Cocculus hirsutus (L.) W.Theob. (Menispermaceae): A Review on Traditional Uses, Phytochemistry and Pharmacological Activities

Background:Cocculus hirsutus (L.) W.Theob. (Menispermaceae) is a perennial climber distributed mostly in tropical and subtropical areas. The main aim of this article is to collect and analyze the scientific information related to traditional uses, bioactive chemical constituents and pharmacological activities. Methods: Scientific information on C. hirsutus was retrieved from the online bibliographic databases (e.g. MEDLINE/PubMed, SciFinder, Web of Science, Google Scholar and Scopus). Information regarding traditional uses was also acquired from secondary resources including books and proceedings. Results: Different plant parts of C. hirsutus were reported to be used for the treatment of fever, skin diseases, stomach disorders and urinary diseases. Alkaloids such as jasminitine, hirsutine, cohirsitine and their derivatives along with a few flavonoids, triterpene derivatives and volatile compounds were reported from whole plant or different plant parts. Extracts were evaluated for their antimicrobial, antidiabetic, immunomodulatory and hepatoprotective activities among others. Conclusion: Although widely used in traditional medicines, only a few studies have been performed related to chemical constituents. Most of the biological activity evaluations were carried out using in vitro evaluation methods and only a few studies were carried out in animal models. In the future, properly designed in vivo and clinical studies are necessary to evaluate the pharmacological activities of C. hirsutus along with bioassay-guided studies to isolate and identify the active constituents.

Various plant parts of C. hirsutus are widely used in South Asia for the treatment of fever, rheumatism, skin disorders and visceral diseases and also as a detoxifier [9]. In Africa, stems are used to make baskets and the purple berries are eaten or used to dye basket materials. Leaves are used by Tsonga people as an important herb in their diet [1]. Regarding the pharmacological activity evaluations, extracts obtained from the plant parts of C. hirsutus have shown potent diuretic, laxative Medicines 2020, X, x FOR PEER REVIEW 2 of 10 areas [3]. In Asia, it is reported from India, Myanmar, Nepal and Pakistan and southern China [1,[4][5][6]. In Africa, it is reported from Egypt, Sudan and Eritrea, Angola and south-west and southern Africa [1,6]. The detailed distribution in Asia and Africa is given in Figure 2 [7]. It is known by various names in local languages such as: Broom creeper (English); Huyer (Bengali); Farid buti, Jamti ki bel (Hindi); Kaage Mari (Kannada); Farid buti (Urdu); Paathaalagarudakkoti (Malayalam), Kaanse laharo (Nepali); Garudi, Patalagarudi (Sanskrit), Chipuru-tiga (Telegu); Kattu-k-koti (Tamil) among others [3,4,8].  hirsutus in Asia and Africa. (Source: GBIF, https://www.gbif.org/species/7930800 [7]).
Various plant parts of C. hirsutus are widely used in South Asia for the treatment of fever, rheumatism, skin disorders and visceral diseases and also as a detoxifier [9]. In Africa, stems are used to make baskets and the purple berries are eaten or used to dye basket materials. Leaves are used by Tsonga people as an important herb in their diet [1]. Regarding the pharmacological activity evaluations, extracts obtained from the plant parts of C. hirsutus have shown potent diuretic, laxative Various plant parts of C. hirsutus are widely used in South Asia for the treatment of fever, rheumatism, skin disorders and visceral diseases and also as a detoxifier [9]. In Africa, stems are used to make baskets and the purple berries are eaten or used to dye basket materials. Leaves are used by Tsonga people as an important herb in their diet [1]. Regarding the pharmacological activity evaluations, extracts obtained from the plant parts of C. hirsutus have shown potent diuretic, laxative [10], analgesic and anti-inflammatory activities [11]. Mainly alkaloids and some other compounds are reported from whole plant or different plant parts, however the detailed chemical analysis has not been performed yet.
A current pandemic outbreak of Coronavirus diseases-2019 (COVID-19) has affected 216 countries and territories and more than 47 million cases were reported till 3 November 2020 (https://www. worldometers.info/coronavirus/). Along with the research on the development of vaccines and antiviral drugs, many researchers are also focusing on the plant-derived natural products as potential sources of therapeutic agents. According to Clinical Trials Registry-India [12], an open label, randomized, comparative, multi-center, parallel group, controlled clinical study has been started in India to evaluate the effect and safety of aqueous extract of Cocculus hirsutus (AQCH) tablets in treatment of Coronavirus infection (CTRI Number: CTRI/2020/05/025397, registered on 28 May 2020). Previously, a randomized, Phase-I, double-blind, placebo-controlled, dose-escalation study to evaluate safety and tolerability of AQCH tablets in healthy adult human subjects was registered (CTRI Number: CTRI/2019/12/022297, registered on 10 December 2019). To the best of our knowledge, the outcomes of these studies have not been published yet.
For a medicinal plant species that has been widely used in traditional medicines and is also being considered as a source for the development of therapeutic agents for various diseases, it is important to critically analyze and understand the available scientific information about the chemical constituents and pharmacological activities. Thus, the aim of this article is to compile and analyze the available scientific information about these reported aspects of C. hirsutus. Persistent gaps in research and future perspectives on research and utilization of C. hirsutus are also discussed in detail.

