Natural products from medicinal plants: non-alkaloidal natural constituents of the Thalictrum species

Contents

• 1 Introduction

• 2 Brief botanical characteristics of the Thalictrum genus

• 3 Secondary metabolites of the Thalictrum genus
  • 3.1 Triterpenoid glycosides and their genins
  • 3.2 Biological activity of the saponins of the Thalictrum genus
  • 3.3 Flavonoids and their glycosides
  • 3.4 Cyanogenic glycosides
  • 3.5 Hydrocarbons and high molecular weight alcohols
  • 3.6 Phenolic compounds
  • 3.7 Fatty acids
  • 3.8 Phytosterols and their glycosides

• References

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Abstract

Covering: 1959 to 2005

Thalictrum is an important plant genus that is widely used in traditional medicine. In this review considerable attention has been given to triterpenoid saponins in connection with their specific distribution in the Thalictrum genus and with their biological activity. All other non-alkaloid compounds isolated from the Thalictrum genus are also reviewed; these metabolites are discussed in relation to their structural features and to their role in the plants.

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Elena A. Khamidullina

Dr Elena A. Khamidullina studied chemistry at Irkutsk State University, Russia. She completed her PhD in the A. E. Favorsky Institute of Chemistry, Siberian Division of RAS and her work dealt with triterpenoids and saponins from Siberian plants. Her current research interests include biologically active compounds derived from plants, and the design of biologically active compounds based on natural products.

Alexandra Gromova

Dr Alexandra Gromova studied chemistry at the Irkutsk State University, Russia. She obtained her PhD degree from the Novosibirsk Institute of Organic Chemistry RAS. She then worked in the A. E. Favorsky Institute of Chemistry Siberian Division of RAS as a Senior Research Fellow. She is currently working at the Irkutsk State Technical University as a Senior Researcher. She has authored more than 110 scientific publications in international journals. Her research field is the isolation and elucidation of natural products with biological properties: e.g. phenols, flavonoids, saponins and alkaloids.

Vladislav Lutsky

Dr Vladislav Lutsky graduated from the Irkutsk State University. He received his PhD degree from the Novosibirsk Institute of Organic Chemistry RAS and carried out his postdoctoral work in the A. E. Favorsky Institute of Chemistry, Siberian Division of RAS (until 2001). In 1999 he was invited to Brigham Young University (Provo, Utah, USA) and worked for a year at the Department of Chemistry and Biochemistry as a visiting scientist. Currently he is a senior lecturer at the Irkutsk State Technical University. He has authored more than 130 scientific publications and his main research interests involve the isolation and structure elucidation of biologically active natural products from plants.

Noel Owen

Dr Noel Owen is a graduate of the University of Wales, with a PhD from Cambridge University and postdoctoral experience at Harvard University. He is a spectroscopist having worked in the area of vibrational, rotational and nuclear magnetic resonance spectroscopy. His current research interests involve the vibrational spectra of wood and wood products and the structure determination of natural products extracted from plants and plant endophytes.

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1 Introduction

For centuries plants of the Thalictrum genus have been used extensively as herbal medicines—in Russia (Siberian region) to treat gastric-intestinal diseases, female disorders, various neoplasms and pulmonary tuberculosis,^1–4 and in Japan (Nagano Prefecture) to treat stomach disorders.⁵ In China Thalictrum plants have a long history as folk medicines in the treatment of many kinds of diseases. At least 43 species of Thalictrum have been used because of their special and proven therapeutic effects.⁶ Until recently, studies on the chemical constituents of the Thalictrum genus have been mainly confined to the alkaloids. More than two hundred alkaloids have been isolated from this genus; this information has been summarized elsewhere.^7–11 Authors⁶ have attempted to relate pharmacological activities of these plants to the composition of the alkaloids and have shown that species containing alkaloids of different types possess different therapeutic effects. Our research¹² has revealed that the biological activity of these plants is not restricted to the alkaloid-containing species. We have investigated selected species, which did not contain alkaloids and have found interesting and potentially important biological activities.

In this review considerable attention has been given to triterpenoid saponins in connection with their specific distribution in the Thalictrum genus and with their biological activity. For the present instance the term “specific distribution” implies a dependence of chemical composition of the plants on ecological factors and geographical conditions. The original discovery of triterpenoid saponins in plants of the Thalictrum genus was made by some of the authors of this review, and the first few articles^13–17 describing these compounds were published in 1981–1983. Further investigations by Japanese, Chinese and Korean research groups have shown a great diversity of triterpenoid saponins in plants of this genus, caused both by changes in the genin structure as well as in the composition of the carbohydrate chain. To date more than 60 triterpenoids and their glycosides have been discovered in the genus, and most of them are new compounds. Those saponins, which have been isolated from plants of the Thalictrum genus, have considerable biological activity. This review includes the original results regarding anti-cancer activity and the effect of these compounds on mammalian reproductive systems.

In addition to triterpenoids and their glycosides, all other non-alkaloid compounds isolated from the Thalictrum genus are reviewed; these include flavonoids, cyanogenic glycosides, hydrocarbons, high molecular weight alcohols, fatty acids, phenolic compounds and sterols. These metabolites are discussed in relation to their structural features and to their role in the plants.

The relevant literature published since 1959 to the present is reviewed, with 120 cited references.

