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REVIEW ARTICLE An Overview of polyphylla, a Highly Vulnerable Medicinal Herb of Eastern Himalayan Region for Sustainable Exploitation

Arcadius Puwein1,* and Shiny C. Thomas2

1Department of Biotechnology, Assam Don Bosco University, Guwahati, India; 2Department of Biochemistry, Assam Don Bosco University, Guwahati, India

Abstract: Paris polyphylla is a member of the family of (earlier Trilliaceae or Lili- aceae). It is known as “Love Apple” in English. This erect and herbaceous is found mostly in South East and was documented for the first time in 1985 in the Chinese pharmacopeia. It is a traditional medicinal herb known to have many medicinal properties like anticancer, antimicrobial, A R T I C L E H I S T O R Y anti-tumor, cytotoxicity etc. The objective of this paper is to highlight the major research works on this miraculous herb and include updated information on its developments that have been rapidly Received: February 15, 2018 Revised: May 07, 2018 taking place in recent years. There have been new approaches in an effort to classify the plant using Accepted: May 14, 2018 high-throughput RNA-Seq technologies, mass propagation and conservation through tissue culture,

DOI: understanding the mechanism of the action of steroidal saponins as a major anticancer activity, and 10.2174/2210315508666180518081208 new bioactive compounds have been added in phytochemistry. The current article attempts to enu- merate an extensive overview of P. polyphylla on classification, morphological characteristics, habi- tat, reproductive phenology, traditional uses, distribution and availability, propagation, phytochemis- try, biological activities, molecular analyses, and conservation. Keywords: Paris polyphylla, herb, steroidal saponins, , bioactive compounds, biological activities.

1. INTRODUCTION of Paris polyphylla published in 2012, chemical constituents of from the Genus Paris published in 2014, and distri- The pursuit of plant-based medicines is growing rapidly bution and phytomedicinal aspects of Paris polyphylla from worldwide. In recent decades, the natural products have the Eastern Himalayan Region published in 2015. All the gained importance in the field of pharmaceuticals [1]. Paris three reviews accentuated on a particular topic only (on me- polyphylla Sm. is one of the medicinal plants which is part of dicinal properties, chemical constituents, distribution and this momentum of growth and recognition in the biological phytomedicinal). Moreover, within two or three years, there activities. It is one of the many medicinal herbs of Asia. It is has been lots of development in research on this herb. This an erect and herbaceous plant. The herb has a spider-like paper compiles almost all the major works on this plant such flower that throws out long, thread-like, yellowish green as classification, morphological characteristics, habitat, re- throughout most of the warm summer months and into productive phenology, traditional uses, distribution and the autumn. The genus name is derived from ‘pars’ referring availability, propagation, phytochemistry, biological activi- to the symmetry of the plant. The species polyphylla means ties, molecular analyses, and conservation. New biotechno- many ; ‘poly’ meaning many and ‘phyla’ meaning logical approaches have been introduced and updated in the leaves [2]. P. polyphylla which is known in English as “love utilization of this plant. Therefore, this paper enumerates apple” belongs to the family of Melanthiaceae (earlier Trilli- research reported in index journals, updates the new ap- aceae or ) [3]. The herb is mostly found in India, proaches and investigates the future possibilities of the plant , , , , , , and Viet- [2]. nam [4]. It was documented in the “Chinese pharmacopeia” in 1985 for the first time. It was first recorded in “Shennong Herb” named as Zaoxiu. It appeared with the same name in 2. CLASSIFICATION Li Shizhen’s “Compendium of Materia” [5]. P. polyphylla is extremely polymorphic. This implies that Being known as a wonder herb, a number of works have its classification is very complicated. Its classification is been carried out in the last few years. The three reviews pub- therefore simplified using Table 1. The classification of lished recently are worth mentioning - Medicinal properties Paris has long been in dispute and still unresolved. Many authors classified the genus Paris into sub-genus and sec- *Address correspondence to this author at the Department of Biotechnology, tions (Table 1). Based on floral and merosity, Hara Assam Don Bosco University, Guwahati, India; Tel: +91857586804; (1969), Li (1984, 1998) and Mitchell (1987, 1988) recog- Fax: +917005139926; E-mail: [email protected] nized Paris as a single genus [6]. Hara further divided the 14

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Table 1. The Classification of Paris polyphylla according to Ji Y.H et al.

Genus Parameters Sub-Genus Sections Author (s)

Hara, Li H and Paris Floral and leaf merosity Single genus - Mitchell

Paris Paris Fruit and seed Single genus (14 species) Kinugasa Hara Euthyra

Paris Type of fruit, the shape of an , seed Paris Kinugasa - Takhtajan morphology, and shape of Daiswa

Kinugasa (1 species) Paris (11 species) Paris (5 species) Axiparis (5 species)

Paris Comprehensive taxonomy Dunnianae (1 species) Ji H Y.H et al. Marmoratae (2 species) Daiswa (13 species) Euthyra (8 species ) Farge sianae (1 species) Thibeticae ( 1species)

Paris Paris DNA sequence Kinugasa - Ji Y.H et al. Daiswa known species at the time into three sections: Paris, Kinu- contains the single stem-like structure and the 2-3 whorls of gasa, and Euthyra, based on fruit and seed characters [7]. In leaves are present on the nodes. The flowering period may 1983, Takhtajan divided Paris into three major genera: last up to three months. It has odd flowers with long yellow Paris, Kinugasa, and Daiswa. radiating anthers [2]. The morphological characteristics of the herb are given below: But in the most recent comprehensive classification, Ji Y.H et al., recognized subgenus Paris consists of 11 species Habit: aerial, herbaceous, rhizomatous, erect plant. and subgenus Daiswa consists of 13 species. Subgenus Paris Stem: unbranched, smooth, 50-100 cm tall, 1-2.5 cm was divided into sections Kinugasa (one species), Paris (five thick. species) and Axiparis (five species), whereas subgenus Daiswa was divided into sections Dunnianae (one species), Leaf: Simple, arranged in whorls, petiolate, lanceolate, Euthyra (eight species), Marmoratae (two species), Farge reticulate with three primary veins, smooth margin, above sianae (one species) and Thibeticae (one species) [5]. How- the green leaves are yellow-green spider-like flowers. ever, from recent phylogenetic studies based on DNA se- Inflorescence: It forms a closed whorl at the initial quence, data support the classification of Paris into three growth, covered by the enclosing the , the an- genera as shown and simplified in Table 1 (Daiswa, Kinu- gasa, and Paris). P. polyphylla belongs to the sub-genus thers and the stigma. Daiswa and section Euthyra. P.polyphylla is furthered di- Flower: The flowers bloom at the terminal and they are vided into 11 different varieties -1) Paris polyphylla Smith solitary, yellowish green, contain both male and female 2) Paris polyphylla var. polyphylla 3) Paris polyphylla var. gametes within the same flower (monoecious), sepaloid yunnanensis 4) Paris polyphylla var. chinensis 5) Paris outer is larger and inner is smaller, 3-5. polyphylla var. nana 6) Paris polyphylla var. alba 7) Paris polyphylla var. stenophylla 8) Paris polyphylla var. minor 9) Androecium: 6-11 and are free. Paris polyphylla var. latifolia 10) Paris polyphylla var. : 1 pistil, 3-5 carpels, syncarpous (carpels pseudothibetica 11) Paris polyphylla var. kwangtunesis. fused), ovary superior. Among these 11 varieties, the ones found in Uttarakhand and Northeastern of India along with other neighbor- Seed: Reddish orange, a mature fruit contains 50-60 ing countries are Paris polyphylla Smith, Paris polyphylla seeds [8]. var. polyphylla, Paris polyphylla var. yunnanensis and Paris polyphylla var. stenophylla. The most common variety col- 4. HABITAT lected and identified from Northeast India is Paris poly- P. polyphylla is a shade loving plant and grows under the phylla Smith [6]. canopy closure of more than 80%, at an altitude of 1300 - 2500 m above sea level. It grows mainly in a forest with 3. MORPHOLOGICAL CHARACTERISTICS bamboo groves, grassy or rocky slopes, stream-sides, mixed P. polyphylla is a perennial, rhizomatous, herbaceous conifer forests and scrub thickets [4]. It is a slow germinating plant with green and unbranched aerial part. The aerial part herb which takes about seven months to sprout from the An Overview of Paris polyphylla, a Highly Vulnerable Medicinal Herb The Natural Products Journal, 2019, Vol. 9, No. 1 3

Table 2. Traditional Uses of P. polyphylla.

Country Plant Parts Ailments References

Rhizomes Vermifuge, anti-helminthic [11]

Nepal Juice of rhizomes Gastric, menstruation pain [12]

Paste of rhizomes Cuts, wounds and to remove worms [12]

The powdered Diarrhoea [13] India Rhizomes Antidote for snake & insect poison [14]

Haemostatic, anti-inflammatory agent, traumatic injuries, snake bites, Rhizomes [15] abscess, parotitis, mastitis, bone fractures and anti-tumor

Rhizomes Detumescent, demulcent, anti-febrile, alexipharmic [16] China Rhizomes Reducing swelling, relieving pain, sore throat, snake bites and bruises [17]

Rhizomes Tumor, hemostasis and inflammation counteraction [18]

