Untiring Researches for Alternative Resources of Rhizoma Paridis

Rhizoma Paridis (RP, 重楼), a traditional Chinese medicine, is the rhizoma of Paris polyphylla var. yunnanensis (PPY) or P. polyphylla var. chinensis which are widely used as important raw materials for several Chinese patent drugs. However, the wild resources of these herbs have become less and less due to their slow-growing characteristics and previously excessive excavation. This review covers untiring investigations on alternative resources of RP by our research group over the past decades, including non-medicinal parts of PPY as well as other plants of Liliaceae and Liliflorae families. The arial parts of PPY and the whole plants of Trillium kamtschaticum might be alternative resources for RP based on the fact that they shared the same or similar saponins and bioactivities.


Introduction
The genus Paris (Liliaceae) comprises approximately 32 plant species throughout the world and with 26 species found in Southwest China. [1][2][3][4][5][6][7]. Among them, the dried rhizoma of Paris polyphylla var. yunnanensis (PPY) and P. polyphylla var. chinensis (PPC), both called Rhizoma Paridis (RP) in China, have long been recorded in Chinese Pharmacopoeia as a traditional Chinese medicine to treat furuncle, snakebite, injuries from falls and convulsion, epilepsy, and sore throat [8]. Because of their remarkable medicinal functions, PPY and PPC have been a hot topic within the medicinal chemistry and drug discovery community since the 1970s. Previous studies revealed that PPY and PPC were rich sources of spirostanol (diosgenin and pennogenin) saponins [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] responsible for various pharmacological effects, such as cytotoxic and antitumor [13][14][15][16][17][18][19][20], antifungal [21,22], and haemostatic bioactivities [23,24]. The available supplies of PPY and PPC are facing increasing shortage based on the fact that their rhizomes can only be harvested until they have grown more than 7 years and the consumption by the pharmaceutical industry of these herbs have increased sharply in recent years. Thus, it is really imperative to search for other saponins or resources that might be substitutes for RP. Over the past 34 years, in order to find valid and alternative resources of RP, our research group have made great effort to phytochemically investigated on the non-medicinal parts of PPY as well as other plants of Liliaceae and Liliflorae families according to their genetic and phylogenetic relationships, which led to the isolation of identical or similar bioactive constituents with those of RP. As a result, a total of 184 saponins and including 120 new ones were obtained and identified, some of which showed interesting bioactive effects as those of RP. This paper mainly describes our untiring researches that can provide active ingredients for alternative resources of RP.

Steroidal Sapogenins and Saponins
According to the fact that the steroidal saponins are the bioactive constituents of RP, the steroidal sapogenins and saponins of non-medicinal parts of PPY and other Paris, Ypsilandra, Trillium, and Tacca plants have been investigated, which led to the isolation of 17 new steroidal sapogenins and 103 steroidal saponins, along with 64 known analogues.

Ypsilandra Species (Liliaceae)
Ypsilandra (Liliaceae), a small genus including only five species, is widely distributed in Southwest China and Myanmar [34]. We speculate that Ypsilandra species should produce similar steroidal derivatives as those of Paris due to their genetic and phylogenetic relationships. Although Y. thibetica has been used as a folk medicine for treating uterine      [45], and ypsilactosides A (71) and B (72) [46]. These new saponins were usually the oxygenated derivatives at C-6, C-7, C-11, and C-12 of those known analogues and some of these isolates had unpredicted aglycones. To be more specific, saponins 44 and 45 represented the first example with a novel 5(6→7) abeo-steroidal aglycone, whereas 59-61 were unusual 23-spirocholestane derivatives and 67 possessed a rare 6/6/6/5/5 fused-rings cholestanol skeleton.

Trillium Species (Liliaceae)
The Trillium genus consists of approximately 49 species throughout the world. However, only three species, T. kamtschaticum, T. tschonoskii, and T. govanianum, are found in Hubei, Sichuan, Yunnan, and Xizang Provinces of China. The rhizomes of T. kamtschaticum, called "Toudingyikezhu" in Chinese, have been traditionally use by Chinese minorities (Tujia and Miao people) for the treatment of traumatic hemorrhage [47,48]. In addition, some pennogenin saponins have been reported from Trillium species [49,50] and the crude extract of the whole plants of T. kamtschaticum displayed significant induced-platelet aggregation activity at a concentration of 1.5 mg/mL as revealed by our initiatory test. All these information strongly inspired us to investigated the hemostatic constituents of the whole plats of T. kamtschaticum, resulting in the isolation of 18 new steroidal saponins ( Fig. 3; Table 3), named trillikamtosides A-R (74-91) [51,52]. Interestingly, some of them were determined to have rare aglycone moieties. For instance, the aglycones of 73-75 had unique 3β,17α-dihydroxyspirostanes featuring a double

Tacca Species (Taccaceae)
Compared with the genera of Liliaceae family, the Tacca plants are very limited. In order to discuss/explore whether the Tacca species possess the same steroidal constituents as that of RP, our group investigated the phytochemicals of two Tacca species (T. plantaginea and T. subflabellata).

Known Sapogenin and Saponins
Obtained from the Non-medicinal Parts of PPY and Other Paris, Ypsilandra, Trillium, and Tacca Plants Apart from the above mentioned new saponins, 1 known sapogenin and 63 known saponins were also identified from the aforementioned species ( Fig. 5; Table 5). Compared with those new isolates, these known compounds usually shared the aglycones with lower oxidation degrees.

Bioactivities
Based on the fact that RP is traditionally used as hemostatic, antimicrobial, and antitumor agents, the hemostatic, antimicrobial, and cytotoxic activities of obtained compounds were evaluated to initially confirm that whether the plants could be alternative resources of RP. Our studies revealed that most of the bioactive compounds were spirostanol saponins with only one sugar chain at OH-3.

Hemostatic Effect
Both the total steroidal saponin moieties and purified saponins of PPY and T. kamtschaticum exhibited hemostatic effects. The 70% EtOH eluted fraction of T. kamtschaticum crude extract obtained from a macroporous resin column showed 76% maximal platelet aggregation rate at a concentration of 1.5 mg/mL [51]. Subsequently, three  [51]. The results also suggested that the hydroxy group at C-17 in pennogenin saponins was indispensable for their hemostatic effects, whereas the introduction of different functional groups in the A, B, or F-ring of pennogenin glycosides could make the hemostatic effect weak or disappear. Interestingly, the total saponin moieties from the above-ground parts and the rhizomes of PPY showed equivalent maximal platelet aggregation rates of 45 and 43% at a concentration of 1.5 mg/mL, respectively [61]. This indicated that the above-ground parts can be an alternative and more sustainable sources for RP. Additionally, two diosgenin-type saponins, ypsilandroside M (49), ypsiparoside C (54), and paris saponin II (133) isolated from Y. parviflora, exhibited MPARs of 43, 44 and 55% at the concentration of 0.3 mg/mL, respectively [41]. This indicated that the carbonyl group at C-12 or the sole α-L-rhamnopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→ 4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranosyl moiety at OH-3 was essential for the hemostatic effect of diosgenin saponins.    [43]. Moreover, the total saponin moieties from the both rhizomes and above-ground parts of PPY showed cytotoxicities against HL-60, A549, SMMC-7721, MCF-7, and SW480 cells [61]. To be more specific, the former displayed cytotoxicities against above-mentioned cancer cells with IC 50 values of 1.77, 1.75, 5.23, 6.62, and 3.49 μM, whereas the latter was less cytotoxic with IC 50 values of 9.54, 9.30, 12.61, 8.12, and 11.25 μM, respectively.

Conclusion
In summary, our continuous effort to search for alternative resources of RP led to the isolation of 184 steroidal derivatives, including 120 new ones. More importantly, several compounds of them displayed remarkable hemostatic, cytotoxic, and antimicrobial effects. Our studies disclosed that the non-medicinal parts of PPY, as well as other plants of Paris, Ypsilandra, Trillium, and Taccaceae family are also resources rich of steroidal saponins similar to those of RP, especially those recorded in Chinese Pharmacopoeia, namely, paris saponins I (131), II (133), VI (135), and VII (141). However, the investigations on the total content of these saponins, the related bioactivities of total saponin moieties of the studied species compared with those of RP, and their security capability are quite indispensable to confirm that whether the non-medicinal parts of PPY and other species from Paris, Ypsilandra, and Tacca genera could be safe and dependable alternative resources of RP. The arial parts of PPY and the whole plants of T. kamtschaticum might be alternative resources for RP based on the fact that they shared the same or similar saponins and bioactivities. The continuous studies on the saponin constituents of non-medicinal parts of RP and other plants will be carried out in our laboratory which may led to the discovery of more alternative resources for RP.   P. verticillata Aerial parts [32] T. kamtschaticum Whole plants [51] Y. parviflora Whole plants [48] Untiring Researches for Alternative Resources of Rhizoma Paridis 275 P. verticillata Aerial parts [32] P. luquanensis Rhizomes [62] PPY Seeds [63] T. kamtschaticum Whole plants [51] Y. parviflora Whole plants [41] Y. thibetica Whole plants [39] 142 Isonuatigenin 3-O-α-L-rhamnopyranosyl-(1-2)-β-D-glucopyranoside PPY Stems and leaves [25] 143 Disoseptemloside H PPY Stems and leaves [25]    T. plantaginea Whole plants [54] T. subflabellata Whole plants [55] 166 Proto-dioscin PPY Stems and leaves [25] 167 Methylprotodioscin PPY Stems and leaves [25] 168 Proto-paris saponin II P. verticillata Aerial parts [32] Y. parviflora Whole plants [41] Y. thibetica Whole plants [43]