Advances in the phytochemistry and pharmacology of plant-derived phthalides

Abstract

Phthalides are a class of unique compounds such as ligustilide, butylphthalide and butyldenephthalide, which have shown to possess multiple bioactivities in new drug research and development. Phthalides are naturally distributed in different plants that have been utilized as herbal treatments for various ailments with a long history in Asia, Europe and North America. Their extensive biological activity has led to a dramatic increase in the study of phthalide compounds in recent years. This review summarizes the latest research progress of plant-derived phthalides, and a total of 133 phthalide compounds are described based on the characteristics of chemical structures. Pharmacological properties of plant-derived phthalides are associated with hemorheological improvement, vascular function modulation and central nervous system protection. Potential treatments for a variety of diseases mainly including cardio-cerebrovascular disorders and neurological complications such as Alzheimer's disease are also concluded. In addition, key metabolic pathways have been clearly elucidated. Further investigations on the molecular mechanisms involved in biological activity are recommended for offering new insights into profound comprehension of phthalide applications.

1. Introduction

Phthalides are one class of abundant and bioactive constituents primarily existed in plant essential oils and are used as pivotal markers for quality assessment of botanical drugs [1]. In 1963, Mitsuhashi and colleagues firstly isolated six monomeric phthalides such as ligustilide, cnidilide and neocnidilide from Umbelliferae plants [2]. Then in 1984, three hydroxy phthalides such as 3-butylidene-7-hydroxyphthalide and one dimeric phthalide wallichilide were found in the rhizome of Ligusticum wallichii [3]. In the past two decades, a series of new natural phthalides were identified, such as 3-carboxyethyl-phthalide [4], ligusticoside A [5] and chuanxiongnolide A and B [6]. Z-ligustilide was reported to represent 20.2 % of essential oil from Ligusticum porter [7,8]. Recently, trimeric phthalides like triangeliphthalides A-D and triligustilides A-B were identified from Angelica sinensis [9,10] and neophathalides A and B with new skeleton were isolated from Ligusticum chuanxiong [11]. Many natural phthalides have shown to possess a wide range of biological activities such as cardiovascular and cerebrovascular function modulation, actions on central nervous system, organ protection, anti-cancer, anti-migraine and anti-inflammation [[12], [13], [14], [15], [16], [17]]. Considerable experimental studies together with advanced analysis techniques have facilitated the research progress on plant-derived phthalides in the past few years.

At present, the majority of reported naturally occurring phthalides has been found in different plants. The leading plant source belongs to Umbelliferae family including Angelica sinensis, Ligusticum chuanxiong, Apium graveolens, and other species. More than 95 phthalides have been separated from Ligusticum chuanxiong, ranked as the most abundant species in the plant [18]. Therapeutic features of Ligusticum chuanxiong for migraine might be attributed to bioactive ingredients such as phthalides and aromatic acids [19]. Angelica sinensis as popular food flavoring and dietary supplement worldwide is also a major plant source of phthalides. Ligustilide could remarkably attenuate oxidative injuries and apoptosis in fish erythrocytes, which represents the protective role of Angelica sinensis extract against trichlorfon stress [20]. Celery (Apium graveolens) was reported to have high content of butylphthalide at 13.1 % in essential oil [21]. In Compositae family, two new glucosidic phthalide were found in Helichrysum mcrophyllum ssp. tyrrhenicum [22], and gnaphalide A-C were isolated from Gnaphalium adnatum [23]. Two new phthalide derivates matteucen C and D separated from the rhizomes of Matteuccia orientalis in Onocleaceae family [24]. Lee et al. discovered Setigerumine I and α-noscapine in Papaver setigerum from Papaveraceae family [25]. Besides, many phthalide derivatives have also been identified in fungi and liverworts. For instance, mycophenolic acid was identified in the endophytic fungus Phomopsis longicolla [26], and 5-hydroxy-7-methoxy-4-methylphthalide was isolated from Penicillium crustosum [27].

In 2017, Leon's work particularly summarized natural distributions, derivative activity, synthetic process and bioactivity in terms of phthalides [28]. The rapid progress of analytical technologies have also promoted the discovery of novel phthalides such as trimeric phthalides (i.e. triangeliphthalides A-D) [10]. Phthalides are recently reported with pharmacological properties for cardio-cerebrovascular and neurological complications [17]. Therefore, this study aims to provide the most up-to-date advances in the research field of plant-derived phthalides. Literatures were searched and screened in the SCIFinder database by using “Phthalide” and “1(3H)-Isobenzofuranone” as keywords, which met the inclusion criteria: 1) published from 1980 to 2023, 2) related to plant sources, 3) written in English, 4) provided structure determination or experimental studies or clinical trials. A total of 469 studies were finally screened to meet the above criteria. A total of 133 plant-derived phthalides are listed, and their biological activities, potential mechanisms of action and different metabolic pathways are summarized for further discussion.

2. Phytochemistry of phthalides

2.1. Structure of phthalides

Generally, the core structure of phthalides is 1(3H)-isobenzofuranone that contains a benzene ring (ring A) combined with a γ-lactone (ring B) between carbon atom 1 and 3 (Fig. 1) [1]. The structure of phthalide derivatives commonly has different substitutions at carbon atom 3 in ring B, and various groups at carbon atom 3a-7a in ring A. A total of 133 phthalide compounds isolated from plants have been identified after screening process and information collection. These reported phthalides can be classified into three main types: monomeric phthalides (1–27), hydroxy phthalides (28–81) and polymeric phthalides (82–133).

Fig. 1.

Core skeleton of phthalides: 1(3H)-isobenzofuranone.

2.2. Classification of phthalides

2.2.1. Monomeric phthalides

The majority of natural phthalides belongs to monomeric phthalides that account for a large proportion of ingredients in plants. Their representative skeleton is 1(3H)-isobenzofuranone. These compounds could be distinguished by various methyl or ethyl substituents at C-10 and C-11, as well as carbon-carbon double bond locations in ring A and a side chain at C-3, while few also occur in ring B. The structures of monomeric phthalides were illustrated in Fig. 2.

Fig. 2.

Chemical structures of monomeric phthalides.

Five phthalides sharing two C–C double bonds between 6,7 and 3a,7a in ring A and a butyl side chain, included Z-ligustilide (1), E-ligustilide (2), senkyunolide A (3), 4,5-dihydro-3-butylphthalide (4) and validene-4,5-dihydrophthalide (5). Compound 1 [29] possessed Z-form of C–C double bond between C-3 and C-8 in butyl side chain, while compound 2 [30] had E-form, which was rare and labile in such a skeleton. Senkyunolide A (3) [31] formed a methyl group at C-11 of a butyl side chain, and so was its stereoisomer, which named 4,5-dihydro-3-butylphthalide (4) [32]. As for validene-4,5-dihydrophthalide (5) [33], there was an ethyl group substituted at C-11. Likewise, there were two other phthalides with three C–C double bonds between 5,6 and 7,7a in ring A and 3,8 in a side chain, namely, Z-iso-butylidene-3a,4-dihydrophthalide (6) [34] with an iso butylidene group, and Z-validene-3a,4-dihydrophthalide (7) [35] with a pentenyl substitution. Compound 5 and compound 7 were stereoisomers of each other. In addition, compounds 8–12 not only presented a benzene ring, but also formed different substitutions at C-10. Among them, only butylphthalide (8) [21] contained a butyl group at C-3 of ring B. Z-butylidenephthalides (9) [36,37] and E-butylidenephthalides (10) [30] were a pair of stereoisomers, both with the highest degree of unsaturation in terms of monomeric phthalides. Compared with propylidene-phthalide (11) [38] formed a methyl moiety at C-10, 3-(3-Methylbutylidene)phthalide (12) [39] was uniquely substituted isopropyl group at C-10. Moreover, Sedanolide (13) [40], 3-butylhexahydro-phthalide (14) [41] and Hexahydro-3-butylphthalide (15) [42] were distinguished by various positions of the C–C double bonds between 3,3a in ring B and 7,7a in ring A. Sedanonic acid lactone (16) [43] was a stereoisomer of compound 14. There was one other compound showed structural similarities to compound 1 but a unique side chain with another carbon bond occurred at C-3 and C-8, namely neoligustilide (17) [44]. Earlier, one other stereoisomer of compound 13 was confirmed to be cnidilide (18) [45]. 10-angeloylbutylphthalide was found in Z-form (19) and E-form (20) [46]. Likewise, 11-angeloylsenkyunolide F (21) was another configuration of angeloyl ester of senkyunolide F (22) [47]. As for phthalides combined with glycoside, celephthalide A (23) [48] shared the same structure of butylphthalide (8) but a glycosidic bond substituted at C-10. Compared with compound 23, 7-hydroxy-3-butylidenephthalide 7-O-glucoside (24) [49] possessed C–C double bond at C-3,8 and a glycosidic bond occurred at C-7. Z-6,7-epoxyligustilide (25) [50] was reported to form a four-membered moiety in ring B. 7-methoxy-3-propylidenephthalide (26) [51] had a methoxy group at C-7 in ring A, while 5,7-dimetoxy-6-(3-methylbut-2-enyl)-isobenzofuran-1(3H)-one (27) [52] had two methoxy groups at C-5 and C-7 in ring A. Monomeric phthalides was defined as a series of 1(3H)-isobenzofuranone-based compounds with unsaturation in ring A and various substitutions at a butyl side chain of C-3 in ring B. However, the limited numbers of monomeric phthalides in plants might be attributed to the lack of related analytical studies.

2.2.2. Hydroxy phthalides

Hydroxy phthalides are one of the most important categories of natural chemicals in plants. The representative structure of these compounds was still 1(3H)-isobenzofuranone, but possible multiple hydroxyl groups would occur at different positions of the core skeleton. All the chemical structures of hydroxy phthalides were shown in Fig. 3.

Fig. 3.

Chemical structures of hydroxy phthalides.

Compounds 28–35 are hydroxy derivatives of Z-butylidenephthalides (9). Among them, a hydroxyl group was located at C-4 of senkyunolide B (28) [50], C-5 of senkyunolide C (29) [53,54], C-9 of senkyunolide E (30) [47], C-7 of 3-butylidene-7-hydroxyphthalide (31) [55], respectively. Only 3-butylidene-4,5-dihydroxyphthalide (32) [56] formed two hydroxyl substitutions at C-4 and C-5 in benzene ring. Three other compounds 33–35 without C–C double bond between C-3 and C-8 shared the hydroxyl moiety at C-3, 4 and 7, named as 4-hydroxy-3-butylphthalide (33) [57,58], 3-hydroxy-3-butylphthalide (34) [58] and 4,7-dihydroxy-3-butylphthalide (35) [58]. Differently, compounds 36–39 shared basic skeleton of Z-ligustilide (1) with various hydroxyl positions at C-3, 4 and 9. Only senkyunolide F (36) [59] with a hydroxyl group at C-9 formed a C–C double bond at C-3,8. Two hydroxyl groups occurred at C-3 and C-9 of 3,9-dihydroxy-ligustilide (37) [60], while senkyunolide G (38) [50] and senkyunolide K (39) [61] as a pairs of stereoisomers formed one hydroxyl moiety at C-3 and C-4, respectively. Structurally, two compounds shared an uncommon configuration with two C–C double bonds at C-3a,4 and C-7,7a, and monohydroxyl group in C-6, which named 3-butylidene-6-hydroxy-5,6-dihydrophthalide (40) and 3-butylidene-6-hydroxy-5,6-dihydro-butylphthalide (41) [62]. But only the latter has another C–C double bond at C-3,8. Similar to sedanonic acid lactone (16), another cluster of compounds 42–51 with different configuration of hydroxyl or carbonyl groups were named as followed: senkyunolide H (42) [59], senkyunolide I (43) [59], senkyunolide J (44) [63], senkyunolide N (45) [63], 3,6,7-trihydroxy-4,5,6,7-tetrahydro-3-butylphthalide (46), senkyunolide R (47) [64], senkyunolide S (48) [64], senkyunolide L (49) [62], senkyunolide M (50) [62] and senkyunolide Q (51) [62]. It needs mentioning that compounds 42–43, 44–45, 47–48 and 50–51 are four pairs of stereoisomers, respectively. Uniquely, a chloro-group was formed at C-7 of compound 49. Recently, series of compounds 52–57 with one C–C double bond between C-7 and C-7a in ring A, were distinguished by different configurations and substitutions at C-4, C-6 and C-9, which confirmed to be (3S, 3aR, 6S)-(−)-6-hydroxysedanolide (52), (3S, 3aR, 4R)-(−)-4-hydroxysedanolide (53), (3S, 3aR, 10R)-(−)-10-hydroxysedanolide (54), (3S, 3aR)-3-hydroxyethyl-3a, 4, 5, 6-tetrahydrophthalide (55), (3S, 3aR, 6S)-(−)-6-hydroxysedanolide-10-one (56), (3S, 3aR)-3-(2-carboxyl) ethyl-3a, 4, 5, 6-tetrahydrophthalide (57) [65]. One is (3R, 6R)-3-butyl-6-hydroxy-4, 5, 6, 7-tetrahydrophthalide (58) [65] without a C–C double bond at C-3,8, and the other 6-hydroxy-7-methoxydihydro-ligustilide (59) [50] with an oxygen bridge at C-7 position. Based on various configurations and substitutions at C-4, 5, 6, 7 and 8, compounds 60–67 with a benzene ring and an alkyl side chain at C-3 could be classified as followed: (S)-3-ethyl-7-hydroxy-6-methoxyphthalide (60) [66], (Z)-3-ethylidene-7-hydroxy-6-methoxyphthalide (61) [66], (Z)-3-ethylidene-6,7-dimethoxyphthalide (62) [66], (S)-3-ethyl-5,6,7-trimethoxyphthalide (63) [66], (S)-3-ethyl-7-hydroxy-5,6-dimethoxyphthalide (64) [66], 6-formyl-7-hydroxy-5-methoxy-4-methylphthalide (65) [66], 1,3-Dihydro-3-oxo-1-isobenzofuranacetic acid (66) [62] and 4,6-dimethoxy-phthalide (67) [67]. Among them, only compounds 61–62 formed a C–C double bond between C-3 and C-8. Specifically, compound 63 had three oxygen bridges at C-5, 6 and 7, but compound 65 had one carbonyl group at C-6, as well as compound 66 had one carboxyl group at C-9 of a side chain. It should be mentioned that arenophthalide B (68) and arenophthalide C (69) were a pair of stereoisomers with xylose [68]. However, compounds 70–71 were listed individually due to their unique structures. Compared with compound 35, Senkyunolide D (70) [50] formed a carbonyl group at C-8 position. Only 3,7-dihydroxyligustilide (71) [59] owned p-hydroxy substitution at C-3a,7a. Recently, neophathalide A (72) had a special structure differing from other phthalides, whereas an oxygen bridge was located at C-6 and C-7 of neophathalide B (73) [11]. 3-butylidene-6α-hydroxy-7α-ethoxy-phthalide (76) [69] had a ethoxy group at C-7 and a hydroxy group at C-6 in ring A. 7-hydroxy-3-butylidenephthalide 7-O-(6′-malonylglucoside) (74) [49], celephthalide B (75) [48], ligusticoside A (77) [5], celephthalide C (78) [48], ligusticosides C (79) [70], ligusticosides D (81) [70] and ligusticosides E (80) [70] all represented as O-glycosides. Hydroxy phthalides are characterized by different configurations and substitutions like hydroxyl and carbonyl groups. However, the number and stereostructure of these compounds need further investigations, and the analytic determination of glycosidic phthalides is insufficient, which also requires more studies to address.

