The Chemical and Pharmacological Research Progress on a Kind of Chinese Herbal Medicine, Fructus Malvae

Since the outbreak of the COVID-19 pandemic, traditional Chinese medicine has played an important role in the treatment process. Furthermore, the discovery of artemisinin in Artemisia annua has reduced the incidence of malaria all over the world. Therefore, it is becoming urgent and important to establish a novel method of conducting systematic research on Chinese herbal medicine, improving the medicinal utilization value of traditional Chinese medicine and bringing great benefits to human health all over the world. Fructus Malvae, a kind of Chinese herbal medicine which has been recorded in the “Chinese Pharmacopoeia” (2020 edition), refers to the dry, ripe fruits of Malva verticillata L. Recently, some studies have shown that Fructus Malvae exhibits some special pharmacological activities; for example, it has diuretic, anti-diabetes, antioxidant and anti-tumor properties, and it alleviates hair loss. Furthermore, according to the reports, the active ingredients separated and identified from Fructus Malvae contain some very novel compounds such as nortangeretin-8-O-β-d-glucuronopyranoside and 1-O-(6-deoxy-6-sulfo)-glucopyranosyl-2-O-linolenoyl-3-O-palmitoyl glyceride, which could be screened as important candidate compounds for diabetes- or tumor-treatment drugs, respectively. Therefore, in this research, we take Fructus Malvae as an example and systematically summarize the chemical constituents and pharmacological activity research progress of it. This review will be helpful in promoting the development and application of Fructus Malvae and will also provide an example for other investigations of traditional Chinese medicine.


Introduction
Since the outbreak of the COVID-19 pandemic, the economic development and public health services in many countries and regions have been influenced and challenged [1]. Traditional Chinese medicine (TCM), with thousands of years of history of use and practice in China, has played an important role in the treatment of diseases and the maintenance of human health. In the fight against the COVID-19 pandemic in China, traditional Chinese medicine (TCM) is widely used and has achieved remarkable success [2]. Additionally, the discovery of artemisinin in Artemisia annua has reduced the incidence of malaria around the world [3]. Therefore, it is becoming urgent and important to establish a novel method of conducting systematic research on Chinese herbal medicine, improving the medicinal value of traditional Chinese medicine and bringing great benefits to human health all over the world. Recently, some studies have found that Fructus Malvae performs some very special pharmacological activities; for example, it has anti-diabetes, antioxidant and anti-tumor properties, it alleviates hair loss, etc. Furthermore, according to the reports, the active ingredients separated and identified from Fructus Malvae contain some very novel compounds, such as nortangeretin-8-O-β-D-glucuronopyranoside and 1-O-(6-deoxy-6-sulfo)glucopyranosyl-2-O-linolenoyl-3-O-palmitoyl glyceride, which could be screened as important candidate compounds for diabetes-and tumor-treatment drugs, respectively. Myristoleic acid was isolated and identified from dichloromethane extract, and linolenic acid was isolated and identified from n-butanol extract, which are the two main active compounds of Fructus Malvae in the treatment of hair loss. Therefore, in this study, we take Fructus Malvae as an example and systematically summarize the chemical constituents and pharmacological activities of it. This review will be helpful in promoting the development and application of the medicinal value of Fructus Malvae and will also provide an example for the investigation of other traditional Chinese medicines.

Acid Compounds
For the acid compounds identified from Fructus Malvae, caffeic acid [7,[11][12][13][14][15] and ferulic acid [7,[15][16][17] are two phenolic acids and they are often screened as marker compounds of Fructus Malvae for qualitative or quantitative analysis in the associated drug standards or literature. In terms of extraction methods, in the Chinese Pharmacopoeia (2020 vision), Fructus Malvae was extracted using 70% ethanol under heating and refluxing, and then, caffeic acid was determined via thin-layer chromatography [4]. In addition, in some other studies, Fructus Malvae was also extracted via water decoction, ultrasonic extraction or Soxhlet extraction; then, the caffeic acid was determined via high-performance liquid chromatography [15]. The extraction, separation and identification methods of ferulic acid are similar to that of caffeic acid [15][16][17]. The stem, leaf and seed mixture of Malva verticillata L. was extracted using 90% ethanol under ultrasound; after that, four fatty-acid compounds (compounds 3-6) were identified from extracts based on UPLC-QTOF-MS/MS analysis [31]. In another study, Fructus Malvae was extracted using ethanol, and then, the extraction was successively extracted using dichloromethane, ethyl acetate, water, n-hexane and other solvents. Finally, linolenic acid and oleic acid were isolated and identified from n-butanol extract [21] and myristoleic acid (compound 8) was separated and identified from dichloromethane extract [22], which are three polyunsaturated fatty-acid compounds contained in Fructus Malvae. Additionally, palmitic acid, a kind of saturated fatty acid, was also identified using GC/MS from the water extract of Fructus Malvae [23]. The information and chemical structures of the acid compounds identified from Fructus Malvae are shown in Table 1 and Figure 2. The seed of Malva verticillata L. [23] Fructus Malvae refers to the fruit or seed of Malva verticillata L.

