Phytochemistry, Food Application, and Therapeutic Potential of the Medicinal Plant (Withania coagulans): A Review

Herbal plants have been utilized to treat and cure various health-related problems since ancient times. The use of Ayurvedic medicine is very significant because of its least reported side effects and host of advantages. Withania coagulans (Family; Solanaceae), a valuable medicinal plant, has been used to cure abnormal cell growth, wasting disorders, neural as well as physical problems, diabetes mellitus, insomnia, acute and chronic hepatic ailments. This review provides critical insight regarding the phytochemistry, biological activities, and pharmacognostic properties of W. coagulans. It has been known to possess diuretic, anti-inflammatory, anti-bacterial, anti-fungal, cardio-protective, hepato-protective, hypoglycemic, anti-oxidative, and anti-mutagenic properties owing to the existence of withanolides, an active compound present in it. Apart from withanolides, W. coagulans also contains many phytochemicals such as flavonoids, tannins, and β-sterols. Several studies indicate that various parts of W. coagulans and their active constituents have numerous pharmacological and therapeutic properties and thus can be considered as a new drug therapy against multiple diseases.


Introduction
Plants are potential sources of medicinal compounds and have been used as ancient healing systems since antiquity. Some medicinal plants are enriched with diverse bioactive constituents. These bioactive constituents have been reported as beneficial to prevent and treat various disorders for maintaining a healthy life. One of the most important medicinal plants of genus Withania is Withania coagulans in the Ayurvedic medicinal system owing to its effective nutraceutical as well as pharmaceutical attributes. It is grown in various regions of the world such as in the Mediterranean region and from North Africa to South Asia [1]. Among twenty-three identified species of genus Withania, only two (W. coagulans and W. somnifera) have economic significance [2]. This plant is widely used to coagulate milk due to the presence of an enzyme in its berries, which is commonly known as an Indian cheesemaker [3]. Figure 1 depicts the leaves, stems, and fruit of the W. coagulans plant. The fruit, roots, and leaves have various therapeutic effects. The main constituents in berries include essential oils, esterases, amino acids, and alkaloids [4]. The healing properties of the plant are attributed to steroid derivative compounds "Withanolides". There are several withanolides such as coagulin F, coagulanolide, withacoagulin, and

Phytochemistry
Withania species have been studied extensively by several researchers that subsequently led to the identification, characterization, and isolation of bioactive compounds in different parts of a plant. It includes several steroidal lactones, tannins, flavonoids, and alkaloids [24][25][26]. Ten new phytoconstituents were identified from air-dried W. coagulans fruit extracted with methanol and their structures were based on their chemical and spectral data [27]. Various constituents of W. coagulans were estimated in three different extracts namely, methanolic, hydroalcoholic, and chloroform. It was reported that total phenolic content (55.9 mg/g), total tannins (76.6 mg/g), total flavonoids (0.88 mg/g), and total flavanol (0.25 mg/g) were higher in the methanolic extract as compared to hydroalcoholic and chloroformic [28].

Application in the Food Industry
Berries of W. coagulans are well known for their milk coagulating potential. Keeping this property in view, the milk coagulating activity of protease from the plant was assessed. A temperature of 70 • C and pH 4 were found to be optimal for enzymatic activity. However, 60 • C was a stable temperature for the activity of the enzyme; SDS-PAGE showed a 66 kDa band [59]. Furthermore, the fruit extract of W. coagulans was utilized to assess the milk coagulating potential which demonstrated the highest impact at pH of 4 and temperature of 65 • C. Additionally, the time for rennet congealing of the extract was observed to be in direct relation with concentrations of NaCl or inversely with enzyme concentrations (protease inhibitors). Moreover, pepstatin-A (aspartic-protease inhibitor) completely inhibited the enzymatic potential of the berry extract [60].
An enzyme aspartic protease was isolated by using fractional ammonium-sulfate precipitation and cation-exchange chromatography from W. coagulans fruit. Furthermore, SDS-PAGE revealed the existence of a monomeric protein with a molecular weight of 31 kDa. The proteolytic activity of the protease enzyme was assessed using casein revealed K m (1.29 mg/mL) and V max (0.035 µmol Tyr/min) values for the protease enzyme. Skim milk was utilized to assess the milk coagulating potential of W. coagulans crude fruit extract. Consequently, mass spectrometry and inhibition assays revealed that aspartic protease is the only enzyme involved in milk coagulation. Additionally, the increasing salts concentrations (NaCl, CaCl 2 ) gradually reduced the enzyme activity. Thus, it was concluded that this enzyme may be apt to produce the low salt cheese [61]. The protease was extracted from the berries of the plant and used for the production of white cheese. It was documented that cheese from W. coagulans was more acidic than cheeses prepared from other rennet sources [62].
Buffalo milk mozzarella cheese was developed by using fruits of W. coagulans as milk coagulants. Thus, an aqueous fraction of W. coagulans may be a suitable option for cheese production [63]. Buffalo milk cheese was developed by using an extract of the fruit of W. coagulans and was evaluated in terms of storage conditions (5 months). The highest content of ash, fat, crude protein as well as total solids was observed in cheese prepared with lyophilized berry extract [64]. Cheese preparation was done by using alcoholic and aqueous fractions of W. coagulans at different levels (0.5, 1, and 1.5%) containing plant proteinase [65].
Furthermore, the preparation of cottage cheese from an aqueous fraction of plant showed significantly higher moisture content as well as pH, however, no difference in ash, fat, and crude protein was observed in cheese prepared from calf rennet and W. coagulans [66]. An acceptable quality white cheese can be developed by the utilization of 0.5% alcoholic extract of the plant. The soy milk coagulating potential of W. coagulans extract was assessed in tofu preparation and compared with calcium-sulfate tofu. Sensory analysis revealed no difference between both types of tofu. However, yield as well as moisture content was lower in W. coagulans's tofu [67].

