extract, the extract was tested in antimicrobial properties. Numerous chemical Salmonella typhimurium reverse mutation experiments and TLC studies revealed the assay with five histidine-requiring strains of occurrence of sterols, glycosides and Salmonella typhimurium (TA1537, TA1535, phenolic compounds when tested on various TA98, TA100 and TA102). The test was extracts of rhizomes of P. kurroa. The main done in two autonomous experiments (viz., chemical components found in P. kurroa Dose Selection Study/Cytotoxicity Study and include iridoid glycosides and cucurbitacins Mutagenicity Study) in the absence and (triterpenoids). The effects of aqueous and presence (10% S9 v/v) of metabolic methanolic extracts on pathogenic fungal activation system in triplicates. In the dose (including Candida albicans, Aspergillus range finding study, the extract was tested at niger, Pseudomonas aeruginosa) and the dose levels of 78.13, 156.25, 312.5, 625, bacterial strains (including Staphylococcus 1250, 2500 and 5000 μg/ml in the absence aureus, Bacillus subtilis, Micrococcus luteus, and presence (10% S9 v/v) of metabolic Escherichia coli) indicated that the plant part activation system. The results of solubility, can be used to treat infections caused by these precipitation of test item and cytotoxicity bacteria and fungi. The aqueous and study/dose range finding study, root extract methanolic extracts showed antibacterial of Picrorhiza kurroa was verified in the activity but the significant antimicrobial mutagenicity assay at the subsequent dosage activity was shown by methanolic extract levels. Mutagenicity Study with metabolic only, against P. aeruginosa and S. aureus. activation: 312.5, 625, 1250, 2500 and 5000 The aqueous extract was not much effective μg/ml. P. kurroa root extract did not induced for anti-microbial activity and also it showed a significant dose-linked rise in the quantity no activity against fungal strains. The of revertant (his+) colonies in each of the five efficacy of the extracts was less than tester strains of Salmonella typhimurium in ciprofloxacin, which is the conventional the absence and presence (10% S9 v/v) of antibiotic [13]. metabolic activation system. It was The antimicrobial potential of acetone, concluded that Picrorhiza kurroa root extract aqueous, ethanol, hexane and methanol is non-mutagenic in the Salmonella

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Masood et al. typhimurium reverse mutation assay (Ames Anti-inflammatory effect of P. kurroa extract test). Thus it verifies the Anti-mutagenic (PK) by l3-adrenergic blockade was activity of P. kurroa root extract [15]. confirmed, which suggests alteration in cell- Oral administration of ethanolic extract of surface biology by PK action. Obstruction of Picrorhiza kurroa rhizome at a dose of protein synthesis by cycloheximide pre- 20mg/kg of the body weight, for 10 treatment reduced PK effect suggesting consecutive days, was found to increase the protein mediation. Dinitrophenol which is a healing speed on Indomethacin-induced metabolic inhibitor, repressed inflammatory abdominal ulcer in mice, compared to the oedema likewise in control and PK treated mice having ulcer but without treatment. The animals, and masking of PK effect was level of peroxidised fat, in terms of concluded. Selective PK effect on membrane thiobarbituric acid reactive species related activation procedures in (TBARS), in abdominal tissue, was increased inflammatory effector cells could be the in ulcerated mice which was restored to origin of anti-inflammatory and perhaps normal on treatment with ethanolic extract. other biological activities [18]. The specific activity of in-vivo antioxidant The antioxidant properties of P. kurroa were enzymes i.e. SOD, catalase and total tissue evaluated in-vitro using different radical sulfhydryl group ,which were distinctly scavenging methods. Moreover, the reduced in ulcerated group ,were found to be antioxidant activity of extract was tested by considerably raised (p< 0.05), on treatment using liver slice culture system and ethanol with the above extract, at the specified dose, was used as a hepatotoxin to generate compared to the indomethacin-induced oxidative stress. Discharge of intracellular ulcerated group without treatment. So it marker enzymes such as glutamate suggests that ethanolic extract of P. kurroa oxaloacetate transaminase, lactate rhizome,at the dose of 20mg/kg, enhanced dehydrogenase and glutamate pyruvate the healing of stomach wall of indomethacin transaminase indicated hepatotoxicity. induced gastric ulcerated rats by an in-vivo Ethanol induced oxidative stress and its free radical scavenging mechanism [16]. modulation in the presence of P. kurroa The ethanolic extract of rhizome of P. kurroa extract was verified by measuring the was checked for nephroprotective and quantities of antioxidant enzymes such as nephrocurative activity in female Wistar catalase, glutathione reductase, glutathione mice against Cisplatin induced peroxidase, glutathione-S-transferase, nephrotoxicity, by estimating blood urea and superoxide dismutase and of antioxidant serum creatinine levels. One of the molecules i.e. uric acid and reduced Ayurvedic preparations i.e. Arogyawardhini, glutathione that were quantified along with comprising P. kurroa as a major ingredient lipid peroxidation. The results clearly prove was also studied for the nephrocurative and that aqueous extract of P. kurroa with high nephroprotective effects against Cisplatin antioxidant activity, as confirmed using induced nephrotoxicity. The preparation was different radical scavenging methods, was standardized for the existence of total active in reducing the harmful effects of polyphenols. Treatments with the ethanol ethanol. Addition of P. kurroa aqueous extract of the rhizome in the dose of 600 extract along with ethanol reestablished the mg/kg could considerably decrease the high activities of antioxidant enzymes and serum levels of blood urea and creatinine. So significantly suppressed lipid peroxidation the preparation was found to have better [19]. effect as compared to the rhizome [17].

