Study on chemical constituents and some bioactivities of lumnitzera littorea (combretaceae) and bruguiera cylindrica (rhizophoraceae) growing at can gio mangrove forest ho chi minh city

LITERATURE REVIEW 1.1 LUMNITZERA GENUS – BOTANIC DESCRIPTION

Lumnitzera racemosa Willd

Lumnitzera racemosa is a medium-sized, evergreen tree native to eastern tropical Africa, Madagascar, South Asia, Malaysia, Southeast Asia, southern China, northern Australia, and Polynesia, but is scarce along the Indian Ocean coasts of Southeast Asia This tree can reach heights of up to 10 meters, featuring grey, fissured bark in older specimens Its leaves are small, measuring 3–7 cm long and 2–3 cm wide, with a succulent, obovate shape and slightly wavy margins The flowers are small and erect, characterized by a green, tube-like calyx with five lobes and five white petals arranged alternately to the sepals The reproductive structure includes ten free stamens organized in two whorls The tree does not exhibit vivipary or cryptovivipary Its fruit is vase-shaped, 1–2 cm long, yellowish-green, glossy, corky, buoyant, and dispersed by water currents, containing a single ovoid-oblong seed.

Lumnitzera littorea (Jack) Voigt

Lumnitzera littorea is a native mangrove tree found from East Africa to tropical Asia, extending throughout Southeast Asia to northern Australia and Polynesia It is recorded in countries such as Myanmar, Cambodia, Thailand, Vietnam, Malaysia, Singapore, the Philippines, East Timor, Brunei, Indonesia, and Papua New Guinea, although it is rare along the Java Sea coasts in Indonesia This tree can grow up to 25 meters tall, featuring slender, knee-shaped pneumatophores and dark brown, fissured bark with a trunk diameter of up to 50 cm Its leaves are slightly fleshy and leathery, narrowly obovate-elliptic, measuring 2–8 by 1–2.5 cm, and are typically clustered at the twig ends The red, bisexual flowers are strongly scented, abundant in nectar, and occur in terminal clusters, with flower stalks up to 3 mm long and stamens that are twice the length of the petals.

The plant features compressed structures measuring 8–12 mm in length, with two ovate leaflets at the base, each 1 mm long Its calyx lobes are broadly ovate and also measure 1 mm The corolla lobes are elliptic and smooth, ranging from 4–6 mm in length and 1.5–2 mm in width Additionally, the fruit is ellipsoidal, somewhat corky, slightly compressed, ribbed, and measures between 9–20 mm in length and 4–5 mm in width.

Figure 1.2 Lumnitzera littorea (Jack) Voigt.

BRUGUIERA GENUS – BOTANIC DESCRIPTION

Bruguiera is the largest genus in the family Rhizophoraceae which extends from

East Africa to Australia and the West Pacific

In Vietnam, there are four species belonging to the Bruguiera genus, which include Bruguiera cylindrica, Bruguiera gymnorrhiza, Bruguiera parviflora, and Bruguiera sexangula, as noted by Pham Hoang Ho.

1.2.1 Bruguiera sexangula (Lour.) Poir (Figure 1.3)

Tree to 20 m, often buttressed, bark light brown to grey Leaves elliptic or oblong- elliptic, acute; lamina 8–16 cm long, 3–6 cm wide, flat; petiole usually reflexed, 1.5–

The plant features stipules measuring 3.5 to 4 cm in length, displaying a green or yellow-green hue Its solitary flowers are approximately 3 cm long at anthesis, with pedicels ranging from 6 to 12 mm The hypanthium is about 1.5 cm long and distinctly ribbed There are 10 to 12 linear sepals that vary in color from yellow to rusty brown The petals, measuring 1.5 cm long, are 2-lobed and pubescent at the base and along the margins, with reflexed tips Each sinus typically contains one bristle, with additional bristles often found near the tips of the lobes The anthers are 4 to 5 mm long and lack a mucro The hypocotyl is somewhat angular, measuring between 6 to 12 cm long and 1.5 to 2 cm wide, tapering at both ends.

Figure 1.3 Bruguiera sexangula (Lour.) Poir

The tree can reach heights of up to 20 meters, featuring dark grey bark and abundant knee roots Its leaves are elliptic and acute, measuring 8–20 cm in length and 3–9 cm in width, with petioles that are 2–4 cm long and often reddish stipules The flowers are solitary, approximately 3.5 cm long at anthesis, with a recurved pedicel measuring 1.0–2.5 cm and showing red coloration on at least one side The hypanthium is often reddish and may be ribbed or indistinctly ribbed near the top It has 10–16 linear-subulate sepals that are thick and range in color from pale pink to bright red The petals are about 1.5 cm long, 2-lobed, white turning brown, with pubescent margins and base, and soft bristles at the lobe tips and in the sinus The anthers are approximately 4 mm long and mucronate.

25 cm long, c 1.5 cm wide, straight, terete or slightly ribbed, rounded at apex [13]

The tree can reach heights of up to 20 meters and features small stilt roots at its base, with abundant knee roots and grey bark Its leaves are elliptic and acute, measuring 5–17 cm in length and 2.5–8 cm in width, with a petiole length ranging from 1 to 4.5 cm and stipules of 2.5–3.5 cm The inflorescence consists of three flowers, with a 5 mm long peduncle and 2 mm long pedicels, displaying cream to green flowers The hypanthium measures 4–6 mm in length and 2 mm in width, accompanied by typically eight reflexed sepals that are at least half the length of the hypanthium The petals are 3–4 mm long, sparsely hairy along the margins, with lobes approximately 0.5 mm long and significantly longer apical bristles Anthers are 0.5 mm long, shorter than the filaments, and taper upwards, while the fruit measures 10–.

12 cm long Hypocotyl 8–15 cm long, 5 mm wide, usually curved, not ribbed [13]

1.2.4 Bruguiera parviflora (Roxb.) W.& Arn ex Giff (Figure 1.6)

The tree can grow up to 20 meters tall and features abundant knee roots with smooth, grey bark Its leaves are elliptic and acute, measuring between 4 to 13 cm in length and 2.5 to 4 cm in width, with a petiole length of 1.5 to 2 cm and stipules approximately 4.5 cm long The inflorescence consists of 3 to 10 flowers on a peduncle about 2 cm long, with pedicels ranging from 6 to 13 mm The flowers are yellow-green, and the hypanthium measures 7 to 9 mm in length, displaying slight to distinct ribbing.

The fruit measures 1–2 cm in length, with a hypocotyl ranging from 7 to 20 cm long and 5 mm wide, exhibiting a non-ribbed structure that detaches along with the fruit The persistent fruit wall remains attached to the seedling, with the sepals oriented downwards The erect petals, measuring 1.5–2 mm long and featuring bearded edges near the base, have lobes approximately 0.5 mm long, each adorned with three terminal bristles and one in the sinus, which are similar in length to the lobes Additionally, the anthers are oblong and noticeably shorter than the filaments.

Figure 1.6 Bruguiera parviflora (Roxb.) W.& Arn ex Giff

LUMNITZERA GENUS – CHEMICAL STUDIES

There are a lot of studies on chemical components of differrent parts of

Lumnitzera racemosa Its constituents can be divided into some major groups: triterpenoids, flavonoids, tannins, lignans, phenolics, steroids and other compounds

In 1980, Majumdar and Patra [14] characterized some compounds from leaves and barks of the Indian L racemosa as triacontanol (1), taraxerol (2), -amyrin (3), betulin (4), -sitosterol (5) and friedelin (6)

In 1993, Ta Chen Lin et al isolated eleven tannins from the leaves of Taiwanese L racemosa, including castalagin, corilagin, chebulagic acid, chebulinic acid, and punicalagin Notably, castalagin, corilagin, and chebulinic acid emerged as promising candidates for antihypertensive compounds, highlighting their potential health benefits.

Moreover, from stems and leaves of the Chinese Beihai L racemosa ten compounds were identified, including 2-methyl-1,3-dihydroxy-5-tridecylbenzene

(18), 1,3-dihydroxy-5-undecylbenzene (19), ergosta-7,22-dien-3-ol (20), emodin

(21), kaempferol (22), quercetin (23), quercitrin (24), isoquercitrin (25), (-)- epigallocatechin (26) and gallic acid (27) [14]

In 2003, Ammanamanchi et al [16] isolated a new aromatic ester, 3-(4- hydroxyphenyl)-propyl 3-(3,4-dihydroxyphenylpropionate) (28) with betulinic acid

(29), betulin (4) and friedelin (6) from the stems of plants growing in Bhiravapalem Indian

In 2010, Lisette D’Souza et al [17] reported that twigs of L racemosa contained many flavonoid derivatives and biflavonoids, namely quercetin (23), quercitrin (24), quercetin 3-O-glucoside (isoquercitrin) (25), myricetin (30), quercetin 3-O-

11 galactoside (31), kaempferol 4-methyl ether (32), kaempferol 3,4-dimethyl ether

(33), bi-isorhamnetin (34), myricetin-7-O-methylether-(38)-quercetin 3-O- rhamnoside (35)

In 2015, Nguyen Phuong Thao et al [18] isolated 36 compounds from L racemosa leaves, namely quercetin (23), quercitrin (24), 1,5,6-trihydroxy-3-methoxyxanthone

(37), 5,6-dihydroxy-2,4-dimethoxyxanthone (38), perforaphenonoside A (39), acetylannulatophenonoside (40), (6S,9R)-9-hydroxy-4,7-megastigmadien-3-one 9-

O-[β- D -apiofuranosyl-(1→6)-β- D -glucopyranoside] (36), (6S,9R)-6-hydroxy-3- oxo-α-ionol 9-O-β- D -glucopyranoside (41), (6S,9R)-vomifoliol 9-O-β-apiofuranosyl- (1→6)-O-β- D -glucopyranoside (42), (+)-pinoresinol (43), polystachyol (44), (+)- lyoniresinol 3α-O-β-D-glucopyranoside (45), (-)-lyoniresinol 3α-O-β-D- glucopyranoside (46), 1,6-di-O-p-coumaroyl-β- D -glycopyranoside (47), alangilignoside C (48), polygalatenoside E (49), 3-(4β- D -glucopyranosyloxy-3- methoxy)-phenyl-2E-propenol (50), lawsoniaside B (51), icariside F2 (52), adenosine

(53), quercetin 4-O-β-D-glucopyranosyl 3-O-β- D -glucopyranoside (54), myricetin 3- arabinoside (55), linolenic acid (56), (2S)-1-O-α-linolenoyl-2-O-[(7Z,10Z,13Z)- hexadeca-7,10,13-trienoyl]-3-O-β-D-galactopyranosyl-sn-glycerol (57), (2S)-1,2-di-

O-[(9Z,12Z,15Z)-octadeca-9,12,15-trienoyl]-3-O-β- D -galactopyranosyl-sn-glycerol

(58), methyl gallate (59), ipuranol (60), ergosta-4,6,8(14),22-tetraen-3-one (61), ginsenoside Re (62), ginsenoside Rg1 (63), 20(29)-lupen-3-ol (64), hederagenin 3-

O-α- L -arabinopyranoside (65), tormentic acid (66), kaji-ichigoside F1 (67), benzyl- α- L -arabinopyranosyl-(1→6)-β- D -glucopyranoside (68) and stigmasterol (69)

In 2017, Nguyen Hoai Phuong et al [19] isolated a new glycoside, 2-O-galloyl-α-

L-rhamnopyranosyl-(34)-3-O-galloyl-α- L-rhamnopyranoside (70) as well as nine known compounds, namely gallic acid (27), kaempferol (22), quercetin (23), myricetin (30), myricetin 3-O-α- L-rhamnopyranoside (myricitrin) (71), 3-O- methylellagic acid (74), myricetin 3-O-(2-O-galloyl-α- L -rhamnopyranoside) (72), myricetin 3-O-(3-O-galloyl-α- L-rhamnopyranoside) (73) and (3S,5R,6S,7E)-3,5,6- trihydroxymegastigm-7-en-9-one (75) from L racemosa leaves

In 2018, Szu-Yin Yu and colleagues identified a novel neolignan named racelactone A, alongside seven established compounds, from the methanol extract of L racemosa's leaves and twigs The known compounds included betulin, methyl gallate, myricitrin, and stigmasterol.

(69), kaempferol (22), 3,4,3-tri-O-methylellagic acid (77) and isoguaiacin (78)

In 2018, Pham Thi Huyen et al [21] isolated eleven compounds from leaves and stems of L littorea, including β-sitosterol (5), quercetin (23), stigmasterol (69), 1,3- dihydroxy-2-methyl-5-tridecylbenzene (79), 1,3-dihydroxy-5-nonadecylbenzene

(80), 1,3-di-O-acetyl-2-methyl-5-tridecylbenzene (81), astragalin (82), 1-acetyl-D- mannitol (83), D-mannitol (84), hexa-O-acetyl-D-manitol (85), 2,3,4,6-tetra-O-acetyl- α- D -glucopyranosyl-2,3,4,6-tetra-O-acetyl-β- D -glucopyranosyl (86) and 1,3,4,5- tetra-O-acetylfructopyranose (87)

COMPOUNDS ISOLATED FROM LUMNITZERA GENUS

LUMNITZERA GENUS – PHARMACOLOGICAL STUDIES

Traditionally, the sap of Lumnitzera racemosa is used to treat cutaneous pruritus, herpes, scabies and thrush [22] The fruits of this plant are a curative for skin disorders

In India, L racemosa was used to treat asthma, diabetes, hypertension and snake bite [23]

L racemosa leaves extract exhibited antihypertensive [15] , hepatoprotective, antioxidant [24] , antimicrobial [17] , anticoagulant [25] , in vitro antiplasmodial activity [26] against chloroquine-sensitive Plasmodium falciparum (one of the species of

L racemosa exhibits significant biological activities, including an IC50 of 110.93 µg/mL against Plasmodium, the malaria-causing agent in humans Additionally, it shows anticancer effects on MCF-7 cells and induces cytotoxicity and apoptosis in Hep G2 cancer cells Furthermore, components isolated from the methanol extract of its leaves and twigs demonstrate antiangiogenic and anti-inflammatory properties.