Flavonoids
For the flavonoids identified from Fructus Malvae, 21 flavonoids (compounds 10-30) been determined so far. A total of 14 flavonoid compounds (compounds 10-23) were id fied via UPLC-QTOF-MS/MS from a 90% ethanol extract of the stem, leaf and seed mixtu Fructus Malvae [31]. In another study, the stem, leaf and seed mixture was extracted using methanol at room temperature for 24 h, and then, successively extracted using water, acetate and n-butanol. A total of six flavonoid compounds, including nortangeretin-8-O glucuronopyranoside (compound 25), isoscutellarein 8-O-β-D-glucuronopyranoside ( pound 26), hypolaetin8-O-β-D-glucuronopyranoside (compound 27), herbacetin-8-O-β-D curonopyranoside (compound 29) and isoscutellarein 7-O-β-D-glucopyranoside (comp 30), were separated and identified from the water extract [32]. Among the six flavon ortangeretin-8-O-β-D-glucuronopyranoside is a new compound. The 5,6,7,8-tetrahyd group and the 8-O-glucuronide attached to the A ring of the flavonoid moiety are rare ported in plants [32]. Additionally, the study also proved that 8-O-glucuronide attached t flavonoid moiety was crucial for the antioxidant activity of Fructus Malvae [32]. The flavo rutin was separated and identified from the EtOAc extract of Fructus Malvae using UV, IR and some other technologies [18]. The chemical names, molecular formulas and che structures of all the identified flavonoids are shown in Table 2 and Figure 3.

Flavonoids
For the flavonoids identified from Fructus Malvae, 21 flavonoids (compounds 10-30) have been determined so far. A total of 14 flavonoid compounds (compounds 10-23) were identified via UPLC-QTOF-MS/MS from a 90% ethanol extract of the stem, leaf and seed mixture of Fructus Malvae [31]. In another study, the stem, leaf and seed mixture was extracted using 80% methanol at room temperature for 24 h, and then, successively extracted using water, ethyl acetate and n-butanol. A total of six flavonoid compounds, and isoscutellarein 7-O-β-D-glucopyranoside (compound 30), were separated and identified from the water extract [32]. Among the six flavonoids, ortangeretin-8-O-β-D-glucuronopyranoside is a new compound. The 5,6,7,8-tetrahydroxy group and the 8-O-glucuronide attached to the A ring of the flavonoid moiety are rarely reported in plants [32]. Additionally, the study also proved that 8-O-glucuronide attached to the flavonoid moiety was crucial for the antioxidant activity of Fructus Malvae [32]. The flavonoid rutin was separated and identified from the EtOAc extract of Fructus Malvae using UV, IR, MS and some other technologies [18]. The chemical names, molecular formulas and chemical structures of all the identified flavonoids are shown in Table 2 and Figure 3.

Sterols
For sterols separated from Fructus Malvae, there are only three sterol compounds identified from Fructus Malvae. They are β-sitosterol, verticilloside and daucosterol. Fructus Malvae was ultrasonically extracted using ethanol for 6 h, and then, the extraction was successively extracted using dichloromethane, ethyl acetate and water. Sterols including β-sitosterol, verticilloside and daucosterol were identified from the dichloromethane extraction [22]. The above sterol compounds could also be separated using other extraction methods [18,23,24]. The information on and chemical structures of the sterol compounds identified from Fructus Malvae are shown in Table 3 and Figure 4. The stem, leaf and seed mixture of Malva verticillata L. [32] Fructus Malvae refers to the fruit or seed of Malva verticillata L. The stem, leaf and seed mixture of Malva verticillata L. [32] 29 The stem, leaf and seed mixture of Malva verticillata L. [32] 30 The stem, leaf and seed mixture of Malva verticillata L. [32] Fructus Malvae refers to the fruit or seed of Malva verticillata L.

Sterols
For sterols separated from Fructus Malvae, there are only three sterol compounds identified from Fructus Malvae. They are β-sitosterol, verticilloside and daucosterol. Fructus Malvae was ultrasonically extracted using ethanol for 6 h, and then, the extraction was successively extracted using dichloromethane, ethyl acetate and water. Sterols including β-sitosterol, verticilloside and daucosterol were identified from the dichloromethane extraction [22]. The above sterol compounds could also be separated using other extraction methods [18,23,24]. The information on and chemical structures of the sterol compounds identified from Fructus Malvae are shown in Table 3 and Figure 4. The seed of Malva verticillata L. [18,22] Fructus Malvae refers to the fruit or seed of Malva verticillata L.  The seed of Malva verticillata L. [18,22] Fructus Malvae refers to the fruit or seed of Malva verticillata L.

