Traditional Uses, Active Ingredients, and Biological Activities of Paris polyphylla Smith: A Comprehensive Review of an Important Himalayan Medicinal Plant

to 2023 using 116 research publications. Major steroidal saponins such as Paris saponin I, V, VI, VII, and H have been found substantially efective in anticancer activity, abnormal uterine bleeding, dysfunctional uterine bleeding, and menorrhagia. Traditional breeding and propagation techniques cannot keep up with the world’s growing demand for herbal drugs. Terefore, it is critically necessary to take conservation measures and develop novel techniques for growing and cultivating this economically signifcant and highly valuable therapeutic herb. Te advanced biotechnological approaches like micropropagation and genetic analysis introduced long back are either rare or lacking in the case of P. polyphylla . It contains a wealth of information that will serve as a baseline data source for various stakeholders, researchers working on various research aspects, and policymakers to defne appropriate utilization and conservation plans for a high-value commercial medicinal plant called P. polyphylla . Te review provides an updated overview and critical assessment of secondary data regarding the past and recent applications and interventions of P. polyphylla .


Introduction
Medicinal and aromatic plants (MAPs) have signifcantly improved human health since the ages. Tese plants are crucial in meeting the demands of the traditional medicine markets across the globe. According to WHO (World Health Organization), about 80% of the world's population depends on herbal medicines for primary health care [1]. Herbal plants account for over 25% of the world's drugs [2,3]. Te immense potential of medicinal plants used in various traditional systems has been evident through increased focus on plant research in recent times [4], especially the physiology and pharmacology of medicinal plants through pharmacological investigation [5,6]. Uncontrolled harvesting of natural fora, including those found in forests, has satiated the rising demand for the vast majority of medicinal plants. As a result, habitat and genetic diversity have sufered catastrophic losses [7,8]. Small population size, restricted distribution, human disturbance [9], and unmanaged harvesting [10,11] lead to poor regeneration of the species [12], and failure of seed development [13] is responsible for the threat to the species. Tis results in the lack of availability of quality plant material for the future [8,14]. Tus, this is causing concern for the long-term sustainability of the natural resource, which can be met by mass multiplication and sustainable conservation of important medicinal plants. Recently, new agrotechniques have evolved for the sustainable cultivation of medicinal plants. However, there is an urgent need for biodiversity management by which medicinal plant resources could be conserved and will help to develop sustainable rural livelihoods [15][16][17].
Considering the increasing demand for medicinal herbs by pharmaceutical industries, the local market, and the traditional health care system followed by overexploitation, illegal trade, and unorganized cultivation, Himalaya has gained priority towards the conservation and sustainable utilization of its herbal wealth [17]. Indian Himalayas has been known for their rich cultural, spiritual, and biodiversity values all across the globe. Te cure for various ailments and the use of medicinal herbs in the Himalayan region are well described in Indian sacred texts. Te 3000-years-old Ayurvedic system in India has evidence of medicinal plant collection from the Himalayan region. Having more than 10% of countries geographical area, Himalaya harbors more than 8000 vascular plants, including 1748 medicinal plants [18]. Te potential of the medicinal wealth of the Himalayas is so immense that about 90% of wild plant species belonging to the alpine grassland of the Himalayas are used in various herbal industries. Moreover, the herbal industries in India are getting 90% of medicinal plant raw material (including for export) from natural habitats [19].
Among various medicinal plants used to prepare medicines, Paris polyphylla from the genus Paris has its unique identity and recognition. P. polyphylla, a high-value medicinal plant, is native to the Indian subcontinent and China and is extensively distributed in the Yunnan-Guizhou plateau of China [20], Manipur in India [21], and Pokhara and Kathmandu in Nepal. Apart from China, India, and Nepal, subspecies and varieties of P. polyphylla are well distributed in Myanmar, Bhutan, Vietnam, Tailand, and Laos [22]. All the plants of the genus Paris are known for various medicinal properties. Tey can be used as medicine [23], such as relieving pain, detoxifying, reducing swelling, and calming the liver. P. polyphylla has received increasing recognition and attention due to its medicinal properties in recent years, which have generated intense ethnopharmaceutical interest in the pharmaceutical industries [24]. Furthermore, due to high demand from pharmaceutical industries, P. polyphylla has been overexploited from its natural resources, which has caused signifcant habitat loss and put this species on the edge of extinction in the last few decades. In addition, this species' slow growth and reproduction have put it in endangered status, resulting in a sharp rise in price [25]. In the past studies, the authors have reviewed the population density, trade, cultivation and propagation, chemical and biological perspectives, pharmacological uses and efects, reproductive biology, genetic diversity, and management of P. polyphylla [5,6,26,27], and there has been no systematic study to understand multiple attributes together. Despite the fact that only a small number of authors have reviewed the bioactive compounds [5], traditional uses [6,28], microbiological aspects [29], distribution, ethnobotanical uses, molecular characterization, and species cultivation [6], there is a dearth of knowledge regarding the distribution of medicinal plants, their suitability for diferent habitats, the ratio of demand to supply, and the efects of climate change. Moreover, attributes like ecology, biotechnology, molecular biology, and conservation have yet to be reviewed. Tus, the published literature on a variety of P. polyphylla-related topics, such as its ecological status, commercial value, ethnopharmacology, pharmacology, phytochemistry, in vitro propagation, conservation status, molecular analysis, and future possibilities, has been reviewed in this study to fll this knowledge gap. Considering the abovementioned issues, the present review is prepared to provide a comprehensive knowledge, which underlines the current scenario and future prospects of this species.

Methodology
Consideration was given to the signifcance of review papers as a thorough literature search was conducted. Various search engines viz., Web of Science (https:// webofknowledge.com), ScienceDirect (https://www. sciencedirect.com), ACS publications, Taylor and Francis Online, SpringerLink, Scopus (https://www.scopus.com), Web of Science, PubMed (https://pubmed.ncbi.nlm.nih. gov/), National Library of Medicine (https://www.nlm.nih. gov/), and Wiley online library databases were used. Te data on P. polyphylla were compiled via research papers, review articles, books, book chapters, reports, and conference and seminar sessions. Te main focus was on the bioactive principles of P. pollyphylla along with keywords like Paris, Paris habitat, Paris geography, Paris ethnopharmacology, Paris pharmacology, Paris ecology, Paris diversity, and Paris trade to investigate the distribution, traditional use, ecological, biotechnological, molecular, phytochemicals, pharmaceutical, ethnopharmacological, pharmacological, and toxicological aspects of P. polyphylla. Tis review was created using 116 references and a thorough database search on 118 diferent aspects of P. polyphylla from 1979 to 2023.
Kashmir, Arunachal Pradesh, Manipur, Nagaland, Meghalaya, and Mizoram are the main locations of P. polyphylla [31]. Tis is one of the most important medicinal herbs across Himalaya, which has been a major source of raw material used in the health care system [32] to cure various ailments like cancer, tumors, fungal infections, back pain, fractures, bleeding, poisons, and skin allergy [31]. Te whole plant is used as febrifuge; rhizome as an expectorant and vermifuge, anthelmintic, antispasmodic, and digestive stomachic [33]; and roots as antitussive and depurative, antiphlogistic analgesic, and antipyretic [34]. Te perennial herb Paris is a temperate genus with twenty-four species. Te plant carries bisexual fowers, berry-like fruits with too many seeds, an erect stem, and thickened rhizome. Stem is thick (1-2.5 cm), tall (50-100 cm), and unbranched. Simple leaves are arranged in whorls, petiolate, and lanceolate, with reticulate venation and smooth margins. Inforescence consists of yellow-green fowers forming a closed whorl at initial growth. Solitary fowers are monoecious (male and female gamete within the same fower); outer sepals are larger and inner smaller, followed by 3-5 petals. Stamens are free and 6-11 in number, and gynoecium consists of one pistil and 3-5 fused carpel (syncarpous). A mature fruit contains 50-60 reddish orange seeds [35]. Germination in P. polyphylla occurs in April, and fowering starts in late April-May. Seed formation starts in late September and becomes mature in October. At the end of October to mid-November, aboveground parts die, and seeds spread in the environment. Between the seed maturation and the death of the plant, new bud sprouts from the rhizome and remains underground due to unfavorable conditions of the environment. Once the favorable conditions (sprouting season) start, these sprouts get germinates and come out from the ground. Te rhizome remains dormant for fve months. Seed germination of P. polyphylla in the natural condition is very low. Madhu et al. [34] also reported poor wild germination of P. polyphylla seed; even if the seed germinates, it takes a long period for fowering and many years to produce seed in a sufcient amount. It has been observed that there is no uniform pattern of sprouting, fowering, fruiting, or senescence of this plant; even, when the plants are taken out from the natural habitat and grow in earthen pots or nurseries in an artifcial condition, plants show variation in senescence, sprouting, fowering, and fruiting behavior [36]. Te plant in the habitat and its underground useable parts are depicted in Figure 1

Ecological Status: Habitat and Occurrence
P. polyphylla prefers a more than 80% closure canopy, altitude between 1300 and 1800 masl. It prefers deep forests and association with Arisaema costatum, Cyrtomium anomophyllum, Daphne bholua, Sarcococca species, and Viburnum erubescens for seed germination [36]. It requires streamside slopes (grassy or rocky) and mixed conifer forests in the habitat to grow properly. In midhills and lower mountains, the moist habitat of rhododendron, oak, and laurel forests provides favorable conditions for growth [34]. Being slow-germinating herbs, human interference sites, and water-logged areas are not suitable for the growth of P. polyphylla. Several problems are associated when it grows in artifcial habitats (cultivation). Lack of inforescence and fowering failure, followed by lack of fruit and seed production, is common in cultivation. However, it can grow well with similar habitat conditions [36].
Recent studies on the distribution and abundance of P. polyphylla indicate that its availability in nature is very low compared to its associated species from the same habitat [37]. However, only some studies on the ecological status of P. polyphylla have been performed in various parts of the Himalayas in the last few years [38][39][40][41]. Most of this ecological research is restricted to Nepal only. Compared with the species density of fve highly demanded medicinal plants of Himalaya, the density range (0.07-3.8 in d·m −2 ) of P. polyphylla was found to be similar. Te status of the species density of P. polyphylla is depicted in Table 1. Tis scenario opens the scope for detailed ecological research in Indian Himalayas.

Trade Value
Apart from its medicinal value, P. polyphylla is known for its high trade value of approximately Rs. 700-4500/kg for fresh and dried rhizomes [31,34,42,43]. Tis species is the most vulnerable species, sharing 24.6% of the total trade value in the Himalayas, Nepal, and China [34]. Te species has been exported to neighboring countries for a long time [42]. In the recent past, P. polyphylla has been traded in bulk amounts in Nepal (Pokhara and Kathmandu) and presently in diferent states in India, including Assam, Arunachal Pradesh, and Uttarakhand. Assam and Manipur have been the main route for the illegal trading of P. polyphylla from northeast India to Myanmar and southeast countries [31]. Basar [44] has reported the illegal export of P. polyphylla rhizomes from Arunachal Pradesh via Myanmar to China. Illegal trade and trafcking of rhizomes from Nagaland, Manipur, Arunachal Pradesh, and Meghalaya to Myanmar have been reported [21]. Currently, in Garhwal Himalaya, the illegal exploitation of this plant is continuing in practice. Due to their commercial value, local people collect these plants from their natural habitat for income. Continuous increases in price cause a tremendous threat to this plant due to overexploitation before maturation. Terefore, this plant is facing the problem of extinction from the wild [43]. Illegal collection of this plant from the Uttarkashi district of Uttarakhand has been highlighted recently [45]. Due to the unorganized and illegal collection and poor viability of seeds in natural conditions, this species has been listed under the endangered category [31]. Nepal has emerged as the highest producer of P. polyphylla rhizome, with over 400 tons of annual production [46]. Te status of trade marketing is depicted in Table 1.

Ethnopharmacology
Ethnopharmacology is the scientifc study of substances used as medicines by various ethnic and cultural groups. P. polyphylla Smith, a signifcant species of the genus, has Journal of Chemistry been called "jack of all trades" as it is used in treating various ailments, from diarrhoea to cancer [47]. Te rhizomes of P. polyphylla Smith are used in traditional Chinese medicines for their haemostatic, anti-infammatory, antitumor, analgesia, and antifungal activity. Only the rhizomes of P. polyphylla Smith var. yunnanensis and P. polyphylla Smith var. chinensis are referred to as medicinal Paris in the Chinese pharmacopoeia [48]. Te Chinese pharmacopoeia mentioned its use as an anti-infammatory, analgesic for sore throat, snake bites, and bruises. Te rhizomes of the species are reported for their use as digestive, expectorant, anticholinergic, and vermifuge. It is helpful in the treatment of heart disease, asthma, and bronchitis. Rhizomes are used as anthelmintic and tonic by the local inhabitants of Garhwal. Also, root powder is used in ethnopediatrics for diarrhea in this region. Rhizoma Paridis (Chonglou) is the main constituent of commercial products of P. polyphylla, namely, Yunnan Baiyao powder, Jidesengshe-yao pain tablets, and Gongxuening capsules which are well-known Chinese herbal medicines. Gongxuening capsules are a commercial product in the Chinese market, widely used for over 10 years [46]. Chinese people use rhizome as  a demulcent, antifebrile, immunity adjustment, analgesia, and anti-infammatory [49,50]. Te rhizome juice relieves gastric and menstrual pain, and rhizome paste is used to treat cuts and wounds and remove worms by ethnic people of western Nepal [51].

Pharmacology
Te primary bioactive chemical components of P. polyphylla, accounting for more than 80% of the total chemicals, are Paris saponins or steroidal saponins. A form of glycoside called a saponin has one or more water-soluble sugar chains, such as glucose, galactose, pentose, or methyl pentose, as well as aglycones like steroids or triterpenoids. Plant saponins have a variety of forms because diferent sugars can be found in various positions and directions. Steroid saponins and polyphyllins are the main constituents among the nearly 100 phytochemicals of therapeutic signifcance that have been discovered in the species [6]. Te anticancer, antioxidative, expectorant, platelet aggregation inhibition, insecticidal, antidiabetic, antifungal/ antiyeast, antiparasitic, antibacterial, antihyperlipidemic, and anti-infammatory properties of steroid saponins are just a few of their medical applications. Te biological activity of steroidal saponins and additional ingredients was investigated against cancer cell lines, bacteria, enzymes, and other parasites that have demonstrated anticancer activity against oesophageal cancer [52], lung cancer, bone cancer, prostate cancer, breast cancer, bladder cancer, liver cancer, and colon cancer [53]. In addition, P. polyphylla rhizome extract and steroidal saponins shown antibacterial and antioxidant action [54]. On Candida albicans, steroidal saponins also demonstrated antifungal properties. Te steroidal saponins had selective antibacterial activity against a variety of bacteria, including Pseudomonas aeruginosa (100%), Staphylococcus aureus (80%), Listeria innocua (65%), Escherichia coli (57%), Salmonella enteric (67%), and Shigella sonnei (47%). Several pure steroidal saponins and crude extracts of P. polyphylla have shown strong efcacy against cancer cell lines, bacteria, and parasites in in vitro and in vivo tests. Some activities of P. polyphylla extract are summarized as follows.
7.1. Anticancer Activity. Te hydroalcoholic extract of P. polyphylla was analyzed and found to contain diosgenyl and pennogenyl saponins having anticancer activity against human A 549 lung cancer cell lines using doxorubicin as a standard. At a 500 microgram/ml concentration, more than 97% increment in cell killing was observed, and the EC 50 was calculated to be 52.34 microgram/ml. In comparison, doxorubicin exhibited 98% killing at a concentration of 1.8559 microgram/ml, and EC 50 was 0.579 microgram/ml [55]. With an IC 50 value of 0.6 and 0.9 micromolar, steroidal saponins from P. polyphylla rhizomes showed cytotoxic action against two human cancer cell lines: HEK 293 and HepG2 [56]. Induced apoptosis has also been reported in breast cancer cell lines and HT-29 and HCT-116 cells. Tis saponin also arrests the cell cycle in the G1 phase. Moreover, autophagy, which can promote apoptosis in MCF-7 and MDA-MB-231 cell lines, was facilitated by XA-2 and inhibited the Akt/mTOR signaling pathway [57]. Because Paris saponins inhibits the activity of IKKβ and p65 translocation, they are recognized as NF-kB activation suppressors. Suppression of mitochondrial fssion has also been reported in Paris saponin by inhibiting the phosphorylation of Erk1/2 and mitochondrial translocation of dynamin-related protein 1 (Drp1) by dephosphorylation Drp1 at Ser616 [58]. Te antiangiogenic efect of polyphyllin D was studied for the implication of a potential drug in cancer treatment, and polyphyllin D was found to suppress the growth of HMEC-1 cells at 0.1-0.4 micromolar without any side efect. Tis concentration inhibited endothelial cell migration and capillary tube formation [59].
Te efect and mode of action of polyphyllin I on human gastric carcinoma cells were examined, and it was found that polyphyllin I inhibited HGC-27 cell proliferation with an IC 50 value of 0.34 ± 0.06 μM after a72-h treatment in vitro. In comparison to paclitaxel, polyphyllin I at 3 mg/kg signifcantly reduced the proliferation of HGC-27 tumour cells in vivo and demonstrated superior safety. By switching LC3-I to LC3-II, polyphyllin I caused cell cycle arrest in HGC-27 cells. It has been reported that analysis of MDC and mGFP-LC3 fuorescence, western blotting, and fow cytometry downregulate cyclin B1 [60]. In the human erythroleukemia cell line K562, polyphyllin D induces apoptosis and diferentiation, which inhibits cell proliferation. Trough the mitochondrial apoptotic pathway, it causes apoptosis [61]. Te efect of polyphyllin I in cisplatinresistant human gastric cancer cell lines was studied, and the result showed that polyphyllin I treatment decreased cell proliferation. It also increased the mRNA and protein expression level of E-cadherin but decreased the expression of vimentin. It was concluded that polyphyllin I can be used as a potent drug candidate for treating cancer invasion and migration [61]. A novel spirostanol steroidal saponin was isolated from P. polyphylla rhizomes. Structure elucidation was done by two-dimensional NMR spectroscopy. Te structure was found to be pennogenin-3-O-{O-α-L-rhamnopyranosyl-(l ⟶; 4)-O-α-L-rhamnopyranosyl-(l ⟶ 4)β-D-glucopyranoside}, which was further evaluated for anticancer activity in human promyelocytic leukemia HL-60 cells. Te results showed that at a 10 μg/ml concentration, more than 50% inhibition in cell growth occurs [62]. Lepcha et al. [40] reported a high concentration of favonoids and phenolic compounds along with DPPH and ABTS scavenging activity in the P. polyphylla plant from a higher altitude. Furthermore, the cytotoxic efect of the extract was determined on three cancer cell lines, HeLa, HepG2, and PC3, by MTT assay, which showed dose-dependent inhibition of HeLa cell growth and moderate inhibition in HepG2 and PC3 cell growth.  [26,43,63]. Diosgenyl saponins isolated from P. polyphylla have a noticeable efect on immunostimulating activity. Tis activity has been investigated in mouse macrophage cells and observed a signifcantly enhanced phagocytic activity. Analysis was observed by phagocytosis, respiratory burst, and nitric oxide production [64].
7.3. Antimicrobial Activity. In order to evaluate P. polyphylla's antibacterial activity, disc difusion assays were performed against two bacterial strains: Escherichia coli and Staphylococcus aureus and as well as two fungus strains: Aspergillus niger and Trichoderma reesei. E. coli, S. aureus, and A. niger showed 95-97% inhibition with signifcant antibacterial activity at a concentration of 5 mg/ml of the sample. In the case of T. reesei, it was 74% [65]. Saponins isolated from the stem and leaves of P. polyphylla showed antimicrobial activity against Propionibacterium acnes, using erythromycin as a standard [66]. P. polyphylla extracts were reported to destroy viral RNA by penetrating the viral capsid. It is also observed that phytoconstituents (Paris saponin, polyphyllin I and II, and diosgenin) of P. pollyphylla could inhibit viral growth by enhanced production of IL-6 cytokine, which augments the antiviral activities [35].

Antioxidant Activity.
Te antioxidant potential of P. polyphylla was evaluated by DPPH assay and by determining the total phenolic content. Prominent inhibition was reported with the ethanolic extract of P. polyphylla. Compared to the standard ascorbic acid inhibition concentration value of 7.8 μg/ml, P. polyphylla showed a higher inhibition concentration value (68 μg/ml) using the DPPH method. Phenolic content was also higher in the ethanol extract (0.68 mg/g catechol) than in the petroleum ether extract (0.47 mg/g catechol). Ethanol extract showed signifcant antioxidant activity due to higher phenolic content [67]. Higher concentrations of plant phenols and favonoids were reported in the plant from high altitudes, but both the high-and low-altitude plants exhibited excellent DPPH and ABTS scavenging activity [40]. 7.5. Antiaging Activity. Te antiaging efect of polysaccharide consisting of L-arabinose and D-galactose in a molar ratio (4.2 : 5.8) was evaluated by D-galactoseinduced mouse aging model revealed prevented malondialdehyde formation and improved antioxidant enzymes levels and total antioxidant capacity in serum and liver [68,69].
7.6. Protective Efect. Te efect of P. polyphylla methanolic extract in ethanol-induced gastric lesions in rats has been reported, and signifcant inhibition was observed. It was also reported that spiroatano structure is essential for the activity, whereas in protective efects against ethanol-induced gastric lesions 17-hydroxyl group plays an important role [70]. Te extract of P. polyphylla var. yunnanesis is an important alternative medicine for abnormal uterine bleeding. In China, it is (P. polyphylla extract, which is nontoxic) used in place of estrogen and progestin, which is used in abnormal uterine bleeding therapeutically [35]. Te spirostanol glycosides obtained from P. polyphylla are reported as contractile agonists for the uterous, and the activity is attributed to glycone and the aglycone part of the compound [71]. 7.7. Toxicity. Te P. polyphylla rhizome extract was investigated for cytotoxic efect on healthy rats over a period of time. Te fndings revealed no sign of mortality, ill health, and toxicity [71]. In rats, the rhizome extract's cytotoxic efect was analyzed over time and observed as nontoxic. All the rats survived well and showed no sign of toxicity or mortality. Although dullness was reported briefy, that could be because of neural suppression. Te extract's cutof median lethal dose (LD 50 ) was also recorded in female rats for 14 days, and it was greater than 5000 mg/kg body weight. From the study, it was clear that the extract showed a cytotoxic efect only for cancerous cells and proved itself a good target for future anticancer drug studies [71]. In another study, cytotoxic activities of triterpenoid saponins isolated from the rhizome of P. polyphylla have been observed against human nasopharyngeal carcinoma epithelial cells [72]. Furthermore, the cytotoxic efect of the extract revealed dose-dependent inhibition of HeLa cell growth while moderate inhibition in HepG2 and PC3 cell growth when determined on three cancer cell lines, HeLa, HepG2, and PC3 [40]. In addition, steroidal saponins from P. polyphylla's rhizomes have been shown to have a cytotoxic efect against the human cancer cell lines HEX 293 and HepG2 [56]. Li et al. [52] found polyphyllin D, a steroidal saponin, in the extract of P. polyphylla. Te author reported viability inhibition and apoptosis induction in treating MCF-7 and MDA-MB-231 cells with polyphyllin D. In vivo studies showed that polyphyllin D starts mitochondrial dysfunction and elicits apoptosis. It has also been demonstrated through in vivo studies that daily administration of polyphyllin D (2.73 mg/kg body weight) reduced 50% of the tumor in terms of weight and size and did not show any toxic efect on the heart and liver of the host Li et al. [52]. Based on the literature cited in this review and available in the public domain, it has been proven that there is no acute or chronic toxic efect of extract of P. polyphylla. Moreover, pharmacological uses and phytoconstituents of P. polyphylla are described in Table 2.

Phytochemistry
Plants are considered biosynthetic laboratories because they synthesize various metabolites. Phytochemistry deals with the secondary metabolites which are produced through the plant defense system. Tese are also known as active principles or active ingredients. Tese phytomolecules help the plant to survive against pests, pathogens, UV exposure, and other environmental hazards. Drug discovery depends entirely on phytochemicals; therefore, understanding these phytochemicals is very important for preparing novel  [75,76] Immunostimulating activity Diosgenylsaponins - [76] Antioxidant activity -DPPH assay [27] Antiaging activity Polysaccharide (consisting of L-arabinose and D-galactose) D-galactose-induced mouse aging model [67] Protective efect
Saponins are biosynthesized in the green part of plants, and then they are transported to the roots for storage and distributed in aerial parts. Tirteen types of saponins are reported in the aerial part of the plant, but some are not isolated because they are either unstable or very minor in concentration. Nautigenin and isonautigenin, a derivative of spirostanol saponin, have been isolated from fresh plants. From P. polyphylla's aerial portions, hydroxy-pennogenins and its tetrasaccharide and pregnane-type saponins have also been discovered. [86]. For the frst time, fve diferent steroidal saponins, namely, Paris saponin I, Paris sapon V, Paris sapon VI, Paris sapon VII, and Paris sapon H have been isolated from P. polyphylla, along with six new oleanane-type triterpenoid saponins, paritrisides A-F (1-6) and nine known triterpenoid saponins (7)(8)(9)(10)(11)(12)(13)(14)(15) [76].
Around 320 chemical components, such as steroidal saponins, C-21 steroids, phytosterols, insect hormones, pentacyclic triterpenes, favonoids, and other compounds, have been identifed up until 2020 [28]. Only four Paris saponins such as Paris saponins I, II, VI, and VII have been formally acknowledged as quality standard components of the Chinese pharmacopoeia, although more than 50 Paris saponins have been identifed from P. polyphylla var. yunnanensis. Diosgenins, pennogenins, and their related glycosides have all been isolated from P. polyphylla in various forms. Parispolyside F and parispolyside G, two new phenylpropanoid glycosides that are phenolic glycoside derivatives, have been identifed from the rhizomes. Isolated from the rhizome of P. polyphylla var. yunnanensis, these two previously identifed favonoid glycosides were named isorhamnetin-3-o-neohesperidoside and isorhamnetin-3-ogentiobioside [78]. Te chemotaxonomic associations of nine species of Paris were collected from various Chinese locations. Infrared Fourier transform (FT-IR) analysis revealed similarities amongst the investigated species. In addition, chemotaxonomic research was carried out on P. pollyphylla. Using ions trap/time-of-fight mass spectrometry and triple quadruple mass spectrometry (QQQ-MS), polyphyllin I, II, V, VI, and VII, and gracillin were identifed in Paris species. [78]. Two simple oligosaccharides, heptasaccharide (M. F. C 42 H 72 O 36 ) and octasaccharide (M. F. C 48 H 82 O 41 ) have been isolated from P. polyphylla var. yunnanensis rhizome water extract. In the hairy root culture of a separate species, P. japonicus var. major, and the shoot culture of P. polyphylla, it was discovered that these oligosaccharides had growth-regulatory functions [79]. Yang et al. [77] created a quick and accurate method to measure 9 P. polyphylla components in rat plasma using LC-MS. Due to its higher sensitivity, this method is benefcial for human clinical study [77]. A UF-LC-MS method with multiple pharmacological targets of TopI, II, and COX-2 enzymes was devised to investigate the mechanism of action of polyphyllins. In vitro, polyphyllin I demonstrated the highest binding afnity and the strongest COX-1, COX-2, and COX-2 inhibitory actions, followed by polyphyllin II, VI, and VII. Te study results imply that polyphyllin I is the main compound responsible for P. polyphylla's anticancer and anti-infammatory efects and that the COX-1 and TOP I enzymes are its targets for these efects. Chen and Guo [58] suggested that polyphyllin I, II, and VI can be used as a marker component for the quality regulator of P. polyphylla by considering the comparative quantities and their inconsistencies in pharmacological activities. Ginsenoside Rh2 was used as the internal standard in developing a quick, precise, and LC-ESI-MS/MS method for the isolation and simultaneous measurement of polyphyllin D and Paris H in rat plasma. A homogenous polysaccharide was found in the leaves of P. polyphylla, which consists of L-arabinose and D-galactose in a ratio of 4.2 : 5.8 [68,69]. For the extraction of polysaccharides from P. polyphylla leaves, extraction conditions were optimized such that temperature, time, and ratio of water to raw material were 90.8°C, 4.8 hrs, and 21.3:1, respectively, which produced the experimental yield of 54.18% [69]. Some important compounds isolated from the rhizome of P. polyphylla and their structures are depicted in Figure 2.

In Vitro Micropropagation
Te plant reproduces naturally by seeds and rhizomes; these techniques are often used for the multiplication and conservation of any endangered species. Plant tissue culture technology is now becoming an important tool for the largescale production of plant species that have medicinal importance and are generally difcult to cultivate [8]. In addition, the in vitro method can multiply this species because it produces genetically comparable plants quickly and can aid in the recovery of rare endangered threatened (RET) Himalayan herbs [80,[87][88][89]. Recently, a procedure using thin cell layer culture and in vitro plant regeneration through direct somatic embryogenesis for this species were described [90,91]. In another study [92], the transverse thin cell layer (tTCL) culture technique was used to regenerate the plant by forming minirhizomes. Minirhizomes were induced from the transverse thin cell layer of basal and middle stem portions, whereas the apical portion failed to produce any growth. Maximum 86.6% mini formation (fresh weight 1.05 ± 0.08 g) was observed on MS media supplemented 0.5 mg/l 6-benzyl amino purine (BAP). Tese minirhizomes produced healthy plantlets after hardening with 95% survival under greenhouse conditions. Active ingredient analysis of these plants showed a 1.41-fold increment in total steroidal saponins. Studies on elicitation using chitosan, salicylic acid, and yeast extract revealed increased steroidal saponin synthesis [92]. Diferent soil compositions also noted the efect of IBA and GA3 hormones (50, 100, and   Journal of Chemistry 150 mg/l) on P. polyphylla sprouting and rooting %. Te results showed that soil texture having soil:loam:sand in a 3: 2 : 1 ratio gave a better response for sprouting and rooting (76.66% and 73.33) [93]. Recently, Jesmi Devi et al. [30] have studied the total steroidal saponins diversity of P. polyphylla in the Northeast region of India. Te elite population for the micropropagation study was identifed based on the highest total saponins content (32.06 mg/g of dry weight). Furthermore, in the same study, MS media supplemented with 0.5 mg/l BAP + 6% sucrose (88.6%) and 1.0 mg/l 2iP + 6% sucrose (89.2%) was found to be the best combination for mini rhizome induction with 1.27 ± 0.02 g fresh weight and 1.36 ± 0.10 g fresh weight, respectively. Te efect of temperature of in vitro culture was observed, which revealed that a temperature range between 14 to 16°C is optimum for culture growth. Low temperature is unfavorable for culture growth, whereas high temperature causes culture contamination with endophytic bacteria [94]. In another study, Jamir et al. [95] split older rhizomes into pieces to raise shoot buds from each rhizome fraction to get a complete plantlet. Tey claimed that just 7% of the plantlets grown from rhizome cuttings were inforescent and displayed fruitful character, with the percentage of regenerated plantlets being 49%. It shows the poor regeneration of the plant. Because of its higher current status due to the illegal collection and economic importance, there is an urgent need to develop cultivation packages for the farmers. Also, the collective and sustainable participation of the farmers to conserve this plant in its natural condition is required.

Molecular Analysis
Studies on genetic diversity are increasingly being employed in conservation planning, particularly with relation to the efective restoration of populations in the wild. Te association between genetic variety and ftness of plant species has been established with support from numerous theoretical and empirical research studies. In plants, a species' geographic and ecological ranges are frequently connected with the genetic diversity within that species. Te quantity and distribution of genetic variation are signifcantly infuenced by a number of variables, including population size and habitat distribution. Population genetic theory states that populations are susceptible to genetic diversity loss if they exhibit any of the following characteristics: (i) genetic drift, (ii) founder efect, (iii) small initial population size, (iv) population bottleneck, or (v) rapid size reduction. Such predicted impacts not only decrease the likelihood of population persistence but also have signifcant efects on the likelihood of extinction of species. Major research issues include comprehending the genetic repercussions of such population structure changes and how they afect conservation value. People have reported many morphological variations in P. polyphylla populations [96]. Te morphological parameters cannot reveal signifcant diferences in the various species of the genus Paris, as some species are very similar at the morphological level. Terefore, it is necessary to use molecular markers to diferentiate the species and discuss their relationship and diversity at the genetic/molecular level. Te genetic homogeneity and diversity of several medicinal plants have been efectively tested using a number of PCR-based RAPD, ISSR, and SCOT molecular markers. Technically, the various molecular markers do not compare equally in terms of price, time, required DNA quantity, labour, and degree of polymorphism. RAPD analysis is easy, quick, and capable of fnding signifcant polymorphisms. Although mismatch annealing causes a certain lack of reliability, it is especially well-suited to DNA fngerprinting. With only a few primer combinations, AFLP analysis is reliable and produces large numbers of repeatable polymorphic bands. Both methods are quick, afordable, and do not need previous sequence knowledge. Several highly informative multiallelic loci make up ISSR markers. Tey are extremely common and ofer highly discriminating information with strong repeatability. By focusing on the conserved region fanking the start codon (ATG) of a functional gene, the SCoT marker overcomes the constraint of conventional DNA markers like RAPD and ISSR, which have limitations since they target a specifc region in the genome. Tese molecular marker approaches produce accurate and repeatable results independent of environmental infuences on plant age, tissue, and developmental stage. To successfully verify the genetic homogeneity of micropropagated trees, arbitrary amplifed markers such as RAPD (random amplifed polymorphic DNA), ISSR (intersimple sequence repeat), and codominant SSR have been used in most cases. Several workers have worked on genetic diversity analysis of the genus Paris, which is listed in Table 3. Zhang et al. [106] investigated the morphological variations in 196 accessions from 8 diferent populations of P. polyphylla. Tey found high diversity between the populations based on qualitative and quantitative characteristics. Results from the study showed that the accessions form the cluster based on their origin, which was refected through their morphological similarity. Te study's results helped do practices for the conservation and sustainable utilization of this medicinal plant [106]. Te genetic diversity analysis of P. polyphylla has been studied in different ecology regions of Vietnam and China using the RAPD technique. RAPD analysis showed that there are 125 DNA fragments were amplifed. Te genetic diversity was found to be high within the studied populations [107].
Zheng et al. [24] investigated the polymorphism analysis produced by 12 microsatellite loci obtained from a P. polyphylla var. chinensis (CT) n-enriched genomic library. Tey took 30 individuals from a natural population and studied polymorphism at each locus. Such studies can be very helpful in further genetic analysis and gene fow analysis of P. polyphylla var. chinensis [24]. In another study, ISSR markers were used to analyze the genetic diversity 3 cultivated and 3 naturally growing populations of P. polyphylla [25]. Fourteen primers were taken in the study, producing 227 polymorphic bands out of 251 bands. Results showed that genetic diversity was low within the population. Also, low genetic diversity was observed in the cultivated population compared to the natural population [25]. Recently, Zhao et al. [108] used SCoT and SRAP markers to study the genetic relationships and diversity among populations of P. polyphylla. Te study used 9 SCoT primers, which produced 134 bands with 100% polymorphism. Using amplifed fragment length polymorphism markers, population genetics of the wild and domesticated populations of P. polyphylla var. yunnanensis have recently been compared [109]. Tey identifed 32 P. polyphylla var. yunnanensis from China and explored spatial patterns in genetic variation in the wild (15) and cultivated (17) populations using AFLV markers.

Conservation Status
Te medicinal plant wealth of Himalaya, as a source of raw material and active compounds of modern medicine and traditional health care system, is under depletion in their natural habitats. Due to habitat fragmentation and illegal collection from the wild, most medicinal plants are under severe threat, and P. polyphylla, a medicinally important endangered plant [31], is not an exception. Depleting this plant in the natural habitat afects plant dispersal, gene fow, and endogamy, and as a result, the capacity of competition and adaptation of a species is infuenced badly [110]. It has been reported that unorganized collection, overexploitation, and illegal trade of the plant part, i.e., rhizome, low viability, and long dormancy of seeds are the major problems that need a proper solution for the conservation and efective utilization of the plant. Species with signifcant economic value are vulnerable to overexploitation. Destructive removal of roots, bark, or whole plants accompanied by livestock trampling may lead to plant death [34,97]. Due to its important medicinal properties, P. polyphylla is now being exploited in the localities, and its occurrence is shrinking. P. polyphylla is an out-crossing species that produce nectarines, is insect-loving, and is thought to have a high population genetic variation [104]. Perennial P. polyphylla grows from the rhizome under ideal conditions following winter defoliation. In addition to the various anthropogenic factors and shifting climatic conditions mentioned above, the plant has a few other distinctive characteristics that afect its conservation statuses, such as long seed dormancy, poor seed viability, less natural regeneration, and variation in annual seed production [79].
Considering the importance of P. polyphylla in the preparation of medicines, it is essential to standardize the method for its conservation through biotechnological tools like in vitro propagation, which will help protect and restore the species on the verge of extinction. Rhizome cultivation has been advocated as the most suitable method for ex situ cultivation and species conservation among the several propagation procedures that have been devised [6]. Despite the fact that the species are widely traded illegally throughout the world, many regions lack conservation and management guidelines. Because of this, medicinal plants can be protected from a variety of hazards by efective in situ and ex situ conservation techniques [6,101]. To conserve this plant, researchers must establish propagation protocols, cultivation practices, and guidelines for the sustainable use of the rhizome. However, few attempts have been made earlier on in vitro propagation of P. polyphylla [30,90,91,93]. Raomai et al. [90] have developed a successful regeneration process for P. polyphylla Sm. using the production of minirhizomes using the transverse thin cell layer (tTCL) culture technique. Half-strength MS medium supplemented BAP (6-benzylaminopurine; 0.5 mg/l) was suitable for maximum mini rhizome formation (86.6%). Regenerated plants showed 95% survival under greenhouse conditions. In another study [93], the rhizome segments of P. polyphylla are responsive to vegetative propagation with noticeable sprouting and rooting in the applications of hormones GA3 and IBA. In another study, Raomai et al. [91] reported that the types and range of cytokinin concentrations infuence minirhizome induction. Rhizomes, once formed, could be easily acclimatized and undergo hardening and can be kept for a long time, about six months, on the same medium without subsequent change in medium. Tus, the production of minirhizomes through in vitro propagation techniques could be potentially used for germplasm conservation and the production of secondary metabolites.
Ex situ conservation of P. polyphylla through cultivation outside of its natural habitats needs to be encouraged and promoted. However, there is a considerably older history of cultivation in China, where Yi healers have historically grown the variation P. polyphylla var. yunnanensis (Franch.) Hand-Mazz. in agroforestry systems in Chuxiong (Yunnan, SW China) [111]. Moreover, the ex situ conservation could Genetic diversity analysis [105] be possible by developing a separate and traceable supply chain to continue cultivating supplies from wild-harvested stocks [97]. Analysis of genetic diversity is one of the major steps for the conservation of the species, as it shows the evolution and competitiveness of the species for long-term survival in the presence of environmental pressure with diferent biotic and abiotic changes [112]. Moreover, developing efective strategies for cultivating species by national and state-level organizations such as Medicinal Plants Boards may lead to long-term conservation and management. Te tactic might involve public education campaigns and widespread engagement in agriculture, which will ease pressure on the wild population. Tis will satisfy consumer demand, strengthen the rural economy, and aid in preserving the species [31]. Te local authority and government agencies should work together to construct a Paris park/ garden in the natural habitats to reproduce and conserve this economically signifcant species [36].