2 Brief botanical characteristics of the Thalictrum genus

The genus Thalictrum belongs to the order Ranunculales, and the family Ranunculaceae. The order Ranunculales consists of 10 families, among which Ranunculaceae is the largest (with ca. 50 genera and more than 2000 species).^18–20 As the family Ranunculaceae is considered one of the most primitive of the angiosperms, it is of significant biological interest, since some of the species are characterized by progressive morphological indications both of vegetative and generative organs.^21,22 The Ranunculaceae family is divided to four subfamilies, according to the structure of the flowers and fruits and cytology data:²² Helleboroideae, Ranunculoideae, Thalictroideae and Coptidoideae.¹⁹ The Thalictroideae subfamily includes the Thalictrum genus. This particular genus is distributed over many parts of the Earth, mainly in the temperate and cold zones of the both hemispheres. It is extremely polymorphous due to the breadth of its ecological–geographical diversity. A careful study of the systematic location of the Thalictrum genus has been given by Bochanceva.²³ At the end of the 1960's Tamura²⁴ suggested intraspecific systematization of the Thalictrum genus, which takes into account the existing genus system. Tamura's systematization is currently considered to be the most advanced.^21,25

The Thalictrum genus consists of about 110 species of herbaceous perennials.²⁶ It should be noted that the independence of some Thalictrum species is not generally accepted and confirmation is needed in specific cases, but on the other hand, some species (e.g. T. minus) have been divided into subspecies.¹

3 Secondary metabolites of the Thalictrum genus

3.1 Triterpenoid glycosides and their genins

The first reports on triterpenoids from the Thalictrum genus appeared in 1981–1983 and involved the isolation of triterpenoid saponins from Thalictrum minus and T. foetidum collected in Eastern Siberia.^13–17

Plants of the Thalictrum genus produce both tetracyclic (lanostane type) and pentacyclic (oleanane type) triterpenoids. Fifty-three saponins have been isolated from 9 species of the Thalictrum genus: 38 new tetracyclic and 15 pentacyclic saponins. The tetracyclic saponins contain 21 new genins. Some of them (2, 11, 12, 15, 28–31, 43) have been characterized as novel compounds. In certain cases artifacts have been obtained during isolation procedures (1a,¹⁶10a,²⁷19a,⁵⁰44²⁸). The other genins have been characterized as part of their respective glycosides.

From the 15 pentacyclic saponins, twelve are new compounds. The novelty of these saponins is determined by the structures of the carbohydrate chains.

Cycloartane type. Cycloartane compounds isolated from the Thalictrum genus have been classified into four groups according to the structural similarities of their side chains.
Cycloartanes with an acyclic side chain (on atom C-17). This type of compound is represented by structures 1–21, and includes saponins and triterpenoids derived from their respective saponins (Table 1). Thalicoside A (1) was the first glycoside isolated from the aerial part of Thalictrum minus.²⁹ Enzymatic hydrolysis (Helix pomatia) of 1 failed to produce any genin, but an acid hydrolysis led to an artifact (1a), which not only did not contain a cyclopropane ring but also had alterations in the side chain. The structure of 1a was elucidated by X-ray analyses.^14,16 The aglycon-thalicogenin (2) was obtained by periodate oxidation of 1 followed by treatment with alkali. The 9,11-cyclopropane ring and the primary hydroxyl group on C-4 were found to be arranged on different sides of the molecular plane on the basis of ¹³C NMR spectral data of 2.^30,31 The configuration of the C-22 asymmetric center of 2 was established as S on the basis of X-ray analysis of 1a and by comparison of the ¹³C NMR chemical shifts of 2 with those of 22(S) and 22(R)-hydroxycholestanols.³¹

Table 1 The structures of triterpenoid compounds with an acyclic side chain (on atom C-17)

R	No.	R1	R2	R3	R4	Ref.
	2	H	CH3	OH	OH	16,29
	1	β-D-Galp	CH3	O-β-D-Glcp	OH	30
	4	α-L-Arap	CH3	O-β-D-Glcp	OH	34
	3	β-D-Galp	CH3	O-β-D-Glcp	OH	33
	5	β-D-Galp	CH3	O-β-D-Glcp	OH	35
	6	β-D-Galp	CH3	O-β-D-Glcp	OH	36
	15	H	CH3	H	H	45,47
	14	β-D-Quip-(1→2)-α-L-Rhap-(1→6)-4-O-Ac-β-D-Glcp	CH3	H	H	45
	16	β-D-Quip-(1→6)-β-D-Glcp-(1→4)-β-D-Fucp	CH3	H	H	47
	17	β-D-Glcp-(1→6)-[α-L-Rhap-(1→2)]-β-D-Glcp-(1→4)-β-D-Fucp	CH3	H	H	47
	18	β-D-Glcp-(1→4)-[α-L-Rhap-(1→2)]-β-D-Fucp	CH3	H	H	49
	7,8	β-D-Galp	CH3	O-β-D-Glcp	OH	36,37
	11	H	CH3	H	OH	41
	9	α-L-Arap	CH3	H	α-L-Rhap-(1→6)-O-β-D-Glcp	39
	12	H	CH2OH	H	OH	42
	10	α-L-Arap	CH2OH	H	α-L-Rhap-(1→6)-O-β-D-Glcp	40
	13	β-D-Xylp-(1→2)-β-D-Xylp	CH2OH	H	OH	43
	19 R5 = Ac	Ac	CH3	β-D-Xylf-(1→6)-[α-L-Rhap-(1→2)]-β-D-Glcp	OH	50
	19a R5 = H	H	CH3	H	OH	50
	20 R5 = H	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	CH2OH	H	H	51
	21 R5 = β-D-Xylp-(1→6)-β-D-Glcp	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	CH2OH	H	H	51

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X-ray analysis of the artifact 1a established the conformation of the side chain which is stabilized by an intramolecular hydrogen bond between the hydroxyl group on C-16 and the O-atom of the tetrahydrofuran (THF) ring as shown on the Newman projection in Fig. 1a. Analysis of the ¹³C NMR chemical shifts of all the thalicogenin structures helped to establish the presence of the hydrogen bond. Comparing the experimental ¹³C chemical shifts with those calculated by the Beierbeck³² method confirmed the presence of an intramolecular hydrogen bond between the hydroxyl groups on C-16 and C-22, and helped to determine that the side chain conformation of the thalicogenin is as shown in Fig. 1b.

Fig. 1

Two other saponins from T. minus, thalicoside C (3)³³ and thalicoside A2 (4),³⁴ both possessing thalicogenin, differed from 2 by the carbohydrate constituents: the first had a trisdesmoside with galactose and two glucose units; the second had arabinose and glucose in two separate carbohydrate chains.

Thalicoside E (5)³⁵ was identified as a new saponin as a result of extensive spectroscopic analyses without degradation of the molecule. Thalicosides G1 (6),³⁶ G2 (7)³⁶ and H2 (8)³⁷ are minor metabolites of T. minus and are isomers of each other. Thalicosides G2 and H2 were shown to be C-24 epimers, but which saponin of this couple is the 24R isomer and which one is the 24S remains unclear. The C-22 stereochemistry of glycosides 6–8 was recognized as S using Seo's process³⁸ and made it possible to establish the stereochemistry of the hydroxyl groups in the chiral secondary alcohols using glycosylation effects observed in ¹³C NMR spectra.