Application in Nanotechnology
Silver nanoparticles (AgNPs) have prodigious potential on behalf of their mechanistic role in biomedical research. Approaches involving green chemistry have gained copious attention recently in plant science for the production of nanoparticles. Keeping in view this fact, leaf extract W. coagulans was utilized for the development of reduced graphene oxide (RGO)/Fe 3 O 4 based nanocomposite with palladium nanoparticles (Pd/RGO/Fe 3 O 4 ) and resulted in the reduction in 4-nitrophenol in the water at ambient temperature [68]. Silver nanoparticles were developed by using W. coagulans leaf extract and characterization of those nanoparticles was conducted by using UV-Vis, scanning electron microscopy, energy dispersive X-ray analysis, transmission electron microscopy, X-ray powder diffraction, and Fourier transform infra-red. The cumulative result indicated the size of particles as 14 nm having a spherical face-centered cubic structure [69].
Encapsulation of the water extract was performed by developing chitosan nanoparticles coated with food-based starch to retard extract release in the stomach. The release was retarded by 2.5 times by this method, hence exerting hypoglycemic potential [70]. W. coagulans was utilized to develop iron oxide nanorods. Iron oxide nanorods with an average size of 16 ± 2 nm and highly crystalline nature was obtained [6]. Furthermore, Keshari [71] also used W. coagulans extract to develop green silver nanoparticles. Those nanoparticles were crystalline, elemental, and spherical which also showed anti-biotic potential.

Therapeutic Potential of W. coagulans
A number of medicinal properties are attributed to W. coagulans such as antifungal, anti-cytotoxic, antidiabetic, hypolipidemic, neuroprotective, anti-inflammatory, anticancerous, anthelmintic, antioxidant activity, and wound healing activity [15]. Various pharmacological and therapeutic activities of W. coagulans are attributed to the various plant parts including roots, leaves, and fruits as shown in Figure 3. The anti-inflammatory mechanism of action of cogulin L isolated from W. coagulans is illustrated in Figure 4. Available literature indicates the therapeutic role of W. coagulans and its withanolides is summarized in Table 3.    The study indicated that biological nanorods are more effective (30% higher activity) than chemically prepared nanorods. W. coagulans nanoparticles showed significant inhibitory potential against P. aeuroginosa and S. aureus that indicates these nanoparticles are more effective than chemically prepared nanoparticles [6] Fruit Aqueous extract In vitro (silver carp fillet) 0.5% extract, 1% extract, 1% chitosan, 1% chitosan with 0.5% extract and 1% chitosan with 1% extract Chitosan coating of extract demonstrated debility in levels of total bacterial counts and psychrophilic total bacterial counts as well as enhanced shelf life of fish fillets [78] Hypoglycemic Potential

Conclusions
W. coagulans possesses considerable therapeutic potential and has been employed as a remedy against various disorders and diseases due to the presence of withanolides. It also possesses esterases, free amino acids, fatty oils, and essential oils. Medicinal properties of W. coagulans such as hepatoprotective, anti-inflammatory, hypoglycaemic, cardioprotective, free radical scavenging, antimicrobial, central nervous system depressant, immunomodulation, antitumor, and cytotoxic activities have been revealed by several pharmacological studies. However, future studies are needed to explore the mechanisms of action of compounds isolated from W. coagulans in higher animals to confirm their protective activity and safety. Crude extracts from different parts of the plant especially from the fruit have significant medicinal potential. Modern medications can be developed after thorough research into the mechanism of action, bioactivity, toxicity, and pharmacotherapeutic potential of plant-derived beneficial chemicals, as well as clinical trials and standardization.

Conflicts of Interest:
The authors declare no conflict of interest.