Phytochemistry, pharmacology, and clinical trials of Morus alba

Chinese Journal of Natural Medicines 2016, 14(1): 00170030

Chinese Journal of Natural Medicines

doi: 10.3724/SP.J.1009.2016.00017

·Review·

Phytochemistry, pharmacology, and clinical trials of Morus alba Eric Wei-Chiang CHAN 1*, Phui-Yan LYE 1, Siu-Kuin WONG 2 1

Faculty of Applied Sciences, UCSI University, 56000 Cheras, Kuala Lumpur, Malaysia;

2

School of Science, Monash University Sunway, 46150 Petaling Jaya, Selangor, Malaysia Available online 20 Jan., 2016

[ABSTRACT] The present review is aimed at providing a comprehensive summary on the botany, utility, phytochemistry, pharmacology, and clinical trials of Morus alba (mulberry or sang shu). The mulberry foliage has remained the primary food for silkworms for centuries. Its leaves have also been used as animal feed for livestock and its fruits have been made into a variety of food products. With flavonoids as major constituents, mulberry leaves possess various biological activities, including antioxidant, antimicrobial, skin-whitening, cytotoxic, anti-diabetic, glucosidase inhibition, anti-hyperlipidemic, anti-atherosclerotic, anti-obesity, cardioprotective, and cognitive enhancement activities. Rich in anthocyanins and alkaloids, mulberry fruits have pharmacological properties, such as antioxidant, anti-diabetic, anti-atherosclerotic, anti-obesity, and hepatoprotective activities. The root bark of mulberry, containing flavonoids, alkaloids and stilbenoids, has antimicrobial, skin-whitening, cytotoxic, anti-inflammatory, and anti-hyperlipidemic properties. Other pharmacological properties of M. alba include anti-platelet, anxiolytic, anti-asthmatic, anthelmintic, antidepressant, cardioprotective, and immunomodulatory activities. Clinical trials on the efficiency of M. alba extracts in reducing blood glucose and cholesterol levels and enhancing cognitive ability have been conducted. The phytochemistry and pharmacology of the different parts of the mulberry tree confer its traditional and current uses as fodder, food, cosmetics, and medicine. Overall, M. alba is a multi-functional plant with promising medicinal properties. [KEY WORDS] Mulberry; Multi-purpose; Medicinal properties; Morus alba

[CLC Number] Q5

[Document code] A

[Article ID] 2095-6975(2016)01-0017-14

Introduction Morus alba L. (mulberry or sang shu) has long been used as fodder and traditional medicine. There has been much research work that has been done since several published reviews on the species [1-4]. Herein, we attempted to provide a comprehensive update on its botany, applications, phytochemistry, medicinal properties, and clinical trials. Morus alba L. of the family Moraceae is native to Chinaand is also widely cultivated in Japan and Korea [5-6]. The species is a fast-growing tree, which can reach up to 20 meters in height. Under cultivation with regular harvesting, pruning, and pollarding, the trees are reduced to a low-growing bush to facilitate the harvesting of leaves or fruits. The bark is dark grey-brown with horizontal lenticels. The leaves are glossy green, alternate, cordate at the base,

[Received on] 04-Feb.-2015 [*Corresponding author] E-mail: [email protected]. These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

and acuminate at the apex, the margins are serrated, and the petioles are long and slender. Varying from 5.0−7.5 cm in length, the leaves are very variable in form. Even on the same tree, some are unlobed while others may be almost palmate. In the temperate and sub-tropical areas, the trees are commonly dioecious (separate male and female plants), but may be monoecious (male and female flowers on the same plants), and sometimes can change from one sex to another. The flowers comprising male and female catkins are inconspicuous, pendulous, and greenish. The fruit consists of many drupes formed by individual flowers to form a sorosis, the characteristic mulberry fruit. The fruit color is green when young, turning orange to red and finally to purplish black when fully ripened. Fig. 1 shows the leaves and fruits of M. alba. Mulberry foliage is valued as the primary food for silkworms, supporting the silk industry for centuries [7-9]. The silkworm eats only mulberry leaves to make its cocoon, which produces the silk, and there is a high correlation between the content of leaf protein and the efficiency of cocoon production [9]. The amino acids (threonine, valine, methionine,

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Fig. 1 Leaves and fruits of Morus alba

leucine, phenylalanine, lysine, histidine, and arginine) found in mulberry leaves are needed for silkworm growth. It is well established that the growth of silkworms as well as the cocoon and raw silk quality depend on the quality of mulberry leaves, which in turn is closely related to the plant varieties, environmental conditions, and cultivation practice [10]. In China, 15−18 kg of mulberry leaves is needed to produce 1 kg of cocoon at the farm level [7]. Mulberry leaves are also used as fodder for livestock. They are nutritious, palatable, and non-toxic and can improve milk production when fed to dairy animals [11]. The high crude protein content and organic matter digestibility of mulberry leaves are superior to most tropical grasses commonly used as cattle feed [12]. In traditional Chinese medicine (TCM), leaves, fruits, and bark of M. alba have long been used to treat fever, protect liver damage, improve eyesight, strengthen joints, facilitate discharge of urine, and lower blood pressure [13]. In Korea and Japan, patients with diabetes consume mulberry leaves as an anti-hyperglycemic supplement [14]. Mulberry leaves are effective against high blood pressure and hangover from alcohol and in lowering blood sugar level related to diabetes [9]. In East and Southeast Asia, the drinking of mulberry tea is gaining popularity. The tea is rich in γ-aminobutyric acid (2.7 mg·g−1 dry weight) which is 10 times higher than that of green tea [6]. The compound is known to lower blood pressure. In Turkey and Greece, trees of M. alba are grown for fruits rather than foliage [15]. The fruits are used to produce mulberry juice, jam, liquor, and canned mulberries. In China, the leaves of M. alba are processed into tea while fruit juice is consumed as a health beverage [7]. Other uses of mulberry include paper and mushroom production [9]. Woodchips of mulberry trees have been used as pulp for paper production and as media for mushroom culture. In India, mulberry

wood is made into sports equipment, furniture, household utensils, and agricultural implements [8]. Phytochemistry Photochemical studies have identified terpenoids, alkaloids, flavonoids (including chalcones and anthocyanins), phenolic acids, stilbenoids, and coumarins in Morus alba. Compounds isolated from the leaf, fruit, root, bark, root bark, twig, and stem of the plant are listed in Table 1. Mulberry fruits yield the most number of compounds. Leaf Three flavonol glycosides, quercetin 3-(6-malonylglucoside), rutin, and isoquercitrin, are identified as the major antioxidant compounds in the ethanol leaf extract of M. alba [14]. Their contents are 9.0, 5.7, and 1.9 mg·g−1 dry weight, respectively. From the ethanol leaf extract of mulberry, four new 2-arylbenzofuran derivatives (moracins V−Y), together with two known compounds (moracins N and P) are isolated [37]. From the butanol leaf extract, two novel prenylflavanes and a glycoside, along with six known compounds, isoquercitrin, astragalin, scopolin, skimmin, roseoside II, and benzyl D-glucopyranoside, are isolated [23]. From the methanol leaf extract, a new and ten known flavonoids are isolated [21] (Fig. 2). From the ethanol leaf extract of M. alba, morachalcones B and C have been isolated [19]. They are unusual chalcones having a five-membered furan ring with an oxygen molecule. Further bioassay-guided fractionation of the extract leads to the isolation of 15 flavonoids, including five new compounds [24]. Fruit Extraction of fresh fruits of M. alba has yielded five anthocyanins [33]. From the ethanol fruit extract of M. alba, bioactivity-guided fractionation has led to the isolation of 25 phenolic compounds, all of which are isolated from the mulberry fruit for the first time [20]. Some of the phytochemicals and their molecular structures are shown in Fig. 3.

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Table 1 Classes and names of compounds isolated from the various parts of Morus alba Compound class and name

Part

Reference

Betulinic acid

Root bark

[30]

Ursolic acid

Root bark

[30]

Uvaol

Root bark

[30]

Calystegins B2, C1

Root

[18]

1-Deoxynojirimycin

Root

[18]

2α, 3β-Dihydroxynortropane

Fruit

[16]

2β, 3β-Dihydroxynortropane

Fruit

[16]

3β, 6exo-Dihydroxynortropane

Fruit

[16]

1, 4-Dideoxy-1, 4-imino-pD-arabinitol

Root

[18]

1, 4-Dideoxy-1, 4-imino-pD-ribitol

Root

[18]

1, 4-Dideoxy-1, 4-imino-(2-O-β-pD-glucopyranosyl)-d-glucopyranosyl)-D-arabinitol

Root

[18]

Fagomine

Root

[18]

Terpenoids

Alkaloids

3-epi-Fagomine

Root

[18]

2-[2-Formyl-5-(hydroxymethyl)-1-pyrrolyl-]3-methyl pentanoic acid lactone

Fruit

[17]

4-[Formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoate

Fruit

[17]

4-[Formyl-5-(methoxymethyl)-1H-pyrrol-1-yl] butanoic acid

Fruit

[17]

2-O-α-D-Galactopyranosyl-1-deoxynojirimycin

Root

[18]

6-O-α-D-Galactopyranosyl-1-deoxynojirimycin

Root

[18]

2-O-α-D-Glucopyranosyl-1-deoxynojirimycin

Root

[18]

3-O-α-D-Glucopyranosyl-1-deoxynojirimycin

Root

[18]

4-O-α-D-Glucopyranosyl-1-deoxynojirimycin

Root

[18]

2-O-β-D-Glucopyranosyl-1-deoxynojirimycin

Root

[18]

3-O-β-D-Glucopyranosyl-1-deoxynojirimycin

Root

[18]

4-O-β-D-Glucopyranosyl-1-deoxynojirimycin

Root

[18]

6-O-β-D-Glucopyranosyl-1-deoxynojirimycin

Root

[18]

2-(5′-Hydroxymethyl-2′-formylpyrrol-1′-yl)-3-(4-hydroxyphenyl) propionic lactone

Fruit

[17]

2-(5′-Hydroxymethyl-2′-formylpyrrol-1′-yl)-3-phenylpropionic acid lactone

Fruit

[17]

2-(5-Hydroxymethyl-2′, 5′-dioxo-2′, 3′, 4′, 5′-tetrahydrobipyrrole) carbaldehyde

Fruit

[17]

2-(5-Hydroxymethyl-2-formylpyrrol-1-yl) isovaleric acid lactone

Fruit

[17]

2-(5-Hydroxymethyl-2-formylpyrrole-1-yl) propionic acid lactone

Fruit

[17]

2-(Hydroxymethyl-2-formylpyrrole-1-yl) isocaproic acid lactone

Fruit

[17]

N-Methyl-1-deoxynojirimycin

Root

[18]

Methyl 2-[2-formyl-5-(methoxymethyl)-1H-pyrrol-1-yl]-3-(4-hydroxyphenyl) propanoate

Fruit

[17]

Methyl 2-[2-formyl-5-(methoxymethyl)-1H-pyrrole-1-yl] propanoate

Fruit

[17]

Morroles B−F

Fruit

[17]

2α, 3β, 4α-Trihydroxynortropane

Fruit

[16]

2α, 3β, 6exo-Trihydroxynortropane

Fruit

[16]

Leaf

[19]

Astragalin

Leaf

[23]

Atalantoflavone

Leaf

[21]

Chalcones Morachalcones B, C Flavonoids

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Eric Wei-Chiang CHAN, et al. / Chin J Nat Med, 2016, 14(1): 1730

