Effects of Mulberry on The Central Nervous System: A Literature Review

Abstract

Background

Mulberry, including several species belonging to genus Morus, has been widely used as a traditional medicine for a long time. Extracts and active components of mulberry have many positive neurological and biological effects and can become potential candidates in the search for new drugs for neurological disorders.

Objectives

We aimed to systematically review the medical literature for evidence of mulberry effects on the central nervous system.

Methods

We conducted a systematic search in nine databases. We included all in vivo studies investigating the effect of mulberry on the central nervous system with no restrictions.

Results

We finally included 47 articles for quality synthesis. Our findings showed that mulberry and its components possessed an antioxidant effect, showed a reduction in the cerebral infarct volume after stroke. They also improved the cognitive function, learning process, and reduced memory impairment in many animal models. M. alba and its extracts ameliorated Parkinson's disease-like behaviors, limited the complications of diabetes mellitus on the central nervous system, possessed anti-convulsant, anti-depressive, and anxiolytic effects.

Conclusion

Mulberry species proved beneficial to many neurological functions in animal models. The active ingredients of each species, especially M. alba, should be deeper studied for screening potential candidates for future treatments.

1. INTRODUCTION

Mulberry is the generic name of species in the genus Morus of the Moraceae family. These plants are mostly found in Asia, Europe, America, and Africa. They grow in various conditions of climate, topography, and type of soil [1]. For a long time, mulberry was widely used in Chinese as a medicinal herbal treating several disorders, and several studies determined certainly its health benefits afterward [2, 3]. Some studies recorded the presence of phenolics, flavonoids, anthocyanins, and carotenoids in deeply colored mulberry, which might be responsible for its several potential effects [4-7]. Amongst these, M. indica root, M. lhou Koidz and flavonoids from these plants could be active compounds causing antioxidant and anti-inflammatory effects, as mice consumed these interventions saw a reduction of oedema and writhing response [8, 9]. Besides, many benefits of mulberry were also reported such as the protection against obesity, diabetic, neurotoxicity and hepatotoxicity [3, 10].

With regards to the neuroprotective effects, polyphenols, anthocyanin, and other phenolic compounds might be attributed to the protection against oxidative damage in the brain resulting in the improvement of brain functions, for example, improving the learning ability via the protection against neurotoxicity and the increase in neuron cells [11]. Nevertheless, the evidence seemed to be inconclusive with the limit of evidence of possible mechanisms of action in vivo models. For instance, the aged-related memory impairment in mice was improved by M. alba fruit powder but this fruit did not entirely show their positive effect in alcoholic mice [12, 13]. Other reports revealed that M. alba leaves possessed the anxiolytic-like activity in mice assessed by elevated plus maze (EPM) and hole-board test via the histaminergic system [14]. Notably, specific compounds that were responsible for these bioactivities have not been presented. This issue led to the arguments about mulberry’s efficiency and its application for potential treatments in the future.

Neurodegenerative diseases are a burden on both human health and finance. Since human has had greater longevity than ever, the numbers of patients with Alzheimer diseases (AD) and Parkinson disease (PD) are sharply increasing. Notably, it is estimated that over 100 million AD patients globally in 2050 [15]. Similarly, the total number of PD patients in 2030 could be doubled compared with their number in 2005 [16]. Unfortunately, there is no cure for both diseases. Besides, pharmacotherapy for AD can only focus on relieving the symptoms [17]. Therefore, it is beneficial to neurodegenerative patients, especially the elderly, to use supplementary food, which can help to enhance their brain activity apart from pharmacological treatments. Interestingly, mulberry is becoming a candidate for the treatment of both AD and PD through improving their symptoms and preventing age-related neurodegeneration. For example, some motor deficits related to PD in mice were improved by consuming M. alba extract [18]. Additionally, artoindonesianin O, mulberrofuran G (MG), albanol B and kuwanon isolated from M. alba were predicted to prevent the amyloid β (Aβ)-peptide plaque via the inihibition of phospho-extracellular signal-regulated protein kinases 1 and 2 (p-ERK1/2) or via the inhibition of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) in vitro models [19, 20].

Although several papers reported the advantages of various species, there is no review of their therapeutic activities on the brain. We conducted a systematic review including papers showing reliable evidence to show a thorough insight into the advantages of mulberry, confirm the effects of the mulberry extract on the brain and nervous system, and suggest the active compounds which can be investigated for further studies of mulberry applications.

2. MATERIALS AND METHODS

2.1. Protocol Registration

We followed the Recommendations of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement to conduct this systematic review, as shown in the PRISMA checklist (Table 1). The protocol could be accessed at PROSPERO (CRD42015026620).

Table 1.

PRISMA checklist.

Section/Topic	Checklist Item	Reported on Page #
TITLE
Title	Identify the report as a systematic review, meta-analysis, or both.	1
ABSTRACT
Structured summary	Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number.	1
INTRODUCTION
Rationale	Describe the rationale for the review in the context of what is already known.	1-2
Objectives	Provide an explicit statement of questions being addressed concerning participants, interventions, comparisons, outcomes, and study design (PICOS).	2
METHODS
Protocol and registration	Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number.	2
Eligibility criteria	Specify study characteristics (e.g., PICOS, length of follow-up), and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving a rationale.	2
Information sources	Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search, and date last searched.	2
Search	Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.	2
Study selection	State the process for selecting studies (i.e., screening, eligibility, included in a systematic review, and, if applicable, included in the meta-analysis).	2
Data collection process	Describe the method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.	2
Data items	List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.	2
Risk of bias in individual studies	Describe methods used for assessing the risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.	2
Summary measures	State the principal summary measures (e.g., risk ratio, the difference in means).	2
Synthesis of results	Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis.	NA
Risk of bias across studies	Specify any assessment of the risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies).	N/A
Additional analyses	Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified.	NA
RESULTS
Study selection	Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram.	5
Study characteristics	For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations.	5
Risk of bias within studies	Present data on the risk of bias of each study and, if available, any outcome-level assessment (see item 12).	5
Results of individual studies	For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.	5-22
RESULTS
Synthesis of results	Present results of each meta-analysis done, including confidence intervals and measures of consistency.	NA
Risk of bias across studies	Present results of any assessment of the risk of bias across studies (see Item 15).	NA
Additional analysis	Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression	NA
DISCUSSION
Summary of evidence	Summarize the main findings, including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers).	22-23
Limitations	Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified research, reporting bias).	23
Conclusions	Provide a general interpretation of the results in the context of other evidence and implications for future research.	24
FUNDING
Funding	Describe sources of funding for the systematic review and other support (e.g., the supply of data); the role of funders for the systematic review.	25

2.2. Selection Criteria

Our inclusion criteria were (1) studies showing neurological effects of the mulberry genus Morus, (2) only including studies of the genus Morus, (3) studies dealing with humans or animals, and (4) no restriction on language, country, gender, age or study design. Exclusion criteria were: (1) unreliably extracted data, (2) overlapped data sets, (3) in vitro studies, (4) articles without available full-text and (5) theses, book chapters, editorials, author responses, conference papers, reviews, posters, letters, and patents.

2.3. Search Strategy

We performed our search in nine electronic databases including MEDLINE (PubMed), Scopus, Google Scholar, ISI Web of Science, POPLINE, the System for Information on Grey Literature in Europe (SIGLE), Global Health Library (GHL), Virtual Health Library (VHL), and the New York Academy of Medicine Grey Literature Report (NYAM) for studies published up to September 18, 2017. Details of the search terms for each database are presented in Table 2. A manual search was also performed by screening the reference of the included studies, the similar studies proposed by PubMed, Google Scholar on the first page, and the references of reviews relevant to our topic.

Table 2.

Detailed search strategy for nine database searches.

No.	Databases (Total 9)	Search Terms	Results Total = 1187
1	PubMed	(mulberry OR Morus) AND (neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system)	118
2	Scopus	(TITLE-ABS-KEY (mulberry OR Morus) AND TITLE-ABS-KEY (neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system)	203
3	ISI (WOS)	(mulberry OR Morus) AND (neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system)	610
4	WHO GHL	(mulberry OR Morus) AND (neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system)	97
5	VHL	(mulberry OR Morus) AND (neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system)	93
6	POPLINE	(mulberry OR Morus) AND (neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system)	0
7	SIGLE	(mulberry OR Morus) AND (neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system)	1
8	Google Scholar	(1) with all of the words: mulberry with at least one of the words: neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system) where words occur: title of the article	44
		(2) with all of the words: Morus with at least one of the words: neurotoxicity OR neurotoxic OR Neuroprotection OR neuroinflammation OR neurodegenerative OR Alzheimer OR Parkinson OR dementia OR Neuroprotective OR neurodegeneration OR Huntington OR memory OR cognitive OR cognition OR learning OR perception OR intelligence OR brain OR CNS OR (central nervous system) where words occur title of the article	21
9	NYAM	(1) Mulberry (2) Morus	0

The search results were then imported into Endnote X7 (Thomson Reuters, USA) software to remove duplications. The references were screened based on the title and abstract with specified criteria by three independent reviewers. The full-texts of the remained papers were downloaded and separately screened for eligibility. We translated the articles in foreign languages into English. Discussions between reviewers and, if necessary, the consultation from the supervisor (NTH) resolved all discrepancies during the screening phases

2.4. Data Extraction

Randomly included studies were used to develop a pilot extraction sheet. Three independent reviewers extracted all data, and the supervisor (NTH) resolved any disagreement related to the data. We extracted articles’ essential characteristics (first author, year of publication, study design) along with essential characteristics of patient/animal characteristics (race, gender, age). Also, information including species, plants, compound, solvent for extraction, the dosage of each experiment were concomitantly retrieved. Additionally, tests of measuring the neurological effect of the mulberry species, as well as their outcomes and times of evaluation, were also reported.

2.5. Quality Assessment

Three independent reviewers assessed the quality of each paper based on SYRCLE tools, which were developed to assess methodological quality in animal experiments [21].

We consecutively evaluated ten domains, including sequence generation, baseline characteristics, allocation concealments, random housing, performance bias blinding, random outcome assessment, detection bias blinding, incomplete outcome data, selective outcome reporting and other sources of bias. We categorized the judgment of each reviewer on each domain as “low risk,” “high risk,” or “unclear risk” of bias. Any disagreement was resolved by discussions between reviewers and by consultation from a supervisor (NTH) to reach a consensus.

3. RESULTS

3.1. Search Results

Our search retrieved 1187 studies. We performed title and abstract screening removed duplicates, screened full texts for inclusion according to our inclusion and exclusion criteria. After that, we performed a manual search in the reference of included studies, and we included 47 studies in the qualitative synthesis. We excluded the rest of the studies with reasons in the PRISMA flow diagram (Fig. 1).

Fig. (1).