As in the case of thalicoside A (1), cyclofoetosides A (9)³⁹ and B (10)⁴⁰ were isolated from T. foetidum at the beginning the 1980's. The isolation of the aglycone was important for their structural elucidation. Both saponins 9 and 10 have novel genins (11 and 12 respectively) and the same carbohydrate chains. Cyclofoetigenin A (11)⁴¹ was obtained by acid hydrolysis of 9 but in low yield, while cyclofoetoside B (10) yielded the artifact 10a during acid hydrolysis. The aglycon-cyclofoetigenin B (12)⁴² resulted from periodate oxidation of 10 followed by alkaline hydrolysis, whilst the α-diol group in the side chain was protected by esterification.⁴² The C-24 configuration of 12 was established as S.

Cyclofoetigenin B was also found to be a constituent of the new glycoside (13) isolated from the roots of T. smithii.^43,44 From the roots of T. foeniculaceum a new trioside—thalifoenoside A (14) was isolated.⁴⁵ Acid hydrolysis of 14 yielded glucose, rhamnose, quinovose and a new genin—3β,22(S),27-trihydroxycycloart-24-en (15). A comparison of ¹³C chemical shifts of 15 with those of 22S- and 22R-hydroxycholestanols⁴⁶ established the C-22 configuration as S. Thalictosides A (16) and C (17) obtained from the aerial part of T. thunbergii⁴⁷ have been reported to contain a new genin—talictogenin A (15). The S-configuration of C-22 in 16 and 17 was established by Mosher's procedure⁴⁸ after acetylating the hydroxyl group on C-26. Talictogenin A (15) and the genin of 14 are likely to be the same compound since their NMR spectra are very similar. The Z-configuration of the double bond in both cases was determined by NOE data. A new trioside (18) from the aerial part of T. squarrosum⁴⁹ also has the same genin 15.

A new glycoside (19)⁵⁰ from the aerial part T. uchiyamai is the only example of a glycoside with an acetylated genin (on C-3 and C-24 atoms) and which is bound by an ester linkage to a carbohydrate chain. Alkaline hydrolysis of 19 produced artifact (19a). The structural assignment of 19 was carried out on the basis of very convincing spectroscopic evidence.

During investigation of the structure of a new genin, a constituent of both thalictosides V (20) and IX (21) from T. Herba,⁵¹ researchers selectively cleaved the ester linkage between the C-21 carboxylic acid group and the carbohydrate chain of 21 using anhydrous LiI and 2,6-lutidine in dry methanol. The product of this reaction turned out to be identical to 20. Glycoside 20 is 3-O-monodesmoside and 21 is 3, 21-di-O-bisdesmoside.

Cycloartanes with a THF ring on C-17 atom. Triterpenoids (28–31) as well as saponins (22–27, 32–38) of this group have been isolated from T. squarrosum, collected in the Baikal region of Russia, and from T. Herba, collected in Japan (Table 2).

Table 2 The structures of triterpenoid compounds with a THF ring on C-17 atom

R	No.	R1	Ref.
	28	H (squarrogenin 1)	53
	22	β-D-Glcp (squarroside A1)	52
	23	α-L-Rhap-(1→6)-β-D-Glcp (squarroside B1)	52
	29	H (squarrogenin 2)	53
	24	β-D-Glcp (squarroside A2)	52
	25	α-L-Rhap-(1→6)-β-D-Glcp (squarroside B2)	52
	30	H (squarrogenin B3)	54
	26	α-L-Rhap-(1→6)-β-D-Glcp (squarroside B3)	54
	31	H (squarrogenin B4)	54
	27	α-L-Rhap-(1→6)-β-D-Glcp (squarroside B4)	54
	32	α-L-Rhap-(1→6)-β-D-Glcp (squarroside C)	55
	33	α-L-Rhap-(1→2)-β-D-Glcp	56
	35	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	57
	34	α-L-Rhap-(1→2)-β-D-Glcp	56
	36	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	57
	37	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	58
	38	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	58

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From T. squarrosum several saponins⁵²—squarrosides A1 (22), B1 (23), A2 (24), B2 (25), B3 (26), and B4 (27)—have been isolated, the genins of which have diastereomeric configuration at the C-21 atom. Squarrogenins 1 (28) and 2 (29),⁵³ both possessing 21-OCH₃, resulted from enzymatic hydrolysis of squarrosides A1, A2, B1, and B2 (22–25). The configuration of the C-21 atom was determined as R in 28 and S in 29 by NOE experiments. Enzymatic hydrolysis of squarrosides B3 (26) and B4 (27) afforded squarrogenins B3 (30) and B4 (31) having hydroxyl groups in positions 21S and 21R respectively.⁵⁴ It should be noted that squarrosides B3 and B4 are chromatographically inseparable isomers. The genin of squarroside C (32)⁵⁵ is squarrogenin B4 and so the 21-hydroxyl group has an α-relative stereochemistry and binds with the carbohydrate chain at the same time. A β-isomer of the trioside has not been found. The 21-O-CH₃-glycosides (22–25) proved to be native, i.e. not modified during the isolation procedure. The absence of methanol during the isolation procedure provided evidence of the above conclusion. When ethanol was used all the expected saponins (22–27) as well as the 21-O-ethoxy derivatives were isolated, confirming the ease with which the alkylation of the hemiacetal hydroxyl group takes place. The possibility of alkylation of the hemiacetal hydroxyl group raises the question of whether compounds of this type are native or not.