Volatile Oils
For the volatile oils, there are 24 volatile oil compounds identified from Fructus Malvae in total. Volatile oils are often extracted via water-vapor distillation. An appropriate amount of Fructus Malvae powder is weighed and soaked in water for 12 h, and finally, a volatile oil extractor is used for steam distillation for 8 h. The effluent is collected and extracted using n-hexane; after that, it is dried using anhydrous sodium

Volatile Oils
For the volatile oils, there are 24 volatile oil compounds identified from Fructus Malvae in total. Volatile oils are often extracted via water-vapor distillation. An appropriate amount of Fructus Malvae powder is weighed and soaked in water for 12 h, and finally, a volatile oil extractor is used for steam distillation for 8 h. The effluent is collected and extracted using n-hexane; after that, it is dried using anhydrous sodium sulfate [19,20]. The information and chemical structures of the 24 volatile oil compounds, identified via GC-MS analysis, are shown in Table 5 and Figure 6.
The fruit of Malva verticillata L. [19,20] Fructus Malvae refers to the fruit or seed of Malva verticillata L.

Polysaccharides
For the polysaccharides, seven polysaccharides and two oligosaccharides have been identified from Fructus Malvae so far. The water extraction and alcohol precipitation methods were used for polysaccharides extraction. Then, the obtained crude polysaccharides were separated and purified via column chromatography [28]. After dealing with periodate oxidation, the smith degradation reaction, methylation analysis, partial acid hydrolysis and an enzymatic reaction, the monosaccharide composition and structural characteristics of the polysaccharides were determined via thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), GC-MS analysis, gel chromatography analysis, nuclear magnetic resonance (NMR) analysis and electrophoresis analysis [26][27][28]. At present, the polysaccharides identified from

Polysaccharides
For the polysaccharides, seven polysaccharides and two oligosaccharides have been identified from Fructus Malvae so far. The water extraction and alcohol precipitation methods were used for polysaccharides extraction. Then, the obtained crude polysaccharides were separated and purified via column chromatography [28]. After dealing with periodate oxidation, the smith degradation reaction, methylation analysis, partial acid hydrolysis and an enzymatic reaction, the monosaccharide composition and structural characteristics of the polysaccharides were determined via thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), GC-MS analysis, gel chromatography analysis, nuclear magnetic resonance (NMR) analysis and electrophoresis analy-sis [26][27][28]. At present, the polysaccharides identified from Fructus Malvae mainly include the neutral polysaccharides MVS-I, MVS-IIA, MVS-IIG, the acidic polysaccharides MVS-IIIA, MVS-IVA, MVS-VI and the peptidoglycan MVS-V [25][26][27][28][29][30]. The monosaccharide composition and molar ratio of the neutral polysaccharide MVS-I were determined to be L-arabinose:D-galactose:D-glucose = 3:6:7 [26,28], and the monosaccharide composition and molar ratio of the acidic polysaccharide MVS-VI were L-arabinose:D-xylose:D-glucose:Lrhamnose:D-galacturonic acid = 30:15:20:3:2:10 [27]. In addition, the oligosaccharides sucrose and raffinose were also isolated and identified from the dichloromethane extract of Malva verticillata seeds [22]. The information and chemical structures are summarized in Table 6 and Figure 7.

Amino Acids
For amino acids, 15 amino acids have been detected from Fructus Malvae at present ( Table 7 and Figure 8). The medicinal powder of Fructus Malvae was soaked in water for 12 h, and then, boiled in hot water for half an hour. It was filtered, the filtrates were combined and the supernatant was taken after centrifugation. After that, the supernatant was eluted using a 732 cation-exchange resin. Finally, a total of 14 amino acids were detected using the amino acid automatic analysis and test system [14,18]. Furthermore, tryptophan was the 15th amino acid detected from n-butanol or 90% ethanol extracts of the Malva verticillata stem, leaf and seed mixture [31,35]. Additionally, tryptophan showed a synergistic antidiabetic effect together with 3,5,6,9-tetrahydroxy-7-megastigmene in [35].

Amino Acids
For amino acids, 15 amino acids have been detected from Fructus Malvae at present ( Table 7 and Figure 8). The medicinal powder of Fructus Malvae was soaked in water for 12 h, and then, boiled in hot water for half an hour. It was filtered, the filtrates were combined and the supernatant was taken after centrifugation. After that, the supernatant was eluted using a 732 cation-exchange resin. Finally, a total of 14 amino acids were detected using the amino acid automatic analysis and test system [14,18]. Furthermore, tryptophan was the 15th amino acid detected from n-butanol or 90% ethanol extracts of the Malva verticillata stem, leaf and seed mixture [31,35]. Additionally, tryptophan showed a synergistic antidiabetic effect together with 3,5,6,9-tetrahydroxy-7-megastigmene in [35].  Figure 8. The chemical structures of amino acids identified from Fructus Malvae.