Rhizomes Immunity adjustment, analgesia, and anti-inflammation [19] seed. P. polyphylla thrives well inside the deep forest where traditionally used P. polyphylla to relieve various ailments. human interference is minimal. It grows in humus-rich and The herb is a well-known Traditional Chinese Medicine well-drained soil. Waterlogging is found to be lethal for this (TCM). The Indian tribes of Arunachal Pradesh, Nagaland, herb. The plant is able to rehabilitate in artificial habitats Manipur, Meghalaya, Uttarakhand, and have been while maintaining similar temperatures and at similar alti- using it as folk medicines, albeit some ethnic communities tudes. Plants that lack inflorescence are usually shorter in are not very conscious about its remarkable medicinal poten- height. The research on its adaptability has displayed that tial [10]. The various uses of the plant are indicated in Table when the whole plant was taken outside its natural habitat, it 2. showed sign of growth at the initial year but failed to flower in an artificial habitat. If the plant flowered, it failed to set 7. GEOGRAPHICAL DISTRIBUTION AND AVAIL- fruit and seed. It was also observed that this species thrives well in the presence of some common associated species ABILITY within 5m perimeter. These common associated species in- Internationally P. polyphylla is found mostly in East clude trees, herbs, and climbers. Further research is required Asia, Himalayan regions and in Europe. It is an extensively to understand their mode of interaction with other species, growing species in China with the - Plateau those that boost its growth [9]. as the center of diversity and thrives well also in Bhutan, Laos, Myanmar, Nepal, Thailand, and [4]. 5. REPRODUCTIVE PHENOLOGY In India, the presence of P. polyphylla is reported in Knowing the pattern and process of the reproduction in many temperate climatic regions, mainly in the Eastern Hi- plants is one of the important steps for effective conserva- malayan regions. Each ethnic group has its own local name tion. Mass multiplication, either vegetative or in vitro repro- for the plant as shown in Table 3. But some of the local duction, depends on the basic knowledge of reproductive names have not been reported yet. phenology. The rhizome of P. polyphylla remains in dor- mancy for almost 4-5 months. The rhizome sprouts in the 8. PROPAGATION months of February-March. It flowers in the month of March-April and sheds leaves in October-November. Al- P. polyphylla grows well in moist, humus-rich soil, and though reproductive phenology has been studied and re- in full or partial shade. However, its regeneration through ported, the herb does not have an exact uniform pattern of seed is a real challenge due to the prolonged seed dormancy time for flowering, sprouting, and senescence. It was ob- and slow germination. It is propagated by portions of rhi- served that when the herb is taken out of the natural habitats zomes, and so can be called as ‘seed rhizomes.’ Hence rhi- and artificially grown in pots, a slight variation is noticed in zomes from the wild are the only source for propagation as sprouting, flowering, and senescence [9]. This might be due well as for medicinal purposes. This leads to a great decline to many external and internal factors (environment, soil nu- of the herb. It is on the edge of extinction due to excessive trients, habitat, sunlight, human interference, etc). illegal collection for many years [28]. Moreover, this peren- nial plant can only be harvested after growing for 5–7 years, which aggravates the shortage of its resource [29]. To pre- 6. TRADITIONAL USES serve this natural resource and ensure a stable and renewable China, India, Nepal, and the whole region of Indo-Burma source of P. polyphylla for medicinal purposes, successful are endemic to many medicinal plants. These countries have propagation is imperative [30]. This calls for an urgent need 4 The Natural Products Journal, 2019, Vol. 9, No. 1 Puwein and Thomas

Table 3. Geographical Distribution and Availability of P. polyphylla in India.

State District Village/Region Local Name References

Kameng, Subansiri, Kurung Kume, Siang, Arunachal Pradesh - Do-Tala [20] Lohit, Tirap and Changlang

Hengbung, Maram, Purul and Ma- Senapati Manipur kui regions Singpan [13, 21, 7] Tamenglong Puilong Village

Uttarakhand - - Satwa [13]

Himachal Pradesh - - - [22, 23]

Jammu & Kashmir - - - [23]

Mizoram - - - [7, 24]

Sikkim - - - [25]

Tuensang Pangsha village

Phek Chida region Nagaland - [9, 26] Kohima Arudara region

Mokokchung Longkum village

Meghalaya West Khasi Hill Nongstoin region Sohbsein [27]