In 2011, Shahbudin Saad et al [28] indicated the potent antimicrobial activities of the extracts of L littorea leaves against six human pathogenic microbes at a concentration of 0.04 mg/mL

BRUGUIERA GENUS – CHEMICAL STUDIES

Chemical studies on various parts of the Bruguiera genus, including leaves, stems, barks, fruits, hypocotyls, flowers, roots, and branches, have led to the isolation of a diverse range of compounds such as alkaloids, diterpenoids, triterpenoids, steroids, lignans, flavonoids, phenolics, sulfur, and others.

1.5.1.1 From barks of Bruguiera sexangula

In 1969, Loder et al isolated a new alkaloid called brugine from the barks of Bruguiera sexangula, alongside several known alkaloids such as tropine and its derivatives: tropine acetate, tropine benzoate, tropine isobutyrate, tropine isovalerate, tropine n-butyrate, and tropine propionate.

1.5.1.2 From stems of Bruguiera sexangula

In 2005, Shuyun Bao et al [30] isolated and characterized a new dithiobenzoquinone namely (-)-3,4-dihydro-3-hydroxy-7-methoxy-2H-1,5- benzodithiepine-6,9-dione (98), three new diterpenes, namely 17-hydroxy-16- oxobeyer-9(11)-ene-19-al (99), 16,17-dihydroxy-19-nor-ent-9(11)-kaurene-3-one

(100), (16R)-13,17-epoxy-16-hydroxy-ent-9(11)-kaurene-19-al (101), six known diterpenes, including 17-hydroxy-16-oxobeyerane-19-al (102), 16,17-dihydroxy-ent- 9(11)-kaurene-19-al (103), methyl 16,17-dihydroxy-ent-9(11)-kaurene-19-oate

(104), methyl (16R)-13,17-epoxy-16-hydroxy-ent-9(11)-kaurene-19-oate (105), ceriopsin F (106), ent-8(14)-pimarene-1β,15R,16-triol (107), two cyclic disulfides such as brugierol (108), isobrugierol (109), and 2,6-dimethoxy-1,4-benzoquinone

(110) from the stems of Bruguiera sexangula, collected at Haikou, Haiman island, China

In 2007, Shuyun Bao and colleagues identified four new phenolic glycosides, designated as rhyncosides A–D, and two novel lignan derivatives, rhyncosides E and F Additionally, they discovered twelve known phenolic compounds, which included the phenolic glycosides 3,4,5-trimethoxyphenyl-β-D-glucopyranoside and 1-(α-L-).

19 rhamnopyranosyl-(1–6)-β- D -glucopyranosyloxy)-3,4,5-trimethoxybenzene (118), four flavonoids such as tricin (119), rutin (120), nicotiflorin (121) and myricetin 3-

O-rutinoside (122), and six lignans, including lyoniside (123), (+)-lyoniresinol 3α-O- α- L -rhamnopyranoside (124), (+)-5-methoxyisolariciresinol 9-β- D -xylopyranoside

(125) and hedyotisols A–C (126–128) from the stems of Bruguiera sexangula, collected at the coastline closed to Xiamen, Fujian province, China

In 2010, Liang Li et al [32] isolated a new cytotoxic lanostane-type triterpenoid, namely sexangulic acid (129) from the stems of Bruguiera sexangula

1.5.2.1 From the root bank of Bruguiera gymnorrhiza

In 1999, Chitti Subrahmanyam and colleagues successfully isolated steviol and identified five new diterpenes, including 13-hydroxy-16-ent-kaurene-19-al, 16-ent-kaurene-13,19-diol, isopimar-7-ene-15S,16-diol, methyl (16R)-13,17-epoxy-16-hydroxy-ent-9(11)-kaurene-19-oate, and ent-8(14)-pimarene-1β,15R,16-triol.

(135) from the root bark of Bruguiera gymnorrhiza

1.5.2.2 From stems and leaves of Bruguiera gymnorrhiza

In 2004, Li Han et al [34] isolated three new ent-kaurane diterpenoids, named

Recent studies have identified several new and known diterpenoids from the stems of Bruguiera gymnorrhiza Notable compounds include 13,16α,17-trihydroxy-ent-9(11)-kaurene-19-oic acid, 16α,17-dihydroxy-ent-9(11)-kaurene 19-al, and a novel ent-beyerane diterpenoid, (4R,5S,8R,9R,10S,13S)-ent-17-hydroxy-16-oxobeyerane-19-al Additionally, nine known ent-kaurane diterpenoids such as methyl 16α,17-dihydroxy-ent-kaurane-19-oate and 16α,17-dihydroxy-ent-kaurene-19-al were also isolated Furthermore, researchers Yan-Qiu Sun and Yue-Wei Guo discovered a new compound named gymnorrhizol from the chipped stems and leaves of the same species.

In 2005, Li Han et al [36, 37] isolated five new aromatic compounds [37] , namely bruguierol A–C (144–146), 1-(3-hydroxyphenyl)hexane-2,5-diol (147) and 3-(3- hydroxybutyl)-1,1-dimethylisochroman-6,8-diol (148); three new pimaren diterpenoids [36] named ent-8(14)-pimarene-15R,16-diol (149), ent-8(14)-pimarene-

1α,15R,16-triol (150) and (5R,9S,10R,13S,15S)-ent-8(14)-pimarene-1-oxo-15R,16- diol (151); along with three known diterpenoids, ent-8(14)-pimarene-1β,15R,16-triol

(135), isopimar-7-ene-15S,16-diol (133) and isopimar-7-ene-1β,15S,16-triol (152) from the stems of mangrove plant Bruguiera gymnorrhiza

In 2007, Li Han et al [38] isolated two new compounds brugunin A (153), bruguierol D (154) and a known one 2,3-dimethoxy-5-propylphenol (155)

In 2008, Suisheng Shang and Shengjing Long [39] isolated a neolignan dioate brugnanin (160) from stem barks of Bruguiera gymnorrhiza

In 2009, Xiao-Ying Huang et al [40] isolated two novel polydisulfides, trans-

3,3,dihydroxy-1,5,1,5-tetrathiacyclodecane (161), cis-3,3,dihydroxy-1,5,1,5- tetrathiacyclodecane (162), along with five known cyclic disulfides, gymnorrhizol

(143), neogymnorrhizol (163), bruguiesulfurol (159), brugierol (108) and isobrugierol (109) from leaves and stems of Bruguiera gymnorrhiza

In 2011, You-Sheng Cai and colleagues discovered seven new compounds, known as palmarumycins BG1–BG7, along with a new derivative of preussomerin, identified as preussomerin BG1, from the aerial parts of the Bruguiera gymnorrhiza plant.

1.5.2.3 From flowers of Bruguiera gymnorrhiza

In 2006, Sudarat Homhual et al [42, 43] isolated three new dammarane triterpenes, bruguierins A–C (156–158); a new cyclic 4-hydroxydithiosulfonate, namely bruguiesulfurol (159) as well as two known 4-hydroxydithiolane 1-oxides, brugierol

(108) and isobrugierol (109) from flowers of Bruguiera gymnorrhiza

1.5.3.1 From fruits of Bruguiera cylindrica

In 2004, Surat Laphookhieo et al [44] isolated six new pentacyclic triterpenoid esters, namely 3α-(E)-feruloyltaraxerol (172), 3α-(Z)-feruloyltaraxerol (173), 3β-(E)-

(176) and 3α-(Z)-coumaroyltaraxerol (177) together with two known compounds, 3α- taraxerol (178) and 3β-taraxerol (179) from fruits of Bruguiera cylindrica

In 2005, Chatchanok Karalai and Surat Laphookhieo [45] isolated three new pentacyclic triterpenoid esters, namely 3α-(E)-coumaroyllupeol (180), 3α-(Z)- coumaroyllupeol (181) and 3α-(E)-caffeoyltaraxerol (182), six known lupane type triterpenoids including 3β-(E)-coumaroyllupeol (183), 3β-(Z)-coumaroyllupeol

(184), 3β-(E)-caffeoyllupeol (185), 3β-lupeol (186), 3α-lupeol (187) and lupenone

(188) from fruits and hypocotyls of Bruguiera cylindrica

1.5.3.2 From roots and leaves of Bruguiera cylindrica

In 2007, researchers Suchada Chantrapromma and colleagues isolated an ent-kaurane diterpene compound, 16-ent-kaurene-19-al, from the roots of Bruguiera cylindrical Additionally, Abdul Wahab Salae and his team isolated another diterpenoid compound, 16-ent-kaurene-13,19-diol.

In 2018, Nithyamol et al [48] isolated astaraxerol (178), 3β-(E)-coumaroyl taraxerol (190), 3β-(Z)-coumaroyltaraxerol (191), β-sitosterol (5) and eicosanol (192) from leaves of Bruguiera cylindrica

1.5.4 Bruguiera parviflora (Roxb.) W & Arn ex Giff

In 2005, Parinuch Chumkaew et al [49] isolated a new lupane caffeoyl ester, 3β-(Z)-caffeoyllupeol (193), together with five known triterpenoids, lupeol caffeate

(194), 3α-(Z)-coumaroyllupeol (181), dioslupecin A (184), lupeol (187),and lupenone

(188) from fruits of Bruguiera parviflora

COMPOUNDS ISOLATED FROM OF BRUGUIERA GENUS

BRUGUIERA GENUS – PHARMACOLOGICAL STUDIES

B gymnorrhiza fruits serve as a carbohydrate substitute for rice and are utilized in medicinal applications for eye diseases and herpes Additionally, its roots and leaves are effective in treating burns, while the bark is employed as an astringent for diarrhea and malaria.

The genus Bruguiera is notable for its diverse array of bioactive compounds, many of which exhibit significant biological activities, including insect antifeedant, antioxidant, antifungal, cytotoxic, antimalarial, and antibacterial properties To date, over 200 bioactive metabolites have been isolated from true mangroves in tropical and subtropical regions, with a substantial number originating from Bruguiera species.

Bruguiera cylindrica showed anticancer activity, among the six pentacyclic triterpenoid esters isolated from the fruits, 3α-(Z)-feruloyltaraxerol (173) and 3α-(Z)-

27 coumaroyltaraxerol (177) exhibited weak cytotoxic activities against NCI–H187 cell line [44]

Bruguierin A, derived from the flowers of Bruguiera gymnorrhiza, demonstrated significant biological activity by inducing NF-κB luciferase activation in response to phorbol esters, with an IC50 value of 1.4 µM Additionally, it selectively inhibited cyclooxygenase-2 (COX-2) activity, showing an IC50 value of 0.37 µM.

Sexangulic acid, derived from the stems of Bruguiera sexangula, demonstrated moderate in vitro activity against A-549 human lung cancer and HL-60 human leukaemic cell lines at a concentration of 5 μg/mL Additionally, ent-8(14)-Pimarene-1-oxo-15R,16-diol and isopimar-7-ene-15S,16-diol are also noteworthy compounds in this context.

(133) from Bruguiera gymnorrhiza showed moderate activities against L-929 (mouse fibroblasts) and K562 (human chronic myeloid leukemia) with IC50 values of 9.8 and 7.0 g/mL, respectively [36, 37]

The lupane caffeoyl ester, 3β-(Z)-caffeoyllupeol (193), isolated from the fruits of

Bruguiera parviflora, exhibited antimalarial activity with an EC50 value of 8.6

The enzyme PTP1B is crucial in regulating insulin receptor signaling, influencing both physiological and pathological processes Research indicates that PTP1B deficiency leads to increased insulin sensitivity and resistance to diet-induced obesity, making it a potential target for treating type II diabetes and obesity Notably, compounds such as Gymnorrhizol and Bruguiesulfurol from B gymnorrhiza exhibit significant inhibitory effects on PTP1B, with IC50 values of 14.9 and 17.5 µM, respectively.

The in vitro antibacterial assay revealed that the methanol extract of B cylindrica had vibriocidal activity Although B cylindrica had low activity, it inhibited the

28 growth of two bacteria, Vibrio alcaligenes (7 mM) and Vibrio alginolyticus (10 mM) [54]

The extract of Bruguiera cylindrica demonstrated significant lipid-lowering and antioxidant effects in a Triton-induced hyperlipidemia model At a dosage of 200 mg/kg body weight, various fractions of the extract successfully reduced serum lipid levels This lipid-lowering effect is attributed to the inhibition of hepatic cholesterol biosynthesis, increased fecal bile acid excretion, and enhanced plasma lecithin:cholesterol acyltransferase activity.

CHEMICALS AND INSTRUMENTS

Solvents including n-hexane, chloroform, ethyl acetate, methanol, and acetic acid for thin-layer chromatography (TLC) and column chromatography (CC) were sourced from Chemsol in Vietnam and utilized without additional purification α-Glucosidase (EC 3.2.1.20) derived from Saccharomyces cerevisiae (750 UN) and p-nitrophenyl-α-D-glucopyranoside were acquired from Sigma Chemical Co in St Louis, MO, USA Additionally, acarbose and dimethyl sulfoxide were obtained from Merck in Darmstadt, Germany.

Healthy young Swiss albino rats, both male and female, aged 6-7 weeks and weighing 20-25 g, were sourced from the Institute of Vaccine and Medical Biologicals in Nha Trang, Vietnam They were housed in polypropylene cages (25x35x15 cm) under controlled conditions of 25 ± 2 °C, with a 12-hour light and dark cycle The rats were provided with a standard laboratory diet and had access to water ad libitum NMR spectra were obtained using a Bruker Avance III spectrometer, operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR, with residual solvent signals serving as internal references: chloroform-d (δH 7.26, δC 77.16) and dimethyl sulfoxide-d6 (δH 2.50, δC).