2.2.3. Polymeric phthalides

All identified phthalide polymers with various fusion of two or three phthalide units, are also a class of important bioactive constitutes in plants. Their chemical structures were shown in Fig. 4.

Fig. 4.

Chemical structures of polymeric phthalides.

Series of compounds 82–86 with two phthalide moieties conjugated at C-6.6′,7.3a′, were distinguished by different configuration and C–C double bonds, which named senkyunolide O (82) [71], senkyunolide P (83) [72], levistolide A (84) [72], 3,8-dihydro-diligustilide (85) [73] and chuanxiongdiolide A (86) [74]. Chuanxiongnolide A (87) and chuanxiongnolide B (88) were a pair of stereoisomer [6]. Recently, compounds 89–93 featured a linkage of C-3a.3′,6.8′ position, named as tokinolide B (89) [72], Z-ligustilide dimer E−232 (90) [72], angeolide (91) [75], gaobennolide A (92) [75] and gaobennolide B (93) [75]. Similarly, another cluster of compounds 94–92 with combination of a butylidenephthalides (9) and a simple phthalide with one C–C double bond at C-3,8, named as ansaspirolide (94) [46], angesinenolide F (95) [75,76], (3′Z)-(3S,8S,3a′S,6′R)-4,5-dehydro-3.3a′,8.6′-diligustilide (96) [77], (3′Z)-(3S,8R,3a′S,6′R)-4,5-dehydro-3.3a′,8.6′-diligustilide (97) [77] and 4,5-Dehydro-diligustilide (98) [73]. Chuanxiongdiolide R1 (99) was a stereoisomer of chuanxiongdiolide R3 (100) fused at C-3a.3′,6.8’ [77]. There were three compounds 101–103 linked at C-3a.3′,6.8′, namely Z,Z′-3.3′a, 7.7′a-diligustilide (101) [78], Z,Z′-3a.7a′,7a.3a′-dihydroxyligustilide (102) [79] and chuanxiongdiolide R6 (103) [80]. Differently, four other compounds 104–107 shared a conjugation of C-6.3a′,7.7a′, which named as follows: angesinenolides C (104) [76], angesinenolides D (105) [76], tokinolide A (106) [81] and tokinolide C (107) [81]. A pair of stereoisomer named as tokiaerialide (108) [80] and neodiligustilide (109) [82] formed a linkage of C-3.3a′,8.7a′ in their structure. Riligustilide (110) [72] was similar to gelispirolide (111) [10] except for one C–C double bond in six-membered ring. Moreover, compounds 112–116 were distinguished by various connections, named as 3,3′Z-6.7′,7.6′-diligustilide (112) [83], 3′,8′,3′a,7′a-tetrahydro-6.3′,7.7′a-diligustilide-8′-one (113) [71], sinaspirolide (114) [46], chuanxiongdiolide B (115) [74] and wallichilide (116) [3]. Uniquely, compound 115 formed only one C–C single bond at C-7.7a′ position. Structurally, three other compounds named as E,E′-3,3':8,8′-diligustilide (117) [72], angesinenolides E (118) [76] and diangeliphthalide A (119) [10] shared the same conjugation at C-3.3′,8.8′, but different numbers of C–C double bonds. However, one is chuanxiongdiolide R4 (120) with a connection at C-3a.3a′,7a.7a′, and methyl ester of angeolide (122) formed a joint of C-3.3′,6.8’ [80] and a joint of C-7.3a′,7a.7a′ occurred in chuanxiongdiolide R5 (121) [80]. (+)-6-3′a,7–6′-Isowallichilide (123) [84] and (−)-6-3′a,7–6′-isowallichilide (124) [84] were a pair of enantiomeric phthalide dimers with new 6–3′a,7–6′ dimerization sites. 1″,3′-seco-1′-ethoxy-3,3a′, 8,6′-diligustilide (125) [69] was a dimeric phthalide with 3,3a′, 8,6′ dimerization sites. Spiroligustolides A (126) [85] and spiroligustolides B (127) [85] both belonged to enantiomeric phthalide dimers. In addition, compounds 128–133 were composed of three phthalide units. Triangeliphthalides A (128) and triangeliphthalides B (129) are a pair of stereoisomers with the same linkage of C-3.7′,8.6′ and C-3’.7a’‘,8’.3a’’ but various configuration at C-8,9; however, triangeliphthalides C (132) and triangeliphthalides D (133) originated from cycloaddition with one new connection at C-3.7′,8.6′ and C-3’.3″,8’’.8’’ [10]. Compared with triligustilides B (131), triligustilides A (130) formed a C–C double bond at C-3a,7a and different configuration at C-6,7 position [9]. Due to the complex structure of phthalide polymers, more analytic studies are needed in this field. Based on the presence of trimeric phthalides in plants, one possible form of tetramers may require further investigations.

2.3. Extraction and analysis of phthalides

Several methods such as organic solvents extraction, water distillation and supercritical fluid extraction have been utilized to isolate natural phthalides for further analysis. Although organic solvents are expensive, it is still the most used method for the separation technique. On the other hand, steam distillation is gradually abandoned due to crude extracts. Modern extraction technologies usually comprised supercritical fluid extraction and pressurized liquid extraction in the last 1990s [2]. Z-ligustilide (1) as an abundant phthalide in plants, is often considered to be a key marker for quality control. Tang and colleagues conducted the volatile oil extraction by steam distillation, and then determined the content of Z-ligustilide in Angelica sinensis and Ligusticum chuanxiong as 1.104 and 2.096 %, respectively [30]. High Performance Liquid Chromatography (HPLC) and gas chromatography mass spectrometry (GC-MS) methods are generally used for the analysis of chemical constitutes. Z-ligustilide from water and ethanol extracts of Angelica sinensis and Ligusticum chuanxiong was comparatively quantified using HPLC coupled with pulsed amperometric detection [86]. Nevertheless, due to thermo-optically instability, Z-ligustilide and other monomeric phthalides are likely to be transformed to isomers after heating. This feature may explain findings from Yi’ works, where the proportion and content of ligustilide measured by GC-MS was relatively lower than those measured by HPLC [87]. It is noteworthy that the quantitative evaluation of ligustilide in herbal essential oil was easily interfered with senkyunolide A (3), especially in the application of GC-MS technique [86]. Recently, Zou and colleagues identified six new trimers including triangeliphthalides A-D and triligustilides A-B from dried roots of Angelica sinensis through LC-HR-ESI-MS [9,10]. Chuanxiongdiolide R4-R6 with unique linkages were also reported in aerial parts of Ligusticum chuanxiong [80]. Phthalide-related plant sources, extraction, detection and their contents were summarized in Table 1.

Table 1.

Plant origin, extraction, detection and content of representative phthalides.

