Effects of Sparassis Crispa in Medical Therapeutics: a Systematic Review and Meta-Analysis of Randomized Controlled Trials

International Journal of Molecular Sciences

Article Effects of Sparassis crispa in Medical Therapeutics: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

Le Thi Nhu Ngoc 1, You-Kwan Oh 2, Young-Jong Lee 3,* and Young-Chul Lee 1,* ID

1 Department of BioNano Technology, Gachon University, 1342 Seongnam-Daero, Sujeong-Gu, Seongnam-Si, Gyeonggi-do 13120, Korea; [email protected] 2 School of Chemical and Biomolecular Engineering, Pusan National University, 2 Busandaehak-ro, Geumjeong-Gu, Busan 46241, Korea; [email protected] 3 Department of Herbology, College of Korean Medicine, Gachon University, 1342 Seongnam-Daero, Sujeong-Gu, Seongnam-Si, Gyeonggi-do 13120, Korea * Correspondence: [email protected] (Y.-J.L.); [email protected] (Y.-C.L.); Tel.: +82-31-750-5415 (Y.-J.L.); +82-31-750-8751 (Y.-C.L.); Fax: +82-31-750-5416 (Y.-J.L.); +82-31-750-4748 (Y.-C.L.) Received: 31 March 2018; Accepted: 9 May 2018; Published: 16 May 2018

Abstract: In this study, we investigated the therapeutic potential and medical applications of Sparassis crispa (S. crispa) by conducting a systematic review of the existing literature and performing a meta-analysis. The original efficacy treatment of the mushroom extract is considered primarily and searched in electronic databases. A total of 623 articles were assessed, 33 randomized controlled experiments were included after the manual screening, and some papers, review articles, or editorials that did not contain data were excluded. A comparative standard means difference (SMD) and a funnel plot between control and S. crispa groups were used as parameters to demonstrate the beneficial effects of S. crispa for diabetes and cancer treatment, as well as anti-inflammatory, anti-fungal and antioxidant activities. The meta-analysis was carried out using Review Manager 5.1 software. Although for therapeutic diabetes there was heterogeneity in the subgroup analysis (I2 = 91.9%), the overall results showed statistically significant SMDs in major symptoms that decreased serum insulin levels (SMD = 1.92, 95% CI (1.10, 2.75), I2 = 0%), wound rates (SMD = 3.55 (2.56, 4.54), I2 = 40%) and contributions to an increase in nutrient intake content (SMD = 0.32 (−0.15, 0.78), I2 = 0%). Simultaneously, the study confirmed the utility of S. crispa treatment in terms of not only anti-cancer activity (reduction of tumor activity and survival of cancer cells I2 = 42 and 34%, respectively) but also anti-inflammatory, anti-fungal and antioxidant activities (I2 = 50, 44, and 10%, respectively). Our findings suggest that S. crispa extracts are useful for prevention and treatment of human diseases and might be the best candidates for future medicines.

Keywords: Sparassis crispa; diabetes treatment; cancer therapeutic; anti-inﬂammatory; anti-fungal; antioxidant activity; meta-analysis

1. Introduction Medical mushrooms have been approved as cures in traditional East Asian therapies [1,2]. Scientists around the world have veriﬁed the unique properties of compounds extracted from mushrooms in the prevention and treatment of cancer and other chronic diseases [3]. Sparassis crispa (S. crispa) is a species of fungus belonging to the genus Sparassis, known as Cauliﬂower mushroom or Sparassis latifolia; also called by other names such as Hanabiratake in Japanese [2–4]. S. crispa is not only an edible mushroom but also a well-known medicinal

Int. J. Mol. Sci. 2018, 19, 1487; doi:10.3390/ijms19051487 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 1487 2 of 17 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 2 of 17 that has many medical applications [3,5] (e.g., anti-tumor and anti-carcinogenic properties; anti- mushroom that has many medical applications [3,5] (e.g., anti-tumor and anti-carcinogenic properties; inflammatory, antiviral, anti-hypertensive, anti-allergic, anti-diabetic activities, and cytokine anti-inflammatory, antiviral, anti-hypertensive, anti-allergic, anti-diabetic activities, and cytokine induction [1–3,6,7]). Recently, this mushroom has been widely utilized in Japan and Korea [3,7,8]. induction [1–3,6,7]). Recently, this mushroom has been widely utilized in Japan and Korea [3,7,8]. S. crispa contains highly active biological and pharmacological ingredients (e.g., β-glucan, anti- S. crispa contains highly active biological and pharmacological ingredients (e.g., β-glucan, fungal compounds (sparassol, methyl-2,4-dihydroxy-6-methylbenzoate, and methyl- anti-fungal compounds (sparassol, methyl-2,4-dihydroxy-6-methylbenzoate, and methyl- dihydroxymethoxy-methylbenzoate), ergosterol peroxides, and benzoate derivatives) that are useful dihydroxymethoxy-methylbenzoate), ergosterol peroxides, and benzoate derivatives) that are in the treatment of human disease [3,5,9–11]. In particular, β-glucan can prevent and heal common useful in the treatment of human disease [3,5,9–11]. In particular, β-glucan can prevent and heal health problems such as diabetes, cancer, wound healing, as well as immune system and cytokine common health problems such as diabetes, cancer, wound healing, as well as immune system and induction [1,6,12–15]. In addition, phenolic compounds, anti-fungal substances, and other S. crispa cytokine induction [1,6,12–15]. In addition, phenolic compounds, anti-fungal substances, and other extracts may be used as anti-oxidant or anti-fungal agents [3,16–19]. S. crispa extracts may be used as anti-oxidant or anti-fungal agents [3,16–19]. Several studies have indicated, through tests on either mice or human cell lines, that S. crispa is Several studies have indicated, through tests on either mice or human cell lines, that S. crispa a potential natural source of medicinal ingredients that can contribute to the limitations and even is a potential natural source of medicinal ingredients that can contribute to the limitations and even prevention of human disease (e.g., cancer, allergies, and especially diabetic disease) [12,13,19,20] prevention of human disease (e.g., cancer, allergies, and especially diabetic disease) [12,13,19,20] However, in individual studies, scientists have not focused on the overall assessments of the benefits However, in individual studies, scientists have not focused on the overall assessments of the benefits of of S. crispa in human health as a systematic review. Thus, this study reviewed randomized and S. crispa in human health as a systematic review. Thus, this study reviewed randomized and controlled controlled trials, also conducted a systematic review and meta-analysis to evaluate the statistically trials, also conducted a systematic review and meta-analysis to evaluate the statistically significant significant benefits of S. crispa in therapeutic approaches. benefits of S. crispa in therapeutic approaches.

2. Results

2.1. Characteristics of Included Studies Figure 11 showsshows thethe flowflow ofof candidatecandidate andand eligibleeligible articles.articles. OurOur searchessearches inin databasesdatabases yieldedyielded aa total of 623 different publications whose titles an andd abstracts were screened and 270 were considered relevant only by title and abstract.abstract. After After reviewing reviewing these these 270 270 full-text articles on the efficiency efficiency of S. crispa extracts for human-disease treatment, thirty-t thirty-threehree articles were considered eligible and, therefore, includedincluded inin the the quantitative quantitative meta-analysis. meta-analysis. One One of thoseof those articles articles was was written written in Japanese in Japanese [21], [21],seven seven in Korean in Korean [19,22– 27[19,22–27],], and the remainingand the remaining in English. in Among English. them, Among some studiesthem, demonstratedsome studies demonstratedmore than two more healing than effectstwo healing of the effects mushroom of the mushroom [7,26,28–30 [7,26,28–30].]. Simultaneously, Simultaneously, the anti-diabetic, the anti- diabetic,anti-tumor, anti-tumor, anti-inflammatory, anti-inflammatory, anti-fungal, anti-fungal, and antioxidant and antioxidant activities activities of S. crispa of wereS. crispa reported were reportedin seven [in6,12 seven,14,28 [6,12,14,28–31],–31], nine-teen nine-teen [7,13,15,21 [7,13,15,23,24,26,21,23,24,26,28,30,32–41],,28,30,32–41], four [7,22 ,four30,42 [7,22,30,42],], three [19,29 three,43], [19,29,43],and six [8, 25and–27 six,44 [8,25–27,44,45],45] studies, respectively. studies, respectively.

Figure 1. SystematicSystematic screening screening stages of the literature review.