The HR-ESI-MS spectra were obtained using a MicroOTOF-Q mass spectrometer and an LC-Agilent 1100 LC-MSD Trap spectrometer, along with an HR-ESI-MS Ion trap-TOF mass spectrometer on an LC/MS-Shimadzu system Additionally, the α-glucosidase inhibitory assay was conducted using a POWERWAVE HT.

Thin layer chromatography (TLC) was performed on silica gel 60 F254 (Merck) or silica gel 60 RP–18 F254S (Merck) Spots were visualized by spraying with 10%

H2SO4 solution in ethanol, followed by heating Gravity column chromatography was performed on silica gel 60 (0.040–0.063 mm, Merck, Darmstadt, Germany) and Sephadex LH–20 (GE Healthcare Bio-Science AB, Uppsala, Sweden)

The laboratories at the University of Science, National University – Ho Chi Minh City, Ho Chi Minh City Open University, and the Central for Applied Spectroscopy of the Institute of Chemistry are equipped with advanced instruments for research and analysis.

PLANT MATERIALS

In August 2014, leaves of Lumnitzera littorea (Jack) Voigt (No US-B012) and Bruguiera cylindrica (No US-B013) were collected from the Can Gio mangrove forest in Ho Chi Minh City, Vietnam The scientific identification of these species was verified by Dr Pham Van Ngot from the Faculty of Biology at Ho Chi Minh City University of Pedagogy The voucher specimens are currently housed at the Herbarium of the Department of Organic Chemistry, University of Science, Vietnam.

EXTRACTION AND ISOLATION

2.3.1 Extraction and isolation of compounds from Lumnitzera littorea

Fresh leaves were thoroughly washed to eliminate sand and epiphytes, then dried and ground into powder This powder (15 kg) underwent maceration with 40 liters of ethanol at room temperature for two days, repeated ten times Following filtration, the ethanol solution was evaporated to yield a crude residue of 1 kg This residue was then fractionated using the solid phase extraction method with silica gel as an adsorbent, applied to a silica gel column The extracts were eluted in succession with n-hexane, ethyl acetate, and ethanol After evaporation, three distinct extracts were obtained: n-hexane (100 g), ethyl acetate (250 g), and ethanol (450 g).

The n-hexane extract (100 g) was fractionated by silica gel column chromatography using a mixture of n-hexaneethyl acetate (stepwise, 98:2 to 0:100, v/v), and then ethyl acetate–methanol (stepwise, 9:1, 8:2, 1:1 to 0:1, v/v) to yield five fractions (H1–H5)

 The fraction H2 (9.5 g) was applied to a silica gel column and eluted with chloroform–methanol (stepwise, 98:2 to 50:50, v/v), to give six subfractions (H2.1–

The subfraction H2.1, weighing 1.3 g, underwent rechromatography on a silica gel column using a stepwise chloroform–ethyl acetate gradient (98:2 to 0:100, v/v), resulting in the isolation of compounds LL01 (37.0 mg) and LL02 (47.0 mg) Additionally, the subfraction H2.3, with a mass of 2.8 g, was further processed through Sephadex LH–20 chromatography, eluted with a chloroform–methanol mixture (1:1, v/v), yielding compounds LL03 (27.3 mg) and LL04 (15.1 mg).

 The fraction H3 (27.5 g) was further separated on a silica gel column, eluted with chloroform–methanol (stepwise, 9:1 to 0:100, v/v) to yield four subfractions

(H3.1–H3.4) The subfraction H3.2 (7.0 g) was subjected to the silica gel column with chloroform–ethyl acetate (stepwise, 98:2 to 0:100, v/v) to obtain LL05 (12.5 mg) and

 The fraction H4 (13.9 g) was subjected to a silica gel column, using chloroform–methanol (stepwise, 9:1 to 0:100, v/v) to get five subfractions (H4.1–

H4.5) The subfraction H4.1 (200.0 mg) was further separated over a Sephadex LH–

The elution process using chloroform–methanol (1:1, v/v) yielded three compounds: LL07 (17.1 mg), LL08 (78.9 mg), and LL09 (12.7 mg) Additionally, the subfraction H4.3 (134.5 mg) underwent rechromatography on a silica gel column, utilizing a stepwise n-hexane–chloroform gradient (95:5 to 50:50, v/v), resulting in the isolation of LL10 (32.0 mg) and LL27 (13.0 mg).

The ethyl acetate extract (250 g) was fractionated by a silica gel column chromatography using a mixture of ethyl acetate–methanol (stepwise, 98:2 to 0:100, v/v) to yield eight fractions (E1–E8)

The fraction E2 (27.6 g) was subjected to silica gel column chromatography and eluted with a chloroform-methanol gradient (9:1 to 5:5 and 0:100, v/v), resulting in seven subfractions (E2.1–E2.7) Subfraction E2.5 (2.0 g) underwent rechromatography on a Sephadex LH–20 column with chloroform-methanol (1:1, v/v), yielding LL11 (105.2 mg), LL12 (87.2 mg), and LL13 (6.4 mg) Meanwhile, subfraction E2.6 (2.3 g) was chromatographed multiple times on a Sephadex LH–20 column with the same solvent mixture, producing LL14 (5.1 mg) and LL15 (14.2 mg).

 The fraction E4 (14.0 g) was applied to Sephadex LH–20 column with chloroform–methanol (1:1, v/v) as eluent to give five subfractions (E4.1E4.5), then

32 the subfraction E4.2 (4.0 g) was rechromatographed on a silica gel column using ethyl acetate–methanol–water–acetic acid (40:1:1:1, v/v/v/v) to get LL16 (13.7 mg), LL17

 The fraction E6 (23.1 g) was further separated on the Sephadex LH–20 column eluting with chloroform–methanol (1:1, v/v) afforded LL19 (8.7 mg), LL20 (9.3 mg),

LL21 (12.0 mg) and LL22 (10.3 mg)

 The fraction E8 (30.2 g) was chromatographed on Sephadex LH–20 with chloroform–methanol (1:1, v/v) as eluent to obtain LL23 (6.7 mg), LL24 (9.8 mg),

LL25 (8.8 mg) and LL26 (9.5 mg)

The remaining fractions were not chemically studied because their TLC showed many spots that were too close together to be separated

The procedure of extraction and isolation of compounds from Lumnitzera littorea (Jack) Voigt leaves was presented in the Scheme 2.1

2.3.2 Extraction and isolation of compounds from Bruguiera cylindrica

Fresh leaves were thoroughly washed to eliminate sandy particles and epiphytes, then dried and ground into powder An 8 kg batch of this powder was macerated with 20 liters of ethanol at room temperature for two days, a process repeated ten times After filtration, the ethanol solution was evaporated under reduced pressure, yielding a crude residue of 900 g This residue underwent fractionation using the solid phase extraction method with silica gel as the adsorbent, applied to a silica gel column The extracts were eluted sequentially with n-hexane, ethyl acetate, and ethanol, resulting in three final extracts: 100 g of n-hexane, 300 g of ethyl acetate, and 380 g of ethanol.

The n-hexane extract (100 g) underwent fractionation using silica gel column chromatography with a gradient of n-hexane and ethyl acetate (from 98:2 to 0:100, v/v), followed by a mixture of ethyl acetate and methanol (from 9:1, 8:2, 1:1 to 0:1, v/v), resulting in five distinct fractions labeled He1 to He5.

The ethyl acetate extract (300 g) was fractionated by a silica gel column chromatography using a mixture of ethyl acetate–methanol (stepwise, 98:2 to 0:100, v/v) to yield six fractions (EA1–EA6)

 The fraction EA1 (28.3 g) was applied to a silica gel column, and eluted with ethyl acetate–methanol (stepwise, 100:0 to 0:100, v/v) to give five subfractions

(EA1.1–EA1.5) The subfraction EA1.2 (167 mg) was rechromatographed on a silica gel column using n-hexaneethyl acetate (stepwise, 98:2 to 0:100, v/v) to obtain

The subfraction EA1.3, weighing 100 mg, underwent chromatographic separation on a silica gel column, utilizing a stepwise elution with chloroform-ethyl acetate in a gradient from 100:0 to 0:100 (v/v) This process resulted in the isolation of three compounds: LL03 (10.3 mg), LL04 (12.4 mg), and LL06 (15.1 mg).

The fraction EA2 (27.8 g) underwent chromatographic separation on a silica gel column using a stepwise elution of n-hexane–chloroform in varying ratios (9:1, 8:2, 5:5, 0:10, v/v), resulting in four distinct subfractions (EA2.1–EA2.4) Among these, subfraction EA2.2 (80 mg) was further purified through a second silica gel column, employing a similar stepwise elution with n-hexane–chloroform at ratios of 99:1 and 98:2.

95:5, 90:10, 80:20, 50:50, 0: 100, v/v) to afford BC03 (7.3 mg) and BC04 (6.4 mg) The subfraction EA2.3 (128 mg) was rechromatographed on a silica gel column eluting with chloroformethyl acetate (stepwise, 98:2 to 0:100, v/v) to obtain BC05

(9.1 mg), BC06 (7.5 mg) and BC07 (12.4 mg)

The fraction EA3 (33.3 g) underwent silica gel column chromatography, eluted with a chloroform–methanol gradient (9:1 to 5:5 and 0:100, v/v), resulting in four subfractions (EA3.1–EA3.4) The subfraction EA3.1 (250 mg) was further purified using Sephadex LH–20 with chloroform–methanol (1:1, v/v) and subsequently subjected to additional silica gel chromatography, yielding compounds LL10 (8.5 mg) and LL12 (37.2 mg) through a stepwise elution with ethyl acetate–methanol (9:1 to 5:5 and 0:100, v/v).

LL14 (16.4 mg) The subfraction EA3.4 (100 mg) was rechromatographed on a silica gel column and eluted with chloroform–methanol (stepwise, 98:2 to 5:5, v/v) to get

LL13 (9.3 mg), BC01 (12.3 mg) and BC02 (13.7 mg)

 The fraction EA4 (28.5 g) was further separated over a Sephadex LH–20 eluting with chloroform–methanol (1:1, v/v) to give BC08 (7.6 mg)

The procedure of extraction and isolation of compounds from Bruguiera cylindrica leaves was presented in the Scheme 2.2

The chemical structures of isolated compounds from Lumnitzera littorea and Bruguiera cylindrica leaves were elucidated by spectroscopic methods, including

MS, 1D and 2D–NMR as well as comparison with the literature values.

Dried ground material of leaves of Lumnitzera littorea

Macerated with ethanol at room temperature Filtrated, evaporated under reduced pressure

Extracted by solid phase and eluted with n-hexane, ethyl acetate and ethanol Evaporated under reduced pressure n-Hexane extract

Silica gel column chromatography (CC) H:EA (98:2–0:100)

LL07 (17.1 mg) LL08 (78.9 mg) LL09 (12.7 mg)

LL11 (105.2 mg) LL12 (87.2 mg) LL13 (6.4 mg)

LL16 (13.7 mg) LL17 (10.2 mg) LL18 (9.8 mg)

LL19 (8.7 mg) LL20 (9.3 mg) LL21 (12.0 mg) LL22 (10.3 mg)

LL23 (6.7 mg) LL24 (9.8 mg) LL25 (8.8 mg) LL26 (9.5 mg) LL28 (8.2 mg) LL29 (7.6 mg)

Scheme 2.1 Procedure of extraction and isolation of compounds from Lumnitzera littorea leaves

Dried ground material of leaves of Bruguiera cylindrica

Macerated with ethanol at room temperature Filtrated, evaporated under reduced pressure

Extracted by solid phase and eluted with n-hexane, ethyl acetate and ethanol Evaporated under reduced pressure n-Hexane extract

Silica gel column chromatography (CC) H:EA (98:2–0:100)

LL03 (10.3 mg) LL04 (12.4 mg) LL06 (15.1 mg)

LL10 (8.5 mg) LL12 (37.2 mg) LL14 (16.4 mg)

LL13 (9.3 mg) BC01 (12.3 mg) BC02 (13.7 mg)

BC05 (9.1 mg) BC06 (7.5 mg) BC07 (12.4 mg)

Scheme 2.2 Procedure of extraction and isolation of compounds from Bruguiera cylindrica leaves

ACID HYDROLYSIS

The acid hydrolysis of the new compound LL19 was performed to isolate the sugar residue Specifically, 2.0 mg of LL19 was treated with 0.2 M HCl in a dioxane/water mixture (1:1, v/v) at 95°C for three hours Following the reaction, the mixture was cooled and extracted with chloroform (3 x 2 mL) to remove the aglycone component The remaining solution was then evaporated to dryness, and the resulting residue was dissolved for further analysis.

D2O for subsequent 1 H–NMR analysis of the hydrolyzed monosaccharide The anomeric ratios were obtained by manual integration with δ H 5.23 (d, J = 3.5 Hz,

36.4%) and 4.64 (d, J = 8.0 Hz, 63.6%) These values were highly reminiscent of those of glucose [57] (Appendix 17.11).

COMPUTATIONAL DETAILS

All DFT calculations were executed using the Gaussian 09 software package Geometric optimization of the predicted structures was carried out at the B3LYP/6-311++G(2d,p) level in both gas phase and methanol solvent Frequency calculations at the same level confirmed that these structures represent minima on the potential energy surface The relative energies, corrected for zero-point energy (ZPE), were determined by evaluating the differences in total energy among the configurations Theoretical 1H and 13C NMR chemical shifts were derived from isotropic magnetic shielding tensors using the Gauge-Independent Atomic Orbital (GIAO) method at the B3LYP/6-311+G(d,p) level Additionally, modified DP4+ probabilities were utilized to accurately assign the conformer, with an online implementation available for use.

α-GLUCOSIDASE INHIBITORY ASSAY

Diabetes is a chronic condition characterized by elevated blood glucose levels Effective diabetes management focuses on maintaining normal blood sugar levels after meals, as postprandial hyperglycemia significantly contributes to the onset of type 2 diabetes and its associated complications.

To reduce post-meal blood glucose spikes, one effective approach is the inhibition of carbohydrate hydrolyzing enzymes, particularly α-glucosidase This intestinal enzyme is responsible for breaking down α-1,4 linked polysaccharides into α-glucose, which can lead to elevated blood sugar levels Therefore, the development of natural product-derived α-glucosidase inhibitors represents a significant advancement in diabetes treatment.

2.6.1 The principle of the α -glucosidase inhibitory assay

The α-glucosidase inhibitory activity of all extracts and most isolated compounds was assessed using the method established by Apostolidis et al [63], with acarbose serving as the positive control, as illustrated in Figure 2.1.

The α-glucosidase inhibitory assay utilized a spectrophotometric method to evaluate enzyme activity In this process, the enzyme α-glucosidase catalyzed the hydrolysis of the substrate 1-nitro-4-hydroxybenzene α-D-glucopyranoside (pNPG), resulting in the production of α-D-glucose and p-nitrophenol (pNP) The inhibitory activity of α-glucosidase was quantified by measuring the color intensity of the released p-nitrophenol from the substrate.

The α-glucosidase reaction was shown in Figure 2.2

The results were expressed as inhibition percentage and calculated by following equation (1)

 Acontrol is the absorbance recorded for the enzymatic activity without the inhibitor

 Asample is the absorbance recorded for the enzymatic activity in the presence of the inhibitor

Figure 2.2 The α-glucosidase catalyzed reaction using pNPG as a substrate

The inhibitory concentration (IC50) represents the concentration needed to inhibit 50% of enzyme activity This value is determined through regression analysis of a graph that plots the percentage of inhibition against various sample concentrations.

Experiments were performed at Biochemistry laboratory, Faculty of Biotechnology, Ho Chi Minh City Open University, 97 Vo Van Tan, District 3, Ho Chi Minh City, Vietnam

The study utilized plant extracts and isolated compounds derived from Schemes 2.1 and 2.2, which were subsequently dissolved in DMSO at different concentrations The final DMSO concentration in the reaction mixture was maintained at 3%.

The reaction mixture containing 60 L of sodium phosphate buffer (100 mM, pH 6.8), 20 L of the sample at different concentrations and 20 L of -glucosidase

In a controlled experiment, 0.3 IU/mL of enzyme in phosphate buffer was incubated in 96-well plates at 37°C for 10 minutes Subsequently, 100 µL of p-nitrophenyl-α-D-glucopyranoside solution (200 µM) was added to initiate the reaction, followed by another 10-minute incubation at the same temperature To halt the reaction, 100 µL of 50 mM NaOH solution was introduced.

The absorbance was measured at 405 nm by using a POWERWAVE HT (BIOTEK) microplate reader

Negative control experiments containing no samples were also run in parallel Acarbose was used as a positive control.

ACUTE TOXICITY ASSAY

2.7.1 The principle of the acute toxicity assay

Acute toxicity assesses the harmful effects experienced by organisms after exposure to a test substance, whether through a single dose or multiple doses, within a 24-hour period via known routes such as oral, dermal, or inhalation.

Samples were tested for acute toxicity at the Animal Biotechnology Laboratory, located within the Faculty of Biotechnology at Ho Chi Minh City Open University, situated at 97 Vo Van Tan, District 3, Ho Chi Minh City, Vietnam.

The oral acute toxicity testing of ethanol crude extract from Lumnitzera littorea leaves was conducted on Swiss albino rats using single dose levels of 1.0, 2.0, 3.0, 4.0, 8.0, and 13.0 g/kg, following the OECD guidelines' "Up and Down" method The maximum concentration tested was 13.0 g/kg, the highest dosage at which the extract could be dissolved in distilled water for injection.

In a controlled study, male and female Swiss albino rats (n = 10 per dose level) underwent a 12-hour fasting period during which they had unrestricted access to water After fasting, the rats were weighed and received an oral administration of a corresponding volume of the test substance.

Rats were administered an ethanol crude extract from Lumnitzera littorea leaves at a dosage of 40 mL per kg of body weight Following the injection, food was withheld from the rats for 3 hours The subjects were monitored for the first 4 hours post-injection, then observed over the next 72 hours, and subsequently daily for 14 days to assess any effects.

41 symtoms of abnormal behavior, toxic symtoms, weight change, and death were found at any observed timebounds

The LD50 is a statistically determined dosage of a substance that is expected to be lethal to 50% of test rats when administered orally This measurement is expressed in milligrams of the test substance per kilogram of the animal's weight (mg/kg).

Experiments of the oral acute toxicity were set up in Table 2.1

Table 2.1.Oral acute toxicity test of the ethanol crude extract of Lumnitzera littorea leaves on rats

The oral extract volume (40 mL extract / kg weight rats)

RESULTS AND DISCUSSIONS 3.1 CHEMICAL STRUCTURE ELUCIDATION

Chemical structure of steroids (group A)

3.1.1.1 Structure elucidation of β -sitosterol (LL01)

Compound LL01 was obtained from the fraction H2.1 of the Scheme 2.1 of

Lumnitzera littorea as well as from the fraction EA1.2 of the Scheme 2.2 of Bruguiera cylindrica Its essential physical data were described as the following

 1 H, 13 C–NMR spectra (Chloroform–d) (Appendix 1.1 and 1.2): Tables 3.1 and 3.2

The 1 H–NMR spectrum of compound LL01 revealed the presence of six methyl proton signals, including two singlet methyls at H 0.68 (s, H-18) and 1.01 (s, H-19), four doublet methyls at H 0.81 (d, 7.0 Hz, H-26), 0.83 (d, 6.5 Hz, H-27), 0.85 (d, 7.5

Hz, H-29) and 0.92 (d, 6.5 Hz, H-21) The olefinic proton signal at H 5.35 (d, 5.0

The analysis revealed characteristic sterol signals, with the proton signal at δH 3.52 (m, H-3) indicating the presence of a carbon carbinol at C-3 The 13C-NMR spectrum showed a total of twenty-nine carbon signals, including notable peaks at δC 121.9 (C-6) and 140.9 (C-5), which signify a double bond, alongside an oxymethine carbon signal at δC 72.0 (C-3).

The compound LL01 has been identified as β-sitosterol, a naturally occurring phytosterol that is optically active This steroidal compound exhibits a range of pharmacological activities, including gastroprotective, cytotoxic, antimicrobial, anti-inflammatory, and anticancer effects, as supported by existing literature.

Table 3.1 1 H–NMR data of known steroids LL01, LL09, BC05 and BC06

3.1.1.2 Structure elucidation of stigmast-4-ene-3-one (LL09)

Lumnitzera littorea with essential physical data as described below

 APCI–MS spectrum (Appendix 2.1): m/z 413.26 [M+H] + , calcd for C29H48O+H, 413.70

The 1 H–NMR spectrum of LL09 closely resembled that of LL01, including two singlet methyls at H 1.18 (s, H-19) and 0.71 (s, H-18), three doublet methyls at H

The 1H-NMR spectrum revealed distinct signals, including doublets at δH 0.81 (d, 7.0 Hz, H-27), 0.83 (d, 7.0 Hz, H-26), and 0.91 (d, 6.5 Hz, H-21), along with a triplet methyl signal at δH 0.84 (t, 7.5 Hz, H-29) Additionally, the spectrum confirmed the presence of a singlet olefinic proton signal at δH 5.72 (s, H-4) and notably lacked the multiplet proton signal at δH 3.10–3.60 corresponding to H-3, as observed in LL01.

The 13 C–NMR spectrum displayed the presence of twenty nine carbon signals, including the conjugated carbonyl carbon signal at C 199.8 (C-3) and two olefinic carbon signals at C 171.9 (C-5) and 123.9 (C-4)

Besides, the APCI–MS spectrum showed the pseudomolecular ion peak at m/z

Based on the obtained spectral data and comparison with literature ones [72] , the structure of LL09 was confirmed as stigmast-4-ene-3-one

The stigmast-4-ene-3-one exhibited high antitumour promoting activity [73] , significant hypoglycaemic activity [74] and vasodepressor effect [75]

3.1.1.3 Structure elucidation of cholest-4-ene-3-one (BC06)

Compound BC06 was obtained from the fraction EA2.3 of the Scheme 2.2 of

Bruguiera cylindrica with essential physical data as described below

 HR–ESI–MS spectrum (Appendix 3.1): m/z 385.3448 [M+H] + , calcd for C27H44O+H, 385.3470

The NMR spectra comparison between BC06 and LL09 revealed that BC06 features a steroid structure with a carbonyl group at C-3 Notably, the 1H–NMR spectrum of BC06 lacked the triplet proton signal at δH 0.84 corresponding to the H-29 position, indicating that BC06 possesses a cholestane skeleton rather than the stigmastane skeleton found in compound LL09.

The 13 C–NMR spectrum revealed twenty-seven carbon signals, including a carbonyl carbon signal at δC 199.8 (C-3) and two olefinic carbon signals at δC 171.9 (C-5) and 123.9 (C-4), along with additional carbon signals ranging from δC 12 to 57 ppm Furthermore, the HR–ESI–MS spectrum indicated a pseudomolecular ion peak at m/z 385.3448 [M+H]+, which corresponds to the molecular formula C27H44O.

Based on the previous data and comparison with literature ones [76] , the structure of BC06 was confirmed as cholest-4-ene-3-one or more commonly name, cholestenone

3.1.1.4 Structure elucidation of 3 β -hydroxycholest-5-ene-7-one (BC05)

Bruguiera cylindrica with essential physical data as described below

Table 3.2 13 C–NMR data of known steroids LL01, LL09, BC05 and BC06

LL01 LL09 BC05 BC06 LL01 LL09 BC05 BC06

The NMR analysis of BC05 revealed a structure closely resembling that of BC06 Notably, the 1H-NMR spectrum of BC05 displayed an olefinic proton signal at δH 5.69 (s, H-6), along with three doublet proton signals at δH 0.82 (d, 3.0 Hz, H-27), 0.84 (d, 2.5 Hz, H-26), and 0.93 (d, 6.5 Hz, H-21), as well as two singlet proton signals.

The chemical shifts at δH 0.68 (s, H-18) and 1.20 (s, H-19) indicate distinct proton environments, with the notable presence of a multiplet signal at δH 3.67 (m, H-3) in BC05 This signal corresponds to a proton associated with a carbinol group, further supported by the detection of an oxygenated methine carbon signal.

The 13 C–NMR spectrum of BC05 clearly displayed twenty seven characteristic carbons of the cholestane skeleton, including one carbonyl carbon at C 202.5 (C-7), two olefinic carbons at C 165.2 (C-5) and 126.3 (C-6) and other carbons at C 12–55 ppm

All the above data were in good agreement with those in the literature [77] Therefore, the structure of BC05 was determined as 3β-hydroxycholest-5-ene-7-one

3.1.1.5 Structure elucidation of β -sitosterol 3- O - β - D -glucopyranoside (LL05)

Lumnitzera littorea as well as from the fraction EA1.2 of the Scheme 2.2 of Bruguiera cylindrica Its essential physical data were described as the following

 1 H, 13 C–NMR spectra (DMSO–d 6) (Appendix 5.1 and 5.2): Tables 3.3 and 3.4

A detailed analysis of the NMR spectra of LL05 revealed the presence of proton and carbon signals characteristic of a stigmastane skeleton Additionally, the 1H–NMR spectrum confirmed the existence of a β-glucose unit, indicated by a doublet signal at δH.

4.21 ppm with the large coupling constant J=8.0 Hz assigning to the anomeric proton H-1 and multiplet signals from 2.89 to 3.15 ppm assigning for carbinol protons of the sugar moiety

The 13 C–NMR spectrum displayed an anomeric carbon signal at C 100.9 (C-1), an oxygenated methylene carbon signal at C 61.2 (C-6) and four oxymethine carbon signals at C 70.2–77.1 (C-25) of the sugar unit

These data confirmed that compound LL05 was β-sitosterol 3-O-β-D- glucopyranoside [78, 79] β-Sitosterol 3-O-β- D -glucopyranoside had a wide spectrum antibacterial and antifungal activity at the concentration of 200 g/mL against Staphylococcus aureus,

Proteus mirabilis, Salmonella typhi, Klebsiella pneumonia, Escherichia coli and

Bacillus subtilis with the inhibition zone of 24–34 mm and also active against the fungi Candida albicans and Candida tropicalis [80]

Table 3.3 1 H–NMR data of known steroids LL05, LL27 and LL02

3.1.1.6 Structure elucidation of kochioside A (LL27)

The NMR data comparison between LL27 and LL05 revealed that both compounds share a steroid structure featuring a β-glucose unit at the C-3 position A notable distinction lies in the positioning of two methyl groups within LL27's structure Specifically, the 1H–NMR spectrum of LL27 displayed an additional singlet proton signal at δH 0.77 (H-29), which corresponds to a tertiary methyl group at the C-14 position, contrasting with the doublet proton signal observed in LL05.

H 0.82 (d, 7.0 Hz, H-29) as in LL05 Additionally, the presence of one doublet proton signal at H 1.00 (d, 6.5 Hz, H-28) was assigned to the secondary methyl group at C-

The 13 C–NMR spectrum of LL27 revealed thirty five carbon signals of the steroid glycoside, including an anomeric carbon signal at C 101.2 (C-1), sugar carbon signals from 62.0 to 77.0 ppm of C-2C-6 positions, one oxygenated carbon at C

79.3 (C-3), two olefinic carbon signals at C 140.4 (C-5) and 122.1 (C-6) and the other carbon signals from 12.0 to 57.0 ppm

The NMR data analysis of LL27, compared with literature findings, confirmed its chemical structure as kochioside A, marking its first isolation from L littorea.

3.1.1.7 Structure elucidation of stigmast-5-ene 3 β - O -(6- O -hexadecanoyl- β - D - glucopyranoside) (LL02)

 HSQC, HMBC spectra (Chloroform–d) (Appendix 7.3 and 7.4)

Similar to the NMR spectra of LL05, the 1 H NMR and 13 C–NMR spectra of

LL02 indicated that it possessed a similar structure to that of β-sitosterol glucoside

The difference was the presence of signals for a palmitoyl moiety in LL02

The 1 H–NMR spectrum of LL02 showed one methylene proton signal adjacent to a carboxyl group at H 2.35 (t, 7.5 Hz, H-2), the other methylene proton signals at

H 1.20–1.50 characterized for a long aliphatic chain and one terminal methyl proton signal at H 0.88 (d, 7.0 Hz, H-16)

The 13 C–NMR spectrum identified fifty-one carbon signals, comprising twenty-nine carbons from a β-sitosterol unit, fifteen aliphatic carbons in the side chain, six carbons from a glucose unit, and one carboxyl carbon Notably, a hexadecanoyloxy group in the side chain is linked to C-6' of the glucose unit, indicated by a carbon signal at δC 174.9 for the carboxyl carbon, along with methylene carbon signals at δC 29.3–29.7 and a terminal methyl group at δC 14.3.

Besides, the HMBC correlations of methylene protons at H 4.26 (dd, 12.0, 2.0

The spectroscopic analysis revealed signals at Hz, H-6b) and 4.50 (dd, 12.0, 4.5 Hz, H-6a), along with a carboxyl carbon resonance at C 174.9 (C-1), indicating the presence of a palmitoyl moiety attached to C-6 of the glucose unit A comparison of these data with existing literature suggests that LL02 is identified as stigmast-5-ene 3β-O-(6-O-hexadecanoyl-β-D-glucopyranoside).

Table 3.4 13 C–NMR data of known steroids LL05, LL27 and LL02

LL05 b LL02 a LL27 b LL05 b LL02 a LL27 b

Chemical structure of triterpenoids (group B)

3.1.2.1 Structure elucidation of lupeol (LL06)

Lumnitzera littorea as well as from the fraction EA1.3 of the Scheme 2.2 of Bruguiera cyclindrica Its essential physical data were described the following

 APCI–MS spectrum (Appendix 8.1): m/z 409.49 [MH2O+H] + , calcd for C30H50O–H2O+H, 409.72

 HSQC and HMBC spectra (Chloroform–d) (Appendix 8.4 and 8.5)

The 1 H–NMR spectrum of LL06 showed the presence of seven singlet signals in the high shielded region at H 0.7–1.7 Those were characteristics of methyl protons of triterpenoids Besides, there was one proton signal at H 3.18 (dd, 11.5, 5.0 Hz, H-

The analysis revealed characteristic olefinic proton signals at δH 4.56 (s, H-29b) and 4.68 (brs, H-29a), indicating the presence of an exocyclic double bond Additionally, a triplet of doublet proton signal at δH 2.37, associated with H-19, was identified as a distinctive feature of lupeol.

The 13 C–NMR spectrum of LL06 revealed thirty carbon signals which were showed characteristics of a lupane skeleton triterpenoid The deshielded carbon signal at C 79.1 was due to the oxygenated carbon C-3 Two olefinic carbons of the exocyclic double bond appeared at C 109.5 (C-29) and 151.1 (C-20) The chemical structure of LL06 was confirmed by the 2D–NMR spectrum

The APCI–MS spectrum showed the pseudomolecular ion peak at m/z 409.49

[MH2O+H] + corresponding to the molecular formula of C30H50O

Thus, LL06 was determined as lupeol that was consistent with the reported data in the literature [83]

Lupeol is a pentacyclic triterpene present in numerous plants, including fruits, vegetables, and medicinal herbs It exhibits pharmacological effectiveness in treating a variety of diseases due to its anti-inflammatory, antimicrobial, antiprotozoal, antiproliferative, anti-invasive, antiangiogenic, and cholesterol-lowering properties Additionally, lupeol shows promise as a potential treatment for pancreatic cancer.

3.1.2.2 Structure elucidation of 3 α -( E )-coumaroyllupeol (BC03)

Bruguiera cyclindrica with essential physical data as described below

The 13 C–NMR spectrum of BC03 revealed thirty signals characteristic of a lupeol moiety, including olefinic carbon signals at δC 150.3 (C-20) and 110.0 (C-29), corresponding to olefinic proton signals at δH 4.69 (s, H-29a) and 4.58 (brs, H-29b), along with seven singlet methyl proton signals in the range of δH 0.87–1.68 Unlike LL06, BC03 exhibited a coumaroyl moiety, evidenced by doublet proton signals at δH 7.46 (d, 8.5 Hz, H-2′,6′) and 6.85 (d, 8.5 Hz, H-3′,5′) from the 1,4-disubstituted benzene ring, as well as olefinic proton signals at δH 6.35 (d, 15.5 Hz, H-8′) and 7.62 (d, 15.5 Hz, H-7′) The E configuration of the double bond between C-7′ and C-8′ was confirmed by a large coupling constant (J 7′,8′ 15.5 Hz) Additionally, a carboxyl carbon signal at δC 167.1 (C-9′) and other carbon signals from 116.7 to 144.0 ppm were identified A broad singlet oxygenated methine proton signal at δH 4.74 (brs, H-3) indicated proximity to an ester group, differing from LL06's doublet doublet with coupling constants of 11.5 and 5.0 Hz, further confirming the 3α-orientation of the coumaroyl group.

From these data as well as the comparison of its NMR ones with those in the literature [45] , BC03 should be 3α-(E)-coumaroyllupeol

3.1.2.3 Structure elucidation of 3 α -( Z )-coumaroyllupeol (BC04)

Bruguiera cyclindrica with essential physical data as described below

 1 H, 13 C–NMR spectra (CDCl3–d) (Appendix 10.1 and 10.2): Tables 3.5 and 3.7

The 1 H and 13 C–NMR spectral data of BC04 were very similar to those of BC03 except for the appearance of signals for a Z- double bond of a coumaroyl moiety at

BC04 was identified as 3α-(Z)-coumaroyllupeol, characterized by NMR signals at H 5.90 (d, 12.5 Hz, H-8) and 6.82 (d, 12.5 Hz, H-7), indicating a Z-double bond instead of the E-double bond present in BC03 The NMR data of BC04 demonstrated strong alignment with the reference data [45].

3.1.2.4 Structure elucidation of betulin (LL03)

Lumnitzera littorea as well as from the fraction EA1.3 of the Scheme 2.2 of Bruguiera cyclindrica Its essential physical data were described the following

 APCI–MS spectrum (Appendix 11.1): m/z 425.34 [MH2O+H] + , calcd for C30H50O2–H2O+H, 425.71

The molecular formula of LL03 was established through the APCI–MS spectrum with the pseudomolecular ion peak at m/z 425.34 [MH2O+H] + , corresponding to the molecular formula of C30H50O2

The NMR spectra of LL03 were similar to those of LL06, including the proton and carbon signals for the lupane skeleton terpenoid The 1 H–NMR spectrum of

LL03 diﬀered from that of LL06 by having a pair of proton signals at H 3.33 (d, 11.0 Hz, H-28b) and 3.80 (d, 10.5 Hz, H-28a), instead of a methyl proton signal at H

The 13 C–NMR spectrum of LL03 revealed significant signals, including two olefinic carbons from the exocyclic double bond at δC 109.8 (C-29) and 150.6 (C-20), as well as an oxygenated methine carbon at δC 79.1 (C-3) Additionally, the presence of an oxygenated methylene carbon signal at δC 60.7 (C-28) indicated a second hydroxy group at C-28 Comparing these spectroscopic data with existing literature suggested that LL03 is identified as betulin.

Betulin, a pentacyclic triterpene found in birch tree bark at concentrations of 10–35%, possesses a range of health benefits including antiseptic, antiviral, and anti-inflammatory properties It has shown effectiveness against herpes and Epstein-Barr viruses, as well as hepatoprotective effects Additionally, betulin exhibits anticancer activities against lung, breast, prostate, and stomach cancer cells, alongside antiproliferative and antitumor effects.

Table 3.5 1 H–NMR data of known triterpenoids LL03, LL04, LL06, BC03 and BC04

BC03 BC04 LL06 LL03 LL04

3 4.74 (brs) 4.69 (brs) 3.18 (dd, 11.5, 5.0) 3.18 (dd,11.0, 4.0) 3.19 (dd, 11.0, 4.5)

19 2.45 (td, 11.0, 5.5) 2.45 (td, 11.0, 5.5) 2.37 (td, 11.0, 6.0) 2.38 (td, 11.0, 6.0) 3.00 (td, 11.0, 5.0)

3.1.2.5 Structure elucidation of betulinic acid (LL04)

Lumnitzera littorea as well as from the fraction E1.3 of the Scheme 2.2 of Bruguiera cyclindrica Its essential physical data were described the following

 APCI–MS spectrum (Appendix 12.1): m/z 455.38 [MH]  , calcd for C30H47O3–H, 455.69

The molecular formula C30H47O3 of LL04 was confirmed through the APCI–MS spectrum with the pseudomolecular ion peak at m/z 455.38 [MH] 

The NMR spectra analysis revealed that LL04 shares a lupane skeleton similar to LL03 and LL06; however, LL04's 1H–NMR spectrum lacked the proton signals at δH 3.30–3.80 associated with the H-28 position Instead, LL04 exhibited a carboxyl carbon signal at δC 180.5, replacing the oxygenated methylene carbon signal at δC 60.7 found in LL03 Consequently, LL04 was identified as betulinic acid, with its NMR data aligning well with existing literature.

Betulinic acid is a naturally occurring pentacyclic triterpenoid found throughout the plant kingdom, known for its diverse biological properties, including the inhibition of human immunodeficiency virus (HIV), specifically against HIV-1NL4-3.

1JRCSF with IC50 values of 0.04 and 0.002 g/mL, respectively [92, 93] ; cytotoxic

In a study evaluating various activities against human leukemic cell lines U937, HL60, and K562, the observed IC50 values were 13.73, 12.84, and 15.27 mg/mL, respectively Additionally, the research assessed the effects on WI-38 fibroblast cells, VA-13 malignant tumor cells, and Hep-G2 human liver tumor cells, yielding IC50 values of 1.3, 11.6, and 21.00 µM, respectively The findings also highlighted the inhibitory effects on DNA Topoisomerases, indicating potential therapeutic implications.

II activity with the EC50 value of 7.19 M against A549 cancer cell line [96] Besides, it also exhibited activities such as antimalarial, anti-inflammatory, anthelmintic, antinociceptive, anti-HSV-1 and antibacterial [92]

Table 3.6 1 H–NMR data of known triterpenoids LL07, LL08, LL10 and BC07

3.1.2.6 Structure elucidation of corosolic acid (LL07)

 APCI–MS spectrum (Appendix 13.1): m/z 471.43 [MH]  , calcd for C30H48O4H, 471.69

The mass spectrum of LL07 revealed a pseudomolecular ion peak at m/z 471.43, indicating a molecular formula of C30H48O4 Additionally, the 1H-NMR spectrum of LL07 exhibited five singlet methyl signals at δH 0.71 (s, H-24), 0.75 (s, H-26), 0.92 (s, H-23,25), and 1.04 (s, H-).

27), two doublet methyl signals at H 0.82 (d, 6.0 Hz, H-29) and 0.91 (d, 7.0 Hz, H-

The analysis revealed key characteristics of an ursane skeleton triterpenoid, with an olefinic proton signal identified at δH 5.15 (td, 14.5, 3.6 Hz, H-12) and a methine proton signal at δH 2.11 (d, 11.5 Hz, H-18) Additionally, two oxygenated methine proton signals were observed at δH 2.74 (d, 9.5 Hz, H-3) and 3.41 (m, overlapping with the solvent signal, H-2).

The 13 C–NMR spectrum of LL07 with the appearance of one carboxyl carbon at

C 178.4 (C-28) suggested the presence of a carboxylic acid functional group In addition, two oxygenated carbon signals were observed at C 67.2 (C-2) and 82.3 (C-

3) along with two olefinic carbon signals at C 124.5 (C-12) and 138.3 (C-13) Therefore, the structure of LL07 was suggested to be corosolic acid or 2α- hydroxyursolic acid due to its NMR spectra showed a good agreement with reported data [78]

Corosolic acid, the main active ingredient in the plant extract known as Glucosol, has been marketed in Japan and the United States for its potential benefits in lowering blood glucose levels and aiding weight loss.

Recently, it had attracted much attention due to its biological activities, such as antitumor [98] , antidiabetes [99] , anti-inflammatory [100] , antiproliferation [101] , protein

60 kinase C inhibition [102] , hypoglycemic [103] and cytotoxic activities against human cancer cell lines HL-60 (leukemia carcinoma), MCF-7 (breast carcinoma) and Hep- G2 (hepatic carcinoma) [99]

Table 3.7 13 C–NMR data of known triterpenoids LL03, LL04, LL06, LL07, LL08, LL10, BC03, BC04 and BC07

LL03 b LL04 a LL06 a BC03 a BC04 a LL07 a LL08 a BC07 a LL10 a

3.1.2.7 Structure elucidation of oleanolic acid (LL08)

Lumnitzera littorea with essential physical data were described the following

Chemical structure of flavonoids (group C)

3.1.3.1 Structure elucidation of lumnitzerone (LL19) (new compound)

Compound LL19 was obtained from the fraction E6 of the Scheme 2.1 of

 HR–ESI–MS spectrum (Appendix 17.1): m/z 501.1398 [M+H] + , calcd for C25H24O11+H, 501.1397

 1 H, 13 C–NMR spectra (DMSO–d 6) (Appendix 17.2 and 17.3): Table 3.8

 COSY, HSQC, HMBC and NOESY spectra (DMSO–d 6) (Appendix 17.4, 17.5, 17.6, 17.7, 17.8, 17.9 and 17.10)

 1 H–NMR spectrum (D2O) of the hydrolyzed sugar of LL19 (Appendix 17.11)

Its molecular formula was established as C25H24O11 through the pseudomolecular ion peak in the HRESIMS spectrum at m/z 501.1398 [M+H] + (calcd for

The 1 H and 1 H 1 H COSY NMR spectra of LL19 revealed signals of AA'BB'- type aromatic protons at δH 7.96 (2H, d, 8.5 Hz,) and 6.94 (2H, d, 8.5 Hz), and two meta coupled protons at δH 6.43 (1H, d, 2.0 Hz) and 6.78 (1H, d, 2.0 Hz) which were assigned to H-2/H-6, H-3/H-5, H-6 and H-8, respectively (Figure 3.1) In addition, an olefinic proton at δH 6.87 (1H, s, H-3), a chelated hydroxy proton and a phenolic proton at δH 12.96 (1H, s, 5-OH) and 10.40 (1H, s, 4-OH), respectively, were observed All these data suggested the presence of an apigenin aglycone

Figure 3.1 Key COSY and HMBC correlations of compound LL19

The 1 H-NMR spectrum analysis revealed a hexopyranose unit characterized by an anomeric proton at δH 5.13 (d, 7.5 Hz, H-1) and additional sugar proton signals ranging from δH 3.15 to 5.50 A detailed examination of the chemical shifts, multiplicities, and coupling constants in the 1 H-NMR spectrum, complemented by the 1 H-1 H COSY and 13 C-NMR spectra of LL19 (refer to Table 3.8), confirmed that the sugar structure is a β-anomer.

D-glucopyranose unit The nature of the sugar was further confirmed from the anomeric proton data of the sugar obtained after acid hydrolysis [57] The attachment of this sugar to C-7 in the apigenin nucleus was confirmed by the HMBC cross-peak of the anomeric proton at δH 5.13 (1H, d, 7.5 Hz) to carbon C-7 (δC 162.6)

Furthermore, characteristic signals of an (E)-propenyl group were observed at δH

5.83 (1H, dd, 15.5, 1.5 Hz, H-2), 6.85 (1H, dq, 15.5, 7.0 Hz, H-3) and 1.64 (3H, d, 7.0 Hz, H-4) A large coupling constant of 15.5 Hz of the two proton H-2 and

The (E) configuration of the double bond at C-2 was assigned based on the multiplicity of protons H3-4, H-3, and H-2, which was confirmed by the 1H-1H COSY spectrum indicating their contiguous arrangement Additionally, HMBC experiments revealed cross-peaks for the propenyl protons at δH 5.83 (H-2) and 6.85 (H-3), along with methylene protons at δH 4.41 (H-6a) and 4.08 (H-6b) connecting to the carbonyl carbon C-1.

(δC 165.3) suggesting that the butenoyloxy group was attached at C-6 of the D - glucose

Table 3.8 The NMR data of the new compound LL19

* overlapped with the solvent signal

The 13 CNMR spectrum of LL19 revealed twenty five carbon signals, including signals of two benzene rings of the apigenin aglycone at δC 164.3 (C-2), 103.1 (C-

3), 182.0 (C-4), 161.4 (C-5), 99.4 (C-6), 162.6 (C-7), 94.7 (C-8), 156.9 (C-9), 105.4 (C-10), 121.0 (C-1), 128.6 (C-2/C-6), 116.0 (C-3/C-5) and 161.1 (C-4), of a hexopyranose unit at δC 99.5 (C-1), 72.9 (C-2), 76.2 (C-3), 70.0 (C-4), 73.8 (C-

5) and 63.4 (C-6) and of a butenoyl group at δC 165.3 (C-1), 122.0 (C-2), 145.3

Accordingly, LL19 was identified as apigenin 7-O-[6-(E)-butenoyl--D- glucopyranoside] for which the trivial name lumnitzerone was proposed

3.1.3.2 Structure elucidation of naringenin (LL20)

 COSY, HSQC, HMBC spectra (DMSO–d 6) (Appendix 18.3, 18.4 and 18.5)

The 1 H–NMR spectrum of compound LL20 with one singlet signal of two protons at H 5.87 (s, H-6 and H-8) showed the pattern of 5,7-dihydroxyflavonoid of the A ring The ortho coupled doublet signals at H 7.31 (d, 8.5 Hz, H-2 and H-6) and the others at H 6.78 (d, 8.5 Hz, H-3 and H-5) were assignable to aromatic protons of the B ring In addition, the presence of an ABX system at H 5.43 (dd, 12.5, 3.0 Hz, H-2), 3.25 (dd, 17.0, 12.5 Hz, H-3ax) and 2.68 (dd, 17.0, 3.0 Hz, H-3eq) was characterized for a flavanone skeleton Besides, three singlet proton signals, one at H 12.13 indicated the presence of a chelated hydroxy at C-5 position, two at H

10.86 and 9.63 indicated the protons of hydroxy groups at C-7 and C-4 positions, respectively

The 13 C–NMR spectrum of compound LL20 revealed the presence of fifteen carbon signals including of one carbonyl carbon at C 196.5 (C-4), six quaternary aromatic carbons in the zone of 100–167 ppm, six methine aromatic carbons in the zone of 95–129 ppm and two signals at C 78.5 and 42.0 characteristics of carbon C-

2 and C-3 respectively of a flavanone skeleton

Moreover, the 1 H 1 H COSY NMR spectrum of LL20 showed the correlations of H-2/H-3a/H-3b

This structure was also confirmed via the HSQC and HMBC correlations of H-

3eq proton at H 2.68 (dd, 17.0, 3.0 Hz) with three carbons at C 42.0 (C-3), 101.9 (C-

10) and 196.5 (C-4), of the H-3ax proton at H 3.25 (dd, 17.0, 12.5 Hz) with four carbons at C 42.0 (C-3), 78.5 (C-2), 128.4 (C-2,6) and 196.5 (C-4) and of the H-2 proton at H 5.43 (dd, 12.5, 3.0 Hz) with four carbons at C 78.5 (C-2), 128.4 (C- 2,6), 129.0 (C-1) and 196.5 (C-4) The proton at H 5.43 (H-2), correlated to the carbon signal at C 78.5 (C-2) in HSQC spectrum, with the large coupling constant

J.5 Hz, further confirming the α-configuration of the B ring at C-2 position

Analysis of NMR data for LL20, which aligns well with existing literature, confirms that the structure of LL20 is identified as naringenin.

Naringenin, a prominent flavanone found in citrus fruits like oranges and grapefruits, has garnered attention for its potential health benefits, including cancer prevention and the management of non-cancer diseases Recent years have seen substantial advancements in research exploring the cellular and molecular biological effects of naringenin.

Naringenin possessed various biological activities such as immunomodulatory, DNA protective, hypolipidaemic, hepatoprotective, antidiabetic, antiatherogenic, antidepressant, antioxidant, anticarcinogenic, antitumor and anti-inflammatory [112]

In addition, it had potential to be a useful chemopreventive and therapeutic agent in various types of cancers as skin, breast, colon, liver, lung and pancreatic [113]

3.1.3.3 Structure elucidation of taxifolin (LL25)

 ESI–MS spectrum (Appendix 19.1): m/z 303.30 [MH]  , calcd for C15H12O7–H, 303.25

The mass spectrum analysis of LL25 revealed a pseudomolecular ion peak at m/z 303.30, indicating a molecular formula of C15H12O7 Additionally, the 1H and 13C-NMR data of LL25 closely resembled those of LL20 The 1H-NMR spectrum exhibited double signals at δH 4.96 (d, 11.0 Hz) and 4.48 (d, 11.0 Hz), corresponding to protons at the H-2 and H-3 positions, respectively These signals aligned with oxygenated carbon signals at δC 83.1 (C-2) and 71.6 (C-3), confirming the presence of a hydroxy group at the C-3 position and characterizing LL25 as a flavanonol skeleton.

2,3-trans configuration Besides, the proton signals at δH 6.87 (s, H-2), 6.73 (d, 8.0

Hz, H-5) and 6.75 (d, 8.0 Hz, H-6) indicated an ABX system of 1',3',4'-trisubstituted aromatic ring, one singlet proton signal at H 8.99 assigning to other hydroxy group at C-3' position

Based on the NMR data analysis of LL25 and the good compatibility of its NMR data with those reported in the literature [115] , LL25 was identified as taxifolin

Taxifolin, or dihydroquercetin, is a natural bioactive flavonoid present in numerous foods and herbs, known for its diverse pharmacological properties Research indicates that it possesses antioxidative, antinitrosative, and anti-inflammatory effects, making it a valuable compound for health benefits.

Table 3.9 1 H–NMR data of known flavonoids LL20, LL22, LL24 and LL25

3.1.3.4 Structure elucidation of luteolin (LL21)

The NMR analysis of LL21 revealed a structural similarity to LL20; however, notable differences were observed in the 1 H–NMR spectrum Specifically, LL21 lacked the doublet doublet proton signal at δH 5.43 corresponding to the H-2 position and exhibited a pair of doublet doublet proton signals at δH 3.25 and 2.68 for H-3ax and H-3eq, respectively.

3eq, respectively In addition, the presence of only one singlet proton signal at H 6.66 of H-3 position suggested the flavone skeleton of LL21

Furthermore, the 1 H–NMR spectrum of LL21 showed the presence of a chelated hydroxy group at H 12.97 (s, 5-OH), three meta coupled aromatic doublet signals at

H 6.18 (d, 2.0 Hz), 6.44 (d, 2.0 Hz) and 7.42 (d, 2.5 Hz), each integrated for one proton, assigned to H-6, H-8 and H-2, respectively, one ortho coupled aromatic doublet signal at H 6.89 (d, 8.0 Hz) and one aromatic doublet doublet signal at H

7.40 (dd, 5.5, 2.0 Hz) corresponding to H-5 and H-6 protons, respectively These were characteristics of a 5,7,3,4-tetrasubstituted flavone

The 13 C–NMR spectrum of LL21 showed the presence of fifteen carbon signals, including one carbonyl carbon at C 181.6 (C-4), eight quaternary aromatic carbons in the zone of 103.7–164.1 ppm, six methine aromatic carbons in the zone of 93.8– 118.9 ppm

Thus, LL21 was identified as luteolin whose structure was confirmed by the comparison of its NMR data with the ones in the literature [117]

Luteolin is a widely recognized flavonoid found in various fruits, vegetables, and medicinal herbs It is known for its diverse biological activities, including anti-allergy, antioxidant, anti-inflammatory, and cytotoxic effects.

Besides, it was also used as a chemopreventive agent against B(a)P induced lung carcinogenesis Hence, luteolin could be developed as a chemopreventive drug to reduce the risk of human lung cancer [121]

3.1.3.5 Structure elucidation of chrysoeriol (LL22)

 APCI–MS spectrum (Appendix 21.1): m/z 299.24 [MH]  , calcd for C16H12O6–H, 299.26

The mass spectrum of LL22 revealed a pseudomolecular ion peak at m/z 299.24, indicating a molecular formula of C16H12O6 The 1H-NMR spectrum of LL22 displayed characteristic signals of a 5,7,3',4'-tetrasubstituted flavone, with notable peaks at δH 6.87 (s, H-3), 12.96 (s, 5-OH), 6.19 (d, 2.0 Hz, H-6), 6.50 (d, 2.0 Hz, H-8), 6.93 (d, 9.0 Hz, H-5'), and 7.54 (d, 2.0 Hz).

Hz, H-2) and 7.56 (d, 2.0 Hz, H-6) Furthermore, one singlet proton signal at H 3.89 identified the presence of one methoxy group that was absent in the 1 H–NMR spectrum of LL21

Chemical structure of phenolic compounds (Group D)

3.1.4.1 Structure elucidation of gallic acid (LL11)

Compound LL11 was obtained from the fraction E2.5 of the Scheme 2.1 of

The 13 C–NMR spectrum of LL11 revealed seven carbon signals, comprising a carboxyl carbons at C 167.8 (C-7), three oxygenated carbons at C 145.7 (C-3,5) and 138.3 (C-4), two methine carbons at C 109.1 (C-2,6) and one quaternary carbon C

120.8 (C-1) They corresponded to a singlet aromatic proton signal at H 6.94 (H-2,4) in the 1 H–NMR spectrum

Therefore, LL11 was identified as gallic acid through the comparison of its NMR data with those published in the literature [145]

Gallic acid is a polyhydroxylphenolic compound found in various plants, fruits, and foods, known for its diverse biological activities, including cytotoxic, anti-inflammatory, and antitumoral effects Additionally, it demonstrates antibacterial properties against Salmonella typhi and Staphylococcus epidermidis, with minimum inhibitory concentration (MIC) values of 2.50 mg/mL and 1.25 mg/mL, respectively.

3.1.4.2 Structure elucidation of 3,3,4-tri- O -methylellagic acid (LL28)

 ESI–MS spectrum (Appendix 32.1): m/z 345.17 [M+H] + , calcd for C17H12O8+H, 344.27

The 1 HNMR spectrum of LL28 displayed the presence of two singlet aromatic proton signals at δH 7.61 (H-5) and 7.52 (H-5), and three singlet proton signals at δH

The 13C-NMR spectrum of LL28 revealed the presence of an asymmetrical ellagic acid unit along with three methoxy groups attached at positions C-3, C-4, and C-3′ The chemical shifts of these methoxy groups were noted at δH 4.06, δH 4.04, and δH 4.00, respectively, with HMBC correlations confirming the attachment of the proton at δH 4.06 to C-3.

4.04 with C-3 and of proton at δH 4.00 with C-4

Additionally, the ESI–MS spectrum of LL28 showed the pseudomolecular ion peak at m/z 345.17 [M+H] + (calcd for C17H12O8+H, 344.27)

On the basis of these results along with the good compatibility of its NMR data with those in the literature [149] , LL28 was determined 3,3,4-tri-O-methylellagic acid

3.1.4.3 Structure elucidation of 3,3,4-tri- O -methylellagic acid 4- O - β - D - glucopyranoside (LL29)

 HSQC, HMBC spectra (DMSO–d 6) (Appendix 33.3, 33.4 and 33.5)

Table 3.14 The NMR data of known phenolic compounds LL11, LL28 and LL29

The 13 C–NMR spectrum of LL29 displayed similar signals to those of LL28, including twelve carbon signals in the zone from δC 107.6113.7 and 140.9154.3, two carboxyl carbon signals at δC158.1 (C-7) and 158.4 (C-7) of an ellagic acid unit Besides, LL29 possessed one more β-D-glucose unit, which was evidenced by the presence of an anomeric carbon at δC101.3, and a series of five oxygenated carbon signals at δC 77.3 (C-3), 76.5 (C-5), 73.3 (C-2), 69.5 (C-4) and 60.6 (C-6) in the

13C–NMR spectrum as well as a doublet anomeric proton signal at δH5.17 (d, 7.0 Hz), and a series of signals from 3.24 to 3.71 ppm in the 1 H–NMR spectrum This sugar

The compound 91 was linked to the ellagic acid moiety at C-4' through an O-glucosyl connection, as confirmed by HMBC correlations between proton H-1'' at δH 5.17 and carbon C-4' at δC 154.3 Furthermore, the NMR data for LL29 demonstrated strong alignment with previously published findings, leading to the identification of LL29 as 3,3',4-tri-O-methylellagic acid 4'-O-β-D-glucopyranoside.

Chemical structure of other compounds (Group E)

3.1.5.1 Structure elucidation of benzobrugierol (BC01)

Bruguiera cylindricawith essential physical data as described below

 HR–ESI–MS spectrum (Appendix 34.1): m/z 167.0190 [M+H] + , calcd for C8H6O2S+H, 167.0167

 1 H, 13 C–NMR spectra (Acetone–d 6) (Appendix 34.2 and 34.3): Table 3.16

 COSY, HSQC, HMBC spectra (Acetone–d 6) (Appendix 34.4, 34.5, 34.6 and 34.7)

Compound BC01 is a white amorphous powder that appears purple on a TLC plate when viewed under UV light at 365 nm Its molecular formula, C8H6O2S, was confirmed by the pseudomolecular ion peak observed in the HR-ESI-MS spectrum at m/z 167.0190 [M+H]+, with a calculated value of 167.0167, resulting in a millimass error of 2.3.

The 1 HNMR spectra of BC01 indicated one olefinic proton signal at H 8.03 (1H, s, H-3), and four aromatic ones at H 8.15 (1H, dd, 7.0, 2.5 Hz, H-4), 7.51 (1H,

92 dd, 7.0, 2.0 Hz, H-7) and 7.19 (2H, m, H-5 and H-6), corresponding to the carbon signals at C 132.7 (C3), 122.0 (C4), 112.8 (C7) and 123.3 (C5 and C6) in the

The 13C-NMR and HSQC spectra of BC01 revealed two quaternary aromatic carbons at δC 137.8 (C7a) and 127.5 (C3a), along with one oxygenated carbon at δC 166.3 (C2) Additionally, the 1H-1H COSY experiment demonstrated correlations between adjacent aromatic protons H4/H5/H6/H7 These spectral characteristics were similar to those of indole-3-carboxylic acid; however, the molecular formula of BC01 (C9H7O2N+H, 162.0555 amu) did not align with the experimental HR-MS spectrum.

Figure 3.3 Key COSY and HMBC correlations of compound BC01

The integration of 2D-NMR and HR-ESI-MS data indicates that BC01 is likely comprised of a 1,2-disubstituted benzene ring, represented by the structural formula C6H4, which is fused with a specific x-membered ring featuring a distinct partial structural formula.

The structure of C2H2O2S was validated through HMBC correlations, revealing the connections between protons and carbons: the H-4 proton at δH 8.15 correlates with carbons at δC 123.3 (C-5, C-6) and 137.8 (C-7a), while the H-7 proton at δH 7.51 connects to carbons at δC 122 (C-4) and 127.5 (C-3a) Additionally, H-5 and H-6 protons at δH 7.19 correlate with four carbons at δC 112.8 (C-7), 122.0 (C-4), 127.5 (C-3a), and 137.8 (C-7a) A singlet at δH 8.03 further confirms the presence of a hydroxy group at C-2 or C-3 in the five-membered ring containing a S=O group Two potential structures, BC01x and BC01y, were identified that align with all NMR and HR-MS data.

Figure 3.4 Proposed structure of compound BC01

Table 3.15 Cartesian coordinates of predicted structures of compound BC01

In order to assign the correct structure of the isolated compound (BC01x or

The stable geometries of the isomer BC01y were optimized and analyzed, revealing that it is more stable than the other isomer by 1.9 kcal/mol in the gas phase and by 4.35 kcal/mol in methanol Additionally, the DP4 probability calculation indicated a high prediction accuracy of 99.85% for the configuration of BC01y based on its NMR chemical shift parameters.

94 probability Accordingly, 3-hydroxybenzo[b]thiophene-1-dioxide (BC01y) was assigned for BC01 and was named benzobrugierol

Five-membered ring compounds containing a thiolane oxide group, eg brugierol

(108) and isobrugierol (109), had been reported in some plants of this species such as in Bruguiera conjugata [152] ,B cylindrica [153] ,B sexangula [30] and B gymnorrhiza [43]

Table 3.16 The NMR data of the new compound BC01

3.1.5.2 Structure elucidation of bruguierine (BC02)

 HR–ESI–MS spectrum (Appendix 35.1): m/z 425.1143 [M+Na] + , calcd for C23H18O5N2+Na, 425.1113

 1 H, 13 C–NMR spectra (Methanol–d 4) (Appendix 35.2 and 35.3): Table 3.18

 HSQC, HMBC spectra (Methanol–d 4) (Appendix 35.4 and 35.5)

Compound BC02 is a white amorphous powder with a molecular formula of C23H18O5N2, determined through the HR-ESI-MS spectrum, which showed a pseudomolecular ion peak at m/z 425.1143 [M+Na] This value closely matches the calculated mass of 425.1113 for C23H18O5N2+Na, resulting in a minimal error of 3.0 millimass.

The 1 HNMR spectrum of BC02 displays four doublet signals corresponding to twelve protons in the aromatic region, specifically at δH 7.77 (4H, d, 8.5 Hz), 7.23 (2H, d, 8.5 Hz), 6.92 (4H, d, 8.5 Hz), and 6.77 (2H, d, 8.5 Hz), indicating the presence of three symmetrical 1,4-disubstituted benzene rings Additionally, a singlet proton signal at δH 9.76 (2H, s) and a carbon signal at δC 192.8 in the 13 CNMR spectrum suggest an aldehyde group in the molecular structure of BC02.

The analysis revealed five oxygenated aromatic carbon signals at δC 165.2, 158.8, and 116.9, alongside twelve aromatic methine carbon signals at δC 133.4, 129.0, 116.9, and 115.8 Additionally, one methine carbon with two heteroatoms was identified at δC 104.9, and three quaternary aromatic carbon signals were noted at δC 130.5 and 130.3 The integration of NMR and HR-ESI-MS data indicated a comprehensive understanding of the compound's structure.

BC02 could be composed of three 1,4-disubstituted benzene rings (A, B, C rings), one imidazole, one hydroxy and two aldehyde groups

Figure 3.5 Key COSY and HMBC correlations of compound BC02

The HSQC and HMBC correlations of two aldehyde proton signals with C-2,C-3,C-4,C-5,C-6 and C-2,C-3,C-4,C-5,C-6 indicated the attachment of the

The presence of an aldehyde group at C4 of the aromatic B ring and another at C-4' of the C ring is illustrated in Figure 3.5 The HMBC cross-peak analysis shows that H-2 is connected to C-3', C-5', C-2', C-6', and C-4', confirming the linkage between C-1' of the aromatic A ring and carbon C-2 of the imidazole ring.

Figure 3.6 Propose structure of compound BC02

Based on these analyses, there were two structures BC02x and BC02y (Figure

3.6) which could fit all the experimental HRMS and NMR data It was obvious that the geometric structure BC02x possessed an imidazole ring while BC02y having a dioxole one

The results presented in Table 3.17 indicate that BC02x is the more energetically favorable structure compared to BC02y, with a relative energy difference of 16.01 kcal/mol in the gas phase and 18.06 kcal/mol in methanol The PD4 analysis supports this finding, showing a probability of 100% for BC02x Consequently, BC02x is identified as the structure of compound BC02, which is named 4,5-di(4-formylphenoxy)-2-(4-hydroxyphenyl)-2,3-dihydro-1H-imidazole, or bruguierine.

Table 3 17 Cartesian coordinates of predicted structures of compound BC02

Table 3.18 The NMR data of the new compound BC02

3.1.5.3 Structure elucidation of (6 R ,9 R )-9-hydroxy-4-megastigmen-3-one

Compound BC08 was obtained from the fraction EA4 of the Scheme 2.2 of

 1 H, 13 C–NMR spectra (Methanol–d 4) (Appendix 36.1 and 36.2): Table 3.19

 HSQC, HMBC spectra (Methanol–d 4) (Appendix 36.3 and 36.4)

The 1 HNMR spectrum of BC08 revealved an olefinic proton signal at δH 5.82 (s, H-4), an oxygenated one at δH 3.70 (m, H-9), a non-equivalent methylene at δH

2.46 (d, 17.5 Hz, H-2a) and 2.01 (d, 17.0 Hz, H-2b), a doublet methyl one at δH 1.17 (d, 6.5 Hz, H-10) and three singlet methyls at δH 1.10 (3H, s, H-11), 1.03 (3H, s, H-

The 13 CNMR spectrum of BC08 displayed thirteen carbon signals including one carbonyl carbon signal at C 202.2 (C-3), two olefinic carbon signals at C 169.7 (C-

5) and 125.4 (C-4), one oxygenated carbon signal at C 68.8 (C-9) and other carbon signals at C 23.5–52.5 ppm

Additionally, the HSQC and HMBC spectra of BC08 showed that H-4 had

HMBC correlations with carbons at δC 52.5 (C-6), 48.1 (C-2) and 24.9 (C-13) This

99 indicated that the position of the double bond was between C-4 and C-5 The correlations of the proton signal H-13 at δH 2.05 with carbon signals at δC 169.7 (C-

5) and 125.4 (C-4) confirmed that there was one methyl group directly attached to an olefinic carbon at C-5 position The attachment of remaining three methyl groups at C-1 and C-9 were assigned by HMBC cross peaks of protons H-11 and H-12 at δH

1.10 and 1.03, respectively, with carbon at δC 37.3 (C-1), and of proton H-10 with carbon at δC 68.8 (C-9)

Based on all the aforementioned analysis and the comparison of NMR data of

BC08 to those published in the literature [154] , BC08 was determined as (6R,9R)-9- hydroxy-4-megastigmen-3-one

Table 3.19 The NMR data of the known compound BC08

Pos BC08 (Methanol– d 4 ) Pos BC08 (Methanol– d 4 )

3.1.5.4 Structure elucidation of laportoside A (LL26)

 ESI–MS spectrum (Appendix 37.1): m/z 842.32 [MH]  , calcd for C48H93NO10–H, 842.24

 HSQC, HMBC spectra (DMSO–d 6) (Appendix 37.4 and 37.5)

The molecular formula of LL26 was established as C48H92NO10 through the pseudomolecular ion peak in the ESIMS spectrum at m/z 842.32 [MH]  (calcd for

The 1 HNMR spectrum of LL26 exhibited a doublet proton signal at δH 7.51 with the coupling constant J= 9.0 Hz of a labile hydrogen of the NH group, a multiplet proton signal at δH 4.09 (m, H-2) assigning to a nitrogenated methine, two olefinic proton signals at δH 5.55 (d, 5.0 Hz, H-12) and 5.36 (brs, H-13), five oxygenated proton signals at δH 3.85 (dd, 11.0, 5.0 Hz, H-2), 3.81 (dd, 11.0, 7.0 Hz, H-1b), 3.64 (dd, 13.0, 4.0 Hz, H-1a), 3.38 (m, H-4) and 3.35 (m, H-3)

The analysis revealed proton signals indicative of a β-D-glucose unit, including signals at δH 2.94–3.09, anomeric proton at δH 4.14 (d, 7.5 Hz, H-1), and doublet-doublet signals at δH 3.67 (dd, 12.5, 6.0 Hz, H-6a) and 3.44 (dd, 12.0, 6.0 Hz, H-6b) Furthermore, methylene proton signals in the range of δH 1.32–1.92 and 0.85 (t, 6.5 Hz, H-24, H-18) confirmed the presence of two long aliphatic chains.

The 13 CNMR spectrum of LL26 showed the presence of an amide group at δC

The compound LL26 features a nitrogenated methine carbon at δC 49.9 (C-2), along with two olefinic carbons at δC 130.2 (C-13) and 129.6 (C-12) Additionally, it contains four oxygenated carbons at δC 74.1 (C-4), 70.9 (C-2), 70.5 (C-3), and 68.9 (C-1) Notably, LL26 includes a β-D-glucose unit, indicated by an anomeric carbon at δC 103.4 (C-1) and five oxygenated carbons at δC 76.8 (C-5), 76.5 (C-3), 73.4 (C-2), 70.0 (C-4), and 61.0 (C-6) It also possesses two long aliphatic chains ranging from δC 24.4 to 32.0 and at 13.9 (C-24).

18) The above data suggested that the structure of LL26 possessed a ceramide skeleton This was confirmed by the HSQC and HMBC experiments

The HMBC correlations indicate that the oxygenated methylene proton at δH 3.64 (H-1a) is linked to the anomeric carbon at δC 103.4 (C-1), confirming the attachment of the glucose unit at C-1 of the ceramide skeleton Additionally, the anomeric proton at δH 4.14 (H-1) shows a correlation with the carbon signal at δC 103.4 (C-1), characterized by a significant coupling constant.