No.	Compound	Molecular formula	Plant source	Herbal part	Extraction	Detection	Content (w/w)	Reference
1	Z-ligustilide	C12H14O2	Angelica acutiloba (Siebold & Zucc.) Kitag.	Roots	MeOH	HPLC	0.08–0.22 %	[29]
			Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort.	Roots	Water distillation	GC-MS	1.077 % and 2.048 %	[30]
			Cnidium officinale Makino and Ligusticum chuanxiong Hort.	Roots	Water extraction	HPLC-UVD	0.06–0.36 mg/g and 0.1–0.43 mg/g	[31]
2	E-ligustilide	C12H14O2	Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort.	Roots	Water distillation	GC-MS	0.027 % and 0.048 %	[30]
3	Senkyunolide A	C12H16O2	Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort.	Roots	Water distillation	GC-MS	0.002 % and 0.444 %	[30]
			Cnidium officinale Makino and Ligusticum chuanxiong Hort.	Roots	Water extraction	HPLC-UVD	0.54–2.22 mg/g and 0.59–2.9 mg/g	[31]
4	4,5-dihydro-3-butylphthalide	C12H16O2	Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort.	Roots	MeOH	2D-LC	/	[32]
5	Validene-4,5-dihydrophthalide	C13H16O2	Levisticum officinale W. D. J. Koch	Roots	Water distillation	TLC, GC-MS	1.3–4.7 %	[33]
6	Z-iso-butylidene-3a,4-dihydrophthalide	C12H14O2	Ligusticum canbyi J.M. Coult & Rose	Roots	70 % EtOH	TOF-MS	/	[34]
7	Z-validene-3a,4-dihydrophthalide	C13H16O2	Apium graveolens L.	Seeds	Water distillation	GC, IR	/	[35]
8	Butylphthalide	C12H14O2	Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort.	Roots	Water distillation	GC-MS	0.007 % and 0.096 %	[30]
			Apium graveolens L.	Seeds	Water distillation	LC-DAD-MS	13.10 %	[21]
9	Z-butylidenephthalides	C12H12O2	Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort.	Roots	Water distillation	GC-MS	0.03 % and 0.16 %	[30]
			Angelica sinensis (Oliv.) Diels	Roots	Pressurized liquid extraction	GC-MS	0.08–0.15 %	[59]
			Ligusticum chuanxiong Hort.	Roots	Supercritical fluid extraction	GC-MS	0.4–0.51 mg/g	[36]
10	E-butylidenephthalides	C12H12O2	Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort.	Roots	Water distillation	GC-MS	0.005 %	[30]
			Angelica sinensis (Oliv.) Diels	Roots	Pressurized liquid extraction	GC-MS	0.04–0.05 %	[59]
11	Propylidene-phthalide	C11H10O2	Levisticum officinale W. D. J. Koch	Matrix, leaves, stems, seeds	Supercritical fluid extraction	GC-MS	/	[38]
12	3-(3-Methylbutylidene)phthalide	C13H14O2	Apium graveolens L.	Leaves, stalks, roots	Water distillation	GC-MS	/	[39]
13	Sedanolide	C12H18O2	Apium graveolens L.	Seeds	Supercritical fluid extraction	GC-MS	72.24 %	[40]
14	3-butylhexahydro-phthalide	C12H16O2	Petroselinum crispum (Mill.) Nyman ex A. W. Hill	Roots	High-speed countercurrent chromatography	GC-MS	1.1–4.2 %	[41]
15	Hexahydro-3-butylphthalide	C12H20O2	Seseli pallasii Besser	Roots, stems, fruits	Water distillation	GC-MS	0.10 %	[42]
16	Sedanonic acid lactone	C12H16O2	Meum athamanticum Ext.	Aerial parts	Water distillation	GC-MS	0.50 %	[43]
17	Neoligustilide	C12H14O2	Angelica sinensis (Oliv.) Diels	Roots	Water distillation	GC-MS	/	[44]
18	Cnidilide	C12H18O2	Ligusticum chuanxiong Hort.	Roots	MeOH	HPLC-DAD-MS	/	[45]
19	10-angeloyl-3-butylphthalide	C17H20O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	UV, IR, NMR	/	[46]
20	10-Angeloylbutylphthalide	C17H20O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	UV, IR, NMR	/	[46]
21	11-Angeloylsenkyunolide F	C17H20O4	Angelica acutiloba (Siebold & Zucc.) Kitag.	Roots	Hexane-MeOH extraction	HPLC-MS, IR, UV, MS, NMR	/	[47]
22	Angeloyl ester of senkyunolide F	C17H20O4	Angelica acutiloba (Siebold & Zucc.) Kitag.	Roots	Hexane-MeOH extraction	HPLC-MS, IR, UV, MS, NMR	/	[47]
23	Celephthalide A	C18H24O8	Apium graveolens L.	Seeds	MeOH	HPLC, NMR	/	[48]
24	7-hydroxy-3-butylidenephthalide 7-O-glucoside	C18H22O8	Petroselinum crispum (Mill.) Fuss	Roots	MeOH	HPLC, GC-MS, NMR	/	[49]
25	Z-6,7-epoxyligustilide	C12H14O3	Angelica sinensis (Oliv.) Diels Angelica sinensis (Oliv.) Diels	Roots Roots	Pressurized liquid extraction MeOH	GC-MS LC-MS	0.12–0.13 % /	[59] [50]
26	7-methoxy-3-propylidenephthalide	C12H12O3	Levisticum officinale W. D. J. Koch	Roots	n-hexane and ethyl acetate	UHPLC-MS, NMR	/	[51]
27	5,7-dimetoxy-6-(3-methylbut-2-enyl)-isobenzofuran-1(3H)-one	C15H18O4	Peperomia nivalis Miq.	Aerial parts	/	HR-TOF-MS, NMR	/	[52]
28	Senkyunolide B	C12H12O3	Angelica sinensis (Oliv.) Diels Angelica sinensis (Oliv.) Diels	Roots Roots	Pressurized liquid extraction MeOH	GC-MS LC-MS	0.13–0.14 % /	[59] [50]
29	Senkyunolide C	C12H12O3	Levisticum officinale W. D. J. Koch Ligusticum chuanxiong Hort.	Roots Roots	Ethyl acetate 60 % EtOH	UHPLC-MS HPLC, HR-ESI-MS, UV, IR, NMR	/ /	[54] [53]
30	Senkyunolide E	C12H12O3	Angelica acutiloba (Siebold & Zucc.) Kitag. Angelica sinensis (Oliv.) Diels	Roots Roots	Hexane-MeOH extraction MeOH	HPLC-MS, IR, UV, MS, NMR LC-MS	/ /	[47] [50]
31	3-butylidene-7-hydroxyphthalide	C12H12O3	Ligusticum sinense Oliv.	Roots	Column chromatography	MS	/	[55]
32	3-butylidene-4,5-dihydroxyphthalide	C12H12O4	Angelica acutiloba (Siebold & Zucc.) Kitag.	Roots	ET2O extraction	IR	/	[56]
33	4-hydroxy-3-butylphthalide	C12H14O3	Ligusticum chuanxiong Hort. Ligusticum chuanxiong Hort.	Roots Roots	Column chromatography MeOH	NMR, MS HPLC	/ /	[58] [57]
34	3-hydroxy-3-butylphthalide	C12H14O3	Ligusticum chuanxiong Hort.	Roots	Column chromatography	NMR, MS	/	[58]
35	4,7-dihydroxy-3-butylphthalide	C12H14O4	Ligusticum chuanxiong Hort.	Roots	Column chromatography	NMR, MS	/	[58]
36	Senkyunolide F	C12H14O3	Angelica sinensis (Oliv.) Diels	Roots	Pressurized liquid extraction	GC-MS	0.13–0.14 %	[59]
37	3,9-dihydroxy-ligustilide	C12H16O4	Angelica sinensis (Oliv.) Diels	Roots	80 % EtOH	NMR	/	[60]
38	Senkyunolide G	C12H16O3	Angelica sinensis (Oliv.) Diels	Roots	MeOH	LC-MS	/	[50]
39	Senkyunolide K	C12H16O3	Ligusticum striatum DC.	Roots	MeOH	MS, UV, NMR	/	[61]
40	3-butylidene-6-hydroxy-5,6-dihydrophthalide	C12H14O3	Ligusticum chuanxiong Hort.	Roots	70 % MeOH	HRMS	/	[62]
41	3-butylidene-6-hydroxy-5,6-dihydro-butylphthalide	C12H16O3	Ligusticum chuanxiong Hort.	Roots	70 % MeOH	HRMS	/	[62]
42	Senkyunolide H	C12H16O4	Angelica sinensis (Oliv.) Diels	Roots	Pressurized liquid extraction	GC-MS	0.13–0.18 %	[59]
43	Senkyunolide I	C12H16O4	Angelica sinensis (Oliv.) Diels	Roots	Pressurized liquid extraction	GC-MS	0.15–0.45 %	[59]
44	Senkyunolide J	C12H18O4	Apium graveolens L.	Seeds	MeOH	NMR	/	[63]
45	Senkyunolide N	C12H18O4	Apium graveolens L.	Seeds	MeOH	NMR	/	[63]
46	3,6,7-trihydroxy-4,5,6,7-tetrahydro-3-butylphthalide	C12H18O5	Ligusticum chuanxiong Hort.	Roots	70 % MeOH	HRMS	/	[62]
47	Senkyunolide R	C12H16O5	Ligusticum chuanxiong Hort.	Roots	MeOH	UV, IR, NMR	/	[64]
48	Senkyunolide S	C12H16O5	Ligusticum chuanxiong Hort.	Roots	MeOH	UV, IR, NMR	/	[64]
49	Senkyunolide L	C12H15ClO3	Ligusticum chuanxiong Hort.	Roots	70 % MeOH	HRMS	/	[62]
50	Senkyunolide M	C16H22O4	Ligusticum chuanxiong Hort.	Roots	70 % MeOH	HRMS	/	[62]
51	Senkyunolide Q	C16H22O4	Ligusticum chuanxiong Hort.	Roots	70 % MeOH	HRMS	/	[62]
52	(3S, 3aR, 6S)-(−)-6-hydroxysedanolide	C12H18O3	Ligusticum sinense Oliv cv. Chaxiong	Aerial parts	95 % EtOH	HR-ESI-MS, UV, IR, NMR	/	[65]
53	(3S, 3aR, 4R)-(−)-4-hydroxysedanolide	C12H18O3	Ligusticum sinense Oliv cv. Chaxiong	Aerial parts	95 % EtOH	HR-ESI-MS, UV, IR, NMR	/	[65]
54	(3S, 3aR, 10R)-(−)-10-hydroxysedanolide	C12H18O3	Ligusticum sinense Oliv cv. Chaxiong	Aerial parts	95 % EtOH	HR-ESI-MS, UV, IR, NMR	/	[65]
55	(3S, 3aR)-3-hydroxyethyl-3a, 4, 5,6-tetrahydrophthalide	C10H14O3	Ligusticum sinense Oliv cv. Chaxiong	Aerial parts	95 % EtOH	HR-ESI-MS, UV, IR, NMR	/	[65]
56	(3S, 3aR, 6S)-(−)-6-hydroxysedanolide-10-one	C12H16O4	Ligusticum sinense Oliv cv. Chaxiong	Aerial parts	95 % EtOH	HR-ESI-MS, UV, IR, NMR	/	[65]
57	(3S, 3aR)-3-(2-carboxyl) ethyl-3a, 4, 5,6-tetrahydrophthalide	C10H12O4	Ligusticum sinense Oliv cv. Chaxiong	Aerial parts	95 % EtOH	HR-ESI-MS, UV, IR, NMR	/	[65]
58	(3R, 6R)-3-butyl-6-hydroxy-4, 5, 6, 7-tetrahydrophthalide	C12H18O3	Ligusticum sinense Oliv cv. Chaxiong	Aerial parts	95 % EtOH	HR-ESI-MS, UV, IR, NMR	/	[65]
59	6-hydroxy-7-methoxydihydro-ligustilide	C13H18O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	LC-MS	/	[50]
60	(S)-3-ethyl-7-hydroxy-6-methoxyphthalide	C11H12O4	Pittosporum illicioides Makino	Roots	MeOH	UV, IR, NMR	/	[66]
61	(Z)-3-ethylidene-7-hydroxy-6-methoxyphthalide	C11H10O4	Pittosporum illicioides Makino	Roots	MeOH	UV, IR, NMR	/	[66]
62	(Z)-3-ethylidene-6,7-dimethoxyphthalide	C12H12O4	Pittosporum illicioides Makino	Roots	MeOH	UV, IR, NMR	/	[66]
63	(S)-3-ethyl-5,6,7-trimethoxyphthalide	C13H16O5	Pittosporum illicioides Makino	Roots	MeOH	UV, IR, NMR	/	[66]
64	(S)-3-ethyl-7-hydroxy-5,6-dimethoxyphthalide	C12H14O5	Pittosporum illicioides Makino	Roots	MeOH	UV, IR, NMR	/	[66]
65	6-formyl-7-hydroxy-5-methoxy-4-methylphthalide	C11H10O5	Pittosporum illicioides Makino	Roots	MeOH	UV, IR, NMR	/	[66]
66	1,3-Dihydro-3-oxo-1-isobenzofuranacetic acid	C10H8O4	Ligusticum chuanxiong Hort.	Roots	70 % MeOH	HRMS	/	[62]
67	4,6-dimethoxy-phthalide	C10H10O4	Asarum heterotropoides F. Schmidt	Roots	Water distillation	MS	/	[67]
68	Arenophthalide B	C19H24O9	Helichrysum arenarium (L.) Moench	Whole plant	70 % EtOH	IR, UV, NMR	/	[68]
69	Arenophthalide C	C19H24O9	Helichrysum arenarium (L.) Moench	Whole plant	70 % EtOH	IR, UV, NMR	/	[68]
70	Senkyunolide D	C12H14O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	LC-MS	/	[50]
71	3,7-dihydroxyligustilide	C12H16O4	Angelica sinensis (Oliv.) Diels	Roots	Pressurized liquid extraction	GC-MS	0.13–0.14 %	[59]
72	Neophathalide A	C12H16O4	Ligusticum chuanxiong Hort.	Roots	80 % EtOH	IR, UV, NMR	/	[11]
73	Neophathalide B	C12H14O4	Ligusticum chuanxiong Hort.	Roots	80 % EtOH	IR, UV, NMR	/	[11]
74	7-hydroxy-3-butylidenephthalide-7-O-(6′-malonylglucoside)	C21H24O11	Petroselinum crispum (Mill.) Fuss	Roots	MeOH	HPLC, GC-MS, NMR	/	[49]
75	Celephthalide B	C23H32O12	Apium graveolens L.	Seeds	MeOH	HPLC, NMR	/	[48]
76	3-butylidene-6α-hydroxy-7α-ethoxy-phthalide	C14H21O4	Ligusticum chuanxiong Hort.	Roots	EtOH	HR-ESI-MS, NMR	/	[69]
77	Ligusticoside A	C19H30O8	Ligusticum chuanxiong Hort.	Roots	Water	MS, IR, UV, NMR	/	[5]
78	Celephthalide C	C18H28O8	Apium graveolens L.	Seeds	MeOH	HPLC, NMR	/	[48]
79	ligusticosides C	C18H28O8	Cnidium officinale Makino	Roots	EtOH	MS, IR, NMR	/	[70]
80	ligusticosides E	C23H37O12	Cnidium officinale Makino	Roots	EtOH	MS, IR, NMR	/	[70]
81	ligusticosides D	C23H37O12	Cnidium officinale Makino	Roots	EtOH	MS, IR, NMR	/	[70]
82	Senkyunolide O	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	Column chromatography	IR, MS, NMR	/	[71]
83	Senkyunolide P	C24H30O4	Ligusticum chuanxiong Hort.	Roots	MeOH	HPLC-DAD-MS	/	[72]
84	Levistolide A	C24H28O4	Ligusticum chuanxiong Hort.	Roots	MeOH	HPLC-DAD-MS	0.0468–0.0928 mg/g	[72]
85	3,8-dihydro-diligustilide	C24H30O4	Ligusticum chuanxiong Hort.	Roots	EtOH	LC-MS	/	[73]
86	Chuanxiongdiolide A	C24H32O4	Ligusticum chuanxiong Hort.	Roots	95 % EtOH	UV, IR, NMR, HR-ESI-MS	/	[74]
87	Chuanxiongnolide A	C24H28O5	Ligusticum chuanxiong Hort.	Roots	90 % EtOH	HR-ESI-MS, NMR	/	[6]
88	Chuanxiongnolide B	C24H28O5	Ligusticum chuanxiong Hort.	Roots	90 % EtOH	HR-ESI-MS, NMR	/	[6]
89	Tokinolide B	C24H28O4	Ligusticum chuanxiong Hort.	Roots	MeOH	HPLC-DAD-MS	/	[72]
90	Z-ligustilide dimer E−232	C24H28O4	Ligusticum chuanxiong Hort.	Roots	MeOH	HPLC-DAD-MS	/	[72]
91	Angeolide	C24H28O4	Ligusticum sinense Oliv.	Roots	95 % EtOH	UV, ECD, IR, NMR	/	[75]
92	Gaobennolide A	C24H30O4	Ligusticum sinense Oliv.	Roots	95 % EtOH	UV, ECD, IR, NMR	/	[75]
93	Gaobennolide B	C24H30O4	Ligusticum sinense Oliv.	Roots	95 % EtOH	UV, ECD, IR, NMR	/	[75]
94	Ansaspirolide	C24H26O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	UV, IR, NMR	/	[46]
95	Angesinenolide F	C24H26O4	Ligusticum sinense Oliv.	Roots	95 % EtOH	UV, ECD, IR, NMR	/	[75]
96	(3′Z)-(3S,8S,3a′S,6′R)-4,5-dehydro-3.3a′,8.6′-diligustilide	C24H26O4	Ligusticum chuanxiong Hort.	Roots	95 % EtOH	NMR, HR-ESI-MS	/	[77]
97	(3′Z)-(3S,8R,3a′S,6′R)-4,5-dehydro-3.3a′,8.6′-diligustilide	C24H26O4	Ligusticum chuanxiong Hort.	Roots	95 % EtOH	NMR, HR-ESI-MS	/	[77]
98	4,5-Dehydro-diligustilide	C24H26O4	Ligusticum chuanxiong Hort.	Roots	EtOH	LC-MS	/	[73]
99	Chuanxiongdiolide R1	C25H31O5	Ligusticum chuanxiong Hort.	Roots	95 % EtOH	NMR, HR-ESI-MS	/	[77]
100	Chuanxiongdiolide R3	C25H31O5	Ligusticum chuanxiong Hort.	Roots	95 % EtOH	NMR, HR-ESI-MS	/	[77]
101	Z,Z′-3.3′a, 7.7′a-diligustilide	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	MS	/	[78]
102	Z,Z′-3a.7a′,7a.3a′-dihydroxyligustilide	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	LC-MS, NMR	/	[79]
103	Chuanxiongdiolide R6	C24H30O4	Ligusticum chuanxiong Hort.	Aerial parts	95 % EtOH	UV, ECD, IR, HR-ESI-MS, NMR	/	[80]
104	Angesinenolides C	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	HRMS, NMR	/	[76]
105	Angesinenolides D	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	HRMS, NMR	/	[76]
106	Tokinolide A	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	Supercritical fluid extraction	HPLC, NMR	/	[81]
107	Tokinolide C	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	Supercritical fluid extraction	HPLC, NMR	/	[81]
108	Neodiligustilide	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	FT-IR, UV, NMR	/	[82]
109	Tokiaerialide	C24H28O4	Ligusticum chuanxiong Hort.	Aerial parts	95 % EtOH	UV, ECD, IR, HR-ESI-MS, NMR	/	[80]
110	Riligustilide	C24H28O4	Ligusticum chuanxiong Hort.	Roots	MeOH	HPLC-DAD-MS	/	[72]
111	Gelispirolide	C24H26O4	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	LC-HR-ESI-MS	/	[10]
112	3,3′Z-6.7′,7.6′-diligustilide	C24H28O4	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	NMR	/	[83]
113	3′,8′,3′a,7′a-tetrahydro-6.3′,7.7′a-diligustilide-8′-one	C24H30O4	Angelica sinensis (Oliv.) Diels	Roots	Column chromatography	IR, MS, NMR	/	[71]
114	Sinaspirolide	C24H26O4	Angelica sinensis (Oliv.) Diels	Roots	MeOH	UV, IR, NMR	/	[46]
115	Chuanxiongdiolide B	C24H32O4	Ligusticum chuanxiong Hort.	Roots	95 % EtOH	UV, IR, NMR, HR-ESI-MS	/	[74]
116	Wallichilide	C25H33O5	Ligusticum striatum DC.	Roots	Water	UV, IR, NMR, MS	/	[3]
117	E,E′-3,3':8,8′-Diligustilide	C24H28O4	Ligusticum chuanxiong Hort.	Roots	MeOH	HPLC-DAD-MS	/	[72]
118	Angesinenolides E	C24H24O4	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	HRMS, NMR	/	[76]
119	Diangeliphthalide A	C24H26O4	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	LC-HR-ESI-MS	/	[10]
120	Chuanxiongdiolide R4	C25H33O5	Ligusticum chuanxiong Hort.	Aerial parts	95 % EtOH	UV, ECD, IR, HR-ESI-MS, NMR	/	[80]
121	Chuanxiongdiolide R5	C24H32O4	Ligusticum chuanxiong Hort.	Aerial parts	95 % EtOH	UV, ECD, IR, HR-ESI-MS, NMR	/	[80]
122	Methyl ester of angeolide	C25H33O5	Ligusticum chuanxiong Hort.	Aerial parts	95 % EtOH	UV, ECD, IR, HR-ESI-MS, NMR	/	[80]
123	(+)-6-3′a,7–6′-Isowallichilide	C25H32O5	Ligusticum chuanxiong Hort.	Roots	MeOH	HR-ESI-MS, NMR	/	[84]
124	(−)-6-3′a,7–6′-Isowallichilide	C25H32O5	Ligusticum chuanxiong Hort.	Roots	MeOH	HR-ESI-MS, NMR	/	[84]
125	1″,3′-seco-1′-ethoxy-3,3a′, 8,6′-diligustilide	C26H33O5	Ligusticum chuanxiong Hort.	Roots	EtOH	HR-ESI-MS, NMR	/	[69]
126	Spiroligustolides A	C24H26O6	Ligusticum chuanxiong Hort.	Roots	CH2Cl2–MeOH	HR-ESI-MS, IR, NMR	/	[85]
127	Spiroligustolides B	C24H26O6	Ligusticum chuanxiong Hort.	Roots	CH2Cl2–MeOH	HR-ESI-MS, IR, NMR	/	[85]
128	Triangeliphthalides A	C36H40O6	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	LC-HR-ESI-MS	/	[10]
129	Triangeliphthalides B	C36H40O6	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	LC-HR-ESI-MS	/	[10]
130	Triligustilides A	C36H42O6	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	HR-ESI-MS	/	[9]
131	Triligustilides B	C36H44O6	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	HR-ESI-MS	/	[9]
132	Triangeliphthalides C	C36H40O6	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	LC-HR-ESI-MS	/	[10]
133	Triangeliphthalides D	C36H38O6	Angelica sinensis (Oliv.) Diels	Roots	95 % EtOH	LC-HR-ESI-MS	/	[10]