Gap Areas and Future Prospects
Te Indian Himalayan region is a signifcant source of several therapeutic herbs. Due to uncontrolled collection and ever-increasing demand, these medicinal herbs are depleted in their natural habitat. Te natural populations of P. polyphylla, as an exclusive source of supply to meet the ever-increasing demand for its rhizome have been under serious threat of extinction. Te collectors adopt the rhizome as its ofcial part, a destructive harvesting method. Despite that, there is no strategic plan or programme to safeguard or manage rare species populations. Te genus's management, protection, and sustainable usage should receive immediate attention due to its importance and potential for commercial application. Both areas must be explored so that the species can be conserved and available phytochemical and pharmacological data can be used to provide the evidence to start any clinical trial of standardized formulation developed from the plant extracts. To achieve this, there is a need for efective implementation of government policies to control in situ harvesting and illegal trade. However, bans or strict regulations are inefective for high-trade value species. Normally people collect them and trade them one way or another. Te sustainable use and conservation management approach seems more efective than the ban or strict regulations. It will be more efective with the involvement of local people, such as the National Agriculture Innovation Project [113]. Cultivation of medicinal, as a self-employment sector, has emerged as an option for livelihood enhancement [6,23,113]. P. polyphylla cultivation outside of areas with high conservation importance needs to be fostered and encouraged in order to increase productivity. It may be possible to accomplish this by creating distinct, traceable supply chains for grown supplies in order to diferentiate them from wild gathered stocks. Farmers depended solely on the wild collection of medicinal plants and later engaged in cultivation practices under changing socioeconomic scenarios. A series of meetings, training, and workshops under the government's innovative programs, i.e., the National Agriculture Innovation Project, must be initiated to build harmony and understanding between farmers and traders and promote a buy-back medicinal plant system. Such an approach may build opportunities for farmers to enhance their confdence, skills, and knowledge and identify selfsustaining livelihood options by conserving medicinal plants in the wild. For mass scale multiplication under ex situ, there is an urgent need to develop rapid, reproducible and reliable in vitro plant regeneration systems for this species [6,14]. Due to a lack of standard cultivation protocols, its raw materials supply has exclusively depended on its natural populations. Setting up conservation and management plans for rare and endangered plant species depends heavily on biotechnological and ecological approaches. An important step toward the species' conservation will be utilizing tissue culture employing biotechnological and conventional multiplication methods for its reintroduction in natural habitats and specialized areas. Developing synthetic seeds using sodium alginate could be a very efective germplasm conservation, distribution, and exchange method. Another crucial component of the tissue culture process is the evaluation of in vitro-produced plants' genetic fdelity, which allows one to determine whether the plants are genetically related to their mother plant. It is possible to measure how closely the in vitro plants resemble their mother plant using molecular markers, which has a remarkable positive impact on the pharmaceutical industry's expansion. In addition, considering the demand for and status of this species, it is essential to develop conservation awareness programs, agrotechnology, and additional research to solve the issue this species is currently facing. In China and Nepal, P. polyphylla is one of the top most traded species. Even while local demand had a negative efect on the P. polyphylla populations they studied in Nepal, domestic commerce, which is much higher in China and much lower in Nepal and India, was a factor. [34]. Tis scenario may be similar for India. Increased requirements for medicinal plants adversely afect the natural population of medicinal plants. Terefore, Government of Nepal has established integrated trade to conserve these medicinal plants. However, one must reintroduce the saplings to their natural habitat to conserve these medicinal plants. Ecological Niche Modelling, also known as species distribution modelling, is a novel technique that combines point distributional data from species distributions with a collection of environmental raster data to produce predicted maps of species distributions in a geographic area. Using the Maxent species distribution modelling approach, as described for other species, the study linked to the prediction of the ecological niche of P. polyphylla species across the Western Himalayas (in Uttarakhand) must be conducted [14,114]. It is possible to forecast the probable distribution of the species across the Himalayan region using the various bioclimatic characteristics. A recent study reveals that old growth areas and fragmented landscapes shows signifcant decline in Paris species populations [27]. Maintaining the damp microclimate inside the preferred habitat of Paris species is crucial for long-term conservation due to the severe efects of logging and animal trampling on the ecosystem [115]. Such modeling-based analysis may help in defning strategies for long-term monitoring and conservation of commercially important species.
Te Jackknife test is also used to assess the signifcance of environmental variables for predictive modelling, which helps forecast the spatial occurrence of species, particularly habitat suitability or realized niche based on feldwork data about climate and topographic factors (such as slope, elevation, and precipitation [116]. Determining the potential ideal locations is done using a variety of algorithms or principles that often integrate data on species occurrence and the surrounding environment. Apart from the limited research going on in P. polyphylla, several research aspects still need to be identifed and yet to be explored (Figure 3). A close examination of the work summary shown in Figure 3 reveals numerous studies that have previously been completed and other areas that have yet to be explored. Notably, the information on propagation, mass multiplication, agro technology, processing, good collection practices, conversation, and sustainable management of this species could be more comprehensive.

Conclusion
P. polyphylla, an important medicinal plant used in traditional healthcare. It possesses steroidal saponins, favonoid glycosides, sterols, triterpenoid saponins that contribute to its various biological activities, including anticancer properties and treatment of abnormal uterine bleeding, dysfunctional uterine bleeding, and menorrhagia. However, due to overexploitation, this species is classifed as rare endangered threatened (RET). Immediate action is required to implement conservation strategies to ensure its natural sustainability and a consistent supply to pharmaceutical industries. Several threatening factors contribute to the decline of P. polyphylla populations, such as habitat loss, fragmentation, and degradation caused by farming, logging, and cattle trampling. Unsustainable harvesting practices specifc to this species also pose a signifcant risk. To address these challenges, ex situ conservation methods can be employed. Cultivation outside of natural habitats should be encouraged, and separate and traceable supply chains for cultivated material should be developed to diferentiate them from wild-harvested stocks. In vitro propagation techniques  Figure 3: Extent of research on ecological, biotechnological, and pharmacological interventions in Paris polyphylla.

Journal of Chemistry 13
have shown promise in the conservation of P. polyphylla. To encourage ex situ conservation, cultivation outside of natural habitats needs to be encouraged and that can be achieved by developing separate, traceable supply chains for cultivated supplies in order to distinguish them from wild-harvested stocks. Furthermore, in vitro propagation has a success story in terms of producing minirhizomes which produced healthy plantlets (95% survival with 1.41-fold increment in total steroidal saponins) after hardening under greenhouse conditions. Tus, the in vitro propagation techniques could be potentially used for germplasm conservation and to reduce pressure of unorganized collection of plant material from natural habitats. Utilizing ecological niche modelling, an efective conservation programme must be designed and implemented considering the ecological characteristics and population condition of this species. Furthermore, it is also necessary to adopt the government's innovative programs, i.e., National Agriculture Innovation Project for local communities to ensure people's participation in the conservation of this species.

Data Availability
Te data supporting this systematic review are from previously reported studies, which have been cited.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Authors' Contributions
BR, SP, JSB, and JMR conceptualized the study, collected the data, and carried out the redaction. SP, NR, P, RKB, and AT carried out critical revisions from the draft and prepared the fnal version. All authors reviewed the fnal version of the work before being accepted.