Those saponins which have been isolated from T. Herba (Takatogusa) are diastereomeric with reference to C-21, (thalictosides I (33) and II (34)⁵⁶ and thalictosides III (35) and IV (36)⁵⁷), but unlike those isolated from T. squarrosum they have an α-configured 22-hydroxyl group. Interestingly, the same two monosaccharides comprise the disaccharide of both biosides from T. squarrosum and T. Herba, but the order of attachment is different: it is rhamnose(1→6)glucose in squarrosides B1 and B2 and rhamnose (1→2)glucose in thalictosides I and II. The difference between thalictosides XII (37) and XIII (38)⁵⁸ from T. Herba consists of the orientation of the genin's C-24 hydroxyl group, the stereochemistry of which was inferred from spectroscopic data.⁵⁸

Cycloartanes with a THF ring on C-20 atom. This group is small in number consisting of the four glycosides 39–42 isolated from T. minus (Table 3). Thalicosides A1 (39) and A3 (40)³⁴ are based on a new genin—thalicogenin A1 (43). Thalicosides H1 (41)⁵⁹ and H3 (42)³⁷ differ from thalicosides A1 and A3 by an additional hydroxyl group on C-24. Glycosides 39–42 have the 22S-configuration. Thalicoside H1 (41) has an S-configured C-24 atom while thalicoside H3 (42) has an R-configuration, as shown by 2D NMR techniques including NOE experiments.⁶⁰

Table 3 The structures of triterpenoid compounds with a THF ring on C-20 atom

No.	R	R1	R2	Ref.
39	H	β-D-Galp	β-D-Glcp	34
40	H	α-L-Arap	β-D-Glcp	34
41	α-OH	β-D-Galp	β-D-Glcp	59
42	β-OH	β-D-Galp	β-D-Glcp	60
43	H	H	H	34

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Squarrofuric acid (44)^28,61 from T. squarrosum is believed to be an artifact produced from squarrogenins 1–4 (28–31) during acid hydrolysis of a methanolic extract of the plant, but the mechanism of transformation is not known.

Cycloartanes with a cyclopentane ring on C-17 atom. In this group there are three saponins 45–47 isolated from T. thunbergii (Table 4). All the glycosides are bisdesmosides: their carbohydrate chains are bound with C-3 and C-22 hydroxyl groups. Thalictoside D (45) and thalictoside E (46) have the same genin,⁶² their differences lie in the structure of carbohydrate chain, glycoside 45 is a pentaoside while glycoside 46 is a hexaoside. The genin of thalictoside F (47)⁶³ is a stereoisomer of the genin of 45 and 46 with opposite configuration of C-21 and C-24 atoms, 45 and 46 have R-configured C-21 and C-24-asymmetric centers, but 47 has these centers in an S-configuration. All structural features including the stereochemistry of the asymmetric centers have been resolved by extensive spectroscopic analyses using 2D NMR and chemical evidence.

Table 4 The structures of triterpenoid compounds with a cyclopentane ring on C-17 atom

No.	R1	R2	Ref.
45	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	β-D-Glcp-(1→2)-β-D-Glcp	62
46	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	β-D-Glcp-(1→2)-[β-D-Xylp-(1→6)-]-β-D-Glcp	62
47	α-L-Rhap-(1→2)-[α-L-Rhap-(1→6)]-β-D-Glcp	β-D-Glcp-(1→2)-[β-D-Xylp-(1→6)-]-β-D-Glcp	63

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Oleanane type. Pentacyclic saponins (Table 5) of the Thalictrum genus belong to the oleanane type only. It is worth noting that oleanolic acid is the most widely distributed pentacyclic triterpenoid isolated from plants. Free pentacyclic genins have been found in two Thalictrum species, namely oleanolic acid (48) and maslinic acid (49) in T. aquilegifolium⁶⁴ and oleanolic acid in T. minus.⁶⁵ Saponins based on oleanolic acid (Table 6) include a monoside 50,⁶⁵ a bioside 51,⁶⁵ triosides 52 and 53,⁶⁵ tetraosides 54,⁶⁶55,⁴⁹ and pentaosides 56–59^49,65,67,68 and 60⁴⁹; those based on 2α,3β-diacetoxy-30-hydroxyolean-24-en-28-oic acid include a bioside 61⁶⁹; those based on hederagenin include a tetraoside 62,⁵⁷ and a pentaoside 63⁵⁷; while those based on 11α, 12α-epoxyolean-28,13β-olid include a trioside 64.⁷⁰ The saponins' novelty lies in the structures of the carbohydrate chains. Bisdesmosides usually occur among Thalictrum pentacyclic saponins, while tetraosides and pentaosides are widely distributed, having oleanolic acid as the genin.

Table 5 The structures of triterpenoid compounds of oleanane type

No.	R1–R5	Name	Plant	Ref.
48	R1 = H; R2 = OH; R3 = R4 = CH3; R5 = H	Oleanolic acid	T. minus	65
61	R1 = R2 = OAc; R3 = CH3; R4 = CH2OH; R5 = α-L-Rhap-(1→2)-β-D-Glcp	Aquilegifolin	T. aquilegifolium	69
49	R1 = R2 = OH; R3 = R4 = CH3; R5 = H	Maslinic acid	T. aquilegifolium	64
62	R1 = H; R2 = β-D-Glcp-(1→3)-α-L-Rhap-(1→4)-β-D-Xylp; R3 = CH2OH; R4 = CH3; R5 = β-D-Glcp	Thalictoside VI	T. Herba	57
63	R1 = H; R2 = α-L-Rhap-(1→3)-β-D-Glcp-(1→3)-α-L-Rhap-(1→4)-β-D-Xylp; R3 = CH2OH; R4 = CH3; R5 = β-D-Glcp	Thalictoside VIII	T. Herba	57
64	R = α-L-Rhap-(1→2)-β-D-Glcp-(1→4)-α-L-Ara	Thalicoside F	T. minus	70

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Table 6 Glycosides of oleanolic acid (48)