3. Pharmacological activities of phthalides

A wide range of biological activities for natural phthalides have been reported, including regulation of cardio-cerebrovascular system and central nervous system, hemorheology improvement, antioxidation, anti-inflammation, analgesia, anti-cancer, organ and bone-related protection. An overview on cardio-cerebrovascular functional modulation of various phthalides has been summarized in Table 2. Neuroprotection of butylphthalide in different models of brain impairment has also been achieved in Table 3. The collected information is based on animal models, dosages, administrations, pharmacological effects and possible mechanisms.

Table 2.

Cardio-cerebrovascular functional modulation of phthalides.

Cardio-cerebr-ovascular functions	Phthalides	Possible mechanisms	Reference
Angiogenesis or neovascularization	Ligustilide	Increase cerebral vessels number, VEGF and eNOS activation	[17]
	Butylphthalide	Alter Ang-1/Ang-2/Tie-2 signaling axis	[88]
	Butylphthalide	Promote VEFG and Ang-1, and upregulate sonic hedgehog expression	[89]
	Butylidenephthalide	Enhance VEGF and BDNF expressions via activation of AKT/mTOR signal pathway	[90]
Protection on endothelial cells	Ligustilide	Stimulate NRF2/ARE pathway	[91]
	Butylphthalide	Maintain the endothelial PGC-1α expression via regulating eNOS activity	[92]
	Butylidenephthalide	Elevate the pool of PI species, and inhibit the phosphorylation of PLCλ and ERK1/2	[93]
	Levistilide A	Suppress NLRP3 gene expression by blocking the Syk–p38/JNK pathway	[94]
	Z,Z′-3a.7a′,7a.3a′-dihydroxyligustilide	Increase the viability and proliferation of HUVECs, and reduce the apoptosis	[95]
Smooth muscle relaxation, inhibition of VSMCs abnormal proliferation and migration	Ligustilide	Downregulate c-Myc/MMP2 signaling pathway and ROCK-JNK signaling pathway	[96]
	Butylphthalide	Inhibit the β-catenin signaling pathway	[97]
	Butylidenephthalide	Phosphorylate JNK and p38 MAPK, and upregulate Nur77 gene	[98]
Vasodilatation or vasorelaxation activities	Ligustilide	Inhibit [Ca2+] influx and cold-sensing protein TRPM8 and TRPA1	[99]
	Butylphthalide	Increase the regional blood flow by preventing vasoconstriction of the artery and maintaining its diameter at normal level	[100]
	Senkyunolide A	Inhibit RYRs and VDCCs	[101]
Anti-arthrosclerosis effects	Ligustilide and Senkyunolide A	Inhibit CD137 expression through suppressing AP-1 and AKT/NF-κB signaling pathway	[102]
	Ligustilide	Activate Nrf2/HO-1 induction and NO synthesis	[103]
	Ligustilide	Activate Nrf2 and ARE-driven genes	[91]
Anti-thrombotic effects	Butylphthalide	Inhibit human platelet activation via inhibition of cPLA2-mediated TXA2 synthesis and PDE	[104]
	(S)-ZJM-289, a nitric oxide-releasing derivative of 3-n-butylphthalide	Maintain mitochondrial integrity and function, and block mitochondria-mediated cell death possibly by upregulation of Hsp70	[105]
	3-Butyl-2-benzothiophen-1(3H)-one (5d)	Decrease NADPH oxidase activity by promoting Nrf2 nuclear localization through PI3K/Akt signaling pathway	[106]
Anti-myocardial ischemia effects	Ligustilide and Cnidilide	Reduce serum lactic acid and free fatty acid	[107]
	Butylphthalide	Inhibit inflammatory and oxidative stress responses by regulating the Akt/Nrf2 signaling pathway	[108]
	Butylidenephthalide	Target the PI3K/STAT3 axis	[12]

Table 3.

Neuroprotection of butylphthalide in different animal experimental models of brain impairment.

Animal	Model	Dosage	Administration	Effects	Mechanisms	Reference
Male CD-1 mice	Cerebral infarction by permanent MCAO	120 mg/kg	–	Neurologic function deficits, Infarction volume, Edema, BBB permeability, MMP- 9↓	Nrf-2/HO-1	[109]
				Claudin-5, VEGF, GFAP, Ultrastructure in capillary endothelial cells, Nrf-2, HO-1↑
Female SD rats	Cerebral infarction by MCAO	15 mg/kg	i.p.	Neurological score, White region, Apoptosis rate, p38 and MAPK ↓	p38/MAPK	[37]
Male C57BL/6 mice	Cerebral ischemia by transient MCAO or photothrombosis	60 mg/kg	i.g. daily for 3 days	Intercellular adhesion molecule 1, Protease-activated receptor 1, Brain infiltration of myeloid cells↓	Neurovascular inflammation	[110]
				CBF↑
Male Wistar rats	Cerebral ischemia	70 mg/kg	p.o. daily for 14 days	Number and length of crossing corticospinal tract fibers, Synapse-associated proteins including PSD95 and VGlut-1, Behavioral performance↑	Axonal growth and neurogenesis	[111]
Male C57BL/6J mice	CCH by BCAS	80 mg/kg	p.o. daily for 60 days	Spatial memory dysfunction, BBB leakage, Endothelial cells damage, Endothelial tight junctions disruption↓	–	[16]
				Cerebral perfusion, White matter integrity↑
Male APP/PS1 transgenic mice	AD	15 mg/kg	p.o. daily for 90 days	Number of synapses, Apical dendritic thorns, Thickness of PSD, Synapse-associated proteins including PSD95, synaptophysin (SYN), b-catenin, and GSK-3β, Synaptic and Spine function↑	Wnt/β-catenin	[112]
				Aβ plaques and Neuroinflammatory responses↓
Male APP/PS1 transgenic mice	AD	20 and 60 mg/kg	p.o. daily for 150 days	Oxidative stress, Learning and memory deficits, Soluble amyloid beta, Amyloid beta oligomer, ↓	CREB/CBP/Nrf-2	[113]
				Synaptic plasticity, CBP, CREB phosphorylation, Recruitment of CBP to the promoters of best-characterized genes downstream of Nrf-2, NADPH quinone oxidoreductase 1, and g-glutamyl cysteine synthetase modifier subunit↑
Male C57BL/6 mice	PD by MPTP	100 mg/kg	i.p. daily for 9 days	Dopaminergic neurodegeneration, Motor deficits, Microglial activation, Pro-inflammatory mediators↓	MAPK, NF-kB and PI3K/Akt	[15]
Male C57BL/6 mice	PD by LPS	120 mg/kg	i.g. daily for 30 days	Behavioral deficits, Dopaminergic neurodegeneration, Phosphorylation of JNK and p38↓	JNK	[114]
Transgenic (SOD1-G93A) mice	ALS	30 and 60 mg/kg	p.o.	Motor performance, Survival interval, Nrf2, HO-1↑	Nrf2/HO-1/NF-kB	[115]
				Motor neuron loss, Motor unit reduction, Immunoreactivity of CD11b and GFAP, NF-kB p65, TNF-a protein↓
Male SD rats	ICH	25 mg/kg	p.o. daily for 15 days	Longa's motor scores, Neurological defects↓	Angiogenesis	[116]
				Numeric density of blood vessels, VEGF, Ang-2 proteins↑
Male SD rats	ICH	25 mg/kg	i.p. twice daily for 2 days	Neurological function↑	Inflammation	[117]
				Hematoma volume, Brain edema, BBB permeability, TNF-α, MMP-9 ↓
Male Wistar rats	Concussive head injury	40 and 60 mg/kg	i.p.	BBB breakdown, Brain pathology↓	Glial activation	[118]
Male SD rats	Diffuse brain injury	80 and 160 mg/kg	i.p. daily for 3 days	Cerebral ultrastructure damage, BBB age, Cerebral edema↓	Microcirculation disorder	[119]
				Vascular density, CBF, Motor and sensory functions↑
Male ICR mice	Traumatic Brain Injury	100 mg/kg	i.p.	Neurological deficits, Brain water content, Cortical neuronal apoptosis↓	Nrf2-ARE	[120]
				MDA, SOD activity, GPx, Translocation of Nrf2 protein from the cytoplasm to the nucleus, HO-1, NQO1↑
Male C57BL/6 mice	Traumatic brain injury	100 mg/kg	p.o. daily for 7 days	BBB disorder, Mitochondrial apoptosis, Autophagy-related proteins, including ATG7, Beclin1 and LC3II↓	Autophagy	[121]
				Neuronal survival, Tight junction proteins, Locomotor functional recovery↑
Female SD rats	SCI	80 mg/kg	p.o. daily for 7 days	Locomotor recovery↑	TLR4/NF-κB	[122]
				Lesion cavity area of the spinal cord, Apoptotic activity in neurons, Number of TUNEL-positive cell, Activation of microglia, Release of inflammatory mediators, Microglial TLR4/NF-κB↓
Female SD rats	SCI	80 mg/kg	p.o. daily for 7 days	BSCB permeability, Breakdown of AJ and TJ proteins, ER stress, ER stress-associated proteins↓	ER stress	[123]
				Locomotion recovery↑
Male SD rats	VD by CCH	60 and 120 mg/kg	p.o. daily for 28 days	Spatial learning and memory impairment, Loss of neurons in the CA1 region of the hippocampus, Endoplasmic reticulum stress-related markers↓	Shh/Ptch1	[124]
				Plasticity‐related synaptic markers, Shh/Ptch1 pathway↑	ER stress
Male SD rats	VD by CCH	5 mg/kg	i.v. daily for 21 days	Cognitive function, GDNF/GFRa1/Ret signaling, Phosphorylation AKT (p-AKT) and ERK1/2 (p-ERK1/2) signaling, Bcl-2↑	GDNF/GFRa1/Ret	[125]
				Hippocampal neuron apoptosis, Bax, Cleaved caspase-3↓	AKT/ERK1/2
Male SAMR1 and SAMP8 mice	Age-related dementia	40 and 80 mg/kg	p.o. daily for 90 days	Cognitive impairment↓	Brain-derived neurotrophic factor/TRK-B signaling	[126]
				Synaptophysin, Postsynaptic density protein 95, Hippocampal structural synaptic plasticity↑

3.1. Cardiovascular and cerebrovascular system

3.1.1. Angiogenesis

Recently, phthalides were found to mediate angiogenesis in the improvement of vascular disorders. Oral administration of ligustilide (1) (10 mg/kg, daily) for 1 week was observed to enhance angiogenesis in mouse brain microvascular endothelial cells (bEnd.3) and focal transient cerebral ischemic model. Since ligustilide was shown to improve neurological function via raising the quantity of brain vessel, it suggested the effective prevention from ischemia-stimulated cerebral damage. Moreover, its neurological recovery closely correlated with the upregulation of vascular endothelial growth factor (VEGF) level and the activation of endothelial nitric oxide synthase (eNOS) signaling in the ischemic regions [17]. Butylphthalide (8) (i.v., 5 mg/kg, daily) was observed to strengthen neovascularization via time-dependent mediation of angiopoietin and Tie signaling in rats with chronic cerebral hypoperfusion (CCH). Specifically, the treatment with butylphthalide could restore the lowered endothelial cell specific tyrosine kinase receptor-2 (Tie-2) and angiopoietins-1 (Ang-1) levels, as well as elevate angiopoietins-2 (Ang-2) level in the CCH model. Additionally, it further augmented the release of VEGF, cluster of differentiation 34 (CD34) and vasodilated bilateral vertebral arteries during CCH [88]. In another report, butylphthalide treatment (p.o., 80 mg/kg, daily) for 2 weeks could inhibit focal ischemia-induced alterations (body weight loss, brain infarct volume enlargement, and neurobehavioral abnormalities) in rats; meanwhile, it also increased VEGF and Ang-1 levels together with elevated sonic hedgehog expression in astrocytes [89].