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All these thirty-three studies estimated the S. crispa beneﬁts based on rats, diabetic mice, and cancer cell test (see Table1), and provided raw data for a standardized mean difference (SMD) estimation.

Table 1. Summary of the characteristics of the studies included in this work.

S. crispa Characteristic of Medical Reference Extract n Dosage Location Object Therapeutic Compound 100 µg/mL [6] Kwon et al., 2009 Mice β-glucan Anti-diabetic 10 Korea β-glucan 100 µg/mL [32] Kim et al., 2010 Dendritic cell β-glucan Anti-tumor 3 Korea β-glucan [31] Yoshitomi 100 µg/mL Mice β-glucan Anti-diabetic 8 Japan et al., 2011 β-glucan 250 µg/mL [33] Lee et al., 2010 RAW 264.7 cell β-glucan Anti-tumor 3 Korea β-glucan 200 µg/mL [34] Choi et al., 2016 Human fibrinogen Wulfase Anti-tumor 3 Korea Wulfase [15] Harada et al., CD 41 and CD 81 200–250 µg/mL β-glucan Anti-tumor 3 Japan 2002a cell β-glucan [35] Harada et al., 25 µg/mL Mice β-glucan Anti-tumor 4 Japan 2003 β-glucan [28] Yamamoto C57BL/6J Anti-diabetic 160 µg/mL β-glucan 10 Japan et al., 2009 cell/Mice cell line Anti-tumor β-glucan 100 µg/mL [7] Yoshikama Phthalide RAW 264.7 cell Anti-tumor 3–4 phthalide Japan et al., 2010 compounds compound [21] Yamamoto S. crispa 35 µg/mL Sarcoma180 cell Anti-tumor 3 Japan et al., 2007 extract β-glucan [14] Yamamoto S. crispa 100 µg/mL Mice Anti-diabetic 6–8 Japan et al., 2010 extract β-glucan [29] Jeong Anti-diabetic 100 µg/mL Mice β-glucan 12 Korea et al., 2017 Anti-fungal β-glucan 200 µg/mL [22] Choi et al., 2013 RAW 264.7 cell β-glucan Anti-inflammatory 3 Korea β-glucan A529 cell 250 µg/mL [23] Choi et al., 2014 HepG2 cell β-glucan Anti-tumor 12 Korea β-glucan AGS cell Sarcoma 180 cell Anti-diabetic Mice 100 µg/mL [30] Kimura T. 2013 β-glucan Anti-tumor 3–5 Japan Colon cancer cell β-glucan Anti-inflammatory F3444N/Rat S. crispa 200 µg/mL [42] Kim et al., 2012 Mast cell (HMC-1) Anti-inflammatory 3 Korea extract S. crispa extract 250 µg/mL [36] Hu et al., 2016 PC12 cell β-glucan Anti-tumor 6 China β-glucan [44] Puttaraju S. crispa 30 µg/mL Mice Antioxidant 3 India et al., 2006 extract β-glucan S. crispa 100 µg/mL [8] Kim et al., 2008 Mice or cell Antioxidant 3 Korea extract S. crispa extract 100 µg/mL [43] Woodward Antibiotic United Botrytis cinerea Anti-fungal 10 antibiotic et al., 1992 compounds Kingdom compound [13] Ohno S. crispa 250 µg/mL Mice Anti-tumor 10 Japan et al., 2000 extract S. crispa extract [12] Yamamoto 250 µg/mL Mice β-glucan Anti-diabetic 10–18 Japan et al., 2014 β-glucan S. crispa 125 µg/mL [19] Lee et al., 2013a Soybean Anti-fungal 3 Korea extract S. crispa extract 100 µg/mL [24] Kim et al., 2013 Raw 264.7 cell β-glucan Anti-tumor 5 Korea β-glucan [37] Harada 100 µg/mL Mice β-glucan Anti-tumor 5 Japan et al., 2002b β-glucan Int. J. Mol. Sci. 2018, 19, 1487 4 of 17

Table 1. Cont.

S. crispa Characteristic of Medical Reference Extract n Dosage Location Object Therapeutic Compound [38] Harada 100 µg/mL Mice β-glucan Anti-tumor 3 Japan et al., 2004 β-glucan [39] Harada 100 µg/mL Mice β-glucan Anti-tumor 3 Japan et al., 2006 β-glucan [40] Nameda 50 µg/mL Mice β-glucan Anti-tumor 3 Japan et al., 2003 β-glucan 120 µg/mL [41] Yao et al., 2008 Mice β-glucan Anti-tumor 10 China β-glucan 200µg/mL [25] Lee et al., 2016a Soybean β-glucan Antioxidant 3 Korea β-glucan S. crispa 200 µg/mL S. [26] Park et al., 2016 Mice Antioxidant 6 Korea extract crispa extract S. crispa 50 µg/mL [27] Lee et al., 2016b Cell Antioxidant 3 Korea extract β-glucan 200 µg/mL Phenolic [45] Lee et al., 2013b Mice Antioxidant 3 phenolic Korea compounds compounds

2.2. Risk of Bias To explore the validity of eligible randomized studies, the quality of bias assessment of the included studies was determined by evaluating the bias of the random sequence generation, allocation concealment, selective reporting, blinding of participants and outcome assessment, and incomplete outcome data based on three levels following the Cochrane guideline (low and high risk of bias that may indicate either lack of information or uncertainty over the potential for bias) [46]. According to Table2 and Figure2, almost all criteria showed a low risk of bias, especially in studies where homogeneity in the random sequence generation criteria was used. Resulting in an evident enhanced of the statistical signiﬁcance of the meta-analysis.

Table 2. Risk of bias rating of each study.

Random Blinding of Incomplete Allocation Selective Blinding of Study Sequence Outcome Outcome Concealment Reporting Participants Generation Assessment Data [6] Kwon et al., 2009 [32] Kim et al., 2010 [31] Yoshitomi et al., 2011 [33] Lee et al., 2010 [34] Choi et al., 2016 [15] Harada et al., 2002a [35] Harada et al., 2003 [28] Yamamoto et al., 2009 [7] Yoshikama et al., 2010 [21] Yamamoto et al., 2007 [14] Yamamoto et al., 2010 [29] Jeong et al., 2017 [22] Choi et al., 2013 [23] Choi et al., 2014 [30] Kimura T. 2013 [42] Kim et al., 2012 [36] Hu et al., 2016 [44] Puttaraju et al., 2006 [8] Kim et al., 2008 [43] Woodward et al., 1992 [13] Ohno et al., 2000 [12] Yamamoto et al., 2014 [19] Lee et al., 2013a Int. J. Mol. Sci. 2018, 19, 1487 5 of 17

Table 2. Cont.

Random Blinding of Incomplete Allocation Selective Blinding of Study Sequence Outcome Outcome Concealment Reporting Participants Int. J. Mol. Sci. 2018, 19, x FOR PEERGeneration REVIEW Assessment Data 5 of 17 [24] Kim et al., 2013 [37] Harada et al., 2002b [39][ 38Harada] Harada et al., et al., 2006 2004 [40][ Nameda39] Harada et etal., al., 2003 2006 [41][40] Yao Nameda et al., et 2008 al., 2003 [25][ 41Lee] Yao et al., et al.,2016a 2008 [25] Lee et al., 2016a [26] Park et al., 2016 [26] Park et al., 2016 [27][ 27Lee] Lee et al., et al., 2016b 2016b [45][ 45Lee] Lee et al., et al., 2013b 2013b Low risk ofof HighHigh riskrisk of of RiskRisk of bias of bias rating rating UnclearUnclear bias biasbias

Figure 2. Risk of bias in individual studies graph.