Other Substances
In addition to the above substances, oleamide, 1,3-dihydroxyacetone dimer, 5hydroxymethyl furfural, 2-hydroxy-gamma-butyrolactone and other compounds were identified via GC-MS from the water extract of Fructus Malvae [23]. The compound 3,5,6,9tetrahydroxy-7-megastigmene was identified from the n-butanol extract of the Malva verticillata stem, leaf and seed mixture [35]. Additionally, 15 trace elements including K, Na, Ca, Mg, Fe, Mn, Zn, Cu, Cr, Se, Pb, Al, Cd, Mo and Ni were also detected from Fructus Malvae using an atomic absorption spectrophotometer [36]. The chemical name, molecular formula, medicinal parts and chemical structures are summarized in Table 8 and Figure 9.

Research Progress on Pharmacological Activity
Shizhen Li recorded the following in the "Compendium of Materia Medica": "Kui, the smell and taste both are light. The character of light and slippery belongs to yang. Therefore, it could promote lactation, reduce swelling and induce abortion. Its roots and leaves have the same function as the seeds". In the Chinese Pharmacopoeia (2020 edition), it is described as follows: "Fructus Malvae has the effects of clearing heat, inducing diuresis and reducing swelling". In addition, Fructus Malvae also shows some other pharmacological effects such as having anti-diabetes, antioxidant and anti-tumor properties, the alleviation hair loss, etc. As shown in Table 9.

Research Progress on Pharmacological Activity
Shizhen Li recorded the following in the "Compendium of Materia Medica": "Kui, the smell and taste both are light. The character of light and slippery belongs to yang. Therefore, it could promote lactation, reduce swelling and induce abortion. Its roots and leaves have the same function as the seeds". In the Chinese Pharmacopoeia (2020 edition), it is described as follows: "Fructus Malvae has the effects of clearing heat, inducing diuresis and reducing swelling". In addition, Fructus Malvae also shows some other pharmacological effects such as having anti-diabetes, antioxidant and anti-tumor properties, the alleviation hair loss, etc. As shown in Table 9.

Diuretic Effect
The diuretic effect is the typical pharmacological effect of Fructus Malvae, as reported in Chinese Pharmacopoeia (2020 edition) and in the literature. Additionally, it is usually used to treat urinary retention, edema and thirst in a clinical setting [4,31]. Furthermore, heart failure [37,38], liver cirrhosis ascites [39,40], urinary calculi [41,42], hypertensive nephropathy [41] and other diseases often need diuretic drugs for treatment in a clinical setting. With regard to animal experiments, the rat metabolic-cage experiment is often utilized to detect the diuretic effect of drugs [43][44][45]. Additionally, male SD rats [46] or Wistar rats [47] are generally selected. Usually, the rats are fasted for 12-18 h, after pressing the abdomen of the rats to drain the remaining urine in the bladder, 0.9% normal saline or deionized water is administered to form a water-loaded rat model [44,48,49]. Hydrochlorothiazide or furosemide are often selected as positive drugs [47,48], and the urine volume, concentration of electrolytes (sodium ion, chloride ion and potassium ion) and the urine pH are measured to help analyze the diuretic effects [50]. For the rat metabolic-cage diuretic experiment of Fructus Malvae, the medicinal powder was refluxed using 70% ethanol, and then, extracted usinf petroleum ether, ethyl acetate, n-butanol and water, respectively, to obtain different solvent extracts [18]. The medication administration groups were given different solvent extracts, and the positive control group was given hydrochlorothiazide [18]. Compared with the negative control group, the results showed that the petroleum ether extract and ethyl acetate extract of Fructus Malvae could significantly increase the urine volume of rats. Additionally, the petroleum ether extract also significantly increased the urinary potassium excretion of rats, showing a strong diuretic effect [18].
Clinically, diabetes is a kind of chronic metabolic disease [53,54], often accompanied by complications such as diabetic nephropathy, diabetic retinopathy, diabetic foot and cardiovascular disease [54][55][56], which are extremely harmful to human health. Exercise, dietary intervention and drug therapy are generally used for the treatment of diabetes [57,58]. In terms of drug treatment, insulin, metformin, glimepiride and other drugs are generally used clinically, but they would also lead to some adverse reactions such as hypotension, obesity, etc. [58]. The use of Fructus Malvae in the treatment of diabetes may produce synergistic effects with above western drugs, reducing the corresponding toxic and sideeffects. Therefore, further research on the anti-diabetic effect of Fructus Malvae would be of great benefit. In addition, the above findings will also contribute to the discovery of new antidiabetic drugs.  [31,32]. The DPPH free-radical scavenging test [59,60], ABTS free-radical scavenging test [61,62], total-antioxidant capacity assay (FRAP) [63], oxygen-radical absorbance capacity (ORAC) assay [64] and superoxide scavenging activity assay were used to determine the antioxidant activity of Fructus Malvae and its related active ingredients in vitro. DPPH EC50, ABTS EC50, ORAC, SOD EC50, etc. were calculated as the detection indicators.