Continued Compound class and name Benzyl d-glucopyranoside Cyclomorusin

Part

Reference

Leaf

[23]

Leaf, Root bark

[21, 23, 27]

Cyclomulberrin

Leaf

[21]

Dihydrokaempferol 7-O-β-D-glucopyranoside

Leaf

[20]

3′, 8-Diprenyl-4′, 5, 7-trihydroxyflavone

Leaf

[21]

Epigallocatechin

Fruit

[25]

Epigallocatechin gallate

Fruit

[25]

Euchrenone a7

Leaf

[24]

Gallocatechin

Fruit

[25]

Gallocatechin gallate

Fruit

[25]

6-Geranylapigenin

Twig

[31]

8-Geranylapigenin

Leaf

[21]

6-Geranylnorartocarpetin

Twig

[31]

7-Hydroxyl-8-hydroxyethyl-4′-methoxylflavane-2′-O-β-D-glucopyranoside

Leaf

[24]

8-Hydroxyethyl-7, 4′-dimethoxylflavane2′-O-β-D-glucopyranoside

Leaf

[24]

Isoquercitrin

Leaf

[14, 22, 23]

Isorhamnetin glucuronide

Fruit

[25]

Isorhamnetin hexoside

Fruit

[25]

Isorhamnetin hexosylhexoside

Fruit

[25]

Kaempferol

Leaf

[21, 24]

Kaempferol 3-O-β-D-rutinoside

Leaf

[20]

Kaempferol 3-O-β-D-glucopyranoside

Leaf

[20]

Kaempferol glucuronide

Fruit

[25]

Kaempferol hexoside

Fruit

[25]

Kaempferol hexosylhexoside

Fruit

[25]

Kaempferol rhamnosylhexoside

Fruit

[25]

Kuwanons A−C, E, G, H, J, S, T

Leaf, Root bark

[21, 28, 30]

7-Methoxyl-8-ethyl-2′, 4′-dihydroxylflavane-2′′-O-β-D-glucopyranoside

Leaf

[24]

7-Methoxyl-8-hydroxyethyl-2′, 4′-dihydroxylflavane

Leaf

[24]

7-Methoxyl-8-hydroxyethyl-4′-hydroxylflavane-2′-O-β-D-glucopyranoside

Leaf

[24]

Morin

Fruit

[25]

Morusin

Leaf, Root bark

[21, 27]

Naringin

Fruit

[25]

Norartocarpetin Oxydihydromorusin Quercetin Quercetin 3, 7-di-O-β-D-glucopyranoside

Leaf

[24]

Root bark

[28]

Leaf, Fruit, Twig

[20, 24, 25, 31]

Leaf

[20]

Quercetin 3-O-(6′′-O-acetyl)-β-D-glucopyranoside

Leaf

[20]

Quercetin 7-O-β-D-glucopyranoside

Leaf

[20]

Quercetin-3, 7-di-O-β-D-glucopyranoside

Leaf

[22]

Quercetin glucuronide

Fruit

[25]

Quercetin hexoside

Fruit

[25]

Quercetin hexosylhexoside

Fruit

[25]

Quercetin 3-(6-malonylglucoside)

Leaf

[14]

Quercetrin

Fruit

[25]

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Eric Wei-Chiang CHAN, et al. / Chin J Nat Med, 2016, 14(1): 1730

Continued Compound class and name Roseoside Rutin

Part

Reference

Leaf

[23]

Leaf, Fruit

[14, 20, 25]

Leaf, Root bark

[21, 30]

Leaf

[23]

Skimmin

Leaf

[23]

7, 2′, 4′, 6′-Tetrahydroxy-6-geranylflavanone

Root

[26]

5, 7, 3′-Trihydroxy-flavanone-4′-O-β-D-glucopyranoside

Leaf

[20]

5, 7, 4′-Trihydroxy-flavanone-3′-O-β-D-glucopyranoside

Leaf

[20]

Cyanidin 3-O-glucoside

Fruit

[34]

Cyanidin 3-O-rutinoside

Fruit

[34]

Cyanidin 3-O-β-D-galactopyranoside

Fruit

[33]

Cyanidin 3-O-β-D-glucopyranoside

Fruit

[33]

Sanggenons F, J, K Scopolin

Anthocyanins

Cyanidin 7-O-β-D-glucopyranoside

Fruit

[33]

Cyanidin galloylhexoside

Fruit

[25]

Cyanidin hexoside

Fruit

[25]

Cyanidin hexosylhexoside

Fruit

[25]

Cyanidin pentoside

Fruit

[25]

Cyanidin 3-O-(6′′-O-α-rhamnopyranosyl-β-D-galactopyranoside)

Fruit

[33]

Cyanidin 3-O-(6′′-O-α-rhamnopyranosyl-β-D-glucopyranoside)

Fruit

[33]

Cyanidin rhamnosylhexoside

Fruit

[25]

Delphinidin acetylhexoside

Fruit

[25]

Delphinidin hexoside

Fruit

[25]

Delphinidin rhamnosylhexoside

Fruit

[25]

Pelargonidin 3-O-glucoside

Fruit

[34]

Pelargonidin 3-O-rutinoside

Fruit

[34]

Pelargonidin hexoside

Fruit

[25]

Pelargonidin rhamnosylhexoside

Fruit

[25]

Petunidin rhamnosylhexoside

Fruit

[25]

Phenolic acids 3-O-Caffeoylquinic acid

Fruit

[26]

5-O-Caffeoylquinic acid

Leaf, Fruit

[26, 35]

m-Coumaric acid

Leaf, Fruit

[35]

p-Coumaric acid

Leaf, Fruit

[26, 35]

Ellagic acid

Fruit

[26]

Ferulic acid

Leaf, Fruit

[26, 35]

Gallic acid

Leaf, Fruit

[26, 35]

Gentisic acid

Fruit

[26]

Leaf, Fruit

[35]

p-Hydroxybenzoic acid

Fruit

[21, 26]

Hydroxyphenylacetic acid methyl ester

Fruit

[21]

Jaboticabin

Fruit

[21]

Methyl 3-O-caffeoylquinate

Fruit

[26]

Methyl 4-O-caffeoylquinate

Fruit

[26]

Methyl 5-O-caffeoylquinate

Fruit

[26]

Hydroxybenzoic acid

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Continued Compound class and name Methyl dicaffeoylquinate Protocatechuic acid

Part

Reference

Fruit

[26]

Leaf, Fruit

[21, 26, 35]

Protocatechuic acid ethyl ester

Fruit

[21]

Protocatechuic acid methyl ester

Fruit

[21]

Protocatechuic aldehyde

Leaf, Fruit

[35]

Syringaldehyde

Leaf, Fruit

[35]

Syringic acid

Leaf, Fruit

[35]

Vanillic acid

Leaf, Fruit

[21, 26, 35]

Alabafuran A

Root bark

[30]

Artoindonesianin O

Stilbenoids Root bark

[30]

Chalcomoracin

Leaf

[24]

Dihydromorin

Stem

[39]

2, 3-trans-Dihydromorin-7-O-β-glucoside 3′, 5′-Dihydroxy-6-methoxy-7-prenyl-2-arylbenzofuran Moracins C, D, M−P, R, V−Y

Stem

[39]

Root bark

[30]

Leaf, Stem, Root bark

[24, 30, 37, 39]

Mulberrofurans L, Y

Root bark

[30]

Mulberrosides A, B, F

Leaf, Stem, Root bark

[30, 36, 39]

Twig, Stem

[32, 39]

Oxyresveratrol Oxyresveratrol 2-O-β-D-glucopyranoside

Root bark

[30]

Resveratrol

Twig, Stem

[32, 39]

Steppogenin

Stem

[39]

Bark

[38]

Coumarins 5, 7-Dihydroxycoumarin 7-(6-O-β-D-apiofuranosyl-β-d-glucopyranoside) 5, 7-Dihydroxycoumarin 7-O-β-d-apiofuranosyl-(1→6)-O-β-D-glucopyranoside

Root bark

[30]

5, 7-Dihydroxycoumarin 7-O-β-D-glucopyranoside

Root bark

[30]

6, 7-Dihydroxycoumarin 7-(6-O-α-rhamnopyranosyl-β-D-glucopyranoside)

Bark

[38]

Isoscopoletin 6-(6-O-β-apiofuranosyl-β-glucopyranoside)

Stem

[39]

Notes: compounds of each class are in alphabetical order. Anthocyanins are a sub-class of flavonoids.

a

a

3′-Geranyl-3-prenyl-2′, 4′, 5, 7-tetrahydroxyflavone (R1 = prenyl, R2 = H, R3 = geranyl, R4 = OH) 3′, 8-Diprenyl-4′, 5, 7-trihydroxyflavone (R1 = H, R2 = prenyl, R3 = prenyl, R4 = H) Kuwanon S (R1 = H, R2 = H, R3 = geranyl, R4 = H) 8-Geranylapigenin (R1 = H, R2 = geranyl, R3 = H, R4 = H)

b

b

Kaempferol (R1 = OH, R2 = H, R3 = H, R4 = H) Morusin (R1 = prenyl, R2 = OH) Atalantoflavone (R1 = H, R2 = H)

The new flavonoid is 3′-geranyl-3-prenyl-2′, 4′, 5, 7-tetrahydroxyflavone, and the 10 known flavonoids are 3′, 8-diprenyl-4′, 5, 7-trihydroxyflavone, kuwanon S, 8-geranylapigenin, kaempferol, morusin, atalantoflavone, cyclomulberrin, sanggenon J, sanggenon K, and cyclomorusin. Fig. 2

Selected flavonoids isolated from mulberry leaves [21]

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Anthocyanins Cyanidin 3-O-(6′′-O-α-rhamnopyranosyl-β-D-glucopyranoside), Cyanidin 3-O-(6′′-O-α-rhamnopyranosyl-β-D-galactopyranoside), Cyanidin 3-O-β-D-glucopyranoside, Cyanidin 3-O-β-D-galactopyranoside, Cyanidin 7-O-β-D-glucopyranoside

Flavonoids

a

b

a

Quercetin (R1 = H, R2 = H) Quercetin 3-O-β-D-glucopyranoside (R1 = H, R2 = β-D-glc) Quercetin 3-O-(6′′-O-acetyl)-β-D-glucopyranoside (R1 = H, R2 = (6′′-O-acetyl)-β-D-glc) Quercetin 3-O-β-D-rutinoside (R1 = H, R2 = α-L-rha-(1→6)-β-D-glc) Quercetin 7-O-β-D-glucopyranoside (R1 = β-D-glc, R2 = H) Quercetin 3,7-di-O-β-D-glucopyranoside (R1 = β-D-glc, R2 = β-D-glc)

b

Kaempferol 3-O-β-D-glucopyranoside (R = β-D-glc) Kaempferol 3-O-β-D-rutinoside (R = α-L-rha-(1→6)-β-D-glc) Fig. 3

Selected phytochemicals isolated from Morus alba fruits [33]

Root and root bark From the root bark of M. alba, polyhydroxylated alkaloids have been isolated, including 1-deoxynojirimycin (DNJ) and its derivatives [18]. The contents of DNJ in leaves from 132 varieties of nine Morus species are determined [40]. Of them, 58 varieties are from M. alba. The younger leaves have higher DNJ content than the older leaves. The DNJ contents in mature leaves of M. alba vary from 0.13−1.46 mg·g−1 dry weight. Other compounds identified from the root and root bark include terpenoids, flavonoids, stilbenoids, and coumarins. Some compounds isolated from the root bark of M. alba are shown in Fig. 4. Twig and stem From the twig and stem of M. alba, flavonoids, stilbenoids, and coumarins have been reported. Bioactive compounds include mulberroside, morusin, resveratrol, and oxyresveratrol. Pharmacology Antioxidant properties Antioxidant properties of ethanol fruit extract of M. alba have shown significant difference between varieties [13]. Mature fruits are rich in anthocyanins, which are excellent antioxidant agents with stronger free radical scavenging

activity than vitamin C [33]. Based on total phenolic content (TPC), free radical scavenging (FRS), ferric reducing power (FRP), and ferrous ion chelating (FIC), our research has shown that aqueous methanol leaf extracts of M. alba have significantly higher values than that of fruits [41]. The ranking is of the order: developing leaves > young leaves ~ mature leaves > mature fruits. Our recent work on the antioxidant properties of herbs have shown that M. alba displays enhancement effects of drying [42, 43]. Increase in values was up to 27% for oven-drying, 16%−44% for freeze-drying, and up to 91% for microwave-drying. Out of 12 herbal teas assessed, TPC, FRS, and FRP of M. alba tea are in the moderate category [44]. However, its FIC value of 0.8 mg·mL−1, based on CEC50 (50% chelating efficiency concentration), is stronger than that of green, oolong, and black teas. Antimicrobial activity Kuwanon G isolated from the methanol root bark extract of M. alba has been reported to exhibit antibacterial activity against oral pathogens such as Streptococcus mutans, Streptococcus sanguis, Streptococcus sobrinus, and Porphyromonas gingivalis, with the minimum inhibitory concentration (MIC) of 8 µg·mL−1 [45]. From the root bark, mulberrofuran G and

Fig. 4 Selected compounds isolated from the root bark of Morus alba [30]

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albanol B strongly inhibit Salmonella typhimurium, Staphylococcus epidermis, and Staphyloccoccus aureus, with MIC being 5.0−7.5 µg·mL−1 [46]. Besides antibacterial activity, leaf extracts of M. alba also possess antifungal properties [47]. At concentrations of 20, 40, 60, and 80 mg·mL−1, all three sequential leaf extracts (methanol, chloroform, and petroleum ether) strongly inhibit Candida albicans and Aspergillus niger with inhibition zones being 12−28 mm. Of eight flavonoids isolated from the root bark of M. alba, leachianone G shows potent antiviral activity (IC50 1.6 mg·mL−1), whereas mulberroside C shows weak activity (IC50 75 mg·mL−1) against herpes simplex type 1 virus (HSV-1) [48]. Skin-whitening properties Mulberroside F isolated from the methanol leaf extract of M. alba exhibits anti-tyrosinase activity that is 4.5-fold stronger than kojic acid and hasan inhibitory effect on melanin formation in melan-a cells [49]. Oxyresveratrol exhibits an inhibitory activity that is 32-fold stronger than kojic acid [50]. Oxyresveratrol with four OH-groups and resveratrol with three OH-groups are two hydroxystilbenes found in M. alba. At 100 μmol·L−1, their tyrosinase inhibitory effects are 97% and 64%, compared to 77% for kojic acid [51]. The number and position of hydroxyl groups seem to play an important role in the inhibitory effects of these compounds [50] . Among the 15 flavonoids isolated from the ethanol leaf extract of M. alba, norartocarpetin, euchrenone, and quercetin display antityrosinase activity, which is significantly stronger than kojic acid [24]. Their IC50 values are 0.08, 0.26, 0.52, and 15.9 μmol·L−1, respectively. It is noted that the flavonoids having both the 2′-OH and 4′-OH groups are more potent inhibitors that those with only the 4′-OH group. Using HPLC analysis, morin, resveratrol, maclurin, rutin, isoquercitrin, and morin are identified in the ethanol mulberry twig extract [52]. Morin (21%) and resveratrol (16%) are the major compounds, known to be potent tyrosinase inhibitors. Besides skin-whitening activity, resveratrol also possesses many pharmacological properties that are beneficial in treating human diseases such as neurodegenerative disease, cardiovascular disease, diabetes, and cancer [53]. Cytotoxic activity Isolated from the aqueous methanol leaf extract of M. alba, two flavonoids, quercetin-3-O-β-D-glucopyranoside (1) and quercetin-3-7-di-O-β-D-glucopyranoside (2) are found to inhibit the growth of human leukemia HL-60 cells [54]. At 2 × 10-4 mol·L−1 concentration, inhibitory effects of Compounds 1 and 2 are 51% and 57%, respectively. A flavanone (7, 2′, 4′, 6′-tetrahydoroxy-6-geranylflavanone) isolated from the ethyl acetate root extract of M. alba exhibits cytotoxic activity against rat hepatoma dRLh84 cells with an IC50 value being 53 µg·mL−1 [26]. A novel flavanone glycoside isolated from the root bark

of M. alba exhibits anti-proliferative activity [31]. IC50 values of the compound against human ovarian cancer HO-8910 cells are 3.7 and 1.9 μmol·L−1 for 48 h and 72 h, of exposure, respectively. Albanol Aisolated from the root bark of M. alba, is reported to have cytotoxic and apoptotic activities in human leukemia HL-60 cells [55]. The compound shows potent cytotoxic activity with IC50 value being 1.7 μmol·L−1. In addition, albanol A induces early apoptosis with marked reduction in procaspases-3, -8, and -9, and activation of caspase-2. It is postulated that the compound induces apoptotic cell death in HL-60 cells via both the cell death receptor pathway by stimulation of death receptor, and the mitochondrial pathway through caspase-2 activation. The study concludes that albanol A may be a promising compound for developing as an effective drug for treatment of leukaemia. Morusinisolated from the root bark of mulberry, induces apoptosis and suppresses NF-κB in human colorectal cancer HT-29 cells [56]. All the 11 flavonoids isolated from the methanol leaf extract of M. alba display cytotoxic activity against human cancer HeLa, MCF-7, and Hep-3B cells [21]. Based on IC50, the strongest activities are observed with morusin against HeLa cells (0.6 μmol·L−1), 8-geranylapigenin against MCF-7 cells (3.2 μmol·L −1 ) and sanggenon K against Hep-3B cells (3.1 μmol·L −1 ), respectively. Against these cancer cells, deguelin (standard drug) has IC50 values of 6.4, 5.3, and 29 μmol·L−1, respectively. Two new chalcones (morachalcones B and C) isolated from leaves of M. alba have moderate cytotoxic activity against human cancer HCT-8 and BGC-823 cells [19]. A recent study shows that water, aqueous MeOH, and MeOH leaf extracts of M. alba exhibit highly significant inhibitory effects on the proliferation of human hepatocellular carcinoma HepG2 cells [57]. It is postulated that the extracts suppress nuclear factor kappa B gene expression with significant declines in α-fetoprotein, γ-glutamyl transpeptidase, and alkaline phosphatase in the cells. Another recent study also shows the anti-cancer activity of the methanol root bark extract of M. alba [58]. The extract induces cell growth arrest and apoptosis in human colorectal cancer SW480 cells. At 6.25, 12.5, and 25.0 μg·mL−1 of extract, the cell viability is reduced by 43%, 71%, and 83%, respectively. The cytotoxic activity of the extract is associated with ROS-dependent cyclin D1 proteasomal degradation and with ROS/GSK3β-dependent ATF3 expression. Anti-inflammatory activity The methanol root bark extract of M. alba has been reported to possess anti-inflammatory activity [58]. Nitric oxide is measured using the Griess method, and iNOS and proteins regulating NF-κB and ERK1/2 signalling are analyzed by Western blotting. Results show that the anti-inflammatory effect of the extract is mediated via inhibition of NF-κB and activation of ERK1/2. Of the

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compounds isolated from the methanol root bark extract, kuwanons C and G possess anti-inflammatory activity. Similarly, the methanol branch extract of M. alba and its active compound oxyresveratrol also have anti-inflammatory activity [59]. The likely mechanism involves the inhibition of CXCR- 4-mediated chemotaxis and MEK/ERK pathway in T and other immune cells. Anti-diabetic properties Studies have reported the anti-diabetic properties of M. alba leaves and fruits in rat models. Postprandial hypoglycemic effects of aqueous leaf extract and leaf powder of M. alba have been studied using Goto-Kakizaki (GK) and Wistar rats [60]. The effect of a single oral administration of leaf extract at 3.75 g·kg−1 on postprandial glucose responses is determined using maltose or glucose as substrate. With maltose, the extract reduces peak responses of blood glucose significantly in both GK and Wistar rats, supporting the inhibition of α-glucosidase in the small intestine. With glucose, the extract also significantly reduces blood glucose concentrations, measured at 30 min, in both animal models, proposing the inhibition of glucose transport. After the leaf powder (10%) has been administered by inclusion in the diet, fasting blood glucose is significantly reduced at weeks 4 and 5. Overall, the findings indicate that the leaf extract has significant postprandial hypoglycemic effect, possibly through the inhibition of α-glucosidase and glucose transport. Earlier studies have also reported hypoglycemic activities in leaves and root bark of M. alba. A single dose of aqueous extracts of leaves and root bark at 200 mg·kg−1 significantly reduce blood glucose level and increase glucose uptake [61]. The administration of the mulberry root bark extract at 600 mg·kg−1·d−1 to streptozotocin (STZ)-induced diabetic rats for 10 days significantly reduces serum glucose and lipid peroxides, and increases insulin levels [62]. In a recent reported study, Wistar rats were fed with mulberry leaf extract at doses of 400 and 600 mg·kg−1, and after 35 days, blood glucose, glycosylated hemoglobin, triglyceride, blood urea, cholesterol, number of β-cells, and diameter of the islets of Langerhans were measured [63]. Blood glucose level and other parameters (except HDL), elevated in the diabetic group, were brought to the control level in the diabetic group treated with 600 mg·kg−1 of leaf extract. The diameter of the islets and the number of β-cells reduced in the diabetic group, were brought to the control level after treatment with the extract. The study concludes that the mulberry leaf extract, at a dose of 600 mg·kg−1, has therapeutic effects in diabetes-induced rats and can restore the diminished number of β-cells. After administration of 250 or 750 mg·kg−1 of the aqueous ethanol leaf extract of M. alba, a decrease in blood glucose levels of type II diabetic rats has been observed after 11 days [64]. The anti-diabetic activity of the extract is

attributed to chlorogenic acid and rutin present in the extract. After Zucker diabetic fatty rats were fed with mulberry fruit extract at doses of 125 or 250 mg·kg−1 twice daily for five weeks, the glucose levels were significantly lower than that of the control group [65]. At 250 mg·kg−1, the insulin levels did not decline and no discernable changes in the histology of the pancreatic β-cells were observed. Another study has also reported that flavonoids in the ethanol fruit extract (100 and 200 mg·kg−1) significantly decreases blood glucose and serum protein, and increases antioxidant enzymatic activities in STZ-induced diabetic mice [20]. The extract, which also shows potent α-glucosidase inhibition, may be partially responsible for the anti-diabetic activity of the fruit extract. Anti-hyperlipidemic effects Mulberroside A prepared from ethanol root extract of M. alba and its aglycone derivative (oxyresveratrol) produced from mulberroside A by enzymatic conversion have been evaluated for their anti-hyperlipidemic effects in two rat models [66]. Oral pre-treatment with mulberroside A or oxyresveratrol (1−5 mg·kg−1) significantly reduces serum lipids levels in hyperlipidemic rats and in high-cholesterol diet hyperlipidemic rats. Oxyresveratrol also shows more pronounced serum lipid lowering capacity than mulberroside A. Findings of this study further support the hypolipidemic effects of the root bark [67] and hypotriglyceridemic effects of leaves [68] of mulberry. Anti-atherosclerotic effects Studies have shown that leaves and fruits of M. alba have anti-atherosclerotic effects in rodents. The effects of a dietary intake of 1% mulberry leaf (ML) powder on atherogenesis in apolipoprotein E-deficient mice have been reported [69]. After 12 weeks of treatment, a significant increase in the lag time of lipoprotein oxidation has been detected in the ML group compared with the control group. The ML group also shows 40% reduction in atherosclerotic lesion size in the aorta. The results show that mulberry leaves contain antioxidative compounds with strong free radical scavenging and lipoprotein oxidation inhibition that can help prevent atherosclerosis. In a study with New Zealand white rabbits on normal diet or high cholesterol diet (HCD), the animals were fed with or without 0.5% or 1.0% aqueous mulberry fruit extract for 10 weeks [70]. The levels of triglyceride, cholesterol and low-density lipoprotein (LDL) cholesterol in the serum of HCD rabbits fed with the fruit extract were lower than that in the control group. The rabbits fed with 0.5% or 1.0% of the extract significantly reduced atherosclerosis in the aorta by 42%–63%, compared with the controls. In a related study, freeze-dried mulberry fruit powder (5% and 10%) lowered serum and liver total cholesterol, and triglyceride content, inhibited lipid peroxidation, and increased antioxidant

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enzyme activity in rats on a high-fat diet, repressing the development of atherosclerosis in hyperlipidemic rats [71]. Anti-obesity activity The effects of the ethanol leaf extract of M. alba on melanin-concentrating hormone receptor activity and on anti-obesity in diet-induced obese mice have been studied [72]. The results of the hormone receptor assay show that the extract (10−100 μg·mL−1) exhibits a potent inhibitory activity, with IC50 value being 2.3 μg·mL−1. In an anti-obesity study, administration of the extract at 100, 250, and 500 μg·mL−1 for 32 consecutive days resulted in a decrease in body weight and adiposity, and regulated hepatic lipid accumulation in the mice. The anti-obesity effects of the extract might be due to receptor antagonism. Male hamsters on high fat diet when fed with aqueous mulberry leaf extract had significantly lower body weight [73]. Decrease in serum triacylglycerol, cholesterol and free fatty acid concentrations as well as increase in HDL/LDL ratios were also observed. Another recent study showed that the combined leaf and fruit extract of M. alba had positive beneficial effects on obese mice [74]. The results showed that the extract ameliorated cholesterol transfer proteins and reduced oxidative stress in the obese mice fed daily with the extract at 500 mg·kg−1 for 12 weeks. Hepatoprotective activity The protective mechanisms of aqueous mulberry fruit extract in male Wistar rats with CCl4-induced hepatic injury have been elucidated [75]. Oral administration of the extract (0.5%, 1%, and 2%) significantly reduces the lipid peroxidation and inhibits lipid deposition and liver fibrosis. The extract attenuates the expression of pro-inflammatory genes such as cyclooxygenase 2, nuclear factor kappa B, and inducible NO synthase. The results suggest that the extract exhibits protective and curative effects against liver damage and fibrosis via decreased lipid peroxidation and inhibition of pro-inflammatory gene expression. The hepatoprotective effect has earlier been reported with leaves of M. alba. At 800 mg·kg−1 dose, the hydroalcoholic leaf extract exhibits significant hepatoprotective effect in mice with CCl4-induced liver injury [76]. Compared to the CCl4 group, the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are lower, with shorter sleeping time, and there is no evidence of fibrosis or inflammation in the extract administered group. Other pharmacological properties Other pharmacological properties of M. alba include antiplatelet [77], anxiolytic [78], anti-asthmatic [79-80], anthelmintic [47, 81], antidepressant [82], immunomodulatory [83-84], and cardioprotective [85] activities. Clinical trials Hypoglycemic effects In a study conducted at the Minneapolis VA Medical

Center, the participants were 10 healthy control subjects (aged 24–61 years) and 10 type-2 diabetic subjects (aged 59–75 years) who were receiving oral hypoglycemic agents [86]. Changes in the blood glucose concentration of the healthy subjects and the type-2 diabetic subjects after ingestion of 75 g sucrose in 500 mL of hot water with 1 g of mulberry leaf extract or placebo were monitored. The results showed that there was a significant difference in the blood glucose level between mulberry and placebo over the first 120 min for the control and diabetic subjects. Another clinical study on the hypoglycemic effects of M. alba leaf extract on postprandial glucose and insulin levels in patients with type 2 diabetes treated with sulfonylurea hypoglycemic agents was conducted at the Miharadai Hospital in Nagasaki City, Japan [87]. Ten patients (5 males and 5 females) with type 2 diabetes mellitus and ten healthy subjects (4 males and 6 females) participated in this study. The results from this study confirmed that postprandial glucose and insulin levels in type 2 diabetic patients treated with sulfonylurea were markedly suppressed after the ingestion of jelly containing 3.3 g of leaf extract. For the patients, the blood glucose was 148 mg·dL−1 after ingestion of the extract jelly and 209 mg·dL−1 after ingestion of the placebo jelly at 30 min. For the healthy subjects, the blood glucose increment after the ingestion of the extract jelly was 97 mg·dL−1, compared to 125 mg·dL−1 after ingestion of the placebo jelly. The insulin level was also significantly suppressed at 30 min after ingestion of the extract jelly, compared to ingestion of the placebo jelly for both the patients and healthy subjects. 1-Deoxynojirimycin (DNJ), isolated from mulberry leaves, is a potent glucosidase inhibitor that is beneficial in suppressing abnormally high blood glucose levels, thereby preventing diabetes mellitus [88]. A food-grade mulberry powder enriched with DNJ (1.5%) was produced and clinically tested to determine the optimal dose. Healthy volunteers received 0.4, 0.8, and 1.2 g of the powder (corresponding to 6, 12, and 18 mg of DNJ, respectively) followed by 50 g of sucrose. The plasma glucose and insulin levels were determined before and 30−180 min after oral administration of DNJ cum sucrose. The results of the clinical trial showed that a single administration of 0.8 and 1.2 g of DNJ-enriched powder significantly suppressed the elevation of postprandial blood glucose and secretion of insulin. Its effective dose and efficacy in humans suggest that the DNJ-enriched powder can be used as a dietary supplement for treating diabetes mellitus. Hypolipidemic effects The hypolipidemic effects of encapsulated mulberry leaf powder have been evaluated in comparison with glibenclamide, the standard anti-diabetic drug [89]. Conducted at the Anantapur K.M. Hospital in Andhra Pradesh, India, the