No.	Glycosides of oleanolic acid (48)	Name	Plant	Ref.
54	R1 = H; R2 = α-L-Rhap-(1→2)-β-D-Glcp-(1→4)-α-L-Arap; R3 = R4 = CH3; R5 = β-D-Glcp	Thalicoside B	T. minus	66
57	R1 = H; R2 = α-L-Rhap-(1→2)-β-D-Glcp-(1→4)-α-L-Arap; R3 = R4 = CH3; R5 = β-D-Glcp-(1→6)-β-D-Glcp	Thalicoside D	T. minus	67
58	R1 = H; R2 = β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-α-L-Arap; R3 = R4 = CH3; R5 = β-D-Glcp-(1→6)-β-D-Glcp	Foetoside C	T. foetidum	68
59	R1 = H; R2 = α-L-Rhap-(1→3)-β-D-Glcp-(1→3)-α-L-Rhap-(1→4)-β-D-Xylp; R3 = R4 = CH3; R5 = β-D-Glcp	Thalictoside VII	T. Herba	57
55	R1 = H; R2 = β-D-Glcp-(1→4)-α-L-Rhap-(1→2)-β-D-Xylp; R3 = R4 = CH3; R5 = β-D-Glcp	Squarroside II	T. squarrosum	57
60	R1 = H; R2 = β-D-Glcp-(1→4)-[α-L-Rhap-(1→2)]-β-D-Xylp; R3 = R4 = CH3; R5 = β-D-Glcp-(1→6)-β-D-Glcp	Squarroside III	T. squarrosum	57
56	R1 = H; R2 = β-D-Glcp-(1→3)-[β-D-Glcp-(1→3)-α-L-Rhap-(1→2)]-β-D-Xylp; R3 = R4 = CH3; R5 = β-D-Glcp	Squarroside IV	T. squarrosum	57
50	R1 = R5 = H; R2 = α-L-Arap; R3 = R4 = CH3		T. minus	65
51	R1 = R5 = H; R2 = β-D-Glcp-(1→4)-α-L-Arap; R3 = R4 = CH3		T. minus	65
52	R1 = R5 = H; R2 = α-L-Rhap-(1→2)-β-D-Glcp-(1→4)-α-L-Arap; R3 = R4 = CH3		T. minus	65
53	R1 = H; R2 = β-D-Glcp-(1→4)-α-L-Arap; R3 = R4 = CH3; R5 = β-D-Glcp		T. minus	65

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As mentioned above, the first triterpenoid glycosides were isolated from the plants of the Thalictrum genus, collected in Eastern Siberia. Triterpenoid compounds have not been discovered in the same species growing in other climatic areas (e.g. Middle Asia and Europe). This information points to a dependence of chemical composition of plants on ecological and on geographical growth conditions, and so we have suggested assigning Eastern Siberia's species to a Siberian chemorace.¹² Further investigations by Japanese, Chinese and Korean research groups have shown a great diversity of triterpenoid saponins in plants of the Thalictrum genus collected exclusively in Asia. These observed differences in the chemical composition of the plants of the same Thalictrum species, which grow in different ecologo-georgraphic areas should be reflected in the morphology and anatomy of the plants, but that awaits a thorough study by botanists, morphologists, phylogenists, and ecologists, etc.

Having analyzed many cycloartane structures of the Thalictrum genus we would like to note that the same Thalictrum species produces both cycloartane and oleanane triterpenoids. All of the cycloartane compounds of the Thalictrum genus have the substituents at C-3 β-orientated; and in the A-ring substitution often occurs either at C-29 or C-30. This substitution site is rarely found and is not typical of cycloartanes in other species of plants. The CH₂OH at position 29 is glycosylated in some cases (1–8). No glycosides of CH₂OH at position 30 have been found. To our knowledge, besides the Thalictrum genus, only from Mangifera indica have two cycloartanes having 30-CH₂OH been reported.⁷¹ One more position in the polycyclic fragment which can be oxygenated is C-16; those Thalictrum cycloartanes which have a 16-hydroxyl group, have it in the β-orientation. The majority of Thalictrum cycloartanes with an acyclic side chain contain a hydroxyl group at C-22, and where the C-22 configuration is known, it is found to be the S-configuration.

Cycloartane saponins from the Thalictrum genus occur as mono-, bis- and trisdesmosides; the only representative of trisdesmosides is thalicoside C (3). The highest possible number of carbohydrates (5–6) was found in bisdesmosides. The most typical site of glycosylation is on the C-3 of the genin, and in some glycosides the carbohydrate chain is branched. It is worth noting that in 11 and 12 in addition to C-3, the hydroxyl group of C-16, which is sterically hindered, is also glycosylated.

The carbohydrate part of the tetracyclic glycosides has the following sugars: D-glucose, D-galactose, L-rhamnose, D-quinovose, D-fucose, D-xylose and L-arabinose in pyranose form (except xylofuranoside 19 from T. uchiyamai²⁵). An acetyl sugar was revealed in a single glycoside—14.

Pentacyclic saponins of Thalictrum have carbohydrate chains on C-3 and C-28. It is interesting to note that from 15 oleanane glycosides there are just two whose genins have nontrivial structures: diacyl genin in 61 (T. aquilegifolium)⁶⁹ and a derivative of oleanolic acid (64) which possesses a 11α,12α-epoxy ring and 28,13β-γ-lactone ring (T. minus).⁷⁰ The carbohydrate part of the pentacyclic glycosides has the following sugars: D-glucose, L-rhamnose, D-xylose and L-arabinose in pyranose form only.

3.2 Biological activity of the saponins of the Thalictrum genus

Natural triterpenoid glycosides show a wide spectrum of biological activity, including fungicidal, antibacterial, antiphlogistic, antineoplastic, antiallergic and immunostimulating activity.⁷² Saponins have a significant effect on the metabolism as well as on the cardiovascular system of mammals.^73–75 There is also abundant evidence on the impact of triterpenoid glycosides on the fertility of animals.^76–77

It has been suggested that the basis for such diverse medical–biological actions of triterpenoid glycosides is their ability both to influence biological processes taking place in the cell, and to modify structural–functional features of biological membranes and thus to act as bioregulators.^78,79

During their study of the biological activity of the triterpenoid saponins of plants of the Thalictrum genus, Japanese scientists, who have published extensively on the isolation of triterpenoid glycosides from the genus, indicate that the Thalictrum species have been used in folk medicine in Japan for treating stomach diseases. Among glycosides isolated by them there is a series of cycloartane compounds (with a THF ring on C-17) that demonstrate a high LTT activity (lymphocyto transformation test).⁵ Thalicoside A2 (4) showed inhibitory activity against Candida albicans (78.7%) and against Staphylococcus aureus (45.7%), both at a concentration of 1 mg mL⁻¹.³⁴