Antioxidant Effect
For the anti-oxidant study of Fructus Malvae, in the DPPH free-radical scavenging test, the free-radical scavenging activity of 90% ethanol extract of Malva verticillata leaves was 12.62 ± 0.41 mg AAE/g extract, of the stems was 5.15 ± 0.19 mg AAE/g extract and of the seeds was 22.14 ± 0.59 mg AAE/g extract [31]. These results indicated that the seeds of Malva verticillata L. (Fructus Malvae) had a better DPPH free-radical scavenging activity than the stems and leaves. Meanwhile, in the ABTS free-radical scavenging test and total-antioxidant capacity assay (FRAP)-wherein antioxidant activity was evaluated by measuring the absorbance value of the ABTS free-radical working solution at 734 nm and the absorbance value of the FRAP working solution at 539 nm, respectively-the 90% ethanol extract of the leaves showed better antioxidant activity than the stems and seeds [31]. In another study, nortangeretin-8-O-β-D-glucuronopyranoside, In a clinical setting, it is widely accepted that antioxidant effects are mostly related to age-related diseases [65], such as cardiovascular disease [66,67], non-alcoholic fatty liver disease [67], vascular dementia [68], Graves' ophthalmopathy [69], cancer [70,71], diabetes [72], etc. ROS, including hydrogen peroxide (H 2 O 2 ), hydroxyl radical (•OH), singlet oxygen (1O 2 ), superoxide (O 2 2− ), etc., is a group of unstable molecules produced by various cells in the human body. These free radicals could take part in human metabolism, immunity, growth, differentiation and many other homeostatic processes [73][74][75]. The cells and tissues will be damaged oxidatively and stay in a pathological state when ROS is excessively produced in the body [76]. At that time, antioxidative drugs are needed to resist the peroxidative effect of ROS on the human body. The above research and discoveries would be very beneficial and promising for the discovery of new antioxidative drugs.

Antitumor Effect
The ethyl acetate extract, n-butanol extract, water extract and 17 glycerides identified from Fructus Malvae or the stem, leaf and seed mixture of Malva verticillata L. show significant anti-tumor activity [33,34]. They have all been verified at the cellular level. Splenocytes, natural killer (NK) cells, human liver cancer cells (HepG2) [77], human gastric cancer cells (AGS), human colorectal cancer cells (HCT) and human non-small-cell lung cancer calls (A549) [77,78] were selected as the experimental models to verify the anti-tumor activity of Fructus Malvae. Splenocyte proliferation ability, natural killer (NK) cell activity, AGS cell apoptosis percentage, and the expression of the apoptosis proteins PARP, Cleaved APRP, Caspase-3, Cleaved Caspase-3, Bcl-2, Bax, β-actin, etc. were detected as indicators in the study [33,34]. In addition to the above cell experiments, tumor-bearing mice have also been used to verify anti-tumor activity at the animal experimental level in other studies [79][80][81]. Additionally, tumor volume and mass; thymus index; spleen index; the serum cytokines IL-2, IL-4 and TNF-α; IFN-7 levels and tumor histopathology could be detected as detection indicators [82]. Additionally, usually, splenocyte and tumor cells would be cultured in RPMI1640 medium or in DMEM medium, which contains 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin [83,84]. The proliferation ability or the viability of tumor cells can be measured using tetramethylazolyl blue (MTT method) or Cell Counting Kit 8 (CCK8) [83]. The apoptosis of tumor cells can be measured using a Tali apoptosis assay kit. The migration ability of tumor cells can be measured using the scratch method or transwell-migration method. Western blot is generally used to measure the expression of apoptosis, its pathway and other related proteins in tumor cells [85].
In a clinical setting, surgery, chemotherapy, interventional therapy, immunization and targeted therapy are often used for the treatment of cancers/tumors [86,87]. Cytotoxic drugs such as cyclophosphamide, hormonal drugs such as exemestane, and monoclonal antibodies such as rituximab are often used in the treatment process [88]. However, these therapeutic drugs are often accompanied by serious toxic and side effects, easily resulting in liver and kidney damage [89,90], hair loss [91], drug resistance [87], etc. A study developed by He Zhu et al. found that the combination of traditional Chinese medicine and western medicine can produce a synergistic anti-tumor effect and reduce the toxic and side-effects of the drug [92]. Fructus Malvae may be beneficial in such an application. Furthermore, the above findings would also be beneficial for the discovery of new anti-tumor drugs.

Treatment of Hair Loss
Extracts of 95% ethanol, ethanol, n-hexane and dichloromethane of Fructus Malvae showed pharmacological activity in treating hair loss [21,22]. Linoleic acid and myristoleic acid were two active compounds. They were all verified at the cellular level by human dermal papilla cells (DPCs) [21,22]. A cell proliferation ability, the expression of Wnt/βcatenin signaling pathway proteins and cell growth factor were detected as detection indicators. Additionally, mice can also be used. For this, the hair on the back of the mice is removed to build a model. The growth rate and appearance of the hair, the number of hair follicles, the hormone levels in the mouse blood, the growth factors in the mouse skin cells, and the histological morphology can be observed or measured to verify the pharmacological activity in the treatment of hair loss.
For the hair-loss-treatment study of Fructus Malvae, a study confirmed that linolenic acid can activate the Wnt/β-catenin signaling pathway and increase the expression of cyclins such as cyclinD1, CDK2 and the cell growth factors VEGF, IGF-1, etc. in a dosedependent manner [21]. When the administration concentration increased from 10 µg/mL to 30 µg/mL, the HFDPC cell proliferation rate increased by 21.46% [21], while oleic acid showed no relevant pharmacological activity. In addition, another research team found that the dichloromethane extract of Fructus Malvae and the active compound myristoleic acid separated from it also showed a therapeutic effect on hair loss [22]. Similar to the effect of linolenic acid, myristoleic acid can activate the Wnt/β-catenin signaling pathway and promote the proliferation of DPCs cell in the treatment of hair loss [22].
In a clinical setting, due to the advancement of technology, the accelerated pace of life, life pressure, work pressure and unhealthy work and diet habits, hair loss has become an important health problem faced by people. According to a population epidemiological survey, about 70% of the population in China is suffering from hair loss, and the phenomenon of hair loss shows a serious trend in youth [93]. Clinically, alopecia refers to a skin disorder characterized by hair loss, and includes androgenetic alopecia (seborrheic alopecia), alopecia areata and congenital alopecia [93,94]. Minoxidil and finasteride are often used to treat androgenetic alopecia, while steroids and retinoic acid are often used to treat alopecia areata [94]. However, the external use of minoxidil can easily cause dermatitis and increases the amount of body hair; finasteride can easily cause hormonal disorders and sexual dysfunction in the human body; and steroids can easily lead to scalp shrinkage and full-moon face [93,94]. The findings above indicate that Fructus Malvae may become a new choice for the treatment of hair loss.

Other Pharmacological Effects
In addition to the pharmacological effects above, Fructus Malvae also shows the potential ability to treat pathological bone disease [23], enhancing reticuloendothelial system activity and increasing anti-complement activity [25][26][27][28][29][30]. A study identified 14 compounds using GC-MS analysis from the water extract of Fructus Malvae. The results indicated that the water extract could inhibit the RANKL signaling pathway, and further inhibited osteoclastogenesis and bone resorption. Therefore, Fructus Malvae could also be used as a supplementary alternative drug for the treatment of pathological bone diseases [23]. Most of the polysaccharide components reported in Fructus Malvae showed the effect of enhancing reticuloendothelial system activity and anti-complement activity. More detailed information can be found in references [20,[25][26][27][28][29].