J=7.5 Hz, further confirming the β-configuration of the glucosyl unit In addition, the nitrogenated methine proton at δH 4.09 correlated to the carbon signal at δC 68.9 (C-

1) confirming this proton being at H-2 position

Table 3.20.The NMR data of the known compound LL26

α-GLUCOSIDASE INHIBITORY ACTIVITY OF EXTRACTS

All extracts and most of the isolated compounds were evaluated the α-glucosidase inhibitory activity according to the method of Apostolidis et al [63] with acarbose as a positive control

3.2.1 α -Glucosidase inhibitory activity of extracts

The α-glucosidase inhibitory activity was applied to the different extracts of Lumnitzera littorea (Scheme 2.1) as well as Bruguiera cylindrica (Scheme 2.2) The results were presented in Table 3.21

Table 3.21 α-Glucosidase inhibitory activity of different extracts from

Lumnitzera littorea and Bruguiera cylindrica leaves

Ethanol crude residue 17.9  0.4 28.1  0.3 38.4  0.1 51.3  0.2 71.9  0.4 66.5  0.4 n-Hexane 32.7  0.3 42.0  0.1 55.1  0.3 68.3  0.1 79.5  0.3 40.1  0.2 Ethyl acetate 46.3  0.2 49.6  0.1 52.8  0.3 57.7  0.3 68.0  0.1 28.3  0.3 Ethanol 23.8  1.0 43.9  0.3 55.1  0.2 67.3  0.1 81.8  0.3 43.4  0.3

Ethanol crude residue 26.1  0.3 36.5  0.1 56.9  0.4 72.5  0.2 80.1  0.4 87.3  0.4 n-Hexane 29.6  0.3 42.6  0.2 57.0  0.4 70.0  0.3 86.1  0.3 78.2  0.2 Ethyl acetate 31.8  0.4 46.0  0.1 65.9  0.2 79.6  0.4 86.7  0.1 61.8  0.3 Ethanol 4.7  0.2 17.1  0.3 37.9  0.2 52.6  0.1 75.5  0.4 135.8  0.4

The study revealed that, with the exception of the ethanol extract from Bruguiera cylindrica leaves, all extracts from Lumnitzera littorea and Bruguiera cylindrica demonstrated superior inhibitory activity against the α-glucosidase enzyme compared to the positive control, acarbose A dose-dependent increase in inhibitory activity was observed, with the ethyl acetate extract of Lumnitzera littorea leaves exhibiting the highest percentage inhibition, achieving an IC50 value of 28.3 ± 0.3 µg/mL These findings suggest that both Lumnitzera littorea and Bruguiera cylindrica may be beneficial in managing postprandial hyperglycemia.

3.2.2 α -Glucosidase inhibitory activity of new compounds

One new compound, lumnitzerone (LL19) isolated from Lumnitzera littorea and two new ones namely benzobrugierol (BC01) and bruguierine (BC02) isolated from

Bruguiera cylindrica were evaluated the α-glucosidase inhibitory activity The results were presented in Table 3.22

Table 3.22 α-Glucosidase inhibitory activity of three new compounds

Positive control Concentration (g/mL) IC 50

Table 3.22 demonstrates that all new compounds outperformed acarbose, the positive control, in terms of activity Notably, lumnitzerone (LL19) emerged as the most effective inhibitor, exhibiting an IC50 value of 11.3 ± 0.5 µg/mL.

3.2.3 α -Glucosidase inhibitory activity of isolated known compounds

Most of known compounds isolated from Lumnitzera littorea and Bruguiera cylindrica were evaluated for the α-glucosidase inhibitory activity The results were presented in Tables 3.23–3.25

The inhibitory effect of flavonoids against α-glucosidase activity presented in

Naringenin (LL20) demonstrated the highest efficacy with an IC50 value of 1.9 ± 0.2 µg/mL, significantly outperforming luteolin (LL21), which had an IC50 value of 10.1 ± 0.5 µg/mL This finding suggests that the presence of a C-2–C-3 double bond diminishes flavonoid activity, as the absence of this bond allows the B ring to shift out of alignment with the A and C rings.

105 rings, destroying the overall planarity of the compounds, while the molecules with near planar structure easily enter the hydrophobic pockets in the enzyme [157]

Table 3.23 α-Glucosidase inhibitory activity of isolated flavonoids and phenols

Naringenin (LL20) 39.9  1.0 96.8  0.3 > 100 > 100 > 100 1.9  0.2 Quercetin (LL12) 47.0  0.3 52.0  0.5 65.5  0.4 82.9  0.4 100.0  0.3 3.4  0.5 Afzelin (LL23) 33.4  0.2 44.8  0.5 64.8  0.4 75.8  0.5 91.5  0.3 6.3  0.3 Quercitrin (LL13) 28.3  0.5 43.1  0.4 53.7  0.3 74.9  0.4 89.4  0.2 7.7  0.2 Luteolin (LL21) - 23.5  0.5 50.2  0.2 80.4  0.3 >100 10.1  0.3

A comparison of quercetin (LL12) and luteolin (LL21) reveals that the presence of a 3-OH group in the C ring enhances the inhibition of α-glucosidase, as evidenced by their respective IC50 values of 3.4 ± 0.5 µg/mL for quercetin and 10.1 ± 0.5 µg/mL for luteolin This structural feature may facilitate the binding of flavonoids to the α-glucosidase enzyme by forming hydrogen bonds, as reported by Xu H.

The replacement of hydroxyl (−OH) groups with methoxy (−OMe) groups reduced the inhibitory activity of flavonoids such as chrysoeriol (LL22), taxifolin (LL25), and pilloin (LL24) Additionally, the inhibition of quercetin (LL12) and myricetin (LL14), along with their glycosides quercitrin (LL13) and myricitrin (LL15), indicates that O-glycosylation of the −OH groups at the C-3 position slightly diminishes α-glucosidase inhibitory activities.

The IC50 values for myricetin 3-O-(2”-O-galloyl)-α-L-rhamnopyranoside (LL18), myricetin 3-O-(3”-O-galloyl)-α-L-rhamnopyranoside (LL17), and myricetin 3-O-(4”-O-galloyl)-α-L-rhamnopyranoside (LL16) were measured at 89.3 ± 0.5, 99.7 ± 0.5, and 68.5 ± 0.5 µg/mL, respectively These results indicate that substituting the sugar moiety in flavononol glycosides with (O-galloyl)rhamnopyranose significantly enhances the enzyme's inhibitory effect The presence of galloyl moieties, which contain three hydroxy groups, improves enzyme binding through hydrogen bonding.

These results showed good compatibility with the structure–activity relationship studies on flavonoids as inhibitors of α-glucosidase enzyme according to Şửhretoğlu

Hydroxy group at any position  Methyl or glycoside group at any position  Phenolic susbtitution at any position of sugar moiety 

 : decrease α-glucosidase inhibitions of flavonoids

 : increase α-glucosidase inhibitions of flavonoids

Figure 3.7 The structure–activity relationships of α-glucosidase effects of flavonoids

Table 3.24 α-Glucosidase inhibitory activity of isolated triterpenoids

Lupeol (LL06) 30.2  0.5 36.3  0.4 39.4  0.3 41.5  0.8 53.0  0.6 98.0  0.6 Betulin (LL03) 17.8  0.5 46.8  0.7 60.3  0.7 70.8  0.4 > 100 38.7  0.6 Betulinic acid

Positive control Concentration (g/mL) IC 50

Pentacyclic triterpenoids exhibit a significant inhibitory effect on α-glucosidase activity, as demonstrated in Table 3.24 Among the tested triterpenoids, corosolic acid (LL07), oleanolic acid (LL08), and betulinic acid (LL04) displayed the strongest inhibitory effects.

The IC50 values for corosolic acid (LL07), oleanolic acid (LL08), and betulinic acid (LL04) were found to be 17.9 ± 0.4, 18.8 ± 0.6, and 28.1 ± 0.4 μg/mL, respectively, demonstrating superior inhibition compared to acarbose These findings suggest that variations in structural skeletons, substituent groups, and their positions significantly influence inhibitory effects Notably, corosolic acid exhibited stronger inhibition than oleanolic acid, indicating that relocating the C-29 methyl group from C-20 to C-19 enhances α-glucosidase inhibition Additionally, the weaker inhibition of betulinic acid compared to corosolic and oleanolic acids highlights the potential importance of the six-membered E ring Furthermore, the comparative analysis of betulinic acid, betulin, and lupeol suggests that the carboxyl group at the C-17 position and the hydroxy group at the C-28 position are crucial for effective α-glucosidase inhibition.

Table 3.25 α-Glucosidase inhibitory activity of isolated steroids

The inhibitory effect of steroids against α-glucosidase activity was shown in

The study reveals that the presence of an -OH or =O group at the C-3 position is crucial for α-glucosidase inhibitory activity Notably, β-sitosterol (LL01) and stigmast-4-ene-3-one (LL09) exhibited lower IC50 values compared to stigmast-5-ene 3β-O-(6-O-hexadecanoyl-β-D-glucopyranoside) (LL02) and β-sitosterol 3-O-β-D-glucopyranoside (LL05), indicating a stronger inhibitory effect likely due to their bulkier structures Conversely, the addition of a β-glucose unit at C-3 of β-sitosterol 3-O-β-D-glucopyranoside (LL05) and the palmitoyl moiety at C-6' of the glucose unit in stigmast-5-ene 3β-O-(6-O-hexadecanoyl-β-D-glucopyranoside) (LL02) resulted in decreased α-glucosidase inhibitory activity.

The oral acute toxicity of crude ethanol extract from Lumnitzera littorea leaves was evaluated in Swiss albino rats using single dose levels of 1.0, 2.0, 3.0, 4.0, 8.0, and 13.0 g/kg, with results detailed in Table 3.26.

Table 3.26 Oral acute toxicity study of the crude ethanol leaf extract of Lumnitzera littorea in rats

Mortality After 72 hours After 14 days

Table 3.26 shows that no mortality or clinical signs of toxicity were observed in treated rats across all tested doses over a 14-day observation period According to the GHS (Globally Harmonised System for Classification of Chemicals), the crude ethanol extract of Lumnitzera littorea leaves is classified as non-toxic at a concentration of 13.0 g/kg.

4.1 THE CHEMICAL CONSTITUENTS OF LUMNITZERA LITTOREA

A chemical investigation of Lumnitzera littorea and Bruguiera cylindrica from the Can Gio mangrove forest in Vietnam resulted in the isolation of thirty-seven compounds, comprising three novel compounds and thirty-four known ones The determination of the chemical structures of these isolated compounds was achieved through a combination of spectroscopic analysis and comparison with existing literature data.

From leaves of Lumnitzera littorea, twenty nine compounds were isolated, including one new flavonoid (LL19), five known steroids (LL01, LL02, LL05,

The study identifies a total of six triterpenoids (LL03, LL04, LL06, LL07, LL08, LL10), thirteen flavonoids (LL12, LL13, LL14, LL15, LL16, LL17, LL18, LL20, LL21, LL22, LL23, LL24, LL25), three phenolic compounds (LL11, LL28, LL29), and one additional compound (LL26).

LL01, LL05 and LL12, although these compounds were already known in other species, the other twenty six compounds were reported for the first time from

From leaves of Bruguiera cylindrica, seventeen compounds were isolated, including two new compounds (BC01 and BC02), four known steroids (LL01, LL05,

BC05, BC06), seven known triterpenoids (LL03, LL04, LL06, LL10, BC03, BC04 and BC07), three known flavonoids (LL12, LL13 and LL14) and one other known compound (BC08)

Among them, nine compounds (LL01, LL03, LL04, LL05, LL06, LL10, LL12,

LL13 and LL14) were isolated from both Lumnitzera littorea and Bruguiera cylindrica

These findings contributed to the knowledge of the phytochemical properties of Lumnitzera littorea and Bruguiera cylindrica

α-GLUCOSIDASE INHIBITORY ACTIVITIES OF EXTRACTS

Different leaf extracts from Lumnitzera littorea and Bruguiera cylindrica, and most of isolated compounds were evaluated on the inhibition against α-glucosidase enzyme The results were presented in Table 4.1

Table 4.1 The in vitro α-glucosidase inhibition by the studied compounds

5 Quercetin (LL12) 11.2 19 Betulinic acid (LL04) 61.5

6 Afzelin (LL23) 14.6 20 Oleanolic acid (LL08) 41.2

7 Quercitrin (LL13) 17.2 21 Corosolic acid (LL07) 37.9

9 Chrysoeriol (LL22) 170.2 23 Stigmast-4-ene-3-one (LL09) 92.6

10 Taxifolin (LL25) 198.8 24 β-Sitosterol 3-O- β-D- glucopyranoside (LL05) 193.4

Stigmast-5-ene 3β-O-(6-O- hexadecanoyl-β- D - glucopyranoside) (LL02)

3,3,4-tri-O-methylellagic acid 4-O-β-D- glucopyranoside (LL29)

An acute toxicity assay was conducted following OECD guidelines on crude ethanol extracts from Lumnitzera littorea leaves at various doses The findings revealed that the extract was non-toxic at a concentration of 13.0 g/kg, as classified by the Globally Harmonised System for Classification of Chemicals (GHS).

Tiêu đề	Study on Chemical Constituents and Some Bioactivities of Lumnitzera Littorea (Combretaceae) and Bruguiera Cylindrica (Rhizophoraceae) Growing at Can Gio Mangrove Forest – Ho Chi Minh City
Tác giả	Nguyen Thi Le Thuy
Người hướng dẫn	Prof. Dr. Nguyen Kim Phi Phung, Prof. Dr. Poul Erik Hansen
Trường học	Vietnam National University – Ho Chi Minh City University of Science
Chuyên ngành	Organic Chemistry
Thể loại	thesis
Năm xuất bản	2020
Thành phố	Ho Chi Minh City

Định dạng
Số trang	291
Dung lượng	21,82 MB