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Phytochemistry of P. kurroa Other minor constituents include picroside- Picrorhiza kurroa Royle ex Benth.comprises III [Figure 3(c)], veronicoside [Figure 3(i)], of iridoid glycosides i.e. picroside I and II minecoside, phenol glycoside i.e. Picein [Figure 3(a), 3(b)] as key bioactive [Figure 3(o)] and androsin [Figure 3(j)], components. The high pressure liquid cucurbitacin glycosides and 4-hydroxy-3- chromatography (HPLC) method was used to methoxy acetophenone [21]. determine the picroside-I and picroside-II Kutkin, which is one of the active constituent content in rhizome of seven accessions of Picrorhiza kurroa is composed of [Rohtang-3978 m (PKR-1); Marhi-3300 m kutkoside and the iridoid glycoside (PKM-2); Manali-2050 m (PKM-3); (picrosides I, II, and III). Other active Keylong-3350 m (PKK-4); Khoksar-3160 m constituents identified from plant extract are (PKK-5); Manikaran-1737 m (PKM-6) and apocyanin [Figure 3(h)], drosin and nine Chamba-996 m (PKC-7)]. Both components cucurbitacin glycosides. Apocynin is a i.e. picroside I and II have been detected in catechol that can prevent neutrophil oxidative the rhizome of all seven accessions of P. burst, also it is a powerful anti-inflammatory kurroa. After thorough evaluation, it was agent, while the curcubitacins are highly confirmed that the fraction of both analytes cytotoxic and antitumor in action [22-24]. were found to be maximum, that is, The cucurbitacins include triterpenes, known picroside-I (3.5%) and picroside-II (2.0%) in for their bitterness and toxicity. rhizome of P. kurroa collected from Rohtang Cucurbitacins isolated from Picrorhiza area (3978 m). The chemical studies on the kurroa include cucurbitacin B, D [Figure rhizomes of P. kurroa indicated the presence 3(f), 3(g)] and R. The basic nucleus of of iridoids, acetophenones and cucurbitacins. cucurbitacins is 9-methyl-19-norlanosta-5- It is known to contain picroside I and II, as ene and it is also known as 10-cucurbit-5-ene. major bioactive compounds. Both species of Some of the iridoids in P. kurroa are genus Picrorhiza, that is, P. scrophulariiflora picroside I,II, III and V [Figure 3(d)], Pennell and P. kurroa Royle ex Benth are pikuroside, 6-feruloylcatalpol, minecoside rich source of picroside I and II. P. kurroa and kutkosides.The basic nucleus of the also contains phytochemicals including iridoids is cyclopentano-[c]-pyran. P. kurroa pikuroside, veronicoside, phenol glycosides, also contains kutkin and picroliv. Kutkin a number of cucurbitacin glycosides and 4- consists of the picroside I. The kutkoside is hydroxy-3-methoxy acetophenone [Figure present in a ratio of 1:2 along with 3(n)] [20]. glycosides. P. kurroa also has monocyclic A method to determine picroside and phenolic compounds like vanillic acid and kutkoside [Figure 3(e)] in Picrorhiza kurroa apocyanin. Picein and androsin are phenolic and its herbal preparation was high glycosides which are isolated from P. kurroa. performance thin layer chromatography The plant extract also contains some (HPTLC). The amount of picroside content in important chemical constituents like P. kurroa and its formulations were found to carbohydrates (D-mannitol), aromatic acids contain 7.27%, 6.83% and 7.03% like cinnamic acid [Figure 3(m)], vanillic respectively. The amount of kutkoside in the acid [Figure 3(k)] and ferulic acid [Figure same plant extract and its formulation was 3(l)] [25]. found to be 3.22%, 3.17% and 2.97% Ethnobotanical Features of P. kurroa respectively. The plant phytochemicals Picrorhiza kurroa Royle ex Benth.is widely include iridoid glycosides i.e. picroside I and used by locals in curing diseases like kutkoside as major bioactive components. stomachache and high fever. Ten gram