PRISMA flow diagram of the included studies. ISI: Web of Science, SIGLE: The System for Information on Grey Literature in Europe, GHL: Global Health Library, VHL: Virtual Health Library, NYAM; The New York Academy of Medicine Grey Literature Report. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

3.2. Baseline Characteristics Of Included Studies

A summary of the included studies is presented in Table 3. All included studies are in vivo, no clinical trial on humans was found. There was a variety of mulberry species used, amongst which M. alba was the most popular one (33 studies). Other species such as M. nigra, M. atropurpurea, M. laevigata, and M. rubra were reported randomly. Methanol and ethanol extractions were the most regularly used solvents for extraction. The used doses of mulberry used and its active ingredients for neuroprotective effects varied considerably among the included studies from 0.2 mg/kg/day up to 10 g/kg twice a day. The administration was mostly via oral, except ten study treated animals via intraperitoneal injection (i.p). The treatments of mulberry fruits often saw positive effects including cognitive function improvement, anti-oxidant, anxiolytic, anti-depressant, and anti-ischemic activities. Multiple tests consisting of infarct volume measurement, cell viability, Morris water maze, Hole-Board Test, Horizontal Wire Test, Open Field Test, Forced swim test, etc. were performed to support the statements and findings of each study.

Table 3.

Baseline characteristics of included studies.

Author/Year	Plant/ Compounds			Solvent for Extraction		Dose			Positive Control		Study Design					Effect					Tests
Nade/2010 [22]	M. alba leaves			Methanol		100 - 300 mg/kg			N/A		Haloperidol-induced oxidative stress in mice					Antidopaminergic effect Antioxidant effect					Behavioral testing Biochemical analysis
Bauomy/2014 [42]	M. alba leaves			70% ethanol		200; 400; 800 mg/kg			N/A		Mice infected with S. mansoni					Antioxidant effect Neuroprotection against damage from S. mansoni					Biochemical analysis
Rebai/2017 [44]	M. alba leaves			70% acetone		100 μg/mL/kg			N/A		Glyphosate-induced toxicity in brain mice					Antioxidant effect					Biochemical analysis
Choi/2000 [47]	M. alba leaves			NA		100 and 300mg/kg			N/A		Healthy rats					Anti-oxidant effect					Biochemical analysis
Choi/2000 [46]	M. alba leaves			NA		100 - 300 mg/kg			N/A		Healthy rats					Antioxidant effect					Oxygen radical formation
Kang/2006 [49]	M. alba leaves			85% Methanol		1, 10, 50 mg/ml			N/A		MCAO mice					Protection against ischemia					Infarct volume measurement Cells viability
Tamtaj/2016 [51]	M. alba leaves			Alcoholic		100, 200, 400 mg/kg			N/A		Healthy rats					Improve cognitive function					Morris water maze test
Nade/2015 [30]	M. alba leaves			methanol		25, 50, 100 mg/kg			Ondansetron		Scopolamine-induced cognitive deficits mice					Improve cognitive function					Elevated plus maze Morris water maze task
Sattayasai/2008 [55]	M. alba leaves			NA		100, 200, 500 or 1000 mg/kg			Desipramine, diazepam		Healthy mice					Antidepressant- without an anxiolytic-like effect					The chronic forced swimming test The elevated plus-maze The climbing test The coordination test The rota-rod test Sieve test.
Yadav/2008 [28]	M. alba leaves			Methanol		50, 100, 200 mg/kg			Diazepam		Healthy mice					Anxiolytic effect					Hole-Board Test Elevated plus maze test Open Field Test
Lee/2013 [14]	M. alba leaves			85% Methanol		200 or 400 mg/kg			Diazepam		Healthy mice					Anxiolytic effect					Elevated plus maze test Hole-Board Test Horizontal Wire Test Open Field Test,
Yadav/2008 [23]	M. alba leaves			Methanol		50, 100, 200 mg/kg			N/A		Catalepsy model					Anti-dopaminergic effect					Footshock-induced aggression Sleeping time
Kim/2003 [52]	M. alba leaves			NA		10 mg/kg and 100 mg/kg			N/A		Healthy mice					Recovery from the central nervous system complications of diabetes mellitus.					New cell formation
Nade/2010 [41]	M. alba root			Methanol		25, 50 and 100mg/kg			Diazepam		Mice suffered chronic restraint stress					Anti-stress Antioxidant effect					Passive shock avoidance test Elevated plus maze Open field test Biochemical analysis
Nade/2009 [31]			M. alba root		Methanol			25, 50, 100 mg/kg		Diazepam			Healthy mice				Adaptogenic activity Anti-stress activity Antioxidant		Elevated plus maze Biochemical analysis
Lee/2013 [56]			M. alba root		NA			50, 100, and 200 mg/kg		RU486 (mifepristone)			Healthy mice				Antidepressant-like effects		Forced swim test Tail suspension test
Lim/2014 [26]			M. alba root bark		NA			30 and 100 mg/kg		RU486 (mifepristone)			Healthy mice				Antidepressant-like effects		Forced swim test
Ye/2017 [57]			M. alba root bark		NA			10 g/kg		N/A			Diabetes mice				Antidepressant-like effects		Forced swim test Open-field test Locomotor activity assessment
Wattanathorn/2012 [12]			M. alba fruit		NA			2, 10, 50 mg/kg		Donepezil			Cholinotoxin-induced cognitive decline in mice				Improve cognitive function Neuroprotection		Morris water maze
Kaewkaen/2012 [25]			M. alba fruit		Ethyl alcohol			2, 10, 50 mg/kg		Vitamin C, Donepezil			MCAO mice				Improve cognitive function Neuroprotection		Morris water maze
Kaewkaen/2012 [50]			M. alba fruits		NA			2, 10, 50 mg/kg		Vitamin C			MCAO mice				Improve cognitive function Neuroprotection		Morris water maze Hot plate test
Wattanathorn/2012 [13]			M. alba fruit		NA			2, 10, 50 mg/kg		Vitamin C, Donepezil			Alcoholic mice				Improve cognitive function Neuroprotection		Morris water maze
Kaewkaen/2012 [32]			M. alba fruit		Ethyl alcohol			2, 10, 50 mg/kg		Donepezil			MCAO mice				Improve cognitive function Neuroprotection		Morris water maze
Kim/2013 [52]			M. alba fruit		Ethanol			20, 100, 500 mg/kg		Donepezil			Healthy mice				Improve cognitive function		Object recognition test Step-through passive avoidance test
Kim/2015 [53]			M. alba fruit		Ethanol			0.1, 1, 10, 100 microgram/ml		N/A			Alzheimer disease-like models				Improve cognitive function		Novel object recognition test Y maze test
Gu/2017 [18]			M. alba fruit		70% ethanol			250 mg/kg		N/A			Parkinson disease model				Protection against PD-like symptoms		Olfactory test Pole test Open filed test
Kim/2010 [61]			M. alba fruit		70% ethanol			500 mg/kg		N/A			Parkinson disease model				Protection against PD-like symptoms		Behavioral test
Hwang/2004 [63]			M. alba fruit		Not reported			Not reported		N/A			Healthy mice				MAO activity modulation		Biochemical analysis
Khan/2015 [59]			M. alba stem bark		Methanol			100, 200, 250, 500 mg/kg		Diazepam			Healthy mice				Sedative effect		Open field test Hole cross test
Kim/2015 [54]			M. alba leaves and fruit mixture		Ethanol			0.2, 0.5, 1 g/kg/day		N/A			Obese mice				Improve cognitive function		Novel object recognition test
Turgut/2015 [33]			M. nigra leaves		Methanol			50, 100 mg/kg		N/A			ᴅ-galactose-induced aging mice				Improve cognitive function		Morris water maze
Dalmagro/2017 [24]			M. nigra leaves		Hot water			3–100 mg/kg		Fluoxetine			Healthy mice				Antidepressant-like effects		Forced swim test Tail suspension test Biochemical analysis
			Syringic acid					0.1 – 10 mg/kg
Shih/2010 [43]		M. atropurpurea fruit					Methanol					NA		N/A	Aging mice			Improve cognitive function Antioxidant effect		Avoidance response tests. Oxidant status assays
Srikanta/2016 [34]		M. rubra fruit					NA					20 mg/kg		Resveratrol	Streptozotocin-induced diabetic rats			Antioxidant effect		Physicochemical analysis Antioxidant Status
Tubaş/2017 [60]		M. rubra fruit					Not reported					5, 10 mg/kg		N/A	Penicillin-induced epileptiform mice			Anti-epileptic activity		Electrocorticogram records
Barman/1980 [9]		M. indica leaves					Methanol					200 mg/kg		N/A	Healthy mice			Sedative effect		Spontaneous activity Anti-convulsant effect
Samuel/2016 [48]		Mulberry variety AR-14 leaves					NA					100 mg/kg p.o.		Resveratrol	MCAO mice			Protection against focal cerebral ischemia		Neurobehavioral test Histological studies
Samuel/2016 [40]		Nine varieties of M. alba and M. indica					Water					100 mg/kg		N/A	Rotenone- induced oxidative stress			Antioxidant effect		Biochemical analysis
El-baz/2016 [45]		M. alba fruit M. rubra fruit					Ethanol					300 mg /Kg		Donepezil	Alzheimer induced rats			Neuroprotection against Alzheimer disease		8-OHdG/2-dG ratio DHCR24 and FKBP1B genes ROS level Apoptotic related enzymes
Aditya Rao/2012 [29]		M. alba leaves M. laevigata leaves					Petroleum, ether, chloroform, methanol					200 and 400 mg/kg		N/A	Healthy mice			Sedative effect		Locomotor activity
Hong/2017 [39]		Mulberrofuran G the root bark of M. bombycis					NA					0.2, 1, and 5 mg/kg		N/A	MCAO mice			Protection against ischemia		Infarct volume measurement
Kang/2006 [37]		Cyanidin-3-O-beta-ᴅ-glucopyranoside from M. alba					1% HCl–MeOH					10, 20, 30 µg/ml		N/A	MCAO mice			Protection against ischemia		Infarct volume measurement Cells viability
Andrabi/2004 [38]		Oxyresveratrol from mulberry wood					NA					2, 10, 20 and 30 mg/kg		N/A	MCAO mice			Protection against ischemia		Infarct volume measurement Histological analysis
Lim/2016 [35]		Sanggenon G isolated from the root bark of M. alba					Ethyl acetate					5, 10 and 20 mg/kg		Yohimbine	Healthy mice			Antidepressant-like effects		Forced swim test Open-field test
Lim/2015 [36]		Sanggenon G isolated from the root bark of M. alba					Ethyl acetate					30 mg/kg		Imipramine	Healthy mice			Antidepressant-like effects		Forced swim test
Gupta/2014 [58]		Morusin from M. alba stem bark					NA					5, 10 mg/kg		Diazepam	Healthy mice			Sedative effect Anticonvulsant activity		Convulsion model Locomotor activity
Ma/2014 [27]		Mulberry flavonoid from M. alba leaves					NA					0.3 g/kg		Methycobal	Alloxan-induced diabetic rats			Recovery of peripheral nerve injury in diabetic rats		Histopathological examination

MCAO: Middle Cerebral Artery Occlusion, N/A: Not applied.