2.3. Diabetes Treatment 2.3. Diabetes Treatment Anti-diabetic activity was evaluated by seven studies [6,12,14,28–31] (Figure 3 and Table 3). Most of Anti-diabetic activity was evaluated by seven studies [6,12,14,28–31] (Figure3 and Table3). the assays were performed in diabetic rat and mouse cells, and the treatment was assessed in five aspects: Most of the assays were performed in diabetic rat and mouse cells, and the treatment was assessed serum glucose levels (mg/dL), serum insulin levels (mg/dL), nutrition intake (mL), body weight (g) of in five aspects: serum glucose levels (mg/dL), serum insulin levels (mg/dL), nutrition intake (mL), mice or tissues before and after treatment, as well as wound healing ability (%). In addition, some body weight (g) of mice or tissues before and after treatment, as well as wound healing ability (%). individual studies have identified and demonstrated the beneficial effects of S. crispa in several healing In addition, some individual studies have identified and demonstrated the beneficial effects of S. crispa aspects, so they were evaluated in various separate analyses. For instance, Yamamoto et al. showed that in several healing aspects, so they were evaluated in various separate analyses. For instance, Yamamoto S. crispa could prevent human diabetes by reducing serum glucose levels, insulin levels, and increasing et al. showed that S. crispa could prevent human diabetes by reducing serum glucose levels, insulin the body weight of diabetic mice [14]. In addition, Jeong et al. indicated the capability of S. crispa in four levels, and increasing the body weight of diabetic mice [14]. In addition, Jeong et al. indicated the aspects including serum glucose and insulin levels, nutrition intake, and body weight [29]. capability of S. crispa in four aspects including serum glucose and insulin levels, nutrition intake, and body weight [29]. Table 3. Summary of standardized mean difference (SMD) comparison between control and S. crispa groups.

Medical No. of Healing Effect n SMD (95% CI) I2 Total Effect Application Studies Serum glucose level (mg/dL) 33 3.52 (−0.16, 7.21) 94% Serum insulin level (mg/dL) 18 1.92 (1.10, 2.75) 0% SMD = 1.29 Diabetes 7 Nutrition intake (mL) 36 0.32 (−0.15, 0.78) 0% 95% CI (0.47, 2.11) treatment Body weight (g) 46 −0.60 (−1.61, 0.41) 78% I2 = 91.9% Wound closure rate (%) 40 3.55 (2.56, 4.54) 40% Tumor activity 67 2.22 (1.69, 2.75) 42% Cancer 19 Cancer cell survival (%) 58 23.05 (18.02, 28.08) 34% treatment IFN- γ production (ng/mL) 43 −0.34 (−0.37, −0.31) 99% Anti- NO production (mg) 19 4.81 (3.30, 6.33) 50% inflammatory 4 Inflammatory cell survival (%) 11 9.03 (0.80, 17.27) 47% activity Anti-fungal 3 Anti-fungal activity 46 0.20 (−0.23, 0.62) 44% activity Antioxidant Anti-oxidant activity 14 −7.72 (−10.96, −4.49) 0% 6 activity DPPH (%) 14 −26.50 (−38.35, −14.64) 10%

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Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 17 of different S. crispa extracts on a cell type [7]. Therefore, these studies have been evaluated and appeared multiple times in a comparison of this analysis. An individual analysis was applied to two groups for each of those aspects, showing significant inter-group differences (see Figures 4–6).

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Figure 3. Comparison of diabetic symptoms between control and S. crispa groups. ( ): SMD of FigureFigure 4. Comparison 3. Comparison of the of survival diabetic of symptoms cancer cells between between control control and andS. S. crispa crispagroups. groups. ((): ): SMD of Figureindividual 9. Comparison studies; of ( the): summary anti-fungal SMDs activity of the betwcomparison.een control and S. crispa groups. ( ): SMD of of individualindividualindividual studies; studies; ( ():):): summary summary SMDs SMDs of of the the comparison. comparison. A subgroup analysis was conducted to quantify the effect of S. crispa in all therapeutic 2.7.Table Antioxidantapproaches 3. Summary Activity comparing of standardized to the control mean difference group. Anti-diabetic (SMD) comparison activity betweenwas significantly control and higherS. crispa in thegroups. S. crispa group than in the control group, and results showed a significant effect of S. crispa in the AccordingMedical to sixNo. studies of [8,25–27,44,45], the antioxidant activity was performed through DPPH (2, treatment (SMD = 1.29, 95% confidenceHealing Effect interval (CI) (0.47,n 2.11),SMD p < 0.00001), (95% CI) althoughI2 a heterogeneityTotal Effect Application Studies 2-diphenyl-1-picrylhydrazyl)was observed in the subgroup radical analysis scavenging (heterogeneity activity X2 =and 50.24, the p < oxidative-inhibitory0.00001, I2 = 91.9%). However, capacity of Serum glucose level (mg/dL) 33 3.52 (−0.16, 7.21) 94% phenolicwhen compounds was considered derived each from aspect S. crispaof the. diabetesIn both cases,treatment, the S.the crispa comparison group reportedbetween serum a higher insulin level of Serum insulin level (mg/dL) 18 1.92 (1.10, 2.75) 0% oxidativelevels protection and wound than healing the control rates group,showed with significan evidencet homogeneities of improving in antioxidantall reported activitysymptomsSMD (Figures =and 1.29 10 Diabetes treatment 7 Nutrition intake (mL) 36 0.32 (−0.15, 0.78) 0% 95% CI (0.47, 2.11) and 11).presented The inhibitory a large SMD activity between indicated the two a significantgroups ((SMD homogeneity = 1.92, 95% CIwith (1.10, I2 = 2.75), 0% andI2 = 0%)X2 = and 1.10I2 =(SMD 91.9%(SMD = Body weight (g) 46 −0.60 (−1.61, 0.41) 78% = 3.55, 95% CI (2.56, 4.54), I2 = 40%), respectively). Neither nutrition intakes showed heterogeneity −7.72, 95% CI (−10.96, −4.49), Woundp < 0.00001), closure rate while (%) the 40DPPH activity 3.55 (2.56, 4.54)showed a 40%very high statistical SMD (SMD = 0.32, 95% CI (−0.15, 0.78), I2 = 0%). Nevertheless, serum glucose levels and body weights significance in the inter-group comparisonTumor activity of four relevant 67 studies 2.22 (SMD (1.69, 2.75)= −26.50, 95% 42% CI (−38.35, −14.64), 2 2 of rats showed2 high2 heterogeneity (I = 94% and I = 78%, respectively). p < 0.00001,Cancer treatment X = 3.32, I 19 = 10%). Cancer cell survival (%) 58 23.05 (18.02, 28.08) 34% γ − − − 2.4. Cancer Treatment IFN- production (ng/mL) 43 0.34 ( 0.37, 0.31) 99% Anti-inﬂammatory NO production (mg) 19 4.81 (3.30, 6.33) 50% 4 activityFrom nine-teen studiesInﬂammatory [7,13,15,21,23,24,26,28,30,32–41] cell survival (%) 11, seven 9.03 (0.80,reported 17.27) an anti-tumor 47% activity, five Anti-fungalshowed activity an inhibition 3 of cancer Anti-fungal cell viability, activity and nine 46 indicated 0.20 IFN- (−0.23, 0.62)induction of 44% S. crispa extracts. On the other hand, there areAnti-oxidant several reports activity which show 14 ed appropriate−7.72 (−10.96, data−4.49) in many 0% respects [3,7]. In Antioxidant activity 6 addition, when considering the DPPHcapability (%) of healing, 14 researchers−26.50 ( −performed38.35, −14.64) experiments10% to describe the effect of S. crispa extract in various types of cells [3,15,23] or to determine the therapeutic potential

A subgroup analysis was conducted to quantify the effect of S. crispa in all therapeutic approaches comparingFigure 5. Comparison to the control of levels group. of tumor Anti-diabetic activity between activity control was and significantly S. crispa groups. higher ( ): in SMD the ofS. crispa groupindividual than instudies; the control (): summary group, SMDs and results of the comparison. showed a significant effect of S. crispa in the treatment Figure 10. Comparison of oxidative inhibitory capacity between control and S. crispa groups. ( ): (SMD = 1.29, 95% confidence interval (CI) (0.47, 2.11), p < 0.00001), although a heterogeneity was SMD of individual studies; (): summary SMDs of the comparison.

Figure 11. Comparison of DPPH radical scavenging activity between control and S. crispa groups.

( ): SMD of individual studies; (): summary SMDs of the comparison. Figure 6. Comparison of the IFN-γ production potential between control and S. crispa groups. ( ): 2.8. Sensitivity Analysis SMD of individual studies; (): summary SMDs of the comparison. Studies that used the same S. crispa extract, similar dosages and the same experimental objects were included in the sensitivity analysis. Wound healing rates in diabetes treatment and survival of cancer cells after anti-tumor activity were analyzed (Figures 12 and 13). All cases showed a high homogeneity and a high reduction of the percentages of cancer cells (SMD = 16.08 (1.83, 27.32), I2 = 0%), as well as an improved wound healing ability of the objects (SMD = 2.89 (1.87, 3.90), I2 = 13%) after treatment with S. crispa.