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Masood et al. slightly boiled root decoction along with pain, eye disease, gastritis, bile disease, sore honey is given to patient to cure throats, blood, and lung fever [32]. It is stomachache. To cure fever, 10 g root powder considered a bitter tonic, used as a mixed with 1 g black pepper and honey is cholagogue (promoting the flow of bile from given to the adult patient. 0.25g powder of the gall bladder), stomachic (stimulating kutki with mother’s milk is advised for gastric activity) and cathartic (purgative) newborns to cure stomachache [26]. In [33]. In Nepal, Kutki is used both by amchi Kashmir it is also used for veterinary (specialists trained in the Tibetan medical purposes. system or Sowa Rigpa) and by non- In Bhutan, kutki is used as a medicine for specialists, in the latter case largely for coughs, colds and fever. The National treating coughs and colds [34]. Institute of Traditional Medicines and other In Pakistan, Kutki is also used in the local hospitals in Bhutan use the rhizome as Ayurvedic and Greek-Arab systems of an ingredient in developing medicines [27]. medicine, most commonly as a stimulant, In China it is used to cure fever, malnutrition anaromatic, carminative agent, as a remedy due to digestive disorders, jaundice,diarrhoea for coughs, bronchial asthma and diseases of and dysentery [28]. It is also used in blood, liver, kidney and skin. Under the name traditional Tibetan medicine. P. kurroa of Qusttalakh, P. kurroa is used in two herbal rhizome extract can significantly prevent the preparations (Roghane-qust-talakh, an hepatic injury as it has choleretic effect. The essential oil and majoon-e-murravehul- plant is bitter in taste and has cold properties. azwah), which are also used for treatment of The plant species is used in combination with hypothermia, debility, tremors, tetanus and Chinese herbs including Corium Erinacei and gout [35]. navel gland secretions of musk deer for Molecular Studies on P. kurroa treating damp-fire accumulation in the large Different techniques such as randomly intestine, hemorrhoids, and anal fistula. The amplified polymorphic DNA (RAPD) and dosage of plant used must be in the range of simple sequence repeat (SSR) marker were 6-12 gm [55]. used for authentication of Picrorhiza kurroa Kutki is widely used in Ayurvedic and Unani Royle ex Benth. and its adulterant Lagotis traditional medicine systems in India with the cashmiriana. The unique bands obtained in rhizomes valued for their effectiveness as an RAPD amplification clearly differentiated antibiotic. It is regarded as one of the major between the two groups of P. kurroa that had components of Arogyavardhini, an effective similar morphology. On the basis of marker Ayurvedic preparation used to treat liver characteristics of species specific SSR illnesses. Kutki is also used as a substitute primers, P. kurroa is considered genuine only for, kurroo [29, 30]. when the motif (PKssrD2F, PKssrD2R) Pharmaceutical uses of Kutki are also being targeting locus (EU883611) is amplifiable in explored in India. a PCR reaction. Thus RAPD and SSR In Nepal, the rhizome of P. kurroa is widely markers can serve as corresponding tools for used to treat coughs, skin disease, fever, quality control. So the authentication of raw indigestion,liver disease, jaundice, hepatitis material for commercially available herbal and metabolic disorders. Manufacturers in products is easy by using this method [36]. Kathmandu described that rhizome of Another study was performed to detect plantused as purgative and to treat scorpion proteins linked with picroside biosynthesis in bites [31]. Rhizome of the plant is also used Picrorhiza kurroa Royle ex Benth. intreatment of high blood pressure, intestinal Differential protein expression was studied