The sum of the triterpenoid glycosides from T. foetidum (cyclofoetosides A (9), B (10) and foetoside C (58)) in addition to foetoside C (58) alone were separately studied using mammals having an experimental endogenous hypercholesterinemia. The results of introducing the sum of the foetosides and foetoside C (7 days with a dose of 50 mg kg⁻¹) showed that both preparations influenced both lipidic and cholesteric metabolisms. The sum of compounds 9, 10 and 58 decreased the level of cholesterol in blood serum by 25.5 mg%, while compound 58 reduced the level of cholesterol by 60.0 mg% relative to the control data.^80–81

Some individual glycosides, isolated from T. minus and T. foetidum were investigated for antineoplastic activity on mammals.⁸² The most interesting results were obtained for foetoside C (58), thalicoside A (1) and cyclofoetoside B (10). Oleanane pentaoside 58 in a dose of 30 mg kg⁻¹ inhibited the growth of transplantable tumor strains as follows: sarcoma: 45–91%, breast cancer RMK: 1–85%, Pliss malignant lymphoma (lymphosarcoma) and Walker carcino(sarco)ma: 84–86%. Cycloartane bioside 1 gently inhibited the growth of Pliss malignant lymphoma (lymphosarcoma), sarcoma and RMK-1 (73–77%). Sarcoma 45 was sufficiently sensitive to compound 10 that 87% of the tumor growth was inhibited.

Data obtained on the efficiency of compound 58 in experiments with rats using the resistant strains of transplantable tumor are of particular interest. The growth of sarcoma 45, which is resistant to sarcolysine applied in cancer clinics, slowed down under the effect of pentaoside 58 by 90%. The other two substances demonstrated a moderate (1) or weak (10) activity.⁸²

Mats and co-authors⁷⁶ studied in detail the contraceptive activity of triterpenoid glycosides T. minus, T. foetidum and T. squarrosum. Their results indicated that at the subcutaneous post coitum, introduction of the triterpenoid glycosides of T. minus demonstrated the greatest activity, while the glycosides of T. foetidum and T. squarrosum showed a smaller contraceptive effect.

Weakly toxic (LD₅₀ 1800 mg kg⁻¹) cycloartane glycoside 1 of T. minus has shown a high (80–100%) post coitum activity in small doses (0.001–0.1 mg kg⁻¹). Analysis of the mechanism of contraceptive action of thalicoside A indicates that the effect is due to the influence of this compound on the formation of endometrium and on the rate of ovule transport.⁷⁶

Korkhov and co-authors⁷⁷ have investigated the influence of compound 1 on ovulation and on the level of gonadotropins in blood serum of laboratory animals. Their results indicate that introducing glycoside 1 orally for 5 days at a dose of 1 mg kg⁻¹ to the rabbits stimulates ovulation. The same scientists revealed that compound 1 changes the level of gonadotropin yield: at 5 days enteral introduction in a dose of 1 mg kg⁻¹ to rats it reduced the content of luteinizing [interstitial cell-stimulating] hormone and increased the content of follicle-stimulating hormone during active stages of the proestrus–estrus cycle. The detected effect of disrhythmia in incretion of gonadotropins caused by thalicoside A, most likely can be used medicinally for different forms of polycystic ovary accompanied with unbalance of hormones.

These studies of the biological activity of triterpenoid saponins from the Thalictrum genus indicate possible directions of their potential usage, viz. as non-hormonal substances having significant antitumoral activity that have an influence on the reproductive system of mammals, even in small doses.

3.3 Flavonoids and their glycosides

Flavonoids are widely distributed in species of Thalictrum, being mostly present as glycosides of flavones and flavonols (Table 7).

Table 7 The structures of flavonoids and their glycosides

No.	R1, R2, carbohydrate chain (if present)	Plant	Ref.
a The site of attachment of the second glucose is unclear.
65	7-O-β-D-Gal, R1 = R2 = H (talictin)	T. thunbergii	83
66	7-O-diGlc^a, R1 = R2 = H	T. rugosum	84
67	7, 4′-bis-O-β-D-Allop, R1 = R2 = H	T. thunbergii;	85
		T. minus;	86
		T. squarrosum	86
68	7-O-(6″-mono-O-acetyl-β-D-Allop), 4′-O-β-D-Allop, R1 = R2 = H	T. thunbergii;	85
		T. minus;	86
		T. squarrosum	86
69	7-O-(4″,6″-di-O-acetyl-β-D-Allop), 4′-O-β-D-Allop, R1 = R2 = H	T. thunbergii	85
70	7-O-β-D-Glcp, R1 = OH, R2 = H (cinaroside)	T. rugosum	84
71	7-O-(6″-O-α-L-Rhap)-β-D-Glcp, R1 = R2 = H, 4′-OCH3 (linarin)	T. aquilegifolium;	88
		T. simplex;	98
		T. przewalskii;	91
		T. smithii Boivin	99
72	7-O-[6″-O-(4‴-O-acetyl-α-L-Rhap)]-β-D-Glcp, R1 = R2 = H, 4′-OCH3	T. aquilegifolium;	87
		T. przewalskii	91
73	7-O-[6‴-O-(4⁗-O-acetyl-α-L-Rhap)-3″-O-β-D-Glcp]-6″-O-acetyl-β-D-Glcp, R1 = R2 = H, 4′-OCH3	T. przewalskii	91
85	R1 = H, R2 = OH (kaempferol)	T. atriplex	95
86	R1 = H, R2 = OCH3 (isokaempferide)	T. minus (roots)	87
79	R1 = H, R2 = O-(6″-O-α-L-Rhap)-β-D-Glcp	T. rugosum	90
78	R1 = H, R2 = O-β-D-Glcp (astragalin)	T. przewalskii	91
80	R1 = H, R2 = O-[6″-O-(3‴-O-acetyl-α-L-Ara)-β-D-Glcp]	T. atriplex	92
81	R1 = OH, R2 = O-β-D-Glcp	T. atriplex	93
82	R1 = OH, R2 = O-(6″-O-α-L-Rhap)-β-D-Glcp	T. foetidum;	94
		T. minus;	90
		T. rugosum	90
83	R1 = OH, R2 = O-β-D-Glcp, 7-O-CH3	T. foetidum	94
74	6-C-β-D-Glcp, R1 = R2 = H (saponaritin)	T. dasycarpum; T. minus; T. minus race B; T. rugosum	90
75	6-C-β-D-Glcp, R1 = OH, R2 = H (isoorientin)	T. dasycarpum; T. minus; T. minus race B; T. rugosum	90
76	8-C-β-D-Glcp, R1 = R2 = H (vitexin)	T. dasycarpum	90
77	8-C-β-D-Glcp, R1 = OH, R2 = H (orientin)	T. dasycarpum; T. minus; T. minus race B; T. rugosum	90
84	6-C-β-D-Glcp, 5-O-α-L-Rhap, R1 = R2 = H (isovitexin 5-rhamnoside)	T. rugosum	84