3.1.2. Protection of endothelial cells

Phthalides can protect the endothelium from vasomotor dysfunctions. Zhu and his group revealed that Z-ligustilide (1) at a dose of 100 μM ameliorated oxidative injury and cellular damage in EA. hy926 endothelial cells when exposed to tert-butyl hydroperoxide (t-BHP) via the activation of Nrf2 and antioxidant response element (ARE) signaling pathways by elevating their downstream expressions [91]. Butylphthalide (8) at 1.0 μM also protected endothelial cells from oxygen-glucose deprivation (OGD)-caused impairment through improvement of cell viability and vascular proliferation, which was associated with promoted intracellular expression of peroxisome proliferators-activated receptor-γ coactivator-1α (PGC-1α) by mediating eNOS activity without affecting protein levels [92]. Levistilide A (84) with 50 μM alleviated the lipopolysaccharide (LPS)-induced inflammation in human umbilical vein endothelial cells (HUVECs) and its treatment (20 mg/kg/day) for 16 days also restrained vasculitis in rats of thromboangiitis obliterans model. Moreover, the underlying mechanisms were demonstrated to be related to blunted production of inflammatory factors such as interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-32, chemokine C–C motif-2 (CCL-2), vascular cell adhesion molecule 1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1), and dose-dependently lessened expression of NLR family pyrin domain containing 3 (NLRP3) inflammasome through obstructing the phosphorylation of spleen tyrosine kinase (Syk) and inactivating p38 and c-Jun N-terminal kinase (JNK) Kelch-like ECH-associated protein 1 (Keap1) pathways [94].

3.1.3. Inhibition of vascular smooth muscle cell proliferation and migration

Phthalides present inhibitory actions in vascular smooth muscle cell proliferation and migration. The administration of ligustilide (1) (i.p., 10, 20 and 40 mg/kg, daily) for 2 weeks suppressed intimal thickening and vascular smooth muscle cells excessive migration in rats with angiotensin II-stimulated cellular migration model in a dose-dependent manner, possibly through inactivating c-Myc/mitochondrial membrane potential-2 (MMP2) and Rho-associated coiled-coil-containing protein kinase (ROCK)/JNK signaling, as well as promoting the synthesis of phenotypic transformation α-smooth muscle actin (α-SMA) protein. Besides, blood pressure and blood lipid contents were obviously decreased in a rat model of atherosclerosis and hypertension following the delivery of ligustilide [96]. Hu and colleagues confirmed that butylphthalide (8) at a dose of 40 μM dramatically restrained platelet-derived growth factor (PDGF)-BB-triggered vascular smooth muscle cell proliferation through reinforcing the autophagic effects, which was accompanied with the limitation of β-catenin pathway [97].

3.1.4. Vasodilatation

Vasodilative activity is one of the important properties in natural phthalides. The vasorelaxation ability of Z-ligustilide (1) was responsible for therapeutic properties of Angelica sinensis in the alleviation of cold-stimulated vasospasm disturbances, which was involved in dose-dependent downregulation of cold-sensing protein TRPM8 and TRPA1 expressions in human aortic smooth muscle cells [99]. Compared with positive control nimodipine (1 μM), senkyunolide A (3) (10 μM) exerted relatively similar calcium antagonistic activity by suppressing the expression of ryanodine receptors and partially obstructing voltage-operated Ca²⁺ channels, indicating its potential in Suxiao Jiuxin Pill for improvement of vasodilatation function [101]. Qin et al. observed that the treatment with butylphthalide (8) (i.g., 90 mg/kg, daily) for 3 weeks inhibited artery vasoconstriction via retaining the normal diameter of vessels in rats subjected to transient MCAO, suggesting its functioning as a potent vasodilator to promote regional blood flow and diminish vascular constrictions for curing ischemic stroke and thrombosis [100].

3.1.5. Atherosclerosis

Phthalides are also responsible for the amelioration of atherosclerotic progression. The administration of Z-ligustilide (1) (i.p., 20 mg/kg, daily) for 8 weeks was confirmed to display a strong anti-atherosclerotic activity in the aortas of high fat diet (HFD)-treated low density lipoprotein receptor (LDLR)-deficient mice through mitigating plague generation and lipid peroxidation, as well as enhancing anti-oxidation-associated enzyme activities, which were related to the potentiation of Nrf2 levels and ARE-modulated genes expressions [91]. Another study reported that ligustilide (1, 3 and 10 μM) could rehabilitate vascular-related inflammatory response and evoke potential defensive mechanisms in TNF-α-treated HUVECs through dose-dependently reducing HL-60 monocyte adhesion and cell adhesion molecule (CAM) secretion, as well as constraining reactive oxygen species (ROS) production, suggesting its potent therapeutic functions for atherosclerosis. Also, it robustly blunted the expression of NF-κB signaling under the conditions of heme oxygenase 1 (HO-1)/Nrf2 induction and intracellular nitric oxide (NO) production [103]. Lei et al. discovered that ligustilide and senkyunolide A (3) from Suxiao Jiuxin Pill represented anti-inflammatory and immunomodulatory effects on the attenuation of atherosclerosis in apolipoprotein E (ApoE)-deficient mice. Compared with positive control dexamethasone (10⁻⁴ M), these two phthalides showed relatively similar effects to restrict CD137 production dose-dependently by downregulating activator protein-1 (AP-1) level and inactivating of Akt/NF-κB pathways in mouse aortic endothelial cells [102].

3.1.6. Thrombosis

Phthalide compounds show anti-thrombotic activity. For example, butylphthalide (8) treatment (30, 100 and 300 μM) downregulated does-dependently the secretion of thromboxane A2 (TXA2) through obstructing the phosphorylation of anti-phospho-cytosolic phospholipase A2 (cPLA2), and the intracellular Ca2⁺ mobilization, accompanied with reduction in the bioactivity of human platelet phosphodiesterase (PDE) and enrichment in cyclic adenosine monophosphate (cAMP). Therefore, it exhibited therapeutic effects against thrombotic diseases via suppressing platelet aggregation and platelet spreading on immobilized fibrinogen by limiting the production of cPLA2-regulated TXA2 and the expression of PDE [104]. A new butylphthalide derivate, 3-Butyl-2-benzothiophen-1(3H)-one treatment (i.g., 30 and 90 mg/kg, daily) for 3 days concentration-dependently decreased oxidase activity of nicotinamide adenine dinucleotide phosphate (NADPH), activated Nrf2 signaling pathway, and promoted nicotinamide adenine dinucleotide phosphate oxidase-4 (NOX4) release in a rat model of ischemia/reperfusion (I/R), indicating that it might be effective as an attractive anti-thrombotic drug to treat ischemic stroke [106].

3.1.7. Myocardial ischemia

The treatment with butylphthalide (8) (i.p., 80 mg/kg, daily) for 4 weeks greatly recovered the morphology of the heart tissues in rats after acute myocardial infarction, decreased the release of NOX-1, p-Akt and p-Nrf2, and enhanced apoptosis rate, which was accompanied with sharp reduction in the content and transcriptional levels of several inflammatory mediators such as IL-1β and TNF-α. Therefore, it suppressed inflammatory response and oxidative injury in myocardial infarction rats via the modulation of Akt/Nrf2 levels, indicating its cardioprotective property through improvement of apoptosis and myocardial morphology [108]. Administration of butylidenephthalide (9) (i.g., 150 mg/kg, daily) for 4 weeks reversed aging-induced augment in cardiac fibrosis and promoted the phosphorylation and activity of signal transducer and activator of transcription 3 (STAT3) and phosphatidylinositide 3 kinase (PI3K) in rats after myocardial infarction. It also caused an increment in interleukin-10 (IL-10) secretion and the number of M2c macrophage, as well as a descending of myofibroblast infiltration. Hence, its therapeutic activities could be used to relieve aging-triggered myofibroblast disorders during myocardial infarction, through the induction of macrophage differentiation into M2 phenotype under the potentiation of PI3K/STAT3 signaling [12].

3.2. Central nervous system

3.2.1. Protection of blood–brain barrier

Phthalides can protect blood-brain barrier (BBB) from neurogenic disturbances. Z-ligustilide (1) protected the permeability of BBB against OGD-triggered damage via suppressing hypoxia-inducible factor 1-α (HIF-1α), VEGF and AQP4 signaling pathways. Specifically, its treatment alleviated OGD-caused destruction of BBB and improved levels of tight junction proteins concentration-dependently in the co-culture of astrocytes and rat brain microvascular endothelial cells [127]. Butylphthalide (8) at 40 mg/kg prevented BBB disruption in a rat model subjected to cerebral ischemia reperfusion (IR) injury through the mitigation of vascular permeability breakdown by modulating tight junctions-related proteins such as CD-31 and zonula occludens-1 (ZO-1) and suppressing caveolin-1 level. Meanwhile, it effectively alleviated cerebral edema and infarct volume, accompanied with recovered neurological function and blood flow in the brain, thus providing evidence for neuroprotection in ischemic stroke [128].

3.2.2. Cognitive amelioration

Phthalides are capable of alleviating cognitive dysfunction. For example, ligustilide (1) treatment (10 and 20 mg/kg, daily) for 8 weeks was shown to robustly attenuate learning and memory impairment in an aging model of senescence-accelerated mouse prone 8 (SAMP8) mice, mainly via the improvement of neurological degeneration and mitochondrial dysfunction. Specifically, its biological mechanisms were related to dose-dependent diminishment of P-dynamin-related protein 1 expression, and enlargement of mitofusin1 (Mfn1), mitofusin2 (Mfn2), P-5′-adenosine monophosphate-activated protein kinase (AMPK) and adenosine triphosphate levels both in mice and H₂O₂- or rotenone-stimulated HT22 cells. Moreover, it not only showed potent anti-oxidative activity by lowering the release of MDA and SOD, but also prevented neural disorders through restricting neuronal apoptosis and neuro-inflammatory reactions in SAMP8 mice. Hence, these results indicated that ligustilide could be developed as an effective agent for curing neurodegenerative diseases such as Alzheimer's Disease (AD) [129]. Butylphthalide (8) treatment (i.g., 80 mg/kg, daily) for 60 days also remarkably protected the white matter and cognitive function in mice model intervened by CCH, which was linked to the mitigation of memory damage by expanding the brain perfusion and maintaining the white matter integrity, and the maintenance of BBB, endothelial cells and tight junctions. In addition, the activation of astrocytes, inflammation-associated pathways and mitochondrial membrane potential (MMP) release were obstructed during exposure to butylphthalide both in bilateral common carotid artery stenosis (BCAS)-triggered mice and LPS-treated astrocytes [16].

3.2.3. Alzheimer's disease

Phthalides are beneficial for the treatment of Alzheimer's disease (AD). Intragastrical administration of ligustilide (1) (30 mg/kg, daily) for 14 weeks showed neuroprotective effects in double-transgenic (APP/PS1) mice model of AD through preventing neuronal injury caused by amyloid-β, enhancing α-processing in amyloid precursor protein (APP) and anti-aging protein Klotho, as well as scavenging amyloid-β deposition. It further improved the catalytic activity of α-secretase via elevating the expression of a disintegrin and metalloproteinase domain-containing protein 10, along with the inactivation of insulin-like growth factor 1 (IGF-1)/Akt/mTOR pathways as observed in AD mice, HEK293T and SH-SY5Y cells [130]. Zhang et al. reported that butylphthalide (8) treatment (i.g., 15 mg/kg, daily) for 3 months exhibited neuroprotection in APP/PS1 transgenic mice of AD model via the maintenance of synaptic and spine function through obstructing amyloid-β aggregates, attenuating neuroinflammation as well as mediating Wnt/β-catenin signaling. Interestingly, it also raised synapses number and postsynaptic thickness, as well as rebalanced neuronal loss and the spine density in the hippocampus, concomitantly with elevated production of synapse-related proteins such as postsynaptic density protein 95 (PSD95) and synaptophysin, ameliorated amyloid-β accumulation, and suppressed microglial activation in vivo [112]. Oral administration of butylphthalide (p.o., 20 and 60 mg/kg, daily) for 150 days remarkedly prevented neuronal undernutrition of the dendrite and spine, and the synaptic impairment in the cerebrum of APP/PS1 transgenic mice. Additionally, it could promote the expressions of cyclic adenosine monophosphate-response element binding protein (CREB) and CREB-binding protein (CBP), and further activate Nrf2 signaling pathways as well as CBP-related Nrf2 acetylation, along with lowered soluble amyloid-β formation and oxidative injury by decreasing the synthesis and release of ROS in vivo [113].