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Pure Appl. Biol., 4(3): 407-417, September- 2015 under picroside accumulating versus Picrorhiza kurroa Royle ex Benth.is listed as picroside non-accumulating conditions using endangered species due to extensive usage of SDS-PAGE. A total of 19 differentially plant from the wild and lack of organized expressed proteins were identified. Proteins cultivation. Underground parts (roots and involved in miscellaneous jobs were rhizomes) are used for extraction of identified, the most important proteins being picrosides, which are the medicinally 1-aminocyclopropane-1-carboxylate important constituents of P.kurroa in herbal oxidase, glyceraldehyde-3-phosphate medicinal system [39]. dehydrogenase, photosystem I reaction Another study was aimed at developing center subunit V, putative cytochrome P450 propagation methods and ex-situ super-family protein and 2-oxoglutarate conservation for P. kurroa, an endangered ferrous-dependent oxygenase because of medicinal plant of western Himalaya. their role in picroside biosynthesis.These Regeneration using leaves from mature plant identified proteins provide a thorough is beneficial because the source plant is not understanding of biosynthesis of secondary deteriorated. A regeneration practice was metabolites and various other physiological regulated by using leaves from aseptic shoot progressions of P. kurroa [37]. cultures, raised from ex-vitro leaves. Micro- Threats and Conservation shoots with well-developed root system were The plant is at the verge of being endangered obtained in MS basal medium after 4 weeks. due to its overexploitation and collection Incubation of cultures at low temperature from the wild. Samples were taken from (15°C) for ten days enhanced the survival different biodiversity zones of India percent under green house conditions and it including northeast Himalaya, western was correlated with the development of thick Himalaya, western ghats, gangetic plain, cuticle and well differentiated leaf tissues semiarid zone and central highlands. Aseptic (palisade and spongy parenchyma). Flow cultures were grown at morphogenic level of cytometric analysis was performed to check callus, suspension, axillary shoot, multiple the genetic stability of in-vitro plantlets. In a shoot and rooted plants. It was effectively parallel study, seed progenies of these hardened under glasshouse conditions and germplasm were raised under ex-situ successful in-vitro conservation was done conditions. Its reproductive cycle was also [38]. studied for successful domestication [40]. Classification Table 1. Hierarchical Classification of Picrorhiza kurroa Royle ex Benth.

Rank/Taxa Classification Kingdom Plantae Division Dicotiledonae Class Asteride Order Scrophulariales Family Scrophulariaceae Genus Picrorhiza Species Picrorhiza kurroa

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Fig. 1. Leaves of P. kurroa [41] Fig. 2. Rhizome of P. kurroa [42]

Fig. 3. Phytochemicals of P. kurroa [43-54]

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