3.3. Quality Assessments

We evaluated almost all included studies (44/47) as a high risk of bias via SYRCLE tools evaluation. The three categories of selection bias, performance bias, and detection bias were frequently determined as high risk. In particular, all studies did not report a method to randomly divide animals into groups and pick them for assessing outcomes. Only six studies performed the blinding of caregivers and researchers [22-27]. Two studies stating the observators were blind in assessing the outcomes were considered low bias for blinding of detection bias [22, 26].

Additionally, 43 among 47 studies reporting the results of all experiments are rated with the low bias of selective outcome reporting. Table 4 represents the details of each item evaluation.

Table 4.

Quality assessment of included studies by using SYRCLE tool.

Author/ Year	Selection Bias			Performance Bias		Detection Bias		Attrition Bias		Other	Overall Assessment
	Sequence generation	Baseline characteristics	Allocation concealment	Random housing	Blinding	Random outcome assessment	Blinding	Incomplete outcome data	Selective outcome reporting	Other sources of bias
Nade/2010 [22]	+	+	+	+	-	+	-	-	-	-	High risk
Samuel/2016 [40]	+	+	+	+	+	+	+	-	-	?	High risk
Nade/2010 [41]	+	+	+	+	+	+	+	-	-	-	High risk
Turgut/2015 [33]	+	-	+	+	+	+	+	+	?	?	High risk
Bauomy/2014 [42]	+	+	+	+	+	+	+	-	-	?	High risk
Shih/2010 [43]	+	-	+	+	?	+	+	?	-	-	High risk
Rebai/2017 [44]	+	-	+	-	+	+	+	-	-	?	High risk
El-baz/2016 [45]	+	+	+	+	+	+	+	-	-	?	High risk
Wattanathorn/2012 [12]	+	+	+	+	?	+	+	?	?	?	High risk
Kaewkaen/2012 [25]	+	+	+	+	-	+	+	-	-	-	High risk
Choi/2000 [47]	+	+	+	+	+	+	+	-	-	-	High risk
Choi/2000 [46]	+	+	+	+	+	+	+	-	-	-	High risk
Srikanta/2016 [34]	+	-	+	+	+	+	+	+	-	?	High risk
Dalmagro/2017 [24]	+	-	+	+	-	+	+	+	+	-	High risk
Hong/2017 [39]	+	-	+	+	+	+	+	-	-	?	High risk
Samuel/2016 [48]	+	-	+	+	+	+	+	-	-	?	High risk
Kang/2006 [49]	+	+	?	+	?	+	+	+	-	?	High risk
Kang/2006 [37]	+	+	+	+	+	+	+	+	-	?	High risk
Andrabi/2004 [38]	+	-	?	+	?	+	+	+	-	?	High risk
Kaewkaen/2012 [50]	+	+	+	+	+	+	+	?	-	?	High risk
Wattanathorn/2012 [13]	+	+	+	+	+	+	+	-	-	-	High risk
Kaewkaen/2012 [32]	+	-	+	+	?	+	+	?	-	-	High risk
Tamtaj/2016 [51]	+	-	+	?	+	-	+	+	-	-	High risk
Nade/2015 [30]	+	+	+	+	+	+	+	-	-	-	High risk
Nade/2009 [31]	+	+	+	+	+	+	+	-	-	-	High risk
Kim/2013 [52]	+	+	+	+	?	+	+	?	-	-	High risk
Kim/2015 [53]	+	+	+	+	?	+	+	-	-	-	High risk
Kim/2015 [54]	+	+	+	+	?	+	+	-	-	-	High risk
Sattayasai/2008 [55]	+	+	+	+	+	+	+	?	-	?	High risk
Lim/2016 [35]	+	+	+	+	+	+	+	+	?	-	High risk
Lee/2013 [56]	+	+	+	+	?	+	+	?	-	-	High risk
Lim/2014 [26]	+	+	-	?	-	+	-	-	-	-	Low risk
Lim/2015 [36]	+	-	+	+	+	+	+	-	-	+	High risk
Ye/2017 [57]	+	-	+	+	+	+	+	?	-	-	High risk
Yadav/2008 [28]	+	+	+	+	+	+	+	-	-	-	High risk
Lee/2013 [14]	+	+	+	+	+	+	+	+	-	-	High risk
Gupta/2014 [58]	+	+	+	+	+	+	+	-	-	?	High risk
Aditya Rao/2012 [29]	+	-	+	+	+	+	+	-	-	-	High risk
Khan/2015 [59]	+	+	+	+	+	+	+	-	-	?	High risk
Barman/1980 [9]	+	+	+	+	+	+	+	?	-	?	High risk
Tubaş/2017 [60]	+	-	+	?	+	+	+	+	-	-	High risk
Yadav/2008 [23]	+	+	+	+	-	+	+	-	-	-	High risk
Gu/2017 [18]	+	-	+	+	+	+	+	?	-	-	High risk
Kim/2010 [61]	+	+	+	+	+	+	+	?	-	-	High risk
Kim/2003 [52]	+	+	+	+	+	+	+	?	-	?	High risk
Ma/2014 [27]	+	-	?	+	-	+	+	-	-	-	Low risk
Hwang/2004 [63]	+	+	+	+	+	+	+	?	-	?	High risk

+: high risk. -: low risk. ?: unclear.

chromatography column, and finally the residual resin was extracted with chloroform (yield = 0.34% of the wood weight). Finally, MG (a prenylated flavonoid) was isolated from the methanol extract of dried root bark of M. bombycis [39]. The purified process involved in varied solvents including n-hexane, chloroform, and ethyl acetate, then fractionalized by methanol via a chromatography column. Detail of constituents of the extracts in this review is presented in Table 5.

3.4. Phytochemical Screening Of Studied Extracts

The methanol extract of M. alba leaves contains a wide range of phytochemical groups, including phenolic, flavonoid, tannin, sterol, alkaloid, saponin, anthocyanin, anthraquinone, carbohydrate, protein, and amino acid [22, 23, 28, 29]. From this extract, it could be found the presence of tannins, alkaloids, glycosides, and flavonoids in the ethyl acetate soluble fraction (EASF) [30]. Extracting its leaves with nonpolar solvents such as petroleum ether or chloroform, we could observe the presence of steroids and glycosides [29]. However, the petroleum ether leaves extract had saponins, flavonoids and tannins while chloroform leaves extract showed terpenoids, alkaloids and carbohydrates. The EASF of M. alba methanol root extract had fewer phytochemical groups compared to the leaves extract, as only phenolics, flavonoids and alkaloids were reported [31]. For M. alba fruit powder, phenolics and anthocyanidins were found [12]. Extracting M. alba fruit with ethanol, we could obtain a high amount of anthocyanins, a smaller quantity of flavonoids, and phenolics [32].

M. nigra leaves extract mainly contains phenolics [24, 33]. However, the major compound of hot water extract is syringic acid (80.57%), while the methanol extract only has a small concentration of this compound. Instead, the methanol leaves extract of M. nigra has a significant quantity of vanillic acid and chlorogenic acid [33]. Other phenolic acid are gallic, protocatechuic, p-hydroxybenzoic, caffeic, p-coumaric, ferulic, o-coumaric, rosmarinic, and trans-cinnamic acids.

The constituents of M. laevigata leaves were also varied [29]. The methanol extract consists of steroids, terpenoids, saponins, alkaloids, flavonoids, tannins, carbohydrates, proteins and amino acids. In the chloroform extract, there are steroids, terpenoids, alkaloids and carbohydrates while the petroleum ether extract has saponins, flavonoids, tannins, and carbohydrates.

In the wine made from M. rubra fruit, they determined the presence of flavonoids and phenolics, in which several polyphenols were determined namely gallic acid, gallocatechin, catechin, caffeic acid, ferulic acid, p-coumaric acid, cinnamic acid, trans-resveratrol, trans-piceid, cis-resveratrol, cis-piceid [34].

Some purified compounds that showed positive effects in this review could be extracted from mulberry as followed. Sanggenon G was extracted from root or root bark of M. alba by methanol, then fractionalized the parent extract by ethyl acetate [35, 36]. Its concentration was indicated of 0.446 ± 0.007 mg/g. An anthocyanin, cyanidin-3-O-β-ᴅ-glucopyranoside (C3G), could be extracted from M. alba fruit by the isolation from 1% HCl-Methanol extract [37]. Oxyresveratrol (a hydroxystilbene) was obtained from M. alba wood after several steps [38]. First, the M. alba wood was extracted with 96% ethanol, then purified by a mixture of chloroform-methanol via a silica vacuum liquid

3.5. The Anti-Oxidant Effect In The Brain

Different extracts from all parts of types of mulberry (M. alba, M. nigra, M. rubra and M. atropurpurea) showed their antioxidant effect on a wide range of animal models (Table 6).

Table 6.

Antioxidant effect of Morus on brain.