Int. J. Mol. Sci. 2018, 19, 1487 7 of 17 observed in the subgroup analysis (heterogeneity X2 = 50.24, p < 0.00001, I2 = 91.9%). However, when was considered each aspect of the diabetes treatment, the comparison between serum insulin levels and wound healing rates showed signiﬁcant homogeneities in all reported symptoms and presented a large SMD between the two groups ((SMD = 1.92, 95% CI (1.10, 2.75), I2 = 0%) and (SMD = 3.55, 95% CI (2.56, 4.54), I2 = 40%), respectively). Neither nutrition intakes showed heterogeneity SMD (SMD = 0.32, 95% CI (−0.15, 0.78), I2 = 0%). Nevertheless, serum glucose levels and body weights of rats showed high heterogeneity (I2 = 94% and I2 = 78%, respectively).

2.4. Cancer Treatment From nine-teen studies [7,13,15,21,23,24,26,28,30,32–41], seven reported an anti-tumor activity, ﬁve showed an inhibition of cancer cell viability, and nine indicated IFN-γ induction of S. crispa extracts. On the other hand, there are several reports which showed appropriate data in many respects [3,7]. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 17 In addition, when considering the capability of healing, researchers performed experiments to describe the effectInt. J. Mol. of Sci.S.crispa 2018, 19extract, x FOR PEER in various REVIEW types of cells [3,15,23] or to determine the therapeutic potential 7 of 17 Int.of J. differentMol. Sci. 2018 S., 19 crispa, x FOR PEERextracts REVIEW on a cell type [7]. Therefore, these studies have been evaluated 7 of 17 and of different S. crispa extracts on a cell type [7]. Therefore, these studies have been evaluated and ofappeared different multipleS. crispa timesextracts in on a comparisona cell type [7]. of thisTherefore, analysis. these An studies individual have analysisbeen evaluated was applied and to two appeared multiple times in a comparison of this analysis. An individual analysis was applied to two ofappearedgroups different for multiple S. each crispa of times thoseextracts in aspects, a oncomparison a cell showing type of this[7]. sign Therefore,anificantalysis. inter-groupAn these individual studies differences analysis have been was (see evaluatedapplied Figures to and 4–6).two groupsappearedgroups for eachfor multiple each of thoseof timesthose aspects, aspects,in a comparison showing showing of signiﬁcantsign thisificant analysis. inter-group inter-group An individual differences differences analysis (see (see Figures was Figures applied 4–6).4 –to6 ).two groups for each of those aspects, showing significant inter-group differences (see Figures 4–6).

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 17

FigureFigureFigure 4.FigureComparison 4. 4.Comparison Comparison 9. Comparison of ofthe the of the survivalof the survival anti-fungal of of cancer cancerof cancer cells activity cells betweencells between betwbetween controleen control controlcontrol and S. and crispaandS. S. groups. crispa crispa groups. (groups.groups. ): SMD ( ( ): ): SMDSMD of Figureof individual 4. Comparison studies; of( the): summary survival ofSMDs cancer of thecells comparison. between control and S. crispa groups. ( ): SMD SMDof of individual individual studies; ( ):): summary SMDs of the comparison. of individualindividual studies; studies; (): summary( ): summary SMDs SMDs of the ofcomparison. the comparison.

Figure 5. Comparison of levels of tumor activity between control and S. crispa groups. ( ): SMD of Figure 5. Comparison of levels of tumor activity between control and S. crispa groups. ( ): SMD of Figureindividual 5. Comparison studies; ( of): levels summary of tumor SMDs activity of the comparison. between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison. individualFigure studies; 5. Comparison ( ): summary of levels SMDs of tumor of the activity comparison. between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison. Figure 10. Comparison of oxidative inhibitory capacity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.

Figure 6. Comparison of the IFN-γ production potential between control and S. crispa groups. ( ): FigureSMDFigure of 6. individualComparison 11. Comparison studies; of the (IFN-): of summaryγ DPPHproduction radicalSMDs potential of scaven the comparison. betweenging activity control betweenand S. crispa control groups. and ( S. ):crispa groups.

SMD (of individual ): SMD ofstudies; individual (): summary studies; SMDs (): summary of the comparison. SMDs of the comparison.

Figure 6. Comparison of the IFN-γ production potential between control and S. crispa groups. ( ): 2.8. Sensitivity Analysis SMD of individual studies; (): summary SMDs of the comparison. Studies that used the same S. crispa extract, similar dosages and the same experimental objects were included in the sensitivity analysis. Wound healing rates in diabetes treatment and survival of cancer cells after anti-tumor activity were analyzed (Figures 12 and 13). All cases showed a high homogeneity and a high reduction of the percentages of cancer cells (SMD = 16.08 (1.83, 27.32), I2 = 0%), as well as an improved wound healing ability of the objects (SMD = 2.89 (1.87, 3.90), I2 = 13%) after treatment with S. crispa.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 17

of different S. crispa extracts on a cell type [7]. Therefore, these studies have been evaluated and appeared multiple times in a comparison of this analysis. An individual analysis was applied to two groups for each of those aspects, showing significant inter-group differences (see Figures 4–6).

Figure 4. Comparison of the survival of cancer cells between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 17

groups for each of those aspects, showing significant inter-group differences (see Figures 4–6). Int. J. Mol.Figure Sci. 2018 5. Comparison, 19, 1487 of levels of tumor activity between control and S. crispa groups. ( ): SMD of 8 of 17 individual studies; (): summary SMDs of the comparison.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 17

FigureFigureFigure 6.Figure 6.Comparison Comparison 4. Comparison 9. Comparison of the the ofIFN- IFN- the ofγ the γsurvivalproductionproduction anti-fungal of potential cancer potential activity cells between betw between controleen control and S. crispaand S. groups. crispa crispa ( groups.groups. ): ( ( ): ): SMDSMD of SMDSMDof of ofindividual individual individual studies; studies;studies; ( (():):):): summary summarysummary SMDs SMDsSMDs of the ofof thecomparison.the comparison.comparison.

AInt.2.7. comparison J. Mol. Antioxidant Sci. 2018, 19 between, x Activity FOR PEER the REVIEW control and S. crispa group showed a lower tumor cell 8 of 17 activity Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEWp 8 of 17 (SMD = 2.22,According 95% CI (1.69, to six 2.75), studies <[8,25–27,44,45], 0.00001); that the reduction antioxidant was activity relevant was to perf theormed homogeneity through inDPPH (2, seven studieA comparison [7,13,15,24 between,30,34,35 the] (Xcontrol2 = 24.27,and S. crispa I2 = 42%).group showed Additionally, a lower tumor the heterogeneity cell activity (SMD was not = 2.22,2-diphenyl-1-picrylhydrazyl)A 95% comparison CI (1.69, between2.75), p < the0.00001); control radical that and reduction S.scavenging crispa wasgroup relevant showedactivity to athe andlower homogeneity the tumor oxidative-inhibitory cell in activity seven studie(SMD capacity of significant for the survival of cancer cells (X2 = 12.11, I2 = 34%) in five studies [7,23,30,33,36], resulting [7,13,15,24,30,34,35]= 2.22,phenolic 95% CIcompounds (1.69, 2.75),(X2 = 24.27,pderived < 0.00001); I2 = from 42%). that S. Additionally, reduction crispa. In was both the relevant heterogecases, to the theneity S. homogeneity crispawas not group significant in reportedseven for studie thea higher level of in a dramatical decrease in2 the cancer2 cell viability after exposure to S. crispa (SMD = 21.36, 95% CI survival[7,13,15,24,30,34,35]oxidative of cancer protection cells (X (X=than 24.27,2 = 12.11, the I =control I42%).2 = 34%) Additionally,group, in five with studies theevidence heteroge[7,23,30,33,36], ofneity improving was resulting not antioxidant significant in a dramatical for activity the (Figures 10 survival pof cancer cells (X2 = 12.11, I2 = 34%) in five studies [7,23,30,33,36], resulting in a dramatical γ (17.91,decrease 24.81and 11).), in < Thethe 0.00001). cancer inhibitory cell The viability activity SMD between after indicated exposure these a significantto two S. crispa groups (SMD homogeneity did = not 21.36, show 95% with a CI significant I(17.91,2 = 0% 24.81), and of the Xp 2< = IFN- 1.10 (SMD = decrease in the cancer −cell viability after− exposure− to S. crispa2 (SMD = 21.36,2 95% CI (17.91, 24.81), p < induction0.00001).−7.72, aspect 95%The (SMD SMDCI ( − =between10.96,0.34, − 4.49),these 95% CItwop < ( groups0.00001),0.37, did0.31), while not Xshow the= 1168.88, aDPPH significant Iactivity= 99%).of the showed IFN- inductiona very high statistical 0.00001). The SMD between these two groups2 did not 2show a significant of the IFN- induction aspectsignificance (SMD = −in0.34, the 95% inter-group CI (−0.37, comparison −0.31), X = 1168.88, of four Irelevant = 99%). studies (SMD = −26.50, 95% CI (−38.35, −14.64), 2.5. Anti-Inflammatoryaspect (SMD = −0.34, Activity 95% CI (−0.37, −0.31), X2 = 1168.88, I2 = 99%). 2 2 2.5.p

Figure 5. Comparison of levels of tumor activity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison. Figure 10. Comparison of oxidative inhibitory capacity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.