Summary and Discussion
The outbreak of the COVID-19 pandemic has brought great harm and challenges to the economic development and public health services of many countries and regions [95,96]. In China, traditional Chinese medicine is used in the treatment of COVID-19 and has obtained remarkable success [97]. The Lianhua Qingwen capsule is one of the most widely reported [98]. Additionally, the discovery of artemisinin in Artemisia annua has reduced the incidence of malaria around the world [3]. Therefore, it is becoming urgent and important to establish a novel method of conducting systematic research on Chinese herb medicine, to bring great benefits to human health all over the world.
Fructus Malvae, a kind of Chinese herb medicine, refers to the dried and ripe fruit of Malva verticillata L. [4]. So far, certain studies have been performed at home and abroad that are associated with the chemical composition and biological activity of Fructus Malvae. The chemical composition of Fructus Malvae is varied, mainly including 9 acid compounds (phenolic acids and fatty acids), 21 flavonoids, 3 sterols, 17 glycerides, 24 kinds of volatile oil, 9 kinds of polysaccharide, 15 kinds of amino acid and 5 other compounds. The above compounds and different solvent extracts of Fructus Malvae have shown various pharmacological activities according to the reports, including having diuretic, anti-diabetic, anti-oxidative and anti-tumor effects, treating hair loss, etc.
In terms of pharmacological activity, the diuretic activity of Fructus Malvae was studied using the rat metabolic-cage diuretic test [18]. The petroleum ether and ethyl acetate extract of Fructus Malva significantly increased the urine output of water-loaded rats. Additionally, the petroleum ether extract also significantly increased their urinary potassium content [18]. In antidiabetic studies, the ethyl acetate, n-butanol, water and n-hexane extracts of Fructus Malva (or of the mixture of stems, leaves and seeds) showed antidiabetic activity [32,35]. The flavonoids nortangeretin-8-O-β-D-glucuronide, hypolaetin8-O-β-D-glucuronopyranoside, herbacetin 8-O-β-D-glucuronopyranoside and isoscutellarein 7-O-β-D-glucopyranoside were able to significantly increase the size of alloxan-injured zebrafish islets, and the study also confirmed that nortangeretin-8-O-β-D-glucuronide and isoscutellarein 7-O-β-D-glucopyranoside were able to block K+ ions channel in islet β cells to increase the size of alloxan-injured zebrafish islets [32]. In addition, 3,5,6,9-tetrahydroxy-7-megastigmene and tryptophan in Fructus Malva also showed significant synergistic antidiabetic activity [35]. When given 1 µg/mL of two compounds, the size of damaged islets in zebrafish was increased by 22.3% and 18.9%, respectively, while their mixture increased the size of damaged islets by 48.6% [35]. The sterol compound β-sitosterol, the neutral polysaccharide MVS-I, the peptidoglycan MVS-V and the peptidoglycan-enriched fraction MVS-V-CH of the polysaccharides also showed activity in the treatment of diabetes [24,29]. For the experimental animal model, in addition to the alloxan-induced zebrafish islet cell-damage model [32,35], a db/db mouse model of type 2 diabetes was also used [24]. In antioxidant research, the DPPH free-radical scavenging assay, the ABTS free-radical scavenging assay, the FRAP total-antioxidant capacity-measurement experiment, the ORAC oxygen-radical absorption capacity-measurement experiment and the SOD superoxide dismutase scavenging assay were used to determine the antioxidant capacity of the of chemical substances in mallow fruit in vitro [31,32]. The 70% ethanol-extracted fraction and six flavonoids identified from Fructus Malvae showed strong antioxidant activity in vitro [31,32]. In a study of anti-tumor activity, the anti-tumor activity of Fructus Malva was verified using immune cells such as splenocytes, natural killer cells (NK), and human tumor cells such as HepG2, AGS, HCT-15 and A549 [33,34] [34]. In studies of the treatment of hair loss, the 95% ethanol/ethanol, n-hexane and dichloromethane extracts showed a therapeutic effect on hair loss [21,22]. Linolenic acid and myristoleic acid, two kind of fatty acid in Fructus Malva, significantly activated the Wnt/β-catenin signaling pathway and promoted the proliferation of human dermal papilla cell DPCs, which could become a new choice for the treatment of hair loss [21,22].
With the improvement of industrialization, lifestyle changes, unhealthy eating habits, obesity/overweight and other factors, the incidence of diabetes is increasing year by year [99]. Globally, there are 382 million people, about 8.3% of the population, suffering from diabetes. Furthermore, diabetes has become the main cause of death for people under 60 years old [99]. Therefore, it is of great clinical value and prospect to further study the antidiabetes effect of Fructus Malvae. So far, there have been six flavonoids (compounds 25-30) isolated and identified from the water extract of Fructus Malvae, showing potential antidiabetic effects ( Figure 10) [32]. Among them, nortangeretin-8-O-β-D-glucuronopyranoside (compound 25) is a new compound. The 5,6,7,8-tetrahydroxyl and 8-O-glucuronide attached to the A ring of the flavonoid group are novel structures that are rarely reported in plants [32]. Experiments have confirmed that nortangeretin-8-O-β-D-glucuronopyranoside can significantly recover alloxan-induced islet damage and block the K+ channel of islet β-cells in zebrafish. What's more, so dose isoscutellarein 7-O-β-D-glucopyranoside (compound 30) [32] (as shown in Figure 11). They could be selected as important candidate compounds for a diabetes-treatment drug. Regarding methodological design, male SD rats can also be used instead of the zebrafish mentioned above. They should be fed high-fat and high-sugar diets for 4-6 weeks, and 1% streptozotocin or alloxan should be injected via intraperitoneal injection to build a type 2 diabetes rat model [100][101][102]. Furthermore, db/db mice could also be used directly to verify the anti-diabetic effect of Fructus Malvae [103,104].