Refs.		Species	Part Used		Solvent		Dose* (Administration)	Positive Control	Animal Model		Model	Test Duration	Main Results
Nade et al. [22]		M. alba	Leaves		Methanol		100-300 mg/kg/day (p.o)	N/A	Male Wistar strain rats, 170–220g		Haloperidol-induced oxidative stress	21 days	↑ CAT and SOD levels ↓ LPO and NO levels
Choi et al. [47]		M. alba	Leaves		Methanol		100-300 mg/kg/day (p.o)	N/A	Male Sprague Dawley, 160±10g		Healthy rats	6 weeks	↓ BORs levels by 25.1%, IORs levels by 16.5%, LPO levels by 18.1% and OP levels by 14.2%.
Choi et al. [46]		M. alba	Leaves		Methanol		100-300 mg/kg/day (p.o)	N/A	Male Sprague Dawley, 260±20g		Healthy rats	6 weeks	↓ Hydroxyl radical by 21.1%, superoxide radical by 12%, LPO by 12.26%, and OP levels by 13.77%. ↑ Mn-SOD activity by 18.6%, Cu/Zn-SOD activity by 17.7%, and GPx activity by 23.9%.
Bauomy et al. [42]		M. alba	Leaves		Methanol		200, 400, 800 mg/kg/day (p.o)	N/A	9-11 weeks male Swiss albino mice		Mice infected with Schistosoma mansoni	10 days	↑GSH and CAT levels in normal and infected mice in a dose-dependent manner. ↑ TAC in infected mice.
Rebai et al. [44]		M. alba	leaves		Cold acetone		100 μg/kg/day (i.p)	N/A	Female Wistar rats, 180–240g		Glyphosate-induced toxicity in brain mice	15 days	↓ LDH activity, PC and MDA levels ↑ SOD activity
Nade et al.. [41]		M. alba	root		methanol		25, 50 and 100mg/kg/day (p.o)	Diazepam	Male Wistar rats, 150–180g		Chronic restraint stress	10 days	↑ CAT, GSH, SOD level ↓ LPO level
Kaewkaen et al. [32]		M. alba	fruits		Ethanol		2, 10 and 50 mg/kg/day (p.o)	Donepezil	8 weeks male Wistar rats, 300–350g		Vascular dementia	28 days	↓MDA level and ↑ SOD and GSH-Px activity. ↑ CAT insignificantly
Wattanathorn et al. [12]		M. alba	fruits		N/A		2, 10 and 50 mg/kg/day (p.o)	Donepezil	Male Wistar rats, 180-200g		Cholinotoxin-induced cognitive decline in mice	2 weeks	↓MDA level
Turgut et al. [33]		M. nigra	leaves		methanol		50,100 mg/kg/day (p.o)	N/A	8 weeks male BALB/c mice		ᴅ-galactose-induced aging mice	5 days	↓ MDA levels, and ↑SOD, GPx and CAT activities
Dalmagro et al. [24]		M. nigra	leaves		Water		3, 10, 30, 100 mg/kg/day (p.o)	Fluoxetine	Male Swiss mice, 30-40g		Healthy mice	Acute: 1 day Chronic: 7 days	Acute and chronic treatment did not change the levels of TBARS, NPHS levels. ↓ PC level only at 30 mg/kg. ↓ NO level in the brain at 30 and 100 mg/kg with subchronic treatment.
		Syringic acid from M. nigra	N/A		N/A		0.1, 1, 10, 100 mg/kg/day (p.o)						↑ TBARS in the brain ↓ PC and NO levels in the brain
El-baz et al. [45]		M. alba M. rubra	Fruit		Ethanol		300 mg/kg/day (p.o)	Donepezil	Male Albino rats, 180-200 g		Alzheimer induced rats	6 weeks	↑109.54 – 118.09% of LPO levels and 55.17 – 54.6% of GSH levels compared with AD-induced mice
Srikanta et al. [34]	Wine made from M. rubra			fruit		N/A	20 mg/kg/day (p.o)	Resveratrol		8 weeks male Wistar rats, 200g	Streptozotocin-induced diabetic rats	6 weeks	No insignificant change of antioxidant capacity in the brain of diabetic rats
Shih et al. [43]	M. atropurpurea			fruit		Methanol	100; 500 mg/kg/day (p.o)	N/A		6 months male SAMR1 and SAMP8 mice	Senescence-accelerated mice	12 weeks	↑ GST and CAT levels at 100 mg/kg, and further GPx level at 500 mg/kg No significant improvement of GRd was observed
Samuel et al. [40]	Nine varieties of M. alba and M. indica			leaves		Water	100 mg/kg/day (p.o)	N/A		Male Sprague Dawley rats, 200±10g	Rotenone- induced oxidative stress	1 hours (pretreatment)	↓ MDA levels by 50.49% and 41.36% when treating with S-146 and BR-2 extract, respectively ↓ SOD level by 54.01% and 40.18% when treating with S-146 and AR-14 extract

BOR: basal oxygen radical, CAT: catalase, GPx: glutathione peroxidase, GRd: glutathione reductase, GSH: glutathione, GST: glutathione S-transferase, IOR: Induced oxygen radical, i.p.: intraperitoneal injection, LDH: lactate dehydrogenase, LPO: lipid peroxide, MDA: malonyldialdehyde, N/A: not applied, NPHS: non-protein sulfhydryls, NO: Nitrite, PC: Protein carbonyl, p.o: per oral, TAC: total antioxidant capacity, TBARS: thiobarbituric acid reactive substance. *weight of extract per body weight of the animal.

Our study showed that mulberry reversed the disorder of redox system in brain caused by rotenone [40], chronic stress [41], haloperidol [22], ᴅ-galactose [33], Schistosoma mansoni infection [42], aging [43], glyphosate [44], Alzheimer [45], and cholinotoxins [12]. These triggers led to the decrease in levels of antioxidant enzymes in the body including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GRd), and the contents of reduced glutathione (GSH). Also, they increased lactate dehydrogenase (LDH) activity, nitrite (NO), and malonyldialdehyde (MDA) levels, which are formed by the oxidation. There was an exception that SOD, CAT, and peroxidase activities increased in order to respond to stress [41], and the neurotoxicity caused by glyphosate [44]. However, all those changes were almost normalized by the acute or subchronic consumption of mulberry except the case of GRd activity reported by Shih et al. [43]. This study indicated that methanol extract of M. alba fruit insignificantly increased that enzyme even at a high dose (500 mg/kg/day after 12 weeks of treatment). Besides, 10 days of treatment at all doses of M. alba leaves extract improved total antioxidant capacity (TAC) in mice after 46 days infected with S. mansoni [42].

In the model of vascular dementia, mice were pretreated with the ethanol extract of M. alba fruit at 10 and 50 mg/kg 7 days before and 21 after occlusion of the right middle cerebral artery (MCAO). The results showed the enhanced activities of SOD, CAT, and GPx, although this elevation of CAT activity was not remarkable [32]. Interestingly, the antioxidant effect of mulberry was also observed in normal mice. Choi et al. [46, 47] demonstrated that treating 100 and 300 mg/kg/day after 6 weeks of methanol extract of M. alba leaves could reduce hydroxyl radical, superoxide radical, lipid peroxide, basal and induced oxygen levels in both mitochondrial and microsome in the brain. Moreover, the results from this group of authors clarified that M. alba leaves extract rose the activities of both Mn-SOD in brain mitochondrial and Cu/Zn-SOD in brain cytosol [46].

There were only two studies that did not show the positive antioxidant effect of mulberry in the brain. Srikanta et al. [34] observed that 6 weeks of the treatment of wine made from M. rubra fruit did not improve the total antioxidant capacity after the oxidation caused by streptozotocin in diabetic mice. Similarly, Dalmagro et al. [24] also reported that almost all dose of the aqueous extract of M. nigra did not dramatically influence the contents of oxidant markers in the brain, like protein carbonyl (PC), non-protein thiol groups (NPSH), thiobarbituric acid reactive substances (TBARS), and NO level compared to normal mice. The subacute treatment of its primary compound - syringic acid (SA) acted as a pro-oxidant compound illustrated by the downgrade of NO level in the brain.

3.6. Protection Against Ischemia

M. alba leaves and the riched gamma-aminobutyric acid (GABA) leaves, M. alba fruit extract (MLE) are positive candidates to screen for the prevention of ischemic injury.

Regarding the neuroprotective effect of mulberry against ischemia, Samuel et al. [48] compared the strength between MLE-AR-14, a freeze-dried solid leaf extracted from the mulberry variety AR-14, and resveratrol. Using doses of 50 and 100 mg/kg of MLE- AR-14 orally one hour before middle cerebral artery occlusion/reperfusion (MCAO/R) induced a similar reduction of infarct size in mice compared to 50 and 100 mg of resveratrol (34%, 65% vs. 55%, 76%, respectively). This study also pointed out a notable active effect of those two interventions, even on a post-ischemic injury. Namely, after 6 hours of ischemic injury, treatment with MLE-AR-14 (50 mg and 100 mg) provided a neuroprotective effect of about 28% and 54%, respectively; whereas the percentage of ischemic brain reduction was a bit higher, by 53% and 68% for doses of 50 mg and 100 mg by resveratrol, respectively. The effect against neural cell death induced by cerebral ischemia was hypothesized to be involved in the free radical scavenging activity. The authors observed the attenuation of MDA level (an oxidative stress marker) and the upregulation of glutathione levels (an endogenous antioxidant) in the blood in the presence of MLE-AR-14 or resveratrol, although resveratrol showed significantly more effective.

However, Kang et al. reported that oral treatment of methanol M. alba leaves extract (200 mg/kg) 30 minutes after the MCAO did not reduce the infarct volume of the mice brain. The neuroprotective effect against MCAO- induced mice was only enhanced when conducting the accumulation of GABA in M. alba leaves (GAML). GAML shortened the cerebral injury size by 31% using a dose of 200 mg/kg orally compared with the control group [49].

This result was quite similar to the effect of positive control (5 mg/kg intravenously injected edaravone). The mulberry fruit extracted with 1% HCl-MeOH also reduced the ischemic brain volume by 26% [37].

Additionally, purified compounds extracted from M. alba fruit (C3G), M. alba fruit (oxyresveratrol), and M. bombycis root bark (MG) showed neuroprotective effects against cerebral ischemia [37]. Treatment of C3G 30 minutes after MCAO (10 mg/kg per orally), successfully reduced the ischemic brain volume by 18%. Similarly, intraperitoneally injecting 10 and 20 mg/kg of oxyresveratrol (twice: before and after MCAO) decreased the injured brain volume at days 3 after stroke by 54% and 63%, respectively [38]. The intraperitoneal administration of MG (0.2, 1, and 5 mg/kg) 30 minutes before MCAO/R showed a similar effective impact on the reduction of injured brain zone in mice compared to carnosine (25, 50, and 75 mg/kg, i.p.) [39]. The injured brain zone was 39.0 ± 6.4%, 26.0 ± 7.4%, and 19.0 ± 4.3% by the dose of 0.2, 1, and 5 mg/kg, respectively, in MG group; and the cerebral infarct size was 50.6 ± 6.2%, 40.0 ± 6.5%, and 19.6 ± 4.2% at dose 25, 50, and 75 mg/kg, respectively, in carnosine group.

Regarding their probable mechanism, these compounds protected the brain cells after MCAO via varied pathways. C3G and MLE prevented the polymorphonuclear leukocytes from infiltrating into cerebral focal ischemic tissue after stroke, which might be helpful for cell survival [37]. Meanwhile, oxyresveratrol prevented the cell death via the inactivation of apoptotic markers including cytosolic cytochrome c release and caspase-3 [38], and MG potentially inhibited the reactive oxygen species (ROS) generation via the decrease in nicotinamide adenine dinucleotide phosphate oxidase (NOX) enzyme activation and NOX4 protein expression [39].

3.7. Effect On Cognitive Functions

Fourteen articles reported the therapeutic activities of mulberry on cognitive impairment using various models, including the Morris water maze test, object recognition test, passive or active avoidance test, and elevated plus-maze model. Various forms of mulberry such as ethanol extracts of M. alba leaves and fruit, methanol extract of M. alba leaves, M. nigra leaves and M. atropurpurea fruit, M. alba fruit powders and a mixture of M. alba leaves and fruit (2:1) showed their effect on memory improvement at a wide range of dosages (Table 7).

Table 7.

The activities of mulberry on learning and memory.

Refs.		Species		Part Used	Solvent	Dose* (Administration)		Positive Control		Animal Model		Model of Study (Duration)		Main Results			Conclusion
Wattanathorn et al. [12]		M. alba		Fruit	N/A	2,10, 50 mg/kg/day (p.o) x 2 weeks		Donepezil		Male Wistar rats, 180-200g		MMT (4 days)		↓ Escape latency time at all doses ↑ Retention time at 2, 50 mg/kg			Enhancing memory in ageing mice.
Wattanathorn et al. [13]		M. alba		Fruits	N/A	2,10, 50 mg/kg/day (p.o) x 2 weeks		Vitamin C Donepezil		8 weeks male Wistar rats, 180-220g		MMT (14 days)		↓ Escape latency at all doses in single-dose administration and on days 7, 14 No significant change of retention time			Enhance spatial memory in alcoholic mice.
Kaewkaen et al. [50]		M. alba		Fruit	N/A	2, 10, 50 mg/kg/day (p.o) x 2 weeks		Vitamin C		8 weeks Male Wistar rats, 300-350g		MMT (14 days)		↓ Escape latency time at 2, 10 mg/kg in a healthy condition in a single dose and after 7 days. No changes in retention time. ↑Retention time at 2, 10 mg/kg in stroke condition 14 days after stroke. No changes in escape latency.			Protect against memory impairment in MCAO mice and improve neuron density in the hippocampus.
Kaewkaen et al. [25]		M. alba		Fruits	Ethanol	2,10 and 50 mg/kg/day (p.o) x 28 days		Vitamin C Donepezil		8 weeks male Wistar rats, 300-350g		MMT (21 days)		↓ Escape latency time at 50 mg/ kg in single-dose administration in healthy/stroke condition 7 days after stroke ↑ Retention time at 2, 10 mg/kg on a single dose in healthy condition ↑ Retention time at 2, 10, 50 mg/kg stroke condition after days 7 and 14. No change observed in 21 days.			Enhance cognitive functions in the MCAO rats.
Kaewkaen et al. [32]		M. alba		Fruit	Ethanol	2, 10, 50 mg/kg/day (p.o) x 28 days		Donepezil		8 weeks male Wistar rats, 300-350g		MMT (21 days)		↓ Escape latency time at 5 and 10 mg/kg after 21 days. No change in retention time			Enhance memory of MCAO mice
Kim et al. [52]		M. alba		Fruit	Ethanol	20, 100 and 500 mg/kg/day (p.o) x 7 days		N/A		6 weeks male ICR mice, 25–28 g		PAT		↑ Retention time at 100 and 500 mg/kg			Enhance memory via up-regulating nerve growth factor.
												ORT		↑ Recognition time at 100 and 500 mg/kg
Kim et al. [53]		M. alba		Fruit	70% Ethanol	20, 100, and 500 mg/kg/day (p.o) x 14 days		N/A		6 weeks male ICR mice, 25–28 g		NORT		↑ Novel object recognition index in a dose-dependent manner			Protect cognitive function and survival neurons in Alzheimer disease-like models.
												Y-maze test (14 days)		↑ Spontaneous alteration
Tamtaj et al. [51]	M. alba		Leaves		Ethanol		100, 200, 400 mg/kg/day (p.o) x 1 month		N/A		Male Wistar rats, 250 g		MMT (4 days)		↓ Time to find the hidden platform at all doses in the learning stage ↓ Time to find the hidden platform at 400 mg/kg in rehearsal stage	Improve the learning process at all dose Improve spatial memory at 400 mg/kg
Kim et al. [54]	M. alba		Leaves and fruits		70% Ethanol		1 g/kg/day (p.o) x 12 weeks		N/A		4 weeks male C57BL/6 mice, 23-25 g		NORT		↑ Memory index by 78.63%	Recover memory function in obese mice.
Nade et al. [31]	M. alba		Root		Methanol/ Ethyl acetate		25, 50 and 100 mg/kg/day (p.o) x 21 days		Diazepam		Male Wistar rats, 150-180g		EPM (21 days)		↓ Transfer latency on days 7, 10, 21 at all doses	Recover cognitive function in mice suffering chronic footshock stress
Nade et al. [41]	M. alba		Root		Methanol/ Ethyl acetate		25, 50 and 100 mg/kg/day (p.o) x 10 days		Diazepam		Male Wistar rats, 150-180g		EPM (5 and 10 days)		↓ Transfer latency on days 5, 10 at all doses	Recover cognitive function in mice suffering chronic restraint stress
Nade et al. [30]	M. alba		Leaves		Methanol/ Ethyl acetate		25, 50 and 100 mg/kg/day (p.o) x 9 days		Ondansetron		Male Swiss albino mice, 22 - 25 g and male Wistar rats, 120-150 g		ORT		↑ Discrimination index	Improve learning and memory in scopolamine-induced cognitive deficits mice
													EPM (4 days)		↓ Transfer latency
													MMT (4 days)		↑Swimming time in the target quadrant
Shih et al. [43]	M. atropurpurea		Fruit		Methanol		100, 500 mg/kg/day (p.o) x 12 weeks		N/A		6 months male SAMR1 and SAMP8 mice		PAT (7 days)		↑ Latency time on days 3, 7 at 500 mg/kg	Improve memory in aging mice
													AAT (7 days)		↑ Latency time on days 2, 3, 4 at all doses
Turgut et al. [33]	M. nigra		Leaves		Methanol		50, 100 mg/kg/day (p.o) x 8 weeks		N/A		8 weeks male BALB/c mice		MMT (4 days)		↓ Time for escape latency ↑ Time spent to find the target quadrant ↑ Time swimming in the target quadrant ↑ Times crossed the platform location	Improve cognitive deficits in aging mice induce by ᴅ-galactose.

AAT: Active avoidance test, EPM: Elevated plus maze, MCAO: Middle Cerebral Artery Occlusion, MMT: Moris Maze Test, N/A: Not applied, NORT: Novel object recognition test, OTR: Object recognition test, PAT: Passive avoidance test, p.o: per oral. *weight of extract per body weight of the animal.

M. alba fruit powder and M. alba ethanol fruit extract at 2, 10 mg/kg enhanced the learning and memory process in models of MCAO, alcohol intoxication-induced memory impairment, or age-related cognitive impairment induced by cholinotoxin [12, 13, 25, 32, 50]. Almost all results showed that mice using mulberry spent less time to reach the hidden platform, and spent more time in the target quadrant (retention time) in Moris Maze Test. Only alcoholic rats consuming mulberry powder, and MCAO mice consuming mulberry ethanol extract had no change of retention time. The healthy mice also enhanced their retention memory when consuming these forms of mulberry [25, 50].

Several leaves’ extracts from M. alba and M. nigra proved their efficacy on improving retention memory via Moris Maze Test. This was reflected by the reduction of time to find the target quadrant and of time for escape latency as well as by the increase in retention time and times that mice came across the platform location [30, 33, 51].

These improvements were in a dose-dependent manner and differed according to the mulberry form. For instance, in stroke condition, mulberry fruit powder at the high dose of 50 mg/kg exhibited no positive effects on retention time,

whereas it was still active in another model [12, 50]. Also, all doses of M. alba fruit extract increased retention time 14 days after stroke, but 50 mg/kg of M. alba fruit powder failed to show that effect [25, 50].

Apart from Moris Maze test, Nade et al. [30, 31, 41] emphasized the memory enhancement of ethyl acetate soluble fraction (EASF) of M. alba methanol extract by the increase in discrimination index via object recognition test, and by the reduction of transfer latency in EPM test on days 7, 14, 21. The extract might be effective than diazepam against chronic footshock stress, as this drug only showed improvement on the 1^st day [31].

In a similar designed model, Kim et al. [52-54] showed that ethanol extract of M. alba fruit and mixture extract of M. alba leaves and fruit (2:1) improved the time spent to discover the novel object in healthy mice, in obese mice, and Aβ_25–35-injected mice. Particularly, M. alba fruit extract at 100 and 500 mg/kg could increase time spent on a novel object by 66.88 ± 72.57%, and 69.14 ± 72.84%, respectively, compared with Aβ_25–35-injected mice [53]. There was a significant improvement in memory of obese mice by 78.63% as well [54].

Finally, the latency time mice spent to avoid the electrical foot shock in both passive and active avoidance test was raised by treating with EASF of M. alba methanol root extract, M. alba ethanol fruit extract and M. atropurpurea methanol fruit extract showing that the learning process of memory-impaired mice, was improved [41, 43, 52].

The positive effect on the cognitive functions might be chronic with the duration of mulberry exposure ranging from 9 days – 12 weeks. In addition, memory and learning improvement resulted from mulberry activities were associated with neuroprotection. This was shown in the increase of antioxidant capacity in the body [13, 32, 33, 41], the increase in density and differentiation of survival hippocampal cells [12, 13, 50, 52, 54], the inhibition of acetylcholinesterase (AchE) [13, 25], the increase in the cholinergic neuron and the acetylcholine formation [32], and the reduction of apoptotic markers in the hippocampus [32, 53].

There was a suggestion that the molecule mechanism of the anti-apoptotic activity of mulberry in vivo relating to the reduction of glycogen synthase kinase-3β (GSK-3β) pathway-mediated tau phosphorylation. This metabolite resulted in the formation of the neurofibrillary tangles [53] as well as the increase in B-cell lymphoma 2 (Bcl-2) expression in the hippocampus which led to the amplification of signals of apoptotic cascade such as cytochrome c release and the activation of caspase-3 [32, 53]. Therefore, preventing this reaction caused the reduction of apoptosis and the protection of brain cells [53]. Nerve growth factor (NGF) content in the hippocampus was also enhanced in a dose-dependent manner after the treatment of mulberry resulting in the induction of neurite and synapse formation, via the promoting extracellular-signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB) phosphorylation as well as pre- and post-synapse markers formation [52]. As a result, new cells generation caused memory improvement effect.

3.8. Antidepressant, Anxiolytic And Sedative Effects

Previous studies have reported the antidepressant-like effects of mulberry extracts such as sanggenon G from root bark, methanolic extract from leaves, ethyl acetate fraction, and n-butanol fraction from methanol extract of M. alba root, and alcohol extract of M. alba root (Table 8).

Table 8.