FigureFigure 7. Comparison 7. Comparison of NO of NO production production potential potential between between controlcontrol and S.S. crispa crispa groups.groups. ( ( ): SMD): SMD of ofFigure individual 7. Comparison studies; ( of): NO summary production SMDs potential of the comparison. between control and S. crispa groups. ( ): SMD individualof individual studies; studies; ( ): summary (): summary SMDs SMDs of the of the comparison. comparison.

Figure 11. Comparison of DPPH radical scavenging activity between control and S. crispa groups.

Figure( 8. ): ComparisonSMD of individual of inflammatory studies; cells (): survival summary between SMDs control of the and comparison. S. crispa groups. (): SMDFigure of 8. individual Comparison studies; of inflammatory (): summary cells SMDs survival of the comparison.between control and S. crispa groups. (): FigureFigure 8. Comparison 6. Comparison of inﬂammatory of the IFN-γ production cells survival potential between between control control and S. and crispa S. crispagroups. groups. ( ):( ): SMD of individual studies; (): summary SMDs of the comparison. SMD2.8. of Sensitivity individual Analysis studies; ( ): summary SMDs of the comparison. 2.6. Anti-FungalSMD of individual Activity studies; ( ): summary SMDs of the comparison. 2.6. Anti-FungalStudies Activitythat used the same S. crispa extract, similar dosages and the same experimental objects 2.6. Anti-Fungal Anti-fungal Activity compounds are produced by S. crispa in cultures and in wood decomposed naturally, were included in the sensitivity analysis. Wound healing rates in diabetes treatment and survival of as reportedAnti-fungal in three compounds studies [19,29 are produced,43]. According by S. crispa to these in cultures studies, and results in wood of the decomposed meta-analysis naturally, (Figure Anti-fungal9)ascancer therereported was cells ina compounds favorablethree after studies anti-tumor effect are [19,29 in produced the,43]. activityS. crispaAccording by group; wereS. crispa to the analyzedthese numbersin studies, cultures (Figures of re bacteria andsults inof12 and woodthe and fungimeta-analysis decomposed13). were All reduced cases (Figure naturally, inshowed a high as reportedthe9)homogeneity there treatment in was three a favorablegroup studies and compared a [effect 19high,29 in ,reduction43 withthe]. S. According the crispa control of group; the togroup. percentages thesethe numbers A studies, significant ofof bacteria results cancer difference ofand cells the fungi was meta-analysis(SMD werefound = reduced 16.08 between (Figure(1.83, in 27.32),9) I2 = thesethe0%), treatment two as groupswell group as (SMD an compared improved = 0.20, 95% with CIwound the (−0.23, control healing 0.62), group. X2 =ability 8.86, A significant I2 of= 44% the < objects 50%).difference (SMD was =found 2.89 between(1.87, 3.90), I2 = 13%) theseafter two treatment groups (SMD with = S.0.20, crispa 95%. CI (−0.23, 0.62), X2 = 8.86, I2 = 44% < 50%).

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 17

A comparison between the control and S. crispa group showed a lower tumor cell activity (SMD = 2.22, 95% CI (1.69, 2.75), p < 0.00001); that reduction was relevant to the homogeneity in seven studie [7,13,15,24,30,34,35] (X2 = 24.27, I2 = 42%). Additionally, the heterogeneity was not significant for the survival of cancer cells (X2 = 12.11, I2 = 34%) in five studies [7,23,30,33,36], resulting in a dramatical decrease in the cancer cell viability after exposure to S. crispa (SMD = 21.36, 95% CI (17.91, 24.81), p < 0.00001). The SMD between these two groups did not show a significant of the IFN- induction aspect (SMD = −0.34, 95% CI (−0.37, −0.31), X2 = 1168.88, I2 = 99%).

2.5. Anti-Inflammatory Activity Data about anti-inflammatory activities of S. crispa extracts were reported in four studies [7,22,30,42]. According to SMDs (Figures 7 and 8, and Table 3), those results demonstrated that S. crispa reduced inflammatory cells survival (SMD = 9.03, 95% CI (0.80, 17.27), X2 = 3.74, I2 = 47%). The heterogeneity did not exist when was compared the NO production potential between control and S. crispa groups, with a large effect (SMD = 4.81, 95% CI (3.30, 6.33), X2 = 4.01, I2 =50%).

Figure 7. Comparison of NO production potential between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.

Figure 8. Comparison of inflammatory cells survival between control and S. crispa groups. (): SMD of individual studies; (): summary SMDs of the comparison. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 17 2.6. Anti-Fungal Activity of Int.different J. Mol. Sci.S. 2018crispa, 19 ,extracts 1487 on a cell type [7]. Therefore, these studies have been evaluated and9 of 17 Anti-fungal compounds are produced by S. crispa in cultures and in wood decomposed naturally, appeared multiple times in a comparison of this analysis. An individual analysis was applied to two as reported in three studies [19,29,43]. According to these studies, results of the meta-analysis (Figure groups for each of those aspects, showing significant inter-group differences (see Figures 4–6). there9) there was was a favorable a favorable effect effect in thein theS. S. crispa crispagroup; group; the the numbers numbers of bacteria and and fungi fungi were were reduced reduced in in thethe treatment treatment group group compared compared withwith thethe controlcontrol group.group. A A significant signiﬁcant difference difference was was found found between between thesethese two two groups groups (SMD (SMD = = 0.20,0.20, 95%95% CI ( (−−0.23,0.23, 0.62), 0.62), X2 X=2 8.86,= 8.86, I2 = I44%2 = 44%< 50%). < 50%).