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clinical study involved 24 mild type 2 diabetic patients (males and aged 40–60 years) who were divided into two treatment groups. The mulberry patients were each given six capsules, three times a day (two capsules after each meal), amounting to a daily dose of 3 g·d−1 for 30 days. The glibenclamide patients were each given one tablet of 5 mg·d−1 for 30 days. The serum and erythrocyte membrane lipid profiles of the patients were analyzed before and after the treatments. Pre- and post-treatment analysis of blood plasma and urine samples showed that the mulberry therapy significantly decreased the concentration of serum cholesterol (12%), triglycerides (16%), plasma free fatty acids (12%), LDL-cholesterol (23%), VLDL-cholesterol (17%), plasma peroxides (25%), and urinary peroxides 55%, while significantly increased HDL-cholesterol (18%). For the glibenclamide patients, changes in the lipid profile were not statistically significant except for triglycerides (10%), plasma peroxides (15%), and urinary peroxides (19%). Having reported that DNJ-enriched mulberry leaf extract can suppress the elevation of postprandial blood glucose in humans [88], a follow-up study has been conducted at the Medical Corporation Kenshokai Kinki Kenshin Center in Osaka, Japan, to evaluate the effects of the leaf extract on plasma lipid profiles in humans [90]. Ten male subjects aged between 20 and 64 years with initial serum triglyceride (TG) levels ≥200 mg·dL−1, ingested capsules containing 12 mg of the DNJ-rich mulberry leaf extract three times daily before meals for 12 weeks. The mean serum TG level decreased from 312 ± 90 mg·dL−1 to 269 ± 66 mg·dL−1 at week 6 and to 252 ± 78 mg·dL−1 at week 12, but the differences were not statistically significant. No significant changes were observed in total cholesterol, LDL-cholesterol or HDL-cholesterol. A clinical trial on the hypolipidemic effects of mulberry leaf tablets has been conducted in non-diabetic patients with mild dyslipidemia at an outpatient clinic in Thailand [91]. Produced by Kitayamakit Co., Ltd., Kyoto, Japan, each leaf tablet weighed 280 mg and contained 255 mg mulberry leaf powder with 0.37 mg of DNJ as the active ingredient. Twenty-three patients received three tablets three times a day before meals for 12 weeks. Routine blood analyses including lipid parameters and liver function tests were performed monthly. At weeks 4 and 8, triglyceride was significantly decreased by 10% and 13%, respectively. At the end of the study, total cholesterol, triglyceride and LDL were decreased by 4.9%, 14%, and 5.6%, respectively, whereas HDL was significantly increased by 20%. Even though some patients experienced some side effects such as mild diarrhoea, dizziness or constipation and bloating, the tablets were effective in reducing cholesterol levels and enhancing HDL in patients with mild dyslipidemia. Cognitive enhancing effects In a clinical trial conducted in Thailand, the cognitive

enhancing effects of M. alba leaf extract have been tested in 60 healthy middle-aged and elderly volunteers [92]. They were randomly assigned to receive the standardized plant extract at doses of 1 050 mg and 2 100 mg once daily for 3 months. The subjects who consumed the extract at both doses showed working memory and cognitive enhancement without any toxic effects.

Conclusion From being the primary food for silkworms, which supported the silk industry over centuries, M. alba has come a long way to be a multi-purpose plant with uses as animal feed, food, cosmetics, and medicine. Its leaves, fruits, and root bark containing flavonoids, anthocyanins, alkaloids, and stilbenoids, possess pharmacological properties, including antioxidant, antimicrobial, skin-whitening, cytotoxic, anti-inflammatory, anti-diabetic, anti-hyperlipidemic, anti-atherosclerotic, antiobesity, hepatoprotective, and cardioprotective activities. Other pharmacological properties of M. alba include antiplatelet, anxiolytic, anti-asthmatic, anthelmintic, antidepressant, cardioprotective, and immunomodulatory activities. Clinical trials on the hypoglycemic and hypolipidemic effects of M. alba extracts in reducing blood glucose and cholesterol levels, and in enhancing cognitive ability have been conducted. Compounds from M. alba with a wide array of bioactivities can serve as lead compounds for drug development. However, the extracts from mulberry would be difficult to standardize as the species has many varieties and cultivarsand can vary in the contents of chemical constituents, particularly the active principle(s). Without proper procedures for authentication and control measures, obtaining reliable and repeatable results of studies in human subjects would be difficult. This poses additional challenges to clinical trials in the future.

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Cite this article as: Eric Wei-Chiang CHAN, Phui-Yan LYE, Siu-Kuin WONG. Phytochemistry, pharmacology, and clinical trials of Morus alba [J]. Chinese Journal of Natural Medicines, 2016, 14(1): 17-30.

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