Anti-depression, anxiolytic, anti-stress effects of mulberry

Refs.		Species		Part Used			Solvent			Dose* (Administration)			Positive Control			Animal Model			Model of Study			Main Results		Conclusion
Ye et al. [57]		M. alba		Root bark			N/A			10 g/kg twice daily (p.o) x 4 weeks			N/A			2 months male Sprague-Dawley rats			OFT			↑ Number of rearing ↑ Number of line crossing insignificantly		Reserve depressant behaviors in diabetes mice
																			LAT			↑ Locomotor activity insignificantly
																			FST			↓Immobility time
Lee et al. [56]		M. alba		Root bark			Ethanol			50, 100, 200 mg/kg/day (p.o) (p.o) x 5 days			RU486 (mifepristone)			Male Wistar rats, 180–220g			FST			↓Immobility time at 100 and 200 mg/kg ↑Climbing time at 200 mg/kg. No significant change of swimming time		Antidepressant-like effects
																			TST			↓Immobility time at 100 mg/kg
Lim et al. [26]		M. alba		Root bark			Methanol/ EtOAc Methanol/ n-butanol			30, 100 mg/kg/day (p.o) x 7 days			RU486 (mifepristone)			8 weeks Male Wistar rats, 180–210g			FST			↓Immobility time, ↑climbing time, ↑swimming time at 100 mg/kg of EtOAc fraction No change observed with an n-butanol fraction		Antidepressant-like effects
Nade et al. [41]		M. alba		Root			Methanol/ EtOAc			25, 50, 100 mg/kg/day (p.o) x 10 days			Diazepam			Male Wistar rats, 150-180g			OFT			↑ Number of squares crossed at all doses on day 10 ↓ Latency at all doses ↑ Number of rearings at 50 and 100 mg/kg		Anxiolytic effect
Nade et al. [31]		M. alba		Root			Methanol			25, 50, 100 mg/kg/day (p.o) x 28 days			Diazepam			Male Wistar rats, 150-180g			DST (21 days)			↓ Immobility time at day 1, 14, 21		Antidepressant-like effects
Khan et al. [59]		M. alba		Stem bark			Methanol			250, 500 mg/kg/day (p.o)			Diazepam			4 weeks male and female Swiss albino mice, 40-45 g			OFT			↓ Number of movement at all dose after 120 minutes of administration		Sedative effect
																			HCT			↓ Locomotor activity at high dose
Lee et al. [14]		M. alba		Leaves			Methanol			50, 100, 200, 400 mg/kg (p.o)			Diazepam			5 weeks male ICR mice, 23–25 g			LAT			No alternation in locomotor activities or rearing frequencies after 1 hour of administration		Anxiolytic effect
																			EPM			↑ Time spent in the open arms after 1 hour of administration ↑ Entries into open arms after 1 hour of administration
																			HBT			↑ Head-dips at doses of 200 and 400 mg/kg after 1 hour of administration
Aditya Rao et al. [29]		M. alba M. laevigata		Leaves			Petroleum ether, chloroform, methanol			200 and 400 mg/kg/day (p.o)			N/A			Male and female albino mice, 25-30 g			LAT (5 mins)			↓ Locomotor activity after 1 hour of administration		Sedative effect
Sattayasai et al. [55]		M. alba	Leaves			Boiling water			100, 200, 500, 1000 mg/kg (i.p.)			Desipramine, diazepam			Male IRC mice		FST			↓ Immobility time at 100 and 200 mg/kg after 30 minutes of administration				Antidepressant-like effect at low dose (100, 200 mg/kg) Sedative effect at high dose (500, 1000 mg/kg)
																	CT			↓ Climbing activity at 500 and 100 mg/kg after 30 minutes of administration
																	OFT			↓ Time spent in open arms and the number of entry at 500 and 100 mg/kg after 30 minutes of administration
																	RRT			↓ Time spent on the rod after 30 minutes of administration
Yadav et al. [28]		M. alba	Leaves			Methanol			50, 100, 200 mg/kg/day (i.p.)			Diazepam			Male Swiss albino mice, 18-22 g		OFT			↑Square traversed at all doses after 30 minutes of administration ↑Rearing and self-rearing at 100 and 200 mg/kg after 30 minutes of administration				Anxiolytic effect
																	HBT			↑ The number of a head poking at 100 and 200 mg/kg after 30 minutes of administration ↑Duration of a head poking at all doses after 30 minutes of administration
																	EPM			↑Time spent in open arms at 100 and 200 mg/kg after 30 minutes of administration ↓ Time spent in closed arms and ↑ The entries to open arms at 200 mg/kg after 30 minutes of administration
																	LDP			↑Time spent in lightboxes and ↓the time spent in dark boxes at 100 and 200 mg/kg after 30 minutes of administration No change of crossings and transfer latency.
Dalmagro et al. [24]		M. nigra,	Leaves			Water			3–100 mg/kg/day (p.o) x 1 day (for acute test, and x 7 days for subchronic test			Fluoxetine			Male Swiss mice, 30–40 g		FST			↓ Immobility time at all doses in acute test				The antidepressant-like property might occur due to syringic acid
																	TST			↓ Immobility time at 3, 10, 30 mg/kg in acute test, and at 3, 10, 30, 100 mg/kg in subchronic test
																	OFT			No significant changes in the number of crossings, rearing, and fecal boluses in both tests
		Syringic acid	N/A			N/A			0.1 – 100 mg/kg/day (p.o)								TST			↓ Immobility time at 1, 10 mg/kg in both acute and subchronic tests
																	OFT			No significant changes in the number of crossings, rearing, and fecal boluses in both tests
Barman et al. [9]		M. indica	Root			Methanol			200 mg/kg/day (i.p.)			N/A			Male adult albino rats (150-165 g)		SAT			↓ Spontaneous activity by 72.78% after 30 minutes of administration				Sedative effect
Gupta et al. [58]	Morusin		N/A		N/A			5, 10 mg/kg (i.p.)			Diazepam			Wistar albino rats (150–200 g)				LAT			↓ Locomotor activity by 48.82% and 70.20% at 5 and 10 mg/kg, respectively after 30 minutes of administration		Sedative effect
Lim et al. [36]	Sanggenon G		N/A		N/A			3, 10, 30 mg/kg/day (i.p.)			Imipramine			8 weeks male Sprague Dawley rats, 180–210g				FST			↓ immobility time at 30 mg/kg after 60 minutes of administration ↑ swimming time at all dose after 60 minutes of administration No change of climbing time		Antidepressant-like effects mediated serotonergic system.
Lim et al. [35]	Sanggenon G		N/A		N/A			5, 10, 20 mg/kg/day (i.p.)			Yohimbine			8 weeks male Sprague–Dawley rats, 180–210g				FST (6 mins)			↓ Immobility time at 20 mg/kg after 60 minutes of administration		Antidepressant-like effect

CT: Climbing test, DST: Despair swim test, EPM: Elevated plus maze, EtOAc: Ethyl acetate, FST: forced swimming test, HCT: Hole cross test, HBT: Hold board test, HWT: Horizontal Wire Test, i.p.: intraperitoneal injection, LAT: locomotor activity test, LDP: Light/dark paradigm, N/A: Not applied, OFT: Open field test, p.o: per oral, RRT: Rota-rod test, SAT: Spontaneous activity test, TST: Tail suspension test. * weight of extract per body weight of the animal.

In general, all parts of mulberry (M. alba leaves green tea, M. alba root bark, M. nigra leaves, sanggenon G and syringic acid extracted from mulberry) could decrease the immobility time that mice spent in forced swim test (FST) [24, 26, 31, 35, 36, 55-57], and in tail suspension test (TST) [24, 56]. These indicated that mulberry possessed an antidepressant-like effect. The antidepressant-like effect of M. alba green tea extract at 200 mg/kg was even comparable with 10 mg/kg of desipramine [55]. The antidepressant-like effect of mulberry could be acute (measured 30 – 60 minutes after administration) or subchronic (measured after 7 days of administration) or chronic (measured after 28 days). The doses of extracts ranged from 3 mg per day to 10 g twice a day orally, or 3 – 100 mg/day by intraperitoneal injection. Sanggenon G showed effective antidepression at higher doses (20 and 30 mg/kg, i.p.), whereas syringic acid was better at average doses (1 and 10 mg/kg, p.o.) The modulation of the limbic hypothalamic–pituitary–adrenocortical (HPA) axis, which reported by few studies, clarified this effect [26, 31, 36, 56]. Accordingly, ethyl acetate fraction of M. alba methanol root bark extract, sanggenon G extract from the root bark, and ethanol extract of Cortex Mori Radicis (CMR) prevented the promotion of corticosterone response and c-fos immunoreactivity in the dentate gyrus or hippocampus under FST-induced depressive condition. These could be associated with the increase in glucocorticoid receptors (GR) expression in the hippocampus through the promotion of phosphorylation at S232 and S246 of GR [56]. Moreover, the anti-depressive effect of sanggenon G might be mediated by an interaction with the serotonergic system as well, as Lim et al. indicated pretreating with a selective 5-hydroxytryptamine_1A (5-HT_1A) receptor antagonist could reserve this positive effect [36].

Apart from that, several other effects related to anti-depressive like effects were observed. Lim et al. indicated that the practical impact of sanggenon G at 5 - 10 mg/kg was also promoted by the presence of the α₂-antagonist (yohimbine) [35]. Otherwise, M. alba root bark extraction could reserve the depressive-like behaviors in diabetic mice assessed via FST [57].

The anxiolytic effect of mulberry had controversial results via different experiments. Additionally, mulberry could show the acute anxiolytic effect after 30 – 60 minutes of administration, regardless of oral administration or intraperitoneally injection. In open field test (OFT), two extracts of M. alba (M. alba leaves methanol extract and M. alba root methanol extract) decreased the latency time to enter the main area and increased the number of squares crossed as well as the number of rearings in both standards and stressed mice indicating that mulberry extracts had an anxiolytic effect [14, 28, 41]. However, only Lee et al. showed no change in the frequencies of rearing observed in mice treated with M. alba methanol leaves extract [14]. Mulberry extracts also showed their anxiolytic effect via the EPM test, light/dark exploration test, and hold board test. In the EPM test, more time was spent in the open arms along with the short transfer latency to the closed arms [14, 28, 41]. For instance, methanol extract of M. alba leaves increased up to 49.9 ± 3.1% of time spent on the open arms and 63.3 ± 1.3% of the number of entries into open arms compared with the control group [14]. Additionally, mulberry enhanced the exploratory head-dipping behaviors and time spent in lightbox in hold board test, and light/dark exploration test, respectively [14, 28]. However, M. alba root bark only tended to improve the results in diabetic mice insignificantly, and the aqueous extract of M. alba leaves showed no anxiolytic effect at 100 - 200 mg/kg [55, 57]. Lee et al. suggested that the anxiolytic activity might relate to the histaminergic system in the central nervous system, as a histamine H₃ receptor antagonist abolished this effect [14].

The decreased movement of mice in the locomotor activity test showed that M. alba leaves and stem bark methanol extract, M. alba steam bark morusin, several extracts of M. laevigata possessed sedative effect [28, 29, 58, 59]. The methanol extract of M. indica root bark at 1000 mg/kg also showed the same effect, as it decreased the spontaneous motility up to 72.78% [9]. As same as the anxiolytic effect, the sedative effect of mulberry was acute. These outcomes above were observed after 30 – 60 minutes of administration.

On the other hand, the anxiolytic and sedative effects of mulberry were in a dose-dependent manner. No significant anxiolytic effect was shown by M. alba leaves methanol extract at a low dose (below 100 mg/kg) [14, 28, 52], and aqueous extract of M. alba leaves only had a sedative effect at high dose (over 500 mg/kg) [55]. The petroleum ether, chloroform and methanol fractions obtained from aqueous extract of both M. alba and M. laevigata leaves decreased over 50% of locomotor activity. Nevertheless, the petroleum ether fractions exhibited stronger effect compared to methanol fractions, then followed by chloroform fraction in both cases, indicating that solvents for extraction affected the effectiveness of mulberry [29].

3.9. Other Effects

Two studies also evaluated the anticonvulsant effect of mulberry. Tubas et al. showed that intraperitoneal treatment of M. ruba fruit extract at 10 mg/kg significantly decreased the spike frequencies of convulsions in penicillin-induced epileptiform mice from the 80^th minute observed during 120 minutes tested although no change of the amplitude was observed [60]. This result was reflected in the study of Gupta et al. [58] who showed that morusin – a compound extract from M. alba dramatically increased the onset of convulsive time caused by isoniazid, from 306.16 ± 22.16 (s) to 491.42 ± 29.07 (s) (at 5 mg/kg) and 659.10 ± 31.28 (s) (at 10 mg/kg). There was a significant reduction in the duration of convulsions and the percentage of mortality as well. In maximal electroshock-induced convulsion rats, morusin intraperitoneal injected administration at 5 mg/kg led to a decrease in the duration of tonic hind limb extension (seconds) whereas 10 mg/kg of this compound even abolished this reaction. Regarding the mechanism of action, the anticonvulsant effect results from the reduction of MDA levels in erythrocytes and plasma, and the preservation of GABA in the brain [58, 60]. These anticonvulsant effects were acute observed after 30 minutes of treatments. However, a report of Barman et al. showed a different result, as M. indica root failed to stop pentylene tetrazole-induced convulsion in mice [9].

Besides, mulberry also showed its effect on sleep medicines, and this effect was dose-dependent. The sleeping time caused by two barbiturates (pentobarbitone and phenobarbitone) was extended by pretreating (i.p.) with M. indica root and M. alba leaves at 200 mg/kg 30 minutes prior the barbiturates treatments [9, 23]. This extension, however, was not significant when pretreating with M. alba leaves at the dose of 100 mg/kg. On the other hand, the use of 100 and 200 mg/kg of M. alba leaves also reduced the onset time of sleeping while there was no change with 50 mg/kg of this extract.

Regarding PD, Gu et al. [18] studied the effect of 70% ethanol extract of mulberry (M. alba) fruit against PD-like symptoms caused by 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine/probenecid (MPTP/p), which is a neurotoxic agent. They found that the extract at the dose of 250 mg/kg/day inhibited the motor deficits in mice after 38 days of treatment, that proved by longer staying of mice on the rod in the rotarod test, less locomotor activity time when mice descended to the floor in the pole test and thus, improved bradykinesia and increased locomotor activity in the open field test. Also, non-motor deficits were improved, as pellet retrieval time was shortened in the olfactory test in the treated group. This result was concordant with the report of Kim et al. [61] who also showed the improvement of bradykinesia in behavioral tests in mice intraperitoneally administrated 500 mg/kg/day of 70% ethanol extract of M. alba fruit during 15 days period. This protection against bradykinesia was hypothesized to be associated with the protection of dopamine neurons in the substantia nigra pars compacta and striatum through the inhibition of Bax protein (an apoptotic protein) or α-synuclein and ubiquitin levels (factors killed dopaminergic cells), in PD models.

Regarding the protection against diabetes mellitus complications on the central nervous system, Kim et al. reported an increase in new cell proliferation in the dentate gyrus in both normal and diabetic rats after 3 days of intraperitoneal treatments with 100 mg/kg of heat-extracted leaves of M. alba [62]. The increased expression of neuropeptide Y, which relates to the cell division, most likely mediated this effect. Additionally, another study showed that 0.3 g/kg of flavonoids extracted from M. alba in 8 weeks period could chronically recover a severe peripheral nerve injury in diabetic rats. Accordingly, the oral administration of these compounds led to an increase in the myelin sheath area and the myelinated fiber cross-sectional area. The study demonstrated that the extramedullary fiber number, the onion-bulb type myelin destruction, and the degeneration of mitochondria and Schwann cells reduced remarkably [27].

Another effect shown is the effect on catalepsy induced by antidopaminergic agents [23]. Haloperidol (a non-selective D₂ dopamine antagonist) and metoclopramide (dopaminergic blocking agent) inhibited dopamine transmission by blocking its receptor in the striatum, causing catalepsy. The authors showed that there was an increase in catalepsy score at 100 mg/kg of M. alba leaves extract after 28 days treatment period. On the other hand, footshock-induced aggression, which was associated with the increase in dopamine level in the brain, was also attenuated with the use of 50, 100 and 200 mg/kg of mulberry leaves extract proved by the increase in latency to fight and the decrease in fighting attacks. These results indicated that M. alba leaves extract might possess antidopaminergic activity.

Hwang et al. [63] suggested that 5 consecutive days of treatment of M. alba fruit extract caused modulation of MAO (monoamine oxidase) in mice brain after physical stress. The study indicated that there was a recovery of the decreased MAO-A level and the increased MAO-B level to their normal levels after 30 minutes of swimming.

Finally, several abnormalities in the brain that caused by S. mansoni infection in the form of a disturbance in the antioxidant system, the attenuation of GABA level and AchE activity returned to normal levels when treated with 200 – 800 mg/kg of 70% methanol extract of M. alba leaves after 10 exposed days. Also, these extracts induced normal brain parenchymal cells from histological view with fewer abnormalities of neuronal architecture compared to the control group [42]. Regarding neuro-amelioration against AD, El-baz et al. [45] realized that there was an increase in the ratio of 8-OHdG/2-dG, pointing out the DNA damage, and a decrease in levels of the expression of DHCR24 and FKBP1B genes which play an essential role in degenerative disorders in AD mice. These expressions turned to the normal levels compared to mice of the control group by consuming 300 mg/kg of M. alba or M. ruba for six weeks. The authors suggested that these effects were associated with the inhibition of reactive oxygen species (ROS) as well as the apoptotic marker (caspases-8), and the promotion of antioxidant enzyme activity (such as GSH).

The mechanism of activities of M. alba fruit is shown in Fig. (2).

Fig. (2).

Mechanism of positive effects of Morusalba fruit in brain functions Bcl-2: B-cell lymphoma 2, CREB: cyclic AMP response element-binding protein, ERK: extracellular-signal-regulated kinase, GSK: glycogen synthase kinase-3β, ROS: reactive oxygen species. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

4. DISCUSSION

Varied types of mulberry have exhibited their protection of brain functions through in vivo models. M. alba was the most popular tested plant for varied effects. This plant showed the antioxidant effect in the brain, cognitive function improvements, antidepressant effect, sedative effect, antidopaminergic effect, anti-PD-like symptoms effect, MAO modulation, prevention of AD and the complications of central nervous in diabetic mice. Meanwhile, M. nigra was only tested for antioxidant effect, antidepressant effect and memory improvement. M. indica and M. laevigata had a sedative effect at the high doses. Besides, M. indica and M. atropurpurea could inhibit oxidative stress. Also, M. atropurpurea enhanced the memory of aging mice. Especially, ethanol fruit extract of M. rubra could promote the antioxidant defense whereas wine made from this plant had no positive affection on the antioxidant system. Additionally, M. rubra was the sole plant that had the anticonvulsant effect in this review. Almost all these effects were subchronic or chronic with the duration of treatments ranging from 5 days to 12 weeks. Exceptionally, the anticonvulsant effect and antidepressant, anxiolytic and sedative effects were acute which showed their protection against the triggers when pretreating with the plants 30 – 60 minutes before the tests.

Our review recorded large ranges of used doses across included studies. M. alba extracts showed their activities at the doses ranging from 5 mg/kg to 1 g/kg per day per oral, and at 100 µg/kg – 1000/kg mg via intraperitoneal injection. M. nigra had the used doses between 3 – 100 mg/kg. Similarly, M. atropurpurea, M. indica, and M. laevigata were examined at 100 – 500 mg/kg/day while M. rubra was intraperitoneally injected at 10 mg/kg for anticonvulsant effect, and 20 – 300 mg/kg per oral for antioxidant effect. This raised a challenge for further studies because the doses have not been standardized. It appeared that the authors randomly chose the ranges of doses for testing, although they conducted the same experiments with similar plant characteristics. For the small doses (2 – 10 mg/kg) or very high doses (1 or 10 g/kg), we might need more studies to confirm the efficacy of the extracts with these doses because it is quite hard for the active ingredients in the extract with small doses reaching the active concentrations in the body after they undergo the absorption and metabolism process. Besides, if an extract only showed their effect at very high doses (1 or 10 g/kg), it is impossible to apply these extracts in human studies as there is a need for a large number of plants to obtain the sufficient extracts for chronic interventions. C3G showed its efficacy at 10 mg/kg/day per oral in mice. However, its absorption in the body is quite low; thus, this point must be considered in further studies in humans [64]. Turn back to specific effects, mulberry contains the high concentrations of polyphenol, anthocyanin, phenolic, and flavonoid compounds that might be the source of their antioxidant effect [12, 13, 32, 33]. The results obtained were slightly inconsistent might be due to the variation of the compositions extracted from different species or used solvents. For illustration, most of the extracts of M. alba increased antioxidant enzymes’ levels. However, wine from M. rubra and aqueous leaves extract of M. nigra failed to improve to the antioxidant capacity in mice, whereas methanol leaves extract of M. nigra significantly altered the levels of CAT, SOD and GPx [24, 33, 34]. The content of syringic acid might be a reason. This ingredient, which played a role as a pro-oxidant compound, was the primary phenolic compound in aqueous extract of M. nigra while its concentration was minor in methanol extract of this plant [24, 33]. Instead, the major phenolic acids determined in methanol extract of M. nigra were vanillic acid and chlorogenic acid, and syringic acid only existed with a minor percent [33]. Also, the different efficiency might be attributed to the used doses. Wine from M. rubra in this experiment was only 20 mg/kg whereas M. rubra methanol extract showed the antioxidant effect at 300 mg/kg. However, we can generally see the fact that mulberry had a positive effect on keeping antioxidant enzymes’ activities and the concentration of brain oxygen radicals in balance. This activity might result in other effects such as improving memory [13, 32] or protecting against ischemia [13, 48] and brain cells [27, 44].

Regarding the result of Srikanta et al. [34], they hypothesized that the negative effect of M. nigra on antioxidant capacity might also be due to the poor bioavailability of antioxidant compounds as phenolics, or their inadequate consumption into tissues. In a clinical trial, Goldberg et al. found that the bioavailability of three prevalent phenolics (catechin, quercetin, and resveratrol) was too low compared to experimental concentrations to cause a positive effect in the in vitro studies [65]. However, we found that the main phenolics compounds of mulberry were syringic acid, vanillic acid, chlorogenic acid. The extracts of mulberry also had benefits in the in vivo studies. Therefore, we suggest that the effect of individual compounds on antioxidant enzymes should be investigated in the future to confirm whether they are active compounds. Also, the bioavailability could be studied to predict their realistic efficiency.

The lack of oxygen, glucose, and blood to the brain cell is the main caution of cerebral ischemia [66]. The oxygen-glucose deprivation, therefore, is a popular model to study ischemic cell deaths. Via this model, the pre-treatment with C3G and MG increased the percent of cell viability of PC12 cells and SH-SY5Y cells comparing to the control group [37, 39]. The study found that these positive results in the in vitro experiments promised satisfactory results of protection against ischemia in the in vivo study. In our review, we found that mulberry extracts and their compositions significantly decreased the infarct volume in MCAO mice, asserting their effect on the in vivo studies. Besides, the connection between antioxidant effect and the protection against stroke became quite clear. Free radicals such as H₂O₂ could cause the death of brain cells [67]. The results of Kang et al. confirmed this by showing that PC12 cells exposure to H₂O₂ had lower cell viability than the vehicle group [37, 49]. However, pre-treatments of these cells with C3G and GAML enhanced more survival cells showing that free radicals scavenging could protect cell apoptosis [37, 49]. In this case, oxyresveratrol might be a potential candidate for the protection against stroke, as it had a strong antioxidant effect and could inhibit the apoptotic cascades [38, 68]. More important, oxyresveratrol could cross the blood-brain barrier where it could directly show its effects [69].