important candidate compounds for a diabetes-treatment drug. Regarding methodological design, male SD rats can also be used instead of the zebrafish mentioned above. They should be fed high-fat and high-sugar diets for 4-6 weeks, and 1% streptozotocin or alloxan should be injected via intraperitoneal injection to build a type 2 diabetes rat model [100][101][102]. Furthermore, db/db mice could also be used directly to verify the anti-diabetic effect of Fructus Malvae [103,104].  Cancer is the leading cause of death in people before the age of 70. According to the "2020 Global Cancer Statistics" released by the American Cancer Society, there were 19.3 million new cancer cases and 10 million cancer deaths worldwide in 2020. There were 4.136 million new cancer cases in China, accounting for 21.0% of the global new cancer cases. The top five cancers, ranked by mortality rate, are lung cancer, colorectal cancer, liver cancer, important candidate compounds for a diabetes-treatment drug. Regarding methodologi cal design, male SD rats can also be used instead of the zebrafish mentioned above. They should be fed high-fat and high-sugar diets for 4-6 weeks, and 1% streptozotocin o alloxan should be injected via intraperitoneal injection to build a type 2 diabetes rat mode [100][101][102]. Furthermore, db/db mice could also be used directly to verify the anti-diabetic effect of Fructus Malvae [103,104].  Cancer is the leading cause of death in people before the age of 70. According to the "2020 Global Cancer Statistics" released by the American Cancer Society, there were 19.3 million new cancer cases and 10 million cancer deaths worldwide in 2020. There were 4.136 million new cancer cases in China, accounting for 21.0% of the global new cancer cases. The top five cancers, ranked by mortality rate, are lung cancer, colorectal cancer, liver cancer Cancer is the leading cause of death in people before the age of 70. According to the "2020 Global Cancer Statistics" released by the American Cancer Society, there were 19.3 million new cancer cases and 10 million cancer deaths worldwide in 2020. There were 4.136 million new cancer cases in China, accounting for 21.0% of the global new cancer cases. The top five cancers, ranked by mortality rate, are lung cancer, colorectal cancer, liver cancer, gastric cancer and female breast cancer [105]. Therefore, it is necessary to further research the anticancer effect of Fructus Malva. Among the 17 glyceride compounds (compounds 34-50) [33,34] identified from the n-butanol extract of Fructus Malva, compounds 38-50 are glycosylglyceride compounds (Figure 10), which show significant cytotoxicity against the human hepatoma cell HepG2, the human gastric cancer cell AGS, the human colorectal cancer cell HCT-15 and the human non-small-cell lung cancer cell A549 [34]. The glycosylglycerides (2S)-1-O-(6-deoxy-6-sulfo)-α-D-glucopyranosyl-2-O-linolenoyl-3-O-palmitoyl glyceride and (2S)-1-O-(6-deoxy-6-sulfo)-α-D-glucopyranosyl-2,3-di-O-linolenoyl glyceride (compounds 38, 39) were isolated from Fructus Malva for the first time and have rarely been reported in plants before. Both compounds contained a unique chemical structure (6-deoxy-6-sulfo)-α-D-glucopyranosyl, which also rarely occurred before. They showed significant cytotoxicity to AGS tumor cells. They accelerated the apoptosis of AGS cells, and significantly affected the expression of apoptotic proteins such as PARP, caspase-3, Bcl-2, Bax and β-actin [34] (Figure 12). They could be further studied as candidate compounds for antitumor drugs. At the same time, monoacylglycerides compounds 34, 35 and 36 significantly enhanced the proliferation ability of spleen cells and the antitumor activity of natural killer cells, and their activity was stronger than that of the diacylglyceride 1,2-di-O-linoleoyl glyceride (compound 37) [33]. Additionally, for the methodological design, in addition to verification at the cellular level, tumor-bearing mice could also be used in research [106]. By evaluating the tumor volume and mass, thymus index, spleen index, serum cytokine levels, tumor histopathology, etc. [82], the anti-tumor activity of Fructus Malva and its active ingredients could be further verified at the level of animal experiments.
Molecules 2022, 27, x FOR PEER REVIEW 24 of 28 gastric cancer and female breast cancer [105]. Therefore, it is necessary to further research the anticancer effect of Fructus Malva. Among the 17 glyceride compounds (compounds 34-50) [33,34] identified from the n-butanol extract of Fructus Malva, compounds 38-50 are glycosylglyceride compounds (Figure 10), which show significant cytotoxicity against the human hepatoma cell HepG2, the human gastric cancer cell AGS, the human colorectal cancer cell HCT-15 and the human non-small-cell lung cancer cell A549 [34]. The glycosylglycerides (2S)-1-O-(6-deoxy-6-sulfo)-α-D-glucopyranosyl-2-O-linolenoyl-3-O-palmitoyl glyceride and (2S)-1-O-(6-deoxy-6-sulfo)-α-D-glucopyranosyl-2,3-di-O-linolenoyl glyceride (compounds 38, 39) were isolated from Fructus Malva for the first time and have rarely been reported in plants before. Both compounds contained a unique chemical structure (6-deoxy-6-sulfo)-α-D-glucopyranosyl, which also rarely occurred before. They showed significant cytotoxicity to AGS tumor cells. They accelerated the apoptosis of AGS cells, and significantly affected the expression of apoptotic proteins such as PARP, caspase-3, Bcl-2, Bax and β-actin [34] (Figure 12). They could be further studied as candidate compounds for antitumor drugs. At the same time, monoacylglycerides compounds 34, 35 and 36 significantly enhanced the proliferation ability of spleen cells and the antitumor activity of natural killer cells, and their activity was stronger than that of the diacylglyceride 1,2-di-O-linoleoyl glyceride (compound 37) [33]. Additionally, for the methodological design, in addition to verification at the cellular level, tumor-bearing mice could also be used in research [106]. By evaluating the tumor volume and mass, thymus index, spleen index, serum cytokine levels, tumor histopathology, etc. [82], the anti-tumor activity of Fructus Malva and its active ingredients could be further verified at the level of animal experiments.