3.2.4. Parkinson's disease

Butylphthalide (8) could mitigate the development of Parkinson's disease (PD) via the improvement of dopamine neuronal degeneration by restricting microglia-activated inflammatory response. Specifically, dopaminergic apoptosis, behavioral disorders and microglial activation were constrained after the administration of butylphthalide (i.p., 100 mg/kg, daily) for 9 days in mice subjected to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-triggered PD model. Moreover, it also diminished the secretion of inflammation-related factors and the production of NO and ROS in LPS-induced BV-2 cells, and attenuated neurotoxicity caused by microglial activation in the dopaminergic neurons. More importantly, the blockage of MAPK, NF-κB and PI3K/Akt signaling pathways might play a unique role in its anti-inflammatory property [15]. Butylphthalide (i.g., 120 mg/kg, daily for 30 days) also presented neuropathologic alterations and behavioral recovery in LPS-stimulated mouse model of PD via ameliorating dopamine neuronal apoptosis and decreasing the phosphorylation of JNK and MAPK [114]. Chi and his colleagues explored the adipose-derived stem cell therapy combined with butylidenephthalide (9) to alleviate behavioral deficits and dopaminergic neurodegeneration in a mouse model of PD, which were related to recovered motor disorders on a basis of neuronal behavior tests such as beam walking, rotarod and locomotor activity assay, and expanded survival rate of dopamine neurons [131].

3.2.5. Migraine

Intragastric administration of Ligusticum chuanxiong and Cyperus rotundus (4.4 and 6.6 g/kg, daily) for 5 days exhibited significant anti-migraine activities in nitroglycerin-injected rats, mainly via restoring the cerebral blood flow, blunting the transcriptional expressions of calcitonin gene-related peptide (CGRP) and c-Fos protein, modulating the levels of endothelin-1 (ET-1), NOS, NO, 5-hydroxytryptamine (5-HT). Moreover, the UPLC-MS/MS method was established to identify and quantify anti-migraine components such as senkyunolide A (3), 3-butylphthalide (8), Z-ligustilide (1) and Z-butylidenephthalide (9) in the serum and cerebral cortex [19]. Another study also explored the anti-migraine activities of senkyunolide A, 3-n-butylphthalide and Z-ligustilide from herbal combination (6.6 g/kg, daily for 5 days) in nitroglycerin-injected rats through determining migraine-related factors like CGRP, and quantifying these phthalides in various locations, including herbs extract, rat serum and brain cortex by using UPLC-MS/MS [132].

3.2.6. Amyotrophic lateral sclerosis

Oral administration of butylidenephthalide (9) (i.g., 400 mg/kg, daily) significantly extended the survival interval and attenuated the motor neuronal (MN) damage via the inhibition of apoptosis in a SOD1-G93A mouse of ALS, which were related to the suppression of autophagy, neuroinflammation as well as oxidative injury [133]. Another study reported that oral pretreatment with butylidenephthalide at 250 mg/kg not only restored motor performance and MN injury, but also extended the survival time (203.9 ± 18.3 days) in ALS mice. Furthermore, its treatment at 7.5 and 10 μg/mL displayed anti-apoptotic activity in NSC34 G93A-SOD1 neuro-blastoma-spinal cord hybrid cells by lowering the levels of microtubule-associated protein light chain 3-II (LC3-II) and caspase 3, and further recovering cell size and mitochondrial morphology. Hence, these results supported its potential for ALS treatment via inactivating autophagic pathways [134].

3.2.7. Cerebral hemorrhage

Butylphthalide (8) treatment (i.g., 10–25 mg/kg, daily) for 15 days effectively rehabilitated neurological dysfunction in a rat model of ICH via lowering the Longa's motor scores, enlarging the vessel density indicated by CD34, and improving the release of VEGF and Ang-2 in a dose-dependent manner. Therefore, it provided beneficial effects for ICH by enhancing neovascularization and attenuating neurological defects [116]. Another report revealed butylphthalide (i.p., 25 mg/kg) showed preventive potential in ICH rats through the amelioration of inflammatory response and cerebral edema. More specific, it remarkably maintained BBB permeability, reduced brain water content, restrained TNF-α and matrix metalloproteinase-9 (MMP-9) secretions [117].

3.2.8. Cerebral ischemia

Intranasal administration of Z-ligustilide (1) at 12 mg/kg for 3 days protected the rat brain against MCAO-triggered ischemic lesions via the improvement of infarct volume, neurological dysfunction, BBB permeability and brain edema, raising collagen IV, occludin and ZO-1 levels, as well as reducing MMP-2 and MMP-9 expressions. Additionally, the blockage of Nrf2 and Hsp70 signaling pathways could antagonize its prophylactic potential for ischemic stroke [135]. Butylphthalide (8) (p.o., 60 mg/kg) significantly blunted the formation of intercellular adhesion molecule-1 (ICAM-1) and protease-activated receptor 1 (PAR1) in endothelial cells from MCAO mice cerebrum, accompanied with the inhibited cerebral permeability of myeloid cells and the increased blood flow. Butylphthalide also alleviated neurovascular inflammatory response for treating ischemic stroke, whereas its protective actions were reversed via the depletion of Gr1⁺ myeloid cells before the establishment of ischemic mouse model [110]. The intragastric administration of senkyunolide H (42) at 20 and 40 mg/kg was found to dose-dependently reduce neurological deficit scoring, infarct volume as well as neuronal apoptosis in a mouse model of cerebral I/R, accompanied with enhanced PI3K and Akt signaling pathways. On the other hand, its treatment at 100 μM attenuated neuronal apoptosis in OGD-stimulated PC12 cells. Taken together, these findings confirmed its potent anti-inflammatory and anti-apoptotic activities by mediating PI3K and Akt pathways, thereby achieving its neuroprotective effects in cerebral ischemic stroke [136].

3.2.9. Cerebral infarction

Butylphthalide (8) treatment (i.p., 15 mg/kg) remarkedly attenuated cerebral infarction in rats after MCAO by abrogating neuronal apoptosis and inactivating pro-apoptotic pathways such as MAPK, which was relate to the decrement of neurological score and cerebral white region area, and the downregulation of apoptotic rate and MAPK expressions [37]. Butylphthalide at 120 mg/kg also ameliorated neurological dysfunction in CD-1 mice with cerebral infarction by reducing infarct area and brain edema, which was associated with promoted claudin-5, VEGF and GFAP levels and diminished MMP-9 expression [109].

3.2.10. Concussive, diffuse, traumatic cerebral injury

Intraperitoneal injection of butylphthalide (8) at 40 or 60 mg/kg significantly relieved cerebral pathology in rats subjected to concussive head injury (CHI), accompanied with the less extent of BBB disruption and edema generation [118]. Its administration (i.p., 80 and 160 mg/kg) might have prophylactic potential in SD rats suffered from diffuse brain injury via attenuating microcirculation disturbances and lowering BBB permeability and brain edema. Another report demonstrated that butylphthalide (i.p., 100 mg/kg) considerably alleviated neurological lesions, brain edema and neuronal degeneration in ICR mice with traumatic brain injury (TBI) though improvement of oxidative stress and cerebral damage by activating Nrf2/ARE pathways. Additionally, it also maintained MDA content and antioxidant enzymes activity such as SOD and glutathione peroxidase (GPx), and enriched HO-1 and NQO1 levels [120]. Intracerebroventricular injection and then oral delivery of butylphthalide at 100 mg/kg daily for one-week constrained BBB breakdown by enlarging tight junction proteins expressions, as well as suppressed neuronal apoptosis by mediating mitochondrial function in a mouse model of TBI. It also displayed the diminished levels of autophagy-associated proteins such as autophagy related 7 (ATG7), Beclin-1 and LC3-II both in vivo and in vitro; however, rapamycin could dramatically abrogate the neuroprotection [121]. More studies are required to elucidate the exact mechanisms underlying butylphthalide neuroprotection in these cerebral impairments.

3.2.11. Vascular dementia

Butylphthalide (8) could prevent vascular dementia in rats through the improvement of cognitive disorders and neuronal apoptosis via activating PI3K/Akt pathway, which was related to the constrained production of Bax and caspase-3 in the serum and the diminished apoptotic rate of cerebral neurons [137]. Butylphthalide (p.o., 30, 60 and 120 mg/kg, daily for 4 weeks) obviously relieved learning and memory dysfunction and suppressed neurological degeneration in rats subjected to CCH-caused vascular dementia (VD) by lowering the ER stress and activating sonic hedgehog (Shh)/protein patched homolog 1 (Ptch1) pathways. It also promoted the expressions of plasticity-associated synaptic regulators and the formation of Shh/Ptch1 signaling, but downregulated the levels of endoplasmic reticulum (ER) stress-related markers [124]. Another study found its administration (i.v., 5 mg/kg/day, 3 weeks) also attenuated cognitive deficits and neuronal apoptosis in a rat model of CCH-evoked VD by activating Glial cell line-derived neurotrophic factor (GDNF)/GFRα1/receptor tyrosine kinase (Ret) and Akt/ERK1/2 signaling pathways. Moreover, it could restore the Ret inhibitor (NVP-AST487)-induced decrement of GDNF, GFRα1 and Ret levels in the hippocampal neurons, and enhance cellular viability, resulting in the activation of Akt and ERK1/2 phosphorylation, the enlargement of Bcl-2 expression, and the reduction of Bax and cleaved caspase-3 levels [125].

3.2.12. Spinal cord injury

Oral administration of butylphthalide (8) at 80 mg/kg/day for 4 weeks potentiated locomotor abilities, and lowered spinal cord damage cavity area, neuronal cell death and TUNEL-positive cells count in SD rats subjected to traumatic SCI, which was linked to the neuroinflammatory mediation by inactivating TLR-4/NF-κB pathways. On the other hand, it also abolished microglial activation, decreased the release of inflammation-associated cytokines, as well as blocked microglial TLR-4/NF-κB signaling cascade in vivo. Meanwhile, its treatment considerably mitigated LPS-induced cytotoxicity, inhibited microglial activation and inflammatory response in the co-culture of PC12 and BV-2 cells [122]. Butylphthalide (p.o., 80 mg/kg/day, 1 week) remarkedly improved the permeability of blood spinal cord barrier (BSCB) and behavioral functions in a rat model of SCI by lowering ER stress and enhancing adherent junction (AJ) and TJ proteins. In addition, its treatment also inhibited the secretion of ER injury-related proteins and the impairment of TJ and AJ in human brain microvascular endothelial cells [123].

3.3. Hemorheology

3.3.1. Hematopoiesis

The Absorption, Distribution, Metabolism and Excretion (ADME) screening combined with KEGG pathway analysis were conducted to identify senkyunolide A (3) and senkyunolide K (39) from Danggui buxue decoction that might represent hematopoietic progression in rats via blood enriching pathways like PI3K-Akt [13]. The pharmacokinetic investigation on 15 compounds from Xin-Sheng-Hua Granule formula discovered that senkyunolide I (43), senkyunolide H (42), senkyunolide A (3), ligustilide (1) and butylidenephthalide (9) showed remarkable variation between normal and blood deficiency rats, suggesting their potential and possible metabolites in blood enriching activity [138].

3.3.2. Blood circulation

Butylphthalide (8) at 90 mg/kg/day for 7 days markedly ameliorated cerebral lesions in rats subjected to transient MCAO through the inhibition of thrombosis and vasoconstriction by promoting blood circulation and diminishing cerebral infarct and atrophy volume [100]. Another study found that its treatment (i.v, 5 mg/kg) time-dependently improved vascular functions in rats with CCH through restraining the vasoconstriction of bilateral vertebralarteries and expanding diameters, which were related to the mediation of the angiopoietin (Ang) and Tie signaling and the elevation of VEGF and CD34 levels [88].

3.3.3. Antiplatelet aggregation

A novel derivative of butylphthalide (8), namely ferulic acid-glucose trihybrids S2, exhibited a strong anti-platelet activity, with more than 10-fold increase in the suppression of adenosine diphosphate or arachidonic acid-triggered platelet aggregation compared to butylphthalide [139]. A ring-opened derivative originated from butylphthalide carrying NO and hydrogen sulfide (H₂S)-donating moieties 8d dramatically alleviated platelet aggregation caused by adenosine diphosphate in vitro. Furthermore, its treatment (p.o., 80 and 380 mg/kg/day) for 7 days also promoted cardio-cerebrovascular circulation, attenuated brain infarction and edema, and reduced oxidative stress by downregulating MDA content and enlarging SOD and glutathione levels in rats intervened by transient focal cerebral ischemia [140].

3.4. Antioxidant effects

Antioxidation is one the most important activities for natural phthalides. For example, Z-ligustilide (1) at 25 μM remarkably alleviated iron-modulated free radical production, oxidative injury and apoptosis in SH-SY5Y cells after OGD/R, which was associated with the decline of iron exporter protein ferroportin 1 and HIF-1α expressions, and the elevation of ferritin level [141]. Another research found its role in Angelica sinensis extract for mitigating apoptosis and oxidative stress in gills and erythrocytes of fish via inhibiting hemolysis and trichlorfon-stimulated phosphatidylserine exposure. Moreover, its treatment (0.2–0.4 mg/mL) dose-dependently elevated Ca²⁺, superoxide anion and met-haemogolobin contents, enlarged calpain activity, and suppressed oxidative enzyme activities such as SOD, catalase and GPx in the erythrocytes of fish [20].