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Flavone O-glycosides are mostly those of apigenin, luteolin and acacetin 7-O-glycosides (65–73). There are two monoglycosides of apigenin (65, 66)^83,84 and three apigenin diglycosides, being bisdesmosides (67–69),^85–87 where the second carbohydrate chain is bound to the 4′-phenolic hydroxyl group. In glycosides 68 and 69, allopyranose, which is bound to the 7-OH group, is acetylated. Acetyl-sugars are characteristic of acacetin glycosides (72, 73), one of them being a disaccharide (72)⁸⁸ and another—a trisaccharide (73).⁸⁹ It is interesting to note that acetyl-derivatives of flavonoid glycosides have only been isolated from Thalictrum species growing in Asia. The only reported glycoside of luteolin is 7-O-glucoside (70)⁸⁴ from T. rugosum.

Flavone C-glycosides are widespread in nature and those present in Thalictrum are 6-C-glucosides of apigenin (saponaretin) (74),⁹⁰ luteolin (isoorientin) (75),⁹⁰ 8-C-glucosides of apigenin (vitexin) (76),⁹⁰ and luteolin (orientin) (77).⁹⁰ Apigenin 6-C-glucoside, 5-O-rhamnoside (84)⁸⁴ has been isolated from T. rugosum. Flavone C-glycosides have only been isolated from Thalictrum species growing in Europe.

Flavonol glycosides found in Thalictrum are exclusively those of the 3-O-glycosides. There are three kaempferol glycosides known, one of them: monoglycoside (78),⁹¹ the others are diglycosides (79, 80).^89,9280 contains acetyl-arabinose and has been isolated from T. atriplex collected in China. The quercetin glycosides include glucoside (81)⁹³ and rutinoside (82).^90,94 The only glycoside of rhamnetin found in T. foetidum is a glucoside (83).⁹⁴ It should be noted that of the 22 flavonoids represented in Table 7, nine are new glycosides; these include 65,⁸³67–69 from T. thunbergii,⁸⁵T. minus, T. squarrosum,^86,8772⁸⁸ from T. aquilegifolium, 73⁸⁹ from T. przewalskii, 80⁹² from T. atriplex, 82⁹⁴ from T. foetidum, and 84⁸⁴ from T. rugosum.

Kaempferol (85)⁹⁵ itself was found in T. atriplex, its methyl ether (86)⁸⁶ was reported to be found in the roots of T. minus. Kaempferol and quercetin, together with two unidentified flavonoids, have been identified by analytical paper chromatography in the leaves and flowers of T. delavay Franch, T. flavum L., T. foetidum, and T. medium Poir.⁹⁶ By the same method, hydrolyzates of extracts of T. angustifolium Jacq., T. coronellum D.C., T. fendleri Engelm, T. minus var. adiantifolium, T. minus L., T. petaloideum, T. purpurascens D.C., T. sparsiflorum Rurch, T. squarrosum Steph., and T. thuberosum L. have been screened, and apigenin, luteolin, kaempferol and quercetin have been identified in the plants.^84,97 Quercetin has been found in T. aquilegifolium L., and kaempferol in T. calabricum Spreng, while both of them have been found in T. rugosum.⁸⁴ These articles represent the first reports devoted to the investigation of Thalictrum flavonoids.

3.4 Cyanogenic glycosides

In the Thalictrum genus, cyanogenesis has been discovered by van Itallie et al.,¹⁰⁰ who reported that the leaves of T. aquilegifolium liberated HCN (about 0.05% of fresh weight) on distillation after being ground and allowed to stand at 30 °C for a period. In addition to HCN, acetone was also detected in the distillate, so acetone cyanhydrin was postulated to have been the cyanogenic principle. Later Sharples et al.^101–103 established the structures of the cyanogenic glycosides from T. aquilegifolium. The major cyanogenic constituent was found to be the monomethyl ether of isotriglochinin (87), which accounted for 90% of the cyanogenic content of the plant. The structures of the minor glycosides were shown to be p-glucosyloxymandelonitrile (88) and p-glucosyloxymandelonitrile-β-D-glucoside (89), each of them accounting for 5% of the cyanogenic content.

Tryglochinin (90) was discovered in T. polygamum and T. aquilegifolium,^104,105 and the authors of the first mentioned work made the assumption that tryglochinin is an indigenous glycoside of T. aquilegifolium and that its esterification (and probably isomerisation) to monomethyl ether of isotriglochinin took place during extraction and purification performed by Sharples etc.

A cyclic unsaturated cyanogenic glycoside 91 (lithospermoside) has been isolated from T. rugosum Ait., T. revolutum¹⁰⁶ and T. orientale Boiss.,¹⁰⁷ and its 4-epimer 92 (dasycarponin) has been found in T. dasycarpum Fisch. and Lall.¹⁰⁶ The absolute stereochemistry of the asymmetric centers of the molecules was proved convincingly using circular dichroism, and NMR spectral data.

In addition to the above mentioned Thalictrum species, several other species have also been found to be cyanophoric: T. foetidum L. (weakly cyanophoric), T. flexuosum Berng. and T. minus L. (very weakly cyanophoric), T. dipterocarpum Franch.¹⁰⁴ (releases trace amounts of HCN), and T. polycarpum,¹⁰⁸ however the composition of their cyanogenic components has not been established.