(-) not reported to discover alternate resources from which the continuous 8.1.2. Rhizome Fragmentations supply can be obtained. This technique involves the breaking of the masses of 8.1 Planting Material rhizomes into each individual rhizome with many buds and sowing in the prepared bed in the polyhouse. Of the total The vegetative propagation of the plant has been separated rhizomes planted, ~75% rhizomes could sprout achieved through rhizomes and it occurs in masses due to successfully and grow into a plant. Though the rhizomes aggregation of large clumps. The older plant has massive fragment showed a better result than rhizomes cutting in rhizomes with multiple buds. It grows intertwined and en- cultivation, the availability of sufficient material (rhizome tangle each other and has a wavy nature in appearance. fragments) is one of the major challenges in the massive cul- Therefore, the seed rhizomes are used in different ways in tivation of this plant at the moment [32]. Therefore, mass cultivation practices that are given below. production of this herb is a prerequisite for sustainable utili- zation. Hence, the in vitro propagation has immense impor- 8.1.1. Rhizome Cuttings tance in the multiplication of P. polyphylla since very scanty The older rhizomes are split into pieces so that each rhi- research have been conducted so far. zome gives rise to buds and later converted into plants in the raised soil bed within 4-5 months. The percentage of 8.1.3. Micropropagation viable regenerated plantlets from rhizome cuttings were Raomai Shiveirou et al. (2014) have reported direct so- found ~49%, and only 7% of the regenerated plantlets matic embryogenesis and subsequent plant regeneration of P. showed fertile nature that bears the inflorescence [31]. The polyphylla. Their study shows that the highest frequency of percentage of the regeneration is found considerably low somatic embryogenesis, and mean number of somatic em- which is one of the main drawbacks found in the cultivation bryos (SEs) were obtained on ½ MS medium directly with- and propagation of this plant from rhizome cuttings. The out an intermediate callus phase. The highest frequency of effect of plant growth regulators (PGRs) in sprouting and SE germination and seedling to plantlet conversion occurred rooting have also been studied and the results show that in- on ½ MS medium by adding 0.5 mg/l gibberellic acid, 0.05 dole-3-butyric acid (IBA) and gibberellic acid (GA3) at dif- mg/l 6-benzylaminopurine, and 0.1 mg/l α-naphthalene ace- ferent concentrations (0, 50, 100 mg/l) were highly effective tic acid [33]. (83.33% and 73.33 %) as compared to control (46.66 % and 36.66 %). The combination of IBA (100 mg/l) and GA3 (100 This result is followed by another investigation by mg/l) presented the best result in sprouting and rooting of the Raomai Shiveirou et al. in 2015. They developed an efficient rhizomes. The soil combinations (soil:loam:sand) in the ratio regeneration protocol for P. polyphylla. through the forma- 3:2:1were also significant for proper sprouting and rooting tion of mini-rhizomes (MRs) using transverse thin cell layer [32]. (tTCL) culture technique. Elicitation of MRs liquid culture An Overview of Paris polyphylla, a Highly Vulnerable Medicinal Herb The Natural Products Journal, 2019, Vol. 9, No. 1 5 with chitosan, salicylic acid (SA) and yeast extract enhanced chemical investigations on several Paris species which led to the production of steroidal saponins but resulted in reduced the isolation of 126 compounds (not shown in Table 4). In growth rate. Highest total steroidal saponins content (87.66 2016, Ling-Yu Jin et al. isolated two new highly oxygenated ± 1.66 mg/g DW) was achieved in cultures treated with SA spirostanol saponins named as paristenosides A and B from at 50 mg/l after 30 days of elicitation which is 3.6 times the rhizomes of P. polyphylla var. stenophylla [42]. Some of higher than the in vivo rhizome [34]. the phytoconstituents isolated and identified from Paris poly- phylla is summarized in Table 4. Many of these compounds exhibit strong bioactivities but there are still several species 9. PHYTOCHEMISTRY that have received little attention, and many constituents are P. polyphylla produced many bioactive compounds still unknown [43]. Some of the important structure of isolated which are vital for various treatments. The most important compounds from this herb is given in Fig. (1). compounds are alkaloids, flavonoids, saponins, carbohy- drates, cardiac glycosides, terpenoids, sterols, quinones, phe- 9.2. Compounds Isolated from the Stems and Leaves nols, and tannins. Nearly 98 compounds were identified from Isolation of compounds from the stems and leaves are the rhizome, which includes more than 30 steroidal saponins significant as they can regenerate every year. Phytochemical [35]. Most bioactive compounds were isolated from the rhi- analysis of the ethanol extracts of stems and leaves of P. zomes, but recently many chemical constituents were also polyphylla var. yunnanensis has yielded six new spirostanol extracted from the stems and leaves. saponins, named as chonglouosides SL-1-SL-6 (1-6), along with one known sapogenin and 18 known steroidal saponins 9.1. Chemical Compounds Isolated from Rhizomes [49]. When the stems and leaves of the same species were The active components present in P. polyphylla are the further investigated using water, 80% ethanol and 95% etha- steroidal saponins, dioscin, polyphyllin D, and balanitin 7 nol as solvents, three C22-steroidal lactone glycosides were [36]. The Paris saponins account more than 80% of the total isolated. Two of these are new compounds, designated as compounds of P. polyphylla. Other important compounds chonglouoside SL-7 and chonglouoside SL-8 [50]. The des- isolated from this plant are Paris saponin I (diosgenin3-O-α- ignated name and molecular formula of the newly isolated L-rha-(1-2)-[α-L-arab-(1-4)]-β-D-glu), Paris saponin II compounds are given in Table 5. (diosgenin3-O-α-rha-(1-4)-α-L-rha-(1-4)-[α-L-rha-(1-2)]-β- D-glu), Paris saponin III, polyphyllin VI and polyphyllin VII 10. BIOLOGICAL ACTIVITIES [18]. Steroid saponins from P. polyphylla are classified into two main groups: diosgenin (Dio) glycosides and penno- P. polyphylla is reported to exhibit potential biological genin glycosides. Furthermore, D-glycopyranoside, L- activities which are substantiated by several studies. Studies rhamnopyranoside, and L-arabinofuranoside are the main have been done in China on the pharmacology of the herb glycosides linked in the structure of steroid saponins [37]. focusing on anticancer and antimicrobial activities. The rhi- zome of P. polyphylla is used to clear heat, remove toxicity, In 2005, Devkota isolated six compounds from P. poly- cool the liver, relieve swelling and pain, and arrest convul- phylla which he collected from Parbat district, Nepal [38, 39]. These compounds are: i) Saponin-1 ii) Polyphyllin C iii) Poly- sion. The rhizomes of P. polyphylla var.yunannensis and P. polyphylla var. chinensis are used for haemostatic activity phyllin D iv) Przewalskinone B v) Stigmasterol vi) Stigmas- [51], anti-fertility, spermicidal enhancement and sedative terol-3-O- β -D-glucoside [39]. Huang Yun et al. in 2007 ex- [38], anti-tumor, anti-inflammatory, antibacterial, brain and tracted a novel steroidal saponin together with the 12 known kidney protection, uterine contraction, antioxidant, menor- compounds from P. polyphylla var. chinensis. The novel com- rhagia, metrorrhagia, metrostaxis [52], showed anticancer pound was attained as an amorphous solid. The 12 known compounds were identified as steroids and their structures activity on several cell lines [53], and antifungal activities [14,49]. The plant extracts of P. polyphylla showed effective (Table 4) were recognized by 13C NMR spectrum (in pyri- spermicidal activity against human and rat sperms. It pre- dine-d5) [14, 15]. Again in 2007 three new steroidal saponins vents pregnancy up to 60% when tested in the rabbits [38]. were isolated from the rhizome of P. pollyphylla var. yun- Some compounds of the herb exhibit tyrosinase inhibitory nanensis. Two compounds were a mixture of a pair of stereoi- activity and anti-leishmanial activity [39]. Recently, many someric new spirostanol saponins and a new cholestane saponin [40]. Along with 18 known steroidal saponins, two studies of the herb accentuate in its anticancer activities. new furostanol saponins (designated as parisyunnanoside A 10.1. Anticancer Activity and parisyunnanoside B) and one new spirostanol saponin (designated as parisyunnanoside A) were isolated from the The P. polyphylla var. yunnanensis has been extensively rhizome of P. polyphylla var. yunnanensis by Yu Zhao et al. investigated in China. Phytochemical and pharmacological in 2009 [41]. Jin-Chao Wei et al. in 2014 identified various studies identified steroid saponins as the main antitumor ac- compounds, such as steroid saponins, phytoecdysones, phytos- tive components [54, 55, 56]. Paris saponins are a group of terols, phenylpropanoids, and flavonoids, from six Paris spe- plant glycosides consisting of a steroid aglycone, to which cies (P. polyphylla var. yunnanensis, P. verticillata, P. pubes- one or more sugar chains are attached [57]. The aqueous, cends, P. axialis, P. polyphylla var. pseudothibetica, and P. ethanolic and methanolic extracts of P. polyphylla showed fargesii). They found that steroid saponins are the main con- their anticancer activity on several types of cancer cell lines. stituents of the six species, along with many other constitu- The biological activities of different steroid saponins are ents, including flavonoids, phenylpropanoids, ecdysteroids, quite different from each other in exhibiting inhibition in and phenolic glycosides. They conducted extensive phyto- many cancer cell lines as shown in Table 6. 6 The Natural Products Journal, 2019, Vol. 9, No. 1 Puwein and Thomas