3.5. Anti-inflammatory effects

Phthalides often presented anti-inflammatory activity in the treatment of diseases. Ligustilide (1) does-dependently suppressed the synthesis and release of inflammatory factors such as iNOS, cyclooxygenase-2 (COX-2), TNF-α and interleukin 6 (IL-6), in nucleus pulposus cells exposed to IL-1β. Its treatment (i.p., 10 mg/kg/day) for 8 weeks could attenuate IL-1β-evoked apoptosis, extracellular matrix degradation and inflammatory response in rats with intervertebral disc degeneration, which was associated with the blockage of NF-κB pathway [142]. Z-ligustilide does-dependently inhibited the production of various inflammation-associated factors, such as NO, COX-2-dependent PGE₂, interleukins, chemokines (CCL-4/macrophage inflammatory protein 1β (MIP-1β)) and pro-inflammatory enzyme (iNOS) in LPS-stimulated murine RAW 264.7 macrophages, accompanied with the diminished expressions and transcriptional levels of pro-inflammatory cytokines (IL-1α, IL-6 and TNF-α), as well as the restricted release of differentiation factors GM-CSF. Consistently, its treatment at 25 μM also abrogated the secretion of inflammation-related proteins, such as CCL-2/MCP-1, CCL-3/MIP-1α, CCL-4/MIP-1β, C-X-C motif chemokine ligand 10/interferon gamma-induced protein 10, and IL-12p70 in LPS-induced human THP-1 cells and peripheral blood leukocytes. More importantly, anti-inflammatory activity of Z-ligustilide was closely related to the inactivation of NF-κB pathway and the downregulation of inflammatory genes, suggesting its potential to ameliorate acute or chronic inflammatory responses in immunocytes [14].

3.6. Analgesic effects

Phthalides isolated from plants possess anti-nociceptive effects. For instance, intravenous administration of ligustilide (1) at 60 mg/kg markedly alleviated hyperalgesia and inflammatory pain triggered by Complete Freund's adjuvant (CFA) in rats through blocking JNK/c-Jun phosphorylation [143]. Another study reported that its intragastric administration of at 120 mg/kg exhibited anti-mechanical allodynia effects on CFA-caused inflammatory pain in SD rats by restraining spinal ERK1/2 and MAPK signaling, and inhibiting pro-inflammatory cytokines production such as TNF-α and IL-1β [144].

3.7. Anti-cancer effects

Anti-cancer properties of botanical drugs may be attributed to the presence of phthalides. The treatment with Z-ligustilide (1) (50–200 μM) attenuated apoptosis in OVCAR-3 ovarian cancer cells by does-dependently decreasing oxidative damage and modulating mitochondrial function. Meanwhile, it also mediated transcription factor Nrf2 and downstream pathways, but Nrf2 knockdown resulted in the ascending outcome of cell death [145]. Liguistilide and senkyunolide A (3) (18 and 1 μM, respectively) might represent the benefits of Si-Wu-Tang formula in Nrf2 signaling activation against carcinogenesis and display anti-mutagenic activity under mutagen 2-nitrofluorene. Interestingly, their treatment does-dependently protected JB6 P+ cells from epidermal growth factor (EGF)-triggered neoplastic biotransformation possibly via restricting neoplasm-related transcription factor AP-1 expression [146].

3.8. Anti-fibrosis effects

Phthalide compounds are found to show anti-fibrosis properties in recent research. Treatment with ligustilide (1) (i.p., 20, 40 and 80 mg/kg/day, for 4 weeks) protected the lung functions in a rat model of bleomycin-triggered pulmonary fibrosis via dose-dependently potentiating ventilation, restraining hyperplasia and fibrotic progression, as well as diminishing transforming growth factor-β (TGF-β), fibronectin, and α-SMA levels. Moreover, it also alleviated oxidative injury and apoptosis through obstructing TLR4/myeloid differentiation primary response 88 (MyD88)/NF-κB p65 pathways, accompanied with adjusted serum lactate dehydrogenase and tissue SOD activities, lowered caspase-3, Bax and Bcl-2 levels, recovered ratio of Th1/Th2 in peripheral blood mononuclear cells, as well as reduced iNOS and IL-10 release in the serum and lung [147]. Butylidenephthalide (9) as an active phthalide from Angelica sinensis was reported to rebalance myofibroblast activities such as collagen contractile and migration capacities in oral submucous fibrosis via interfering the binding of Snail to α-SMA. Meanwhile, it also does-dependently reduced the expressions of epithelial-mesenchymal transition-associated markers including Twist, Snail and Slug, as well as caused significant decrement in myofibroblast markers like α-SMA and fibronectin in fibrotic buccal mucosal fibroblasts [148].

3.9. Organ protection

Phthalides are reported to prevent renal and gastrointestinal impairment. Z-ligustilide (1) from Ligusticum chuanxiong extract could blunt oxidative injury and inflammatory responses by dose-dependently initializing Nrf2 signaling and blocking NF-κB pathway in vitro, and further attenuated renal lesions and fibrosis in a mouse model of diabetic nephropathy caused by streptozotocin [149]. Butylidenephthalide (9) occupied 20.54 % of Ligusticum porter essential oils, might contribute to gastrointestinal protection against indomethacin-caused gastric injury, which was related to the maintenance of H₂S gastric contents [150].

3.10. Bone-related protection

Phthalides can alleviate the progression of bone-related disorders. Ligustilide (1) at 5 and 10 μM considerably inhibited apoptosis and extracellular matrix degradation in nucleus pulposus cells exposure to IL-1β; furthermore, its administration (i.p., 10 mg/kg/day, for 8 weeks) could lead to the regression of intervertebral disc degeneration in rats [142]. Ligustilide at 25 and 50 μM prevented chondrocytes from sodium nitroprusside (SNP)-induced apoptosis by dose-dependently abolishing cytoskeletal remodeling, decreasing cleaved caspase-3 and Bax levels, as well as elevating Bcl-2 content. Moreover, its intra-articular injection (30 μL of 75 and 150 μM) attenuated cartilage degeneration via enhancing anti-apoptotic signaling and restraining JNK and MAPK pathways in vivo [151]. Ligustilide treatment at 10 μM remarkable recovered prednisolone-suppressed bone formation and ameliorated osteoporosis in zebrafish by activating G-protein coupled receptor 30 (GPR30) signaling and epidermal growth factor receptor (EGFR) and ERK1/2 pathways. In addition, it also does-dependently enhanced the differentiation of pre-osteoblastic cell line MC3T3-E1 and bone marrow mesenchymal stem cells, and protected MC3T3-E1 cells from H₂O₂-evoked apoptosis via augmenting Bcl-2 level [152].

3.11. Anti-bacterial and anti-fungal effects

On note, anti-bacterial and anti-fungal activities are important properties of phthalides. The chemometric study coupled with GC-MS analysis demonstrated that ligustilide (1) might be responsible for anti-candidal property via elevating selectivity index, indicating its great importance in the composition of synergistic anti-candidal substances [153]. Against uropathogenic Escherichia coli, Grube et al. discovered sedanenolide (16) and ligustilide with IC₅₀ values of 790 and 611 μM, respectively, which suggested a novel phytotherapeutic strategy to prevent bacterial urinary tract infections [154].

4. Pharmacokinetics of phthalides

The pharmacokinetic research of plant-derived phthalides has concentrated on ligustilide (1), senkyunolide I (43) and butylphthalide (8), which represented as critical markers in the quality control of botanical drugs.

4.1. Absorption

Owing to gastrointestinal microenvironments, the oral bioavailability (BA) of active phytochemicals is easily altered via biological transformations. Therefore, except for intragastric route, intravenous and intranasal administration, intraportal and intraduodenal permeability trials were performed to obtain better absorption profiles of these medicative phthalides [155,156]. After intragastric administration of Shunaoxin pills, ligustilide (1) was sharply absorbed as achieving highest concentration within 8.7 min. According to pharmacokinetic parameters, α and β values were shown as 0.07 min⁻¹ and 0.002 min⁻¹, supporting its fast distribution and slow clearance. The large value of V (43.59 ± 37.32 L) also indicated its rapid diffusion from plasma to tissue and higher drug concentration in tissues as compared with that of plasma due to strong lipophilicity [156]. Interestingly, rats with blood stasis symptoms displayed elevated values of C_max, AUC_0-t, and AUC₀-_∞, as well as reduced values of MRT_0-t and T_1/2z, which implied the potentiated time and extent of ligustilide absorption [155]. After oral administration of herb pair Angelica sinensis-Carthami flos, the blood stasis rats upregulated C_max, AUC_0-t, and AUC_0-∞, and decreased T_1/2z and MRT_0-t in the pharmacokinetic studies of butylphthalide (8). Since CYP2E1, 2C11 and 3A1/2 expressions were tightly related to the metabolic catalyzation of butylphthalide in vivo, this herb pair was confirmed to affect their mRNA expressions, resulting in dramatical differences between T_1/2z and AUC_0-t values of such a constitute [155].

4.2. Tissue distribution

Ligustilide (1) could easily penetrate the BBB, and showed an ascending in transportation ratio over time, which might contribute to the fast onset of therapeutic actions. After intragastric administration of Shunaoxin pills, the concentration of ligustilide in brain was estimated to be 0.4 μg/g wet tissue within 5 min and maintained at 0.2 μg/g wet tissue for a total of 4 h [156]. After intravenous administration of senkyunolide I (43) at 36 mg/kg, it was distributed quickly and short-term existed in the extra-vascular tissues, with the ascending order of AUC being spleen, thymus, heart, brain, lung, liver, and kidney. The AUC value of kidney was 259 % of that of plasma, referring that kidney was the primary excretion organ of prototype senkyunolide I. More importantly, the AUC value in the cerebrum was 77.9 % of that in the plasma, indicating that senkyunolide I could across the BBB [157].

4.3. Metabolism

Metabolic research aims to identify bioactive phthalides from plant species, their potential metabolites, and relevant metabolic mechanisms. Since pharmacological properties are generally believed to be obtained after the absorption of herbal ingredients, their bio-transformations appears to be of great importance in the efficacy. Seven metabolites of ligustilide (1), such as butylidenephthalide (9), senkyunolide H (42) and senkyunolide I (43), were obtained on the basis of HPLC/MS analysis (Fig. 7a), and its potential metabolic mechanisms in rats were involved in direct oxidation, aromatization, hydration and glutathione conjugation pathways [158]. After oral administration of senkyunolide I (43) (36 mg/kg), 9 possible metabolites were discovered in rat bile by the application of Liquid Chromatographic-Mass Spectrometry (LC-MS)/MS technique (Fig. 7b). Possible metabolic pathways in vivo primarily included methylation, glucuronidation and glutathione conjugation [157].

Fig. 7.

The proposed metabolic pathways of Ligustilide (a) and Senkyunolide I (b).

5. Conclusion and future perspectives

The research progress of plant-derived phthalides regarding phytochemistry, pharmacology, toxicology, and clinical application was integrated and discussed in the review. For a comprehensive understanding of plant-derived phthalides in perspective of modern medical development, scientific gaps should be fulfilled with current information. Several critical points also need to be discussed for the direction of future studies.

This work scientifically reviews a total of 133 plant-derived phthalides. These phthalides have been identified in more than 200 plant species that belong to 23 families [1]. From current works and literature investigations (Table 1), natural phthalides are primarily identified from the genus Angelica, Apium, Cnidium and Ligusticum in the Umbelliferae family. Roots are often used in the extraction of active ingredients, as well as seeds reported in few research [21]. More importantly, Angelica sinensis and Ligusticum chuanxiong are two of critical phthalide-containing herbs. A series of phthalide compounds such as Z-ligustilide (1), senkyunolide A (3), butylphthalide (8), Z-butylidenephthalides (9), neoligustilide (17), senkyunolide H (42) and I (43), Z,Z′-3a.7a′,7a.3a′-dihydroxyligustilide (102), triligustilides A-B (130–131) were isolated and reported from the rhizomes of Angelica sinensis [9,30,44,59,79]. Meanwhile, Z-ligustilide (1), cnidilide (18), 4-hydroxy-3-butylphthalide (33), 3-butylidene-6-hydroxy-5,6-dihydro-butylphthalide (41), levistolide A (84), chuanxiongdiolide R4-R5 (120–121) were also identified from the roots of Ligusticum chuanxiong [18,30,45,57,62,72,80]. And the seeds of Apium graveolens is also reported as an abundant source of natural phthalides like compounds (7, 8, 13, 23, 77–78). Moreover, some other plants sources include Ligusticum acutilobum [29,47], Levisticum officinale [33,38,54], and Ligusticum wallichii [3,61]. Taken together, Angelica sinensis and Ligusticum chuanxiong shared considerable similarity in chemical components, particularly phthalide monomers [30,32]. However, phthalides displayed distinct variation in natural distribution, such as 3-(3-Methylbutylidene) phthalide (12) and sedanolide (13) in Apium graveolens [39,40], senkyunolide K (39) and wallichilide (116) in Ligusticum wallichii [3,61], chuanxiongdiolide R1 (99) and R4 (120) in Ligusticum chuanxiong [77,80], angesinenolides E and diangeliphthalide A (118, 119) in Angelica sinensis [10,76]. Monomeric phthalides are usually distributed in many plants without specificity, but hydroxy phthalides and polymers present in some special herbs. Influence on the natural distribution of phthalides may be attributed to their biogenesis in specific plants. The results above possibly show a near relationship among these plant species, with the family Apiaceae being of significance. Nevertheless, it is difficult to draw the conclusive characteristics of phthalide distribution at current backgrounds. Therefore, the taxonomy of phthalide-containing plants necessitates further investigations.

There were few studies that simply investigate the biogenesis of plant-derived phthalides. The generation of dimeric phthalides was possibly based on the cycloaddition reactions of monomers, and hydroxyl substitutions are formed via oxidation [60]. For instance, compounds 89–91 were composed of two Z-ligustilide (1) through Diels-Alder reaction. Z-ligustilide was transformed to 3,9-dihydroxy-ligustilide (37) by further oxidation and H₂O addition. Furthermore, Deng et al. hypothesized the biosynthetic pathways of ansaspirolide (94) and sinaspirolide (114) that were derived from the combination of Z-ligustilide and Z-butylidenephthalide (9) by π2s cycloaddition and Diels-Alder reaction, respectively [46]. 10-angeloyl-3-butylphthalide (19) was thought to be a potential biosynthetic metabolite that could be incorporated into wallichilide (116) [159]. Recently, the discovery of polymers with new linkages promotes the diversity of natural phthalide family and obtains more interest from synthetic and pharmacological properties for further research. Chuanxiongdiolide R4 (120) was originated from type A cycloaddition of Z-ligustilide and senkyunolide A (3) [80], while three Z-ligustilide connected via cycloaddition of type A plus B for formation of triligustilide A-B (130–131) [9]. And it is also worth noting that monomeric phthalides derived from polyketide biogenetic pathways [28]. Whereas Zou and colleagues uncovered the possible modes of spontaneous formation of polymers such as triangeliphthalides A–D (128–129, 132–133) based on time-dependent density functional theory (TD-DFT) calculations [10]. On the other hand, the chemical synthesis of phthalides provides novel diverse derivatives with more potent bioactivities and low toxicity. For example, Fan et al. designed and synthesized new phthalide derivatives through bromine substitution and etherification for improving antifungal potential [160]. Brimble and Rathwell reviewed phthalide anion annulation reactions for the synthesis of quinoid natural products [161]. The strategy of chemical synthesis combined with biogenesis has promoted the development of innovative botanical drugs. So far, these phthalide polymers linked two or three achiral monomers via cycloaddition show the racemic features. However, as for the phthalide-like constitutes isolated from plants, their corresponding optically pure racemates have not yet been obtained, and absolute configurations are also not established in the work. Consequently, plausible biogenetic pathways of phthalide compounds in this class are still unknown, which needs further studies to validate.