The biosynthesis and subsequent accumulation of cyanogenic glycosides probably represents a defence for the plant from attack by herbivores. Within the Thalictrum genus species; the ability to synthesize cyanogens is expressed differently and appears to correlate with ecologo-geographic conditions of growth and numbers of herbivores.¹⁰⁹

3.5 Hydrocarbons and high molecular weight alcohols

Hydrocarbons and high molecular weight alcohols are common metabolites in higher plants. In the Thalictrum genus these constituents have only been discovered in two species: T. aquilegifolium¹¹⁰ and T. simplex.⁹⁸ The identification and relative quantities of the hydrocarbons were determined by gas liquid chromatography-mass spectroscopy (GC-MS), (the alcohols were preliminary acetylated). In the dried whole plant (including the roots) of T. aquilegifolium 13 n-alkanes and 9 alcohols were found. These included normal saturated hydrocarbons C₂₃–C₃₅, in which n-C₂₉H₆₀ and n-C₃₁H₆₄ were the major components. All identified alcohols had normal saturated structures (C₂₈–C₃₆) and the majority consisted of n-C₃₂H₆₅OAc along with n-C₃₄H₆₉OAc.

From T. simplex all the above-mentioned compounds have been characterized including two other hydrocarbons (C₂₁ and C₂₂). It was reported that the C₂₃–C₂₈ fraction was dominant in this instance.

3.6 Phenolic compounds

Some Thalictrum species represent a source of unusual nitrogen-containing compounds, namely, T. aquilegifolium^110–111 and T. orientale.¹⁰⁷ Besides the cyanogenic glycosides described above, other types of phenolic glycosides containing a nitro group have been isolated from these species. The first representative was thalictoside (93).^110–111 Its structure was established by NMR spectroscopy and synthetic methods. T. aquilegifolium is poisonous for domestic animals. Research into the poisonous compounds of this plant enabled the authors to isolate thalictoside, but a toxicity test, performed by injection of 93 into the abdominal cavities of mice did not show the expected level of toxicity.

A compound (94) isolated from the underground part of T. orientale contained a disaccharide fragment that was bound with the same phenylethyl moiety as (93).¹⁰⁷ The complete assignment of the structure was based on 2D NMR and HR-MALDI mass spectral data.

Three phenolic acids have been isolated from the aerial part of T. atriplex.⁹⁵ These are protocatechuic acid (95), caffeic (96) and p-coumaric acids (97).

From the roots of T. dioicum, used medicinally in Mexico as a diuretic, m-meconina (5,6-dimetoxiftalid) (98) has been isolated.¹¹²

3.7 Fatty acids

The fatty acids of the Thalictrum genus have been studied rather extensively: in 17 plant species studied (including various populations of the same species) 21 saturated and unsaturated acids have been identified. The Thalictrum genus is the only known natural source of a unique class of fatty acids having an isolated trans-double bound at position-5.

The first report of fatty acids from Thalictrum (T. polycarpum) was by Bagby, et al.¹¹³ in which 5-trans-octadecenoic acid (110), and 5-trans,9-cis,12-cis-octadecetrienoic acids (118) were identified, and two other C₁₈ acids which were not characterized (C₁₈-dienoic acid and an unknown C₁₈-acid). Bhatty et al.¹¹⁴ have established that the seed oil of T. venulosum contains acids 110, 118, and the previously unknown 5-trans-hexadecenoic (108) and 5-trans,9-cis-octadecadienoic (116) acids. Traces of 5-trans-pentadecenoic acid (107) and 5-trans-heptadecenoic acid (109) were also detected. Moreover, Bhatty et al. found fatty acids having only a cis-double bond (111–115, 117, 119) as well as other saturated acids (99–106). Thus, T.venulosum oil has been studied with more detail than any other species of Thalictrum.

The composition of seed oils from other studied species of Thalictrum closely resembles that reported from T. polycarpum and T.venulosum.

The presence of four unsaturated 5-trans series fatty acids—108, 110, 116, and 118—has been reported by Rankoff et al. in different species of the Thalictrum genus (Bulgarian populations: T. aquilegifolium, T. adiantifolium, T. glaucum, T. minus Balchik, T. minus Losen, T. minus Sofia),¹¹⁵ by Wu (T. revolutum D.C.),¹¹⁶ by Ucciani (T. flavum and T. morisinii),¹¹⁷ and by Freiman (Uzbekistan population: T. minus, T. flavum, T. foetidum, T. simplex and T. sultanobadens).^118–119 In leaves of T. simplex (Eastern Siberia population) Trofimova et al.⁹⁸ have isolated saturated fatty acids (99–104), in which acids C₁₆ and C₁₈ were dominant. Thus, from 21 fatty acids detected in the Thalictrum genus, 8 acids (99–106) are saturated (5–10%), 9 acids (107–115) are monoenes (12–49%), 2 acids (116, 117) are dienes (22–27%) and 2 acids (118, 119) are trienes (30–56%). The fatty acid composition of seed oils of all investigated species were shown to consist of about 50–60% fatty acids having double bonds in a trans-position. The major component of the oil is 5-trans,9-cis,12-cis-octadecatrienoic acid. The number, the position and the configuration of double bonds in the unsaturated acids was correctly established using chemical (studying the products of oxidation and reduction) and spectroscopic methods (IR-, UV-, MS- and GLC of methyl esters). All acids were identified as the methyl esters by gas chromatography.

3.8 Phytosterols and their glycosides

β-Sitosterol (120) has been isolated from the roots of T. dioicum,¹¹² and the aerial parts of T. aquilegifolium,¹¹⁰T. atriplex⁹⁵ and T. foeniculaceum.⁴⁵ β-Sitosterol 3-O-β-D-glucopyranoside (121) was also obtained from the aerial part of T. aquilegifolium,¹¹⁰T. minus L., T. squarrosum St. ex Willd, and T. foetidum.¹²⁰ From T. simplex a fraction of phytosterol glucosides was isolated.⁹⁸ After acid hydrolysis of this fraction β-sitosterol [M⁺ 414], campesterol [M⁺ 400] (122), and stigmasterol [M⁺ 412] (123) were identified by GS-MS spectral analysis. Glucose was the only carbohydrate in the aqueous part of the hydrolysate.

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