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 17

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 17 FigureInt.Figure J. Mol. 4. Comparison9. Sci. ComparisonComparison 2018, 19, x ofFOR theof PEER the survival anti-fungal REVIEW of cancer activity cells betw betweenbetweeneen control control andand S.S. crispacrispa groups. ( ):): SMD SMD 9 of of 17 of individualindividualFigure 9. studies; Comparison ():): summary of the anti-fungal SMDsSMDs ofofactivity thethe comparison.comparison. between control and S. crispa groups. ( ): SMD of Figure 9. Comparison of the anti-fungal activity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.  2.7. Antioxidant Antioxidantindividual Activity Activity studies; ( ): summary SMDs of the comparison. 2.7. Antioxidant Activity 2.7.According Antioxidant to to sixActivity six studies studies [8,25–27,44,45], [8,25–27,44,45 ],the the antiox antioxidantidant activity activity was was perf performedormed through through DPPH DPPH (2, According to six studies [8,25–27,44,45], the antioxidant activity was performed through DPPH (2, 2-diphenyl-1-picrylhydrazyl)(2, 2-diphenyl-1-picrylhydrazylAccording to six studies [8,25–27,44,45],radical radical scavenging scavenging the antiox activity activityidant activityand and the the was oxidative-inhibitory performed through DPPHcapacity capacity (2, of 2-diphenyl-1-picrylhydrazyl) radical scavenging activity and the oxidative-inhibitory capacity of phenolic2-diphenyl-1-picrylhydrazyl) compounds compounds derived derived from fromradical S. crispaS. scavenging crispa. In. both In both activitycases, cases, the and S. thecrispathe S.oxidative-inhibitory crispagroup groupreported reported a capacityhigher a level higherof of phenolic compounds derived from S. crispa. In both cases, the S. crispa group reported a higher level of oxidativelevelphenolic of oxidative protection compounds protection than derived the control than from the group,S. controlcrispa .with In group, both evidence cases, with theof evidence improvingS. crispa ofgroup improvingantioxidant reported antioxidant aactivity higher (Figureslevel activity of 10 oxidative protection than the control group, with evidence of improving antioxidant activity (Figures 10 (Figuresoxidative 10 and protection 11). The than inhibitory the control activitygroup, with indicated evidence a of signiﬁcant improving homogeneityantioxidant2 activity with2 (Figures I2 = 0% 10 and and and11). 11).The The inhibitory inhibitory activity activity indicated indicated aa significantsignificant homogeneityhomogeneity with with I2 =I 0%= 0% and and X2 =X 1.10 = 1.10 (SMD (SMD = = X2 =and 1.10 11).(SMD The =inhibitory−7.72, 95%activity CI (indicated−10.96, −a 4.49),significantp < 0.00001),homogeneity while with the I2 DPPH= 0% and activity X2 = 1.10 showed (SMD a = very −7.72,−7.72, 95% 95% CI CI(− 10.96,(−10.96, − 4.49),−4.49), p p< <0.00001), 0.00001), whilewhile thethe DPPHDPPH activity activity showed showed a verya very high high statistical statistical −7.72, 95% CI (−10.96, −4.49), p < 0.00001), while the DPPH activity showed a very high statistical significancehighsignificance statistical in the in signiﬁcance the inter-group inter-group in comparison comparison the inter-group of of fourfour comparison relevant studies studies of (SMD four (SMD relevant= −=26.50, −26.50, 95% studies 95% CI (CI−38.35, (SMD (−38.35, −14.64), = −14.64),26.50, significance in the inter-group comparison2 of four relevant2 studies (SMD = −26.50, 95% CI (−38.35, −14.64), p95% < 0.00001,p CI< 0.00001, (−38.35, X2 =X 3.32,2 =− 3.32,14.64), I2 =I2 10%).= p10%).< 0.00001, X = 3.32, I = 10%). p < 0.00001, X2 = 3.32, I2 = 10%).

Figure 5. Comparison of levels of tumor activity between control and S. crispa groups. ( ): SMD of individualFigure studies; 10. Comparison (): summary of oxidative SMDs inhibitoryof the comparison. capacity between control and S. crispa groups. ( ): FigureFigure 10. 10.Comparison Comparison of of oxidative oxidative inhibitory inhibitory capacity between between control control and and S. crispaS. crispa groups.groups. ( ( ): ): FigureSMD 10. of Comparison individual studies; of oxidative (): summary inhibitory SMDs capacity of the comparison. between control and S. crispa groups. ( ): SMD of individual studies; ( ): summary SMDs of the comparison. SMDSMD of individual of individual studies; studies; ( (): ):summary summary SMDs SMDs ofof the comparison.comparison.

Figure 11. Comparison of DPPH radical scavenging activity between control and S. crispa groups. Figure 11. Comparison of DPPH radical scavenging activity between control and S. crispa groups. Figure( 11. ): SMDComparison of individual of DPPHstudies; radical (): summary scavenging SMDs activityof the comparison. between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison. Figure( ): SMD11. Comparison of individual of studies;DPPH radical ( ): summary scavenging SMDs activity of the comparison.between control and S. crispa groups.

2.8.( Sensitivity ): SMD of Analysis individual studies; (): summary SMDs of the comparison. 2.8. Sensitivity Analysis 2.8.Figure SensitivityStudies 6. Comparison Analysis that used of the IFN-sameγ S.production crispa extract, potential similar between dosages control and theand same S. crispa experimental groups. ( objects ): 2.8. SensitivityStudies Analysis that used the same S. crispa extract, similar dosages and the same experimental objects SMDwere of included individual in studies;the sensitivity (): summary analysis. SMDs Wound of the he alingcomparison. rates in diabetes treatment and survival of wereStudies included that usedin the thesensitivity same S. analysis. crispa extract, Wound similarhealing dosagesrates in diabetes and the treatment same experimental and survival objects of cancerStudies cells that after used anti-tumor the same activity S. crispa were extract, analyzed similar (Figures dosages 12 and and 13). the All same cases experimental showed a high objects werecancer included cells inafter the anti-tumor sensitivity activity analysis. were Wound analyzed healing (Figures rates 12 and in diabetes 13). All cases treatment showed and a survivalhigh werehomogeneity included in and the asensitivity high reduction analysis. of the Wound percentages healing of cancer rates incells diabetes (SMD =treatment 16.08 (1.83, and 27.32), survival I2 = of homogeneity and a high reduction of the percentages of cancer cells (SMD = 16.08 (1.83, 27.32), I2 = 0%), as well as an improved wound healing ability of the objects (SMD = 2.89 (1.87, 3.90), I2 = 13%) cancer0%), cells as well after as anti-tumoran improved activity wound werehealing analyzed ability of (Figures the objects 12 (SMDand 13).= 2.89 All (1.87, cases 3.90), showed I2 = 13%) a high after treatment with S. crispa. 2 homogeneityafter treatment and witha high S. crispareduction. of the percentages of cancer cells (SMD = 16.08 (1.83, 27.32), I = 0%), as well as an improved wound healing ability of the objects (SMD = 2.89 (1.87, 3.90), I2 = 13%) after treatment with S. crispa.

Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 17

Figure 9. Comparison of the anti-fungal activity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.

Figure 10. Comparison of oxidative inhibitory capacity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.

Figure 11. Comparison of DPPH radical scavenging activity between control and S. crispa groups. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 17 ( ): SMD of individual studies; (): summary SMDs of the comparison. of different S. crispa extracts on a cell type [7]. Therefore, these studies have been evaluated and 2.8. Sensitivity Analysis Int. J. Mol. Sci. 2018, 19, 1487appeared multiple times in a comparison of this analysis. An individual analysis10 of 17 was applied to two Studies that groupsused the for same each S. of crispa those extract, aspects, similar showing dosages sign ificantand the inter-group same experimental differences objects (see Figures 4–6). were included in the sensitivity analysis. Wound healing rates in diabetes treatment and survival of of cancer cells after anti-tumor activity were analyzed (Figures 12 and 13). All cases showed a high cancer cells after anti-tumor activity were analyzed (Figures 12 and 13). All cases showed a high 2 homogeneityhomogeneity and and a high a high reduction reduction of theof the percentages percentages of cancerof cancer cells cells (SMD (SMD = 16.08 = 16.08 (1.83, (1.83, 27.32), 27.32),I =I2 0%= ), 2 as0%), well as well an improved as an improved wound wound healing healing ability ability of the of objects the objects (SMD (SMD = 2.89 = 2.89 (1.87, (1.87, 3.90), 3.90), I = I 13%)2 = 13%) after treatmentafter treatment with S. with crispa S.. crispa.

Int.Int. J. Mol. J. Mol. Sci. Sci. 2018 2018, 19, 19, x, xFOR FOR PEER PEER REVIEW REVIEW 10 of 10 17 of 17 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 17 Figure 12. SensitivityFigure analysis 4. Comparison of wound rates of the in survival diabetes of treatment cancer cells between between control control and andS. S. crispa crispa groups. ( ): SMD FigureFigure 12. 12. Sensitivity Sensitivity analysis analysis ofof woundwound rates inin diabetesdiabetes treatmenttreatment between between control control and and S. S.crispa crispa Figure 9. Comparison of the anti-fungal activity between control and S. crispa groups. ( ): SMD S MD ofof of individual individual studies; ():): summary SMDs of the comparison. groups.groups. ( ( ): ):S SMD MD of of individual individual studies;studies; ():): summarysummary SMDs SMDs of of the the comparison. comparison. individual studies; (): summary SMDs of the comparison.

2.7. Antioxidant Activity According to six studies [8,25–27,44,45], the antioxidant activity was performed through DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging activity and the oxidative-inhibitory capacity of phenolic compounds derived from S. crispa. In both cases, the S. crispa group reported a higher level of oxidative protection than the control group, with evidence of improving antioxidant activity (Figures 10 and 11). The inhibitory activity indicated a significant homogeneity with I2 = 0% and X2 = 1.10 (SMD = FigureFigure 13. 13. SensitivitySensitivity analysis analysis of ofsurvival survival of cancer of cancer cell in cell cancer in cancertreatment treatment between betweencontrol and control S. crispa and −7.72, 95% CI (−10.96, −4.49), p < 0.00001), while the DPPH activity showedFigure a very 13. Sensitivity high statistical analysis of survival of cancer cell in cancer treatment between control and S. crispa S. crispagroups.groups. ( ): SMD ( of): individual SMD of individual studies; ( studies;): summary ( ): SMDs summary of the SMDscomparison. of the comparison. significance in the inter-group comparison of four relevant studies (SMD = −26.50,groups. 95% ( CI (− ):38.35, SMD of−14.64), individual studies; (): summary SMDs of the comparison. p < 0.00001, X2 = 3.32, I2 = 10%). 2.9. Bias Analysis 2.9. Bias Analysis 2.9. Bias Analysis Funnel plots were drawn to assess the publication bias of studies on the medical applications of Funnel plots were drawn to assess the publication bias of studies on the medical applications S. crispaFunnel. Figures plots were 14 and drawn 15 are to approximately assess the publication symmetrical bias but of studies small studies on the showing medical diabetesapplications and of of S. crispa. Figures 14 and 15 are approximately symmetrical but small studies showing diabetes S. cancercrispa. treatmentFigures 14 effects and 15of S.are crispa approximately remain unpublished. symmetrical In contrast, but small Figure studies 16 estimated showing that diabetes the most and and cancer treatment effects of S. crispa remain unpublished. In contrast, Figure 16 estimated that the cancerimportant treatment studies effects on anti-inflammatory of S. crispa remain activity unpublished. of S. crispa In havecontrast, been Figure missing, 16 so estimated the outcomes that theof the most S. crispa mostimportantanti-inflammatory important studies studies on treatment anti-inflammatory on anti-inflammatory were not highly activity signific activity of S.ant crispa of statistically. have beenhave Even missing, been though missing, so the the funnel sooutcomes the plots outcomes of in the ofFigures the anti-inflammatory 17 and 18 also demonstrated treatment were that notmany highly releva significantnt studies statistically.have not been Even published though yet, the all funnel the anti-inflammatory treatment were not highly significant statistically. Even though the funnel plots in plotsFigurespublished in 17 Figures and data 18 17 were also and demonstratedstatistically 18 also demonstrated significant that many for that relevathe manyanti-fungalnt studies relevant and have studiesantioxidant not been have publishedactivity not been of S.yet, published crispa all the Figure 5. Comparison of levels of tumor activity between control and S. crispa groups. ( ): SMD of yet,publishedextracts. all the publisheddata were datastatistically were statistically significant significant for the anti-fungal for the anti-fungal and antioxidant and antioxidant activity of activity S. crispa of S. crispa individual studies; (): summary SMDs of the comparison. extracts.extracts. Figure 10. Comparison of oxidative inhibitory capacity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison.

Figure 11. Comparison of DPPH radical scavenging activity between control and S. crispa groups. ( ): SMD of individual studies; (): summary SMDs of the comparison. Figure 6. Comparison of the IFN-γ production potential between control and S. crispa groups. ( ): Figure SMD14. Funnel of individual plot of studies studies; evaluating (): summary diabetes SMDs treatment of the of comparison. S. crispa. 2.8. Sensitivity Analysis

Figure 14. Funnel plot of studies evaluating diabetes treatment of S. crispa. Studies that used the same S. crispa extract, similar dosages and the same experimentalFigure 14.objectsFunnel plot of studies evaluating diabetes treatment of S. crispa. were included in the sensitivity analysis. Wound healing rates in diabetes treatment and survival of cancer cells after anti-tumor activity were analyzed (Figures 12 and 13). All cases showed a high homogeneity and a high reduction of the percentages of cancer cells (SMD = 16.08 (1.83, 27.32), I2 = 0%), as well as an improved wound healing ability of the objects (SMD = 2.89 (1.87, 3.90), I2 = 13%) after treatment with S. crispa.

Int. J. Mol. Sci. 2018, 19, 1487 11 of 17 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 11 of 17 Int.Int. J.J. Mol.Mol. Sci.Sci. 20182018,, 1919,, xx FORFOR PEERPEER REVIEWREVIEW 11 11 ofof 1717

Figure 15. FunnelFunnel plot plot of studies assessment cancer treatment of S. crispa. FigureFigure 15.15. FunnelFunnel plotplot ofof studiesstudies assessmentassessment cancercancer treatmenttreatment ofof S.S. crispa.crispa.

Figure 16. Funnel plot of studies assessment anti-inflammatory activity of S. crispa. FigureFigure 16.16. FunnelFunnel plotplot ofof studiesstudies assessmentassessment anti-inﬂammatoryanti-inflammatoryanti-inflammatory activityactivityactivity of ofof S. S.S. crispa. crispa.crispa.

Figure 17. Funnel plot of studies evaluating the anti-fungal activity of S. crispa. FigureFigure 17.17. FunnelFunnel plotplot ofof studiesstudies evaluatingevaluatievaluatingng thethe anti-fungalanti-fungal activityactivityactivity of ofof S. S.S. crispa. crispa.crispa.

Int. J. Mol. Sci. 2018, 19, 1487 12 of 17 Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 12 of 17

Figure 18. Funnel plot of studies evaluatingevaluating antioxidant activity ofof S.S. crispa.crispa.

3. Discussion InIn thisthis systematicsystematic review andand meta-analysismeta-analysis ofof thirty-threethirty-three studiesstudies onon thethe medicalmedical applicationapplication ofof S.S. crispacrispa extractsextracts werewere confirmedconfirmed thatthat S.S. crispacrispa isis notnot onlyonly anan edibleedible mushroommushroom butbut alsoalso aa medicinalmedicinal mushroom thatthat has been increasingly cultivated because of its potential value in traditional traditional medicine. medicine. Indeed,Indeed, S. crispa contains highly physiological active substances (e.g., β-glucan,-glucan, phenolic phenolic compounds, compounds, chloroform extract,extract, andand somesome antibioticantibiotic compounds)compounds) that can support a healthy level of blood-sugar and recovery ofof thethe normal cellular immunization [[33].]. The beneficialbeneficial effects have been demonstrated by anti-diabetic, anti-tumor, anti-inflammatory,anti-inflammatory, anti-fungal,anti-fungal, and anti-oxidant activities [1,3,11,20,47]; [1,3,11,20,47]; almost all of thethe typicaltypical therapeutictherapeutic effects of S. crispacrispa showedshowed significantsignificant differences, relative to the control groups.groups. As an an immunomodulating immunomodulating substance, substance, β-glucanβ-glucan plays playsan important an important role in most role healing in most modalities healing modalities[48]. Mainly, [48 involving]. Mainly, an involving enhance an of enhancethe immune of the re immunesponse against response cancer against and cancerstimulating and stimulating the cells of thethe innate cells of immune the innate system immune [48–51]. system The discovery [48–51]. The of specific discovery receptors of specific for gluc receptorsans in cells, for glucans as well inas cells,interactions as well with as interactions other receptors with othermainly receptors expressed mainly by innate expressed immune by innatecells, have immune been cells,reported have as been the reportedprimary mechanism as the primary of β-glucan mechanism for regulation of β-glucan of anti-tumor for regulation therapy of anti-tumor and some therapyassociated and medical some associatedtreatments medical[51]. Our treatments meta-analysis [51]. indicated Our meta-analysis that S. crispa indicated extracts thathad S.a large crispa influenceextracts hadin reducing a large influencesignificantly in reducingcancer cells significantly viability and cancer tumor cells cells viability suppression and tumor (Figures cells 4 suppression and 5). Although, (Figures here4 and there5). Although,was not a herehigh there homogeneity was not a in high the homogeneity IFN- production in the IFN-aspectγ production (Figure 6).aspect Moreover, (Figure the6). estimate Moreover, as theclinical estimate evidence as clinical for a relationsh evidenceip for between a relationship structure between and activity, structure suggested and activity, the contributions suggested the of contributionsmultiple receptor-ligand of multiple interactions receptor-ligand in glycan-mediated interactions in glycan-mediatedimmunopotentiation. immunopotentiation. Diabetes that has been caused by aa singlesingle highhigh dosedose ofof streptozotocinstreptozotocin isis typicallytypically accompaniedaccompanied by symptomssymptoms suchsuch as as weight weight loss, loss, polyuria, polyuria, hyperglycemia, hyperglycemia, and neuroendocrineand neuroendocrine dysfunction dysfunction [6,14]. On6,14].On the other the other hand, hand, whereas whereas wound wound healing healing progresses progresses at at an an optimal optimal rate rate in in healthyhealthy individuals, patientspatients with diabetes diabetes usually usually exhibit exhibit delayed delayed or orimpaired impaired wound wound healing, healing, which which is a serious is a serious highhigh-blood-glucose-relatedblood-glucose-related clinical clinical problem problem [52]. [The52]. present The present subgroup subgroup analysis analysis did not did report not report any anysignificant significant difference difference (I2 = (I 291.9%)= 91.9%) between between S. crispaS. crispa andand control control groups groups (Figure (Figure 33 andand TableTable3 ),3), suggesting that mushroom extractsextracts have not had complete effects on all the diabetes symptoms. However, anan individualindividual analysisanalysis forfor eacheach aspect, two topical therapy symptoms (incidence of wounds and serum insulin levels levels in in the the blood) blood) were were eliminated eliminated after after treatment treatment with with S. S.crispa crispa. In. Insummary, summary, S. S.crispa crispa showedshowed a aslightly slightly beneficial beneficial influence influence on on di diabetesabetes therapeutics. therapeutics. Meanwhile, Meanwhile, we we expect thatthat additional studiesstudies onon S.S. crispacrispa treatmentstreatments willwill improveimprove thethe accuracyaccuracy ofof thethe analysis.analysis. Additionally, the mushroom has shown an anti-inflammatory activity [1,2,47]. The present analysis estimates (Figures 7 and 8, and Table 3) that studies on anti-inflammatory therapy were