Besides, the increase in NOX4 expressions could result in ROS generation after cerebral ischemia [70]. Thus, the inhibition of the expressions of this protein by MG in vitro was predicted to prevent excessive ROS expression [39]. MG was also demonstrated to suppress the expression of factors involving the apoptotic cascade, namely poly (ADP-ribose) polymerase, caspase-9, and caspase-3, which could be active in vivo and protect against brain cells death [39]. From our results in this review, there was an actual downgrade of these expressions in MCAO mice implying the activity of MG via the ROS scavenging and the prevention of apoptotic signals. Nevertheless, we must emphasize that MG was intraperitoneally administrated at 0.2 – 5 mg/kg in this study. This is a challenge for its application in the future because it could be hard to reach these concentrations in the body via the oral admistration. We recommend pharmacokinetics studies of MG to predict its effect in clinical in the future.

Regarding the protection against the cognitive deficit, included studies showed that different parts and preparations of mulberry could improve the learning ability and memory impairment. There were studies reported that stress oxidant, hippocampal damage, and GSK-3β activation were associated with downgraded brain functions via neural cell death [71, 72]. Therefore, the hippocampal protection and antioxidant could be a critical factor against these affections. Otherwise, the enhancement of ACh in the brain, which plays a role in encoding and retrieval of spatial memory in the hippocampus, could lead to memory improvement [73]. Our study presented the evidence that was concordant with these theories, as the memory and learning process of mice was improved following by the increase in survival brain cells in several hippocampal areas, the elevation of the acetylcholine levels as well as the suppression of MDA level. These findings reinforced the potential effect of several species of Morus genus with an insight into mechanisms of action.

GABA plays a primary role in the transmission in the brain, which mediates the depolarization through K⁺ and Ca²⁺ channels, which is vital for the differentiation of brain cells and the development of the brain [74]. The GABA_A defects might cause idiopathic epilepsy, which correlated with the mutation in genes of voltage-channels and ligand-gated ion-channels [75]. Hence, GABAergic inhibitory transmission is an essential element in the mechanism of both anxiolytic and anticonvulsant effects [76]. Moreover, the decrease in dopaminergic transmission relates to the enhancement of GABA transmission as well [77]. In our study, we found that mulberry extract increased GABA levels in the brain and might possess antidopaminergic activity through dopamine D₂ receptors [23, 58]. These results provided strong evidence of mulberry’s therapeutic potentials relating to GABA receptor and GABA inhibitory transmission. Moreover, flavones are known to bind with the GABAA receptor [77] strongly. In our review, flavonoids could be found in various parts of M. alba (leaves, root, and fruit). From these results, we can see that the GABA receptor and flavones might be clues for the mechanism of anxiolytic and anticonvulsant activities shown in this review, which should be more investigated.

The loss of dopaminergic neurons is well known to be the leading cause of the core motor symptoms in PD patients [78]. In this review, we found that consistent results are showing that ethanol extract of mulberry fruit protected dopaminergic neurons against some neurotoxicity and improved PD-like symptoms in rats [18, 61]. The regulation of ROS generation and some apoptotic signals as Bcl2, Bax protein, or caspase-3 to normal levels indicated that the protection of dopaminergic cells might be related to the modulation of oxidative stress and the apoptosis [61]. These outcomes seem to be incompatible with the antidopaminergic activity of this plant because the dopaminergic blocking could worsen the PD symptoms. That conflict could be attributed to the different used solvents and used parts, as the antidopaminergic activity might result from the presence of flavonoids, tannins, and saponins in mulberry leaves whereas the major compounds of mulberry fruit which improved PD-like behaviors were phenolics, flavonoids and anthocyanins [18, 23, 32]. On top of that, antidopaminergic agents had their adverse reactions in mental health [79]. For example, mulberry treatment enhanced the haloperidol-induced catalepsy as shown in our review [23]. further studies should investigate the compounds in these extracts and their bioactivities to elucidate the active ingredients for each activity to use to suitable intervention and minimize the risk of unexpected effects.

One of our limitations was that we could not compare different species of genus Morus to clarify which species were more beneficial. However, from the general perspective, we realized that many parts from M. alba showed a stronger antioxidant effect than other species. The antioxidant effect of M. nigra depended on used parts and the extracted solvents. Almost all parts of different species of mulberry showed a positive effect on cognitive function improvement, while root bark extract might be responsible for anti-depressive activities. The variable compositions in different extracts could cause a controversial effect, like antidopaminergic action and PD-like behavior improvement. Although there are many studies investigating the effects of mulberry in vivo, there are still no more profound studies investigating the pharmacokinetics of their active ingredients, leading to its limited application in human studies. The doses of these extracts should be considered and standardized to have optimized options for further studies. Finally, we did not include and report the systemic toxicology of mulberry because of our strategy. Other studies should consider this point when conducting their researches with high doses of mulberry extract (1 g or 10 g/kg).

Furthermore, most of the included studies carried out experiments on the whole extracts, but only very few studies identified which compounds were used. In specific cases, MG (a prenylated flavonoid), C3G (an aglycone of anthocyanin) and oxyresveratrol (a stilbenoid) decreased the brain infarct volume. Through these studies, we can only assert that the protection against the stroke caused by these compounds was due to the antioxidant activities and the prevention of the apoptosis suggesting that there would be many compounds that possess this activity. Sanggenon G was the only specific compound that was investigated and showed a useful anti-depressive effect. No active ingredients were used for investigating other activities as an antioxidant effect, PD, and AD-like behavior improvement. It narrows the number of active compounds that further studies should focus on. Instead, we need more studies finding out the active ingredients in specific extracts, making the comparison to determine the most effective candidates. Nevertheless, our study contributes to elucidating the potential activities of the plant-based on evidence, which can promote its application. We recommend further studies to concentrate on investigating compounds from active extracts to provide stronger evidence.

CONCLUSION

Mulberry species proved beneficial to many neurological functions in animal models. M. alba leaves methanol extract; ethyl acetate fraction and n-butanol fraction from methanol extract of M. alba root; and alcohol extract of M. alba root possessed anti-depressant-like effect. The methanol extraction of both M. alba root and M. alba leaves had an anxiolytic effect. Plus, M. laevigata leaves, M. alba methanol extract of leaves and stem bark, as well as its morusin showed the sedative effects at high doses. Varied species exhibited their ability to improve the memory and learning process, including M. alba leaves and fruit, M. nigra leaves, M. atropurpurea fruit. Some specific compounds extracted from mulberry namely MG, oxyresveratrol, and C3G could prevent ischemia in the brain either before or after stroke. Interestingly, M. rubra fruit and morusin increased the onset of convulsive time as well as reduced the convulsive duration. The antioxidant activity is still inconsistent and might be associated with the specific species, solvent for extraction, route of administration, doses, and the main constituents. Anti-Alzheimer disease and anti-Parkinson’s disease activities are intriguing, as the symptoms in mice were improved and the mechanisms were also suggested. However, we need more studies to confirm them. The active ingredients of each species, especially M. alba, should be deeper studied for screening potential candidates for future treatments.

Table 5.

Phytochemical analysis in studied extracts.

Refs.	Species	Part Used	Solvent	Phytochemical Analysis
				Phe	Fla	Ster	Tan	Alk	Sap	Antho	Anthra	CH	Proteins	Amino Acids	Terp	Gly
[22, 23, 28, 29]	M. alba	Leaves	Methanol	+	+	+	+	+	+	+	+	+	+	+
[30]	M. alba	Leaves	Methanol/ EASF		+		+	+								+
[29]	M. alba	Leaves	Petroleum ether		+	+	+		+							+
[29]	M. alba	Leaves	Chloroform			+		+				+			+	+
[31]	M. alba	Root	Methanol/ EASF	+	+			+
[12]	M. alba	Fruit	N/A	+						+
[32]	M. alba	Fruit	Ethanol	+	+					+
[24, 33]	M. nigra	Leaves	Hot water	+
[24, 33]	M. nigra	Leaves	Methanol	+
[29]	M. laevigata	Leaves	Methanol		+	+	+	+	+			+	+	+	+
[29]	M. laevigata	Leaves	Chloroform			+		+				+			+
[29]	M. laevigata	Leaves	Petroleum ether		+		+		+			+
[34]	M. rubra	Fruit	N/A	+	+

Alk: alkaloids, Antho: anthocyanins, Anthra: anthraquinones, EASF: ethyl acetate soluble fraction, Fla: flavonoids, Gly: glycosides, N/A: Not applied, Phe: phenolics, Sap: saponins, Ster: steroids, Tan: tannins, Terp: terpenoids.

list of ABBREVIATIONS

5-HT_1A

5-hydroxytryptamine_1A

AchE

Acetylcholinesterase

AD

Alzheimer diseases

C3G

Cyanidin-3-O-b-ᴅ-glucopyranoside

CAT

Catalase

CMR

Cortex mori radicis

CREB

Cyclic AMP response element-binding protein

EASF

Ethyl acetate soluble fraction

EPM

Elevated plus-maze

ERK

Extracellular-signal-regulated kinase

GABA

Gamma-aminobutyric acid

GAML

GABA in mulberry leaves

GHL

Global health library

GPx

Glutathione peroxidase

GR

Glucocorticoid receptors

GRd

Glutathione reductase

GSH

Glutathione

GST

Glutathione S-transferase

GSK-3β

Glycogen synthase kinase-3β

FST

Forced swim test

HPA

Hypothalamic–pituitary–adrenocortical

LDH

Lactate dehydrogenase

MAO

Monoamine oxidase

MCAO/R

Middle cerebral artery occlusion/reperfusion

MDA

Malonyldialdehyde

MG

Mulberrofuran G

MLE

Mulberry fruit extract

MLE-AR-14

Mulberry leaf extracted variety AR-14

MPO

Myeloperoxidase

MPTP/p

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

NO

Nitrite

NOX

Nicotinamide adenine dinucleotide phosphate oxidase

NPSH

Non-protein thiol groups

NYAM

the New York Academy of Medicine Grey Literature Report

OFT

Open field test

ROS

Reactive oxygen species

PC

Protein carbonyl

PD

Parkinson disease

PRISMA

Preferred Reporting Items for Systematic Reviews and Meta-Analyses

SA

Syringic acid

SIGLE

the System for Information on Grey Literature in Europe

SOD

Superoxide dismutase

SYRCLE

The Systematic Review Centre for Laboratory Animal Experimentation

TAC

Total antioxidant capacity

TBARS

Thiobarbituric acid reactive substances

TST

Tail suspension test

VHL

Virtual health library

Consent for Publication

Not applicable.

Funding

None.

Conflict of Interest

The authors declare no conflict of interest, financial or otherwise.