Table 4. Some Chemical Constituents Isolated from the Rhizomes of P. polyphylla.

Plant Species Isolated Compounds References

Paris saponin I (diosgenin3-O-α-L-rha-(1→2)-[α-L-arab-(1→4)]-β-D-glu) Paris saponin I (diosgenin3-O-α-rha-(1→4)-α-L-rha-(1→4)-[α-L-rha-(1→2)]-β-D-glu)

Paris saponin III (diosgenin 3-O-α-L-rhamnopyranosyl-(1→2)-[ α -L-rhamnopyranosyl-(1→4)]- β-D - P. polyphylla glucopyranoside) [18] Polyphyllin VI (pennogenin-3-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucopyranoside)

Polyphyllin VII (pennogenin-3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)[O-β-D-glucopyranosyl- (1→2)]-β-D-glucopyranoside) Saponin-1 (diosgenin-3-O [α-L-rhamnopyanosyl (1Rha-2Glu)-α-L-rhamnopyranosyl (1Ara-4Glu)]-β- glucopyranoside) Polyphyllin C (diosgenin-3-O [α-L-rhamnopyanosyl(1→3)-α-D-glucopyranoside)

Polyphyllin D (diosgenin-3-O[α-L-rhamnopyanosyl (1Rha-2Glu)-α-L-arabinofuranosyl (1Ara-4Glu)]-β-D glu- P. polyphylla copyranoside) [39] Przewalskinone B (1,5-Dihydroxy-7-methoxy-3-methylanthraquinone) Stigmasterol Stigmasterol-3-O-β-D-glucoside. Diosgenin Pennogenin Diosgenin-3-O-α-L-rhamnopyranosyl (1→2)-β-D-glucopyranoside Pennogenin-3-O-α-L-rhamnopyranosyl(1→2)-β-D-glucopyranoside Diosgenin-3-O-α-L-rhamnopyranosyl(1→2)[-α-L arabinofuranosyl(1→4)] -β-D-glucopyranoside Pennogenin-3-O-α-L-rhamnopyranosyl(1→2)[-α-L arabinofuranosyl (1→4)]-β-D-glucopyranoside

P. polyphylla Diosgenin-3-O-α-L-rhamnopyranosyl(1→2)-[β-D-glucopyranoside(1→3)]-β-D-glucopyranoside var. chinensis [14,15] Diosgenin-3-O-α-L-rhamnopyranosyl (1→4) -α-L rhamnopyranosyl (1→4)[α-L-rhamnopyranosyl (1→2)]-β-D- glucopyranoside Pennogenin-3-O-α-L-rhamnopyranosyl(1→4) -α-L-rhamnopyranosyl (1→4)[α-L-rhamnopyranosyl (1→2)]- β-D- glucopyranoside 3-O-α-L-arabinofuranosyl(1→4)[ α-L-rhamnopyranosyl(1→2)] -β-D-glucopyranoside-β-D-chacotriosyl-26-O-β-D- glucopyranoside 2β, 3β, 14α, 20β, 22α, 25β hexahydroxycholest-7-en-6-one 2β,3β,14α, 20β,24β,25β hexahydroxycholest- 7-en-6-one P. polyphylla 3b, 21-dihydroxy pregnane-5-en-20S-(22,16)-lactone-1-O-α-L-rhamnopyranosyl (1→2) -[β-D-xylopyranosyl (1→3)] var. chinensis -β-D-glucopyranoside. [14] (25R)-spirost-5-en-3b,7b-diol-3-O-α-L-arabinofuranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside P.pollyphylla (25R)-spirost-5-en-3b,7a-diol-3-O-α-L-arabinofuranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside Smith var. [40] yunnanensis 26-O-β-D-glucopyranosyl-(25R)- ∆ 5(6) 17 (20)-dien-16,22-dione-cholestan-3b,26-diol-3-O-α-L-arabinofuranosyl-(1→4)- [α-L-rhamnopyranosyl-(1→2)] -β-D-glucopyranoside 26-O-β-D-glucopyranosyl-(25R)-5-ene-furost-3β,17α, 22α, 26-tetrol-3-O-α-L-arabinofuranosyl-(1→4)-[α-L- rhamnopyranosyl-(1→2)]-β-D-glucopyranoside P.pollyphylla