Int. J. Mol. Sci. 2018, 19, 1487 13 of 17

Additionally, the mushroom has shown an anti-inflammatory activity [1,2,47]. The present analysis estimates (Figures7 and8, and Table3) that studies on anti-inflammatory therapy were statistically significant, achieving a small homogeneity in the analysis. Furthermore, results also demonstrated that S. crispa extracts played an inhibitory role in inflammatory responses via regulation of NO production; suggesting a potential role as a component of inflammatory drugs. Recently, some evidence has suggested that the biological actions of phenolic compounds are associated with their antioxidant capacity based on their ability to chelate metals and lipoxygenase inhibitors [19,26]. The present survey also considered and evaluated antioxidant activities of the medical mushroom by meta-analysis (Figures 10 and 11). According to the coefficient of heterogeneities of the analysis (I2 = 0% and 10%), it was confirmed that phenolic compounds and other S. crispa extracts could significantly contribute to antioxidant properties; explaining the relationship between phenolic compounds and antioxidant activities, as well as anti-fungal ability. Finally, anti-bacterial and anti-fungal compounds have been identified in S. crispa [43], though their utilities were reported in only three studies [19,29,43]. The analysis showed that after exposure to S. crispa extracts the numbers of bacteria and fungi were reduced, indicating that the mushroom, as a component of pharmaceuticals, can protect humans from bacterial and fungal contamination.

4. Materials and Methods

4.1. Methods The study followed the Cochrane Collaboration method [46], as well as the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines [53] to report a systematic review and meta-analysis. Also, this work was based on the protocols and reviews on medical applications of S. crispa. It included all the researches that assessed the ability of S. crispa extracts on human health treatment (i.e., diabetes and cancer treatment, anti-fungal, antioxidant, and anti-inflammatory activity). It excluded ineligible studies such as studies of the effects of mushroom in other application fields and contained inappropriate data for the analysis. It compared and analyzed the statistical significance of individual studies of the same effect of S. crispa for the specific therapeutics using meta-analysis. Outcomes provided an overview and a systematic assessment about the clinical efficacies of S. crispa based on comparing SMDs between control and S. crispa groups, as well as the heterogeneity coefficient (I2) of each analysis [54].

4.2. Literature Search and Data Extraction

4.2.1. Database Research Strategy The searched literature on medical applications of S. crispa extracts was performed in the databases PubMed (National Library of Medicine, Bethesda, MD, USA), EMBASE (Excerpta Medica database, Amsterdam, Netherlands), Elsevier (Information and Analytics, Amsterdam, Netherlands), CENTRAL (Cochrane Central Register of Controlled Trials, New York, NY, USA), and Web of Science (Institute of Scientific Information and Clarivate Analytics, Philadelphia, PA, USA), considering articles published between 1990 and 2018. Also, we made a hand searching for important conference papers, as well as checking reference lists. Combinations of the following keywords were used: S. crispa, Sparassis Latifolia, Cauliflower mushroom, Hanaratake, medical applications, immune stimulating activity, anti-tumor, anti-cancer, anti-microbial, anti-melanin, anti-metastatic, anti-inflammatory, anti-fungal, antioxidant, anti-viral, anti-diabetic, and anti-hypertensive activity. The respective reference lists of the identified papers also were searched. All articles were written in English, Korean or Japanese.

4.2.2. Data Extraction The bibliographic reference list was screened and manually selected from eligible studies from the electronic database for the meta-analysis, according to the criteria of associations between medical Int. J. Mol. Sci. 2018, 19, 1487 14 of 17 applications of S. crispa and human therapeutics. The following information from each article was obtained: article title, the name of ﬁrst author, location, study year, publication year, study design, number of participants, dosage and administration, type of treatment and outcomes. These items were selected based on the presence and short descriptions of important study characteristics (e.g., title, abstract, study design, experimental object, and kind of medical application). The criteria included in the quantitative meta-analysis were empirical data that could be used to calculate the SMDs of treatments.

4.2.3. Exclusion Criteria Articles were excluded based on the following criteria: no data presentation (e.g., review articles and editorials), incomplete data, repeated and similar studies.

4.3. Meta-Analysis For each analysis, we determined the effect size (SMD) and 95% CI for the comparison between control and S. crispa groups. The SMD was obtained by dividing the mean difference between the two groups by the pooled variance, with adjustment for small samples. We considered SMDs about 0.2 or less as small values, 0.5 as moderate values, and 0.8 or greater values as large [54]. We quantiﬁed the extension to which the observed variability between studies was due to true differences between studies using the I2 statistic. Heterogeneity was considered to be small when I2 was less than 25%, moderate when 25–50%, and large when greater than 50% [54]. The subgroup analysis assessed the overall effects in the subgroups according to the model type, kind(s) of treatment(s), and symptoms. The value of p < 0.05 was considered statistically signiﬁcant, and bias was examined using a funnel plot. All these analyses were performed using Review Manager [46] (version 5.3, Copenhagen: The Nordic Cochrane Centre, The Cochrane Collaboration, 2014).

5. Limitation of Study The limitation of this study is the number of individual studies, which are not as large as our expectation in some respect (i.e., anti-inﬂammatory, anti-fungal, and antioxidant activity) leading to estimations not highly signiﬁcant. Especially in the sensitivity assessment, the results might be not high accuracy because of a very small quantity of those that have the same test conditions for consideration of the sensitivity.

6. Conclusions Briefly, this investigation determined that S. crispa is useful in medical therapeutics, each extract showed their properties and specific applications. Particularly, a meta-analysis revealed that β-glucan, which is known as the primary ingredient of S. crispa extract, plays an important part in the treatment of cancer and diabetes. Additionally, the analysis confirmed that β-glucan and other constituents (i.e., phthalide compounds, low-molecular-weight ingredients, and anti-bacterial substances) are used in anti-inflammatory activities, as well as antioxidant and anti-fungal immunotherapies. However, recent studies have focused on the clinical application of S. crispa [3,12,13,26,31,35,44]. To support our analysis, further studies to improve the statistical significance is necessary.

Author Contributions: Y.-C.L. planned the study and contributed the main ideas; L.T.N.N. collected the data and L.T.N.N. and Y.-C.L. were principally responsible for the writing of the manuscript; Y.-C.L., Y.-K.O., and Y.-J.L. commented on and revised the manuscript. Acknowledgments: This work was supported by the Korean Ministry of Environment’s “GAIA project (2015000550006)” and by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2017R1D1A1A09000642). Conﬂicts of Interest: The authors declare that they have no competing interests. Int. J. Mol. Sci. 2018, 19, 1487 15 of 17

Abbreviations

S. crispa Sparassis crispa SMD Standardized mean difference

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