Only few literatures reported the structure activity relationship of natural phthalides. For example, Chen and colleagues comparatively evaluated the anti-inflammatory activities of 31 novel synthetic phthalides derivatives, and found one promising compound that exhibited 95.23 % inhibitory rate at 10 mM with IC₅₀ value of 0.76 mM against LPS-caused NO elevation and regulated inflammation-related pathways including Nrf2/HO-1 and NF-kB/MAPK [162]. Another study performed by Tang et al. discussed the relationship between vasodilatory activity and phthalides including monomers, dimers and enantiomers. Results from this work indicated that polymerization position and enantiomeric configuration showed remarkable impacts on vasorelaxation [80]. Here, the information on reported relationships between phthalide structures and activity provide innovative leads for the design and development of new drugs against neurogenic complications. The compound library therefore requires to be further enriched.

Pharmacological evaluation of natural phthalides has been conducted in vivo and in vitro studies, such as different bioactivities of ligustilide (Fig. 5) and moderating effects of phthalide compounds on hemorheology (Fig. 6). Nevertheless, their molecular mechanisms towards therapeutic properties for various complications are still unknown. Furthermore, it is challenging to establish the intimate relationship between compounds and bioactivities due to multiple potential targets and pathways involved in the pathogenesis of diseases. One thing needs to note is that phthalides need to be individually investigated for therapeutic effects when positive results obtained from plant extract containing phthalide in animal model. To ensure safety, high dose or long-term medication in trials should also be concerned.

Fig. 5.

Schematic diagram suggesting partial pharmacological effects of ligustilide. (a), ligustilide inhibits aging-induced cognitive impairment via modulating mitochondrial-associated disorders in SAMP8 mice [129]. (b), ligustilide protects BBB from OGD-triggered ascending of permeability mainly through the potentiation of tight junction proteins (ZO-1 and occludin) and the blockage of HIF-1α/VEGF signaling pathways [127]. (c), ligustilide attenuates CFA-stimulated mechanical hyperalgesia by the downregulation of JNK/c-Jun pathways in rat model of inflammatory pain [143]. (d), ligustilide enhances autophagic response and suppresses apoptosis caused by OGD/R in PC12 cells, which are related with the activation of LKB1/AMPK/mTOR signaling pathways [163].

Fig. 6.

Possible pharmacological actions of phthalides on hemorheology.

Toxicology of natural phthalides attracted relatively few attentions. Li et al. evaluated the toxicity of potassium 2-(1-hydroxypentyl)-benzoate derived from butylphthalide in beagle dogs (i.v., 108 mg/kg/day, 4 weeks), along with 3 weeks period for recovery. This derivative was quickly metabolized into butylphthalide in the plasma based on UPLC-UV method. However, experimental animals were found a slight to moderate behavioral toxicity during delivery and recovered to normal status until the end of intervention. Significant alterations in blood hematological profiles such as elevated NEUT and TG levels, as well as reduced LYM content were observed in treated dogs, but these abnormalities were restored during recover stage [164]. Zhang and colleagues investigated the safety of ligusticum cycloprolactam modified from ligustilide (1) in KM mice. The weight of all animals gradually raised and reached the same after 14 days, but the weight gain of administration group at doses of 2.0, 3.5 and 5.0 g/kg was lower than that of the normal group. Moreover, the results from organ coefficients based on the weight of heart, liver, spleen, lung and kidney showed no significant difference in all mice. And no histological damage was observed in tissue sections of these organs. Experimental mice exhibited good condition after acute toxicity tests; thus, this derivative was proved to be less toxic or non-toxic [165]. As toxicology findings on natural phthalide is still inadequate, further investigations on the toxicity involved in drug dosage, animal selection, test duration, diverse assessment and possible mechanisms are of great importance in the safety for future clinical applications.

Clinical research was also evaluated comparatively for practical effectiveness, safety and tolerability in patients. Among these natural phthalides, butylphthalide (8) had extensive utility for the treatment of cardio-cerebrovascular disorders and neurological diseases. For instance, its administration (200 mg/capsule, three times daily) for 4 weeks was observed to show beneficial effects on cerebral hypoperfusion in patients with carotid atherosclerotic stenosis by enlarging the regional cerebral blood flow (rCBF) in 4 regions of interest (ROIs) in 48 patients (83.5 % ± 11.4 % vs. 85.8 % ± 12.5 %). Moreover, butylphthalide-treated group exerted higher proportion of ROIs with increased rCBF in the territory of middle cerebral artery (MCA) in contrast to the placebo group (93.1 % vs. 79.2 %) and had greater number of patients who experienced recovered CBF in ipsilateral MCA (Wald-χ2 = 5.247, OR = 3.31) [166]. In another clinical study, butylphthalide administration (25 mg, twice daily) for 7 days ameliorated perioperative neurological deficits after revascularization surgery in 49 patients with moyamoya disease, as signified by less transient neurological deficit, higher disability-free recovery rate, as well as greater number of patients with improved neurological functions [167]. Butylphthalide at a dose of 200 mg three times daily for 24 weeks was found to relieve cognitive impairment in 140 patients with subcortical vascular cognitive impairment without dementia, as verified by the differences in AD assessment scale-cognitive subscale (butylphthalide group −2.46 vs. placebo group −1.39). Additionally, clinician's interview-based impression of change and caregiver input consistently reflected the promoted recovery of cerebral functions, with 80 (57.1 %) vs. 59 (42.1 %) subjects in butylphthalide or placebo group. Notably, mild gastrointestinal symptoms were usually observed as uncommon adverse events caused by butylphthalide [168]. Considering the difference of diseases, patient population, and study design in these clinical applications, the obtained results need to be interpreted with caution. Besides, an important association between drug administration and risk of adverse events should be elucidated in detail. Prospective studies with large sample size and long follow-up duration to verify clinical efficacy of phthalides are essential in future research.

Phthalides with characteristic structure and biological activities become one class of promising chemical scaffold. Moreover, their derivatives synthetized from chemical modification have shown greater therapeutic effects and less toxicity in animal and cell models, which has been developed to be a new trend for modern use of natural products. In recent years, plant-derived phthalides has been further investigated and reported with growing global attentions for biological variability, phytochemistry and pharmacology. Owing to huge advances in new trimeric structures, therapeutic bioactivities and potential mechanisms, in-depth studies with respect to comparisons of phthalides will renew current knowledge and provide a comprehensive reference for future medicinal exploitation and clinical application.

Data availability statement

Data will be made available on request.

CRediT authorship contribution statement

Yulong Chen: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review & editing. QingZhou Cheng: Project administration, Software, Supervision. Site Lv: Formal analysis, Methodology, Software. Zhen Kang: Formal analysis, Resources, Software, Validation, Visualization. Shan Zeng: Funding acquisition, Project administration, Resources, Supervision.

Declaration of competing interest

.The authors declare no competing interests.

Abbreviations

α-SMA

α-smooth muscle actin

AD

Alzheimer's Disease

ADME

Absorption, Distribution, Metabolism and Excretion

AJ

Adherens junction

Akt

Protein kinase B

ALS

Amyotrophic lateral sclerosis

AMPK

5′-adenosine monophosphate-activated protein kinase

Ang

Angiopoietin

AP-1

Activator protein-1

APP

Amyloid precursor protein

ApoE

Apolipoprotein E

ARE

Antioxidant response element

ATG7

Autophagy related 7

Bax

Bcl-2 associated X protein

BA

Bioavailability

BBB

Blood-brain barrier

BCAS

Bilateral common carotid artery stenosis

BCCAO

Bilateral common carotid artery occlusion

Bcl-2

B-cell lymphoma-2

BDNF

Brain derived neurotrophic factor

BSCB

Blood spinal cord barrier

cAMP

cyclic adenosine monophosphate

cPLA2

cytosolic phospholiapase A2

Caspase-3

cysteinyl aspartate specific proteinase-3

CAM

Cell adhesion molecule

CBF

Cerebral blood flow

CBP

CREB-binding protein

CCH

Chronic cerebral hypoperfusion

CCL-2

Chemokine C–C motif-2

CD34

Cluster of differentiation 34

CFA

Complete Freund's adjuvant

CGRP

Calcitonin gene-related peptide

CHI

Concussive head injury

CI

Cerebral infarction

COX-2

Cyclooxygenase-2

CREB

Cyclic adenosine monophosphate-response element binding protein

eNOS

endothelial nitric oxide synthase

EGF

Epidermal growth factor

EGFR

Epidermal growth factor receptor

ET-1

Endothelin-1

ER

Endoplasmic reticulum

ERK

Extracellular signal-regulated kinase

ERK1/2

Extracellular signal-regulated kinase 1/2

GC

Gas chromatography

GC-MS

Gas chromatography mass spectrometry

GDNF

Glial cell line-derived neurotrophic factor

GFAP

Glial fibrillary acidic protein

GFRa1

GDNF family receptor alpha-1

Glu

Glutamate

Gly

Glycine

GO

Gene Ontology

GPx

Glutathione peroxidase

GPR

G-protein coupled receptor

5-HT

5-hydroxytryptamine

H₂S

Hydrogen sulfide

H₂O₂

Hydrogen peroxide

Hsp70

Heat shock protein 70

HFD

High fat diet

HIF-1α

Hypoxia-inducible factor 1-α

HO-1

Heme oxygenase 1

HPLC

High performance liquid chromatography

HUVECs

Human umbilical vein endothelial cells

i.g.

intragastrical

i.p.

intraperitoneal

i.v.

intravenous

iNOS

inducible nitric oxide synthase

ICH

Intracerebral hemorrhage

IGF-1

Insulin-like growth factor 1

IR

Ischemia reperfusion

ICAM-1

Intercellular adhesion molecule-1

LC-MS

Liquid Chromatographic–Mass Spectrometry

LDLR

Low density lipoprotein receptor

IL-1β

Interleukin 1β

IL-6

Interleukin 6

IL-10

Interleukin-10

JNK

c-Jun N-terminal kinase

Keap1

Kelch-like ECH-associated protein 1

KEGG

Kyoto Encyclopedia of Genes and Genome

LC3-II

Microtubule-associated protein light chain 3-II

LKB1

Liver kinase B1

LPS

Lipopolysaccharide

mTOR

mammalian target of rapamycin

MAPK

Mitogen-activated protein kinase

MCA

Middle cerebral artery

MCAO

Middle cerebral artery occlusion

MCP-1

Monocyte chemoattractant protein-1

MDA

Malondialdehyde

Mfn

Mitofusin

MIP

Macrophage inflammatory protein

MMP

Mitochondrial membrane potential

MMPs

Matrix metalloproteinases

MN

Motor neuronal

MPP⁺

1-methyl-4-phenylpyridinium

MPTP

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

MWM

Morris water maze

MyD88

Myeloid differentiation primary response 88

NADPH

Nicotinamide adenine dinucleotide phosphate

NF-κB

Nuclear factor- κB

NLR

Nod-like receptor

NLRP3

NLR family pyrin domain containing 3

NO

Nitric oxide

NOX

Nicotinamide adenine dinucleotide phosphate oxidase

Nrf-2

Nuclear factor erythroid 2-related factor 2

OGD/R

Oxygen-glucose deprivation/reoxygenation

p.o.

per os (oral gavage)

PAR1

Protease-activated receptor 1

PD

Parkinson's disease

PDE

Phosphodiesterase

PDGF

Platelet-derived growth factor

PGC-1α

Peroxisome proliferators-activated receptor-γ coactivator-1α

PGE₂

Prostaglandin E₂

PI3K

Phosphatidylinositide 3 kinase

PLCλ

Phospholipase Cλ

PS1

Presenilin-1

PSD

Postsynaptic density

PSD95

Postsynaptic density protein 95

Ptch1

Protein patched homolog 1

rCBF

Regional cerebral blood flow

Ret

Receptor tyrosine kinase

ROCK

Rho-associated coiled-coil-containing protein kinase

ROIs

Regions of interest

ROS

Reactive oxygen species

RYRs

Ryanodine receptors

Shh

Sonic hedgehog

SAMP8

Senescence-accelerated mouse prone 8

SCI

Spinal cord injury

SNP

Sodium nitroprusside

SOD

Superoxide dismutase

Syk

Spleen tyrosine kinase

STAT3

Signal transducer and activator of transcription 3

t-BHP

tert-butyl hydroperoxide

Tie-2

Endothelial cell specific tyrosine kinase receptor-2

TBI

Traumatic brain injury

TCM

Traditional Chinese medicines

TGF-β

Transforming growth factor-β

TJ

Tight junction

TLR-4

Toll-like receptor-4

TNF-α

Tumor necrosis factor-α

TRK-B

Tropomyosin receptor kinase B

TRP

Transient receptor potential cation channel

TXA2

Thromboxane A2

VCAM-1

Vascular cell adhesion molecule 1

VD

Vascular dementia

VDCCs

Voltage-operated Ca2+ channels

VEGF

Vascular endothelial growth factor

VGlut-1

Vesicular glutamate transporter-1

VSMCs

Vascular smooth muscle cells

ZO-1

Zonula occludens-1