Smith var. 26-O-β-Dglucopyranosyl-(25R)-5, 20 (22)-diene-furost-3β, 26-diol-3-O-α-L-arabinofuranosyl-(1→4)-[α-L- rhamnopyranosyl-(1→2)]-β-D-glucopyranoside [41] yunnanensis (25R)-spirost-5-ene-3β, 12α-diol-3-O-α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-[α-L- rhamnopyranosyl-(1→2)]-β-D-glucopyranoside 24-O-β-D-galactopyranosyl-(23S,24S,25S)-spirost-5-ene-1b,3b,21,23,24-pentol-1-O-α-L-rhamnopyranosyl-(1→ 2)- P. polyphylla [β-D-xylopyranosyl-(1→3)]-β-D-glucopyranoside var. steno- [42] phylla. 21-O-β-D-galactopyranosyl-24-O-β-D-galactopyranosyl-(23S,24S)-spirost-5,25(27)-diene-1b,3b,21,23,24-pentol-1-O- α-L-rhamnopyranosyl-(1→ 2)-[β-D-xylopyranosyl-(1→ 3)] -β-D-glucopyranoside An Overview of Paris polyphylla, a Highly Vulnerable Medicinal Herb The Natural Products Journal, 2019, Vol. 9, No. 1 7

O O OH OHO O

HO 0 HO O HO HO O O O O O HO O O O O HO HO HO O HO HOHO HO OH HO HO OH Polyphyllin VI Polyphyllin VII

O OH CH3 OH H C O HO 3 HO O CH3 O H OH H O O O HO HO OH OH O H O CH3 H O O H H H H O O O O HO OH HO OH O O

HO HO CH3 OH OH Polyphyllin D Polyphyllin I

O

HO O OH OH O H O HO HO O H O O H H H OH O HO O HO O O H HO O O HO OH O O HO O O O O HO O HOHO HO HO HO HO OH OH Paris saponin II Paris Saponin VII

O O

OH O O

R O R O Diosgenin Pennogenin Fig. (1). Some important structure of compounds isolated from P.polyphylla. Polyphyllin VI [44], Polyphyllin VII.

10.2. Antimicrobial Activity tumefaciens, E. coli, Pseudomonas lachrymans, Ralstonia solanacearum, Xanthomonas vesicatoria, B. subtilis, Staphy- This magical herb has been reported to have antibacterial, lococcus aureus and S.haemolyticus [73]. When Enterovirus antifungal and antiviral activities. Few of the compounds 71 (EV71), coxsackievirus B3 (CVB3) and control (ri- isolated from the stems and leaves of P. polyphylla var. yun- bavirin) were inoculated in the medium containing P. nanensis were found to be active against Propionibacterium polyphylla Smith and incubated at 37ºC for 72h, anti-EV71 acnes [51]. The roots of P. polyphylla have shown antibacte- and CVB3 activities were observed. The anti-EV71 activity rial action against Bacillus dysentery, B. paratyphi, B. typhi, was reported to be stronger than that against CVB3. Staphylacoccus aureas, Escherichia coli, Haemolytic strep- Moreover, it was observed that the compound extracts of the tococci, Meningococci. Certain compounds isolated from P. herb penetrated the viral capsid and degraded the viral RNA polyphylla var. yunnanensis showed significant antifungal [44], Polyphyllin D [45], Polyphyllin I [46], Paris saponin II activities against Saccharomyces cerevisiae hansen, Cla- [47], Paris saponin VII [48], Diosgenin [29], Pennogenin dosporium cladosporioides and Candida albicans [14]. The [29]. and so inhibited the viral replication. The bioactive endophytic fungus Pichia guilliermondii Ppf9 which was compounds of P. polyphylla also increased the production of derived from P. pylyphylla var. yunnanensis also demon- IL-6 cytokine which further augments the antiviral activities strates strong antimicrobial activity against Agrobacterium [74]. 8 The Natural Products Journal, 2019, Vol. 9, No. 1 Puwein and Thomas

Table 5. The New Compounds Isolated from the Stems and Leaves of P. polyphylla var. yunnanensis.

Designated Molecular Isolated Compounds References Name Formula

Chonglouoside (25R)-spirost-5-en-3β,7α-diol-3-O-β-D-glucopyranoside C H O [49] SL-1. 33 52 9

Chonglouoside (23S,25R)-spirost-5-en-3β,23 C H O [49] SL2 39 62 14 α,27-triol-3-O-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranoside

Chonglouoside 23-O-β-D-glucopyranosyl-(23S,25R)-spirost-5-en-3β,23α,27-triol-3-O-α-L-rhamnopyranosyl- C H O [49] SL3 51 82 23 (1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside

27-O-β-D-glucopyranosyl-(23S,25R)-spirost-5-en-3β,23 Chonglouoside C H O α,27-triol-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D- [49] SL4 51 82 23 glucoyranoside

(25R)-7-oxospirost-5-en-3β-ol-3-O-α-L-rhamnopyranosyl- Chonglouoside C H O (1→4)-α-L-rhamnopyranosyl-(1→4) [α-L-rhamnopyranosyl- [49] SL5 51 80 21 (1→2)]-β-D-glucopyranoside

Chonglouoside 27-O-β-D-glucopyranosyl-(25R)-spirost-5-en-1β,3β,27-triol-1-O-α-L-rhamnopyranosyl- C H O [49] SL6 50 80 23 (1→2)-[β-D-xylopyranosyl-(1→3)]-β-D-glucopyranoside

chonglouoside (20S)-3β, 16β, 20-trihydroxy-pregn-5-en-20-carboxylic acid (22, 16)-lactone-3-O-α-L- C H O [50] SL-7 40 62 17 rhamnopyranosyl-(1→4)-O-[α-L rhamnopyranosyl-(1→2)]-β-D-glucopyranoside

Chonglouoside 3β, 16β-dihydroxypregn-5, 20-dien-carboxylic acid (22, 16)-lactone-3-O-α-L rhamnopyrano- C H O [50] SL8 40 60 16 syl-(1→4)-O-[α-L-rhamnopyranosyl -(1→ 2)]-β-D-glucopyranoside

10.3. Anti-Abnormal Uterine Bleeding functional diversity based on different annotation methods. Then two small RNA libraries from seeds and seed coats Abnormal Uterine Bleeding includes both dysfunctional respectively were sequenced. Their combining data showed uterine bleeding and bleeding due to structural causes, in- that 263 conserved miRNAs belonging to at least 83 families cluding fibroids, polyps, endometrial carcinoma and preg- and 768 novel miRNAs in 1174 transcripts were found [78]. nancy complications. It can also result from contraception The miRNAs were chosen as candidates for sequencing be- [75]. Drugs such as progestins prostaglandin or a combina- cause they play an important role in gene regulation in the tion of estrogen and progestins greatly decreased menstrual development of many plant tissues and organs [79, 80]. bleeding in patients with abnormal uterine bleeding. How- Many transcription factors such as myeloblastosis-related ever, most women are unwilling to opt for drugs because of proteins (MYB), growth-regulating factor (GRF), squamosa side effects that often make them unsuitable for long-term promoter binding protein-box (SBP- box), Apetala2 (AP2), use. Steroidal saponins extracted from the rhizome of P. and MADS-box gene families, have been shown to be regu- polyphylla var.yunnanesis have been used in China as an lated by miRNAs in plants [81]. This molecular analysis alternative treatment of abnormal uterine bleeding which is helps the researchers to predict that miRNAs were involved comparatively less toxic. The study reported that the steroi- in the cell, metabolism and genetic information processing dal saponins extract reduced hemorrhage by approximately by direct and indirect regulation patterns in dormant seeds of 95%. This includes the treatment of 300 cases of abnormal P. polyphylla var. yunnanensis [78]. uterine bleeding, 122 cases of dysfunctional uterine bleeding, 103 cases of menorrhagia and 75 cases of other causes [76]. The sequencing of the complete chloroplast (cp) genome was driven by the problem of an unresolved fact of classifi- cation of Paris. The complete cp genome was chosen as the 11. MOLECULAR ANALYSIS candidate for sequencing because it offered valuable infor- P. polyphylla was subjected to molecular analysis with a mation for plant phylogenetic analyses and species identifi- view to solving problems related to its sustainable conserva- cation and in the reconstruction of complex evolutionary tion or classification. One of the major problems is that seeds relationships in plants [82, 83]. Albeit, the previous mor- of P. polyphylla var.yunnanesis germinate only 40% after 18 phology-based classification and molecular phylogeny have months or even two years of being in a state of dormancy in been subjected to numerous critical revisions but not without the natural environment [77]. This long dormancy may be controversial issue. So, to enhance the understanding of the due to the seed coat or the seed itself. Thus the transcriptome classification of this genus, the complete cp genomes of 11 data of dormant seeds and their seed coats were sequenced Paris taxa were sequenced [84]. The complete cp genomes and assembled using high-throughput RNA-Seq technolo- of the 11 Paris taxa were compared with the previously re- gies. A total of 146,671 unigenes with an average length of ported cp genome of P. verticillata [85] which ranged from 923 bp (base pair) were identified. These genes showed 157,379 to 158,451bp. The cp genomes consist of a pair of An Overview of Paris polyphylla, a Highly Vulnerable Medicinal Herb The Natural Products Journal, 2019, Vol. 9, No. 1 9

Table 6. Anticancer Activities of P. polyphylla.

Cancer Types Bioactive Compounds Mechanism of Action References

Polyphyllin VII Induces apoptosis via intrinsic and extrinsic pathway [58, 59] Liver Cancer Induces reactive oxygen species (ROS) and oxidative Rhizoma paridis saponins (RPS) [60, 61, 62] damage of DNA

Polyphyllin VI Induces G2/M cell cycle arrest and triggers apoptosis. [44] Lung Cancer Polyphyllin VII Rhizoma paridis saponins (RPS) Inhibits the migration of the tumor cells [44,63]

Human Crude extract Cytotoxicity and apoptosis [64] Chondrosarcoma

Aqueous extract Ovarian Cancer Apoptosis via suppression of peroxisome PGC – 1alpha [65] of P. polyphylla

Cervical Cancer Paris saponin VII Apoptosis through intrinsic apoptotic pathway [48]

Human Chronic Myelogenous Polyphyllin D Induces apoptosis via mitochondrial apoptotic pathway [66] Leukemia

Human Glioma Polyphyllin D Apoptosis via JNK pathway [45]

Apoptosis and increased expression of Connexin26 and Esophageal Cancer P.polyphylla Smith extract (PPSE) [67] formation of gap junction (GJ)

Rhizoma Paridis saponins (RPS) Induces apoptosis via mitochondrial apoptotic pathway [68] Breast Cancer Polyphyllin D Elicits apoptosis through mitochondria dysfunction. [69, 70]

Elicits G2/M phase arrest and apoptosis via mitochon- Gastric Cancer Dioscin [71] drial pathway

A monomer compound Elicits S and G2/M phases arrest and apoptosis via ex- Tongue squamous cell carcinoma [72] named as PP-22 trinsic apoptotic pathway inverted repeated regions (IRs: 27,329–28,373 bp) separated vation of this plant as well as educating the locals on the by a large single-copy region (LSC: 82,726–85,187 bp) and a importance of this plant and its conservation in the region is small single-copy region (SSC: 17,907–18,671 bp). The 12 imperative. The species need to be propagated either through genomes contained 115 genes, including 81 protein-coding conventional mean in the nursery or through the in vitro genes, 4 ribosomal RNA genes, and 30 tRNA genes [84]. technique. The overall cp genomes structure of the 11 Paris taxa Effective conservation strategies both in situ and ex- situ was found to be quite similar. After sequencing, the phylo- of this herb need the cooperation of government and non- genetic analyses derived from the complete cp genomes did government organizations, and ordinary local folks to protect not resolve the issue of classification of Paris as a mono- the species from its extinction. Spreading awareness about phyletic group. However, the work provides evidence sup- the vulnerability of the plant, its high medicinal properties, porting the division of the 12 taxa into two genera: Paris economic importance, and getting the involvement of the sensu strict and Daiswa. One of the most important results of people from various localities of different countries where this research is the finding of the ten rapidly evolving re- the plant thrives could increase its population. Conservation gions that were identified across the cp genomes that could in the higher level could be the inclusion of the species under serve as potential DNA barcodes for species identification in the priority species list of both at the National and State Me- Paris sensu strict and Daiswa [84]. dicinal Plant Boards for cultivation may be helpful for its long-term management and conservation. Meticulous re- search on exploration, documentation, and identification of 12. THREATS AND CONSERVATION its major habitats and distribution regions with the aid of a Based on reports of some anticancer potential of the rhi- tool such as ecological niche modeling is required. After zomes, P. polyphylla has been unsustainably harvested from having known the appropriate regions or selected areas of a the wild of the subtropical forest of Eastern Himalayan re- good viable population of this herb, proper legal protection gions by the local communities. Such unsustainable collec- act could be enacted. At present, biotechnological ap- tion and harvesting practices have pushed the species on the proaches such as clonal propagation of the species through edge of vulnerability [10]. The plant which was abundant in vegetative techniques or through tissue culture are being the last decade has decreased drastically. Therefore, conser- carried out for the conservation and improvement of the 10 The Natural Products Journal, 2019, Vol. 9, No. 1 Puwein and Thomas plant species. When taken out from its natural habitats, the BIMSTEC countries, Dec. 11-Dec.13, 2008, 82-86, Imphal, Ma- herb grows well with associated plants as found in the wild. nipur, India. [9] Deb, C.R.; Jamir, S.L.; Jamir, N.S. Studies on vegetative and re- This is another possibility for the future conservation of this productive ecology of Paris polyphylla Smith: A vulnerable me- herb. Associated plants might possibly act as a powerful dicinal plant. American Journal of Plant Sciences, 2015, 6, 2561- catalyst for mass multiplication [86]. 2568 [10] Tag, H. Mainstreaming conservation and sustainable use of me- dicinal plant diversity in three Indian States. Final Technical Re- CONCLUSION port submitted to UNDP Cell, New Delhi, India: Govt. of India, 2012, 1-124. Medicinal plants such as P. polyphylla have drawn much [11] Pande, P.C.; Tiwari, L.; Pande, H.C. Ethnoveterinary plants of attention in the field of pharmaceutical since they are com- Uttaranchal- A review. Indian J Tradit Know, 2007, 6, 444-458. paratively less toxic than synthetic drugs. The bioactive [12] Mall, B.; Gauchan, D.; Chhetri, R.B. An ethnobotanical study of medicinal plants used by ethnic people in Parbat district of western compounds of the herb have significant biological activities Nepal. Journal of Ethnopharmacology, 2014. such as anticancer, antibacterial, antifungal and antiviral. [13] Tiwari, J.K.; Ballabha, R.; Tiwari, P. New York Science Journal, The compilation of this overview indicates that studies on 2010, 3(4), 123-126. the herb on classification, phytochemistry, and biological [14] Sharma, A.; Kalita, P.; Tag, H. Distribution and phytomedicinal aspects of Paris polyphylla Smith from the Eastern Himalayan Re- activities are being updated and refined every year. These gion: A review. Humanitas Medicine, 2015, 5(3), 15. mentioned research gradually augment the mode of action of [15] Yun, H.; Lijian, C.; Wenhong, Z.; Yuhong, D.; Yongli, W.; Qiang, the compounds derived from the herb and beacons for further W.; Ding, Z. Chemistry of Natural products, 2007, 43(6), 672-677. works. [16] Lee, M.S.; Yuet-Wa, J.C.; Kong, S.K.; Yu, B.; Eng-Choon, V.O.; Nai-Ching, H.W.; Wai, T.M.; Fung, K.P. Cancer Bio Ther., 2005, 4(11), 1248-1254. CONSENT FOR PUBLICATION [17] The Pharmacopoeia Committee of China. Chinese Pharmacopoeia 2010 ed., Beijing: The People's Medical Publishing House, 2010, Not applicable. 243–4. 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