Traditional Uses
Various publications have mentioned the traditional uses of C. hirsutus as practiced by the ethnic people in South Asia. The Koyas use the leaf paste, which is applied on head for its cooling effect [13][14][15][16][17]. The juice of the plant mixed with sesame oil is applied on the head and body to reduce heat. To allay the stomach heat and for the treatment of blood dysentery, the plant-paste is applied over the navel region [16]. The leaves are used to treat prurigo, impetigo, eczema, sores, cuts, wounds and other skin disorders [18,19]. Leaves are also used in the treatment of urine disorders, fever, leucorrhoea and acute gonorrhea [17,[20][21][22]. The leaves and stems are used in the treatment of conjunctivitis and other eye disorders [15,18,23]. The leaf powder is given orally for the treatment of dysentery and diarrhea [24]. The stem is used in the treatment of stomach disorders [16,18].
The roots are bitter, alterative and laxative and are used in the treatment of fever, skin irritation, rheumatism, gout, syphilitic cachexia and also in children for the treatment of stomach-ache [18,21,22,[25][26][27][28][29][30][31]. The extract of stems and roots are used as a sedative, hypotensive, cardiotonic and spasmolytic [25]. The root is made into paste and mixed with water and given orally to reduce stomach pain [32].

Chemical Constituents
Although widely used in traditional medicines and studied well for its pharmacological activities, the chemical constituents of C. hirsutus are not well explored. Earlier studies in 1960s and 1970s have reported the presence of alkaloids through preliminary phytochemical screenings and isolation and identification of a few alkaloids such as trilobine, isotrilobine, coclaurine and magnoflorine [33][34][35] and other compounds i.e., β-sitosterol, ginnol and monomethyl ether of inositol [36]. Few studies performed during or after 1980s, have reported several alkaloids from various plant parts. The list of alkaloids isolated from C. hirsutus is given in Table 1 and their structures are represented in Figure 3. Three flavonoids rutin, liquiritin and quercetin were also reported from the leaves [37]. A triterpene derivative, hirsudiol is also reported from the ethanol extract of whole plant [38]. Similarly, β-sitosterol and 28-acetyl botulin were isolated from the aqueous extract of aerial parts [39]. Many studies have reported the preliminary phytochemical screening of the extracts and presence of carbohydrates, steroids, alkaloids, glycosides, flavonoids, tannins and saponins [40]. Further, gas chromatography-mass spectroscopy (GC-MS) analysis of the extracts have revealed the presence of various compounds [40,41]. Cocsuline-N-2-oxide Whole plant/ ethanol extract [48] Medicines 2020, X, x FOR PEER REVIEW 4 of 10 Further, gas chromatography-mass spectroscopy (GC-MS) analysis of the extracts have revealed the presence of various compounds [40,41].

Pharmacological Activities
Various pharmacological activities have been reported for the extracts and isolated compounds from the different plant parts of C. hirsutus.

Anti-Cancer Activity
De Wet et al. carried out the anti-cancer activity of crude alkaloidal extract of rhizomes of C. hirsutus in three cancer cell lines, i.e., breast (MCF7), melanoma (UACC62) and renal (TK10) cell lines and the extract showed moderate anticancer activity [57]. Thavamani et al. carried out the in-vitro cytotoxic activity of the methanolic extract of C. hirsutus against HeLa cell line and the results showed an IC 50 value of 111 µg/mL [58]. Another study evaluated the anti-cancer activity of the plant C. hirsutus against Dalton's lymphoma ascites (DLA) cells in mice. The methanolic extract of C. hirsutus showed significant cytotoxic activity with an IC 50 value of 84.56 mg/mL in MCF-7 cancer cell line in-vitro. The extract also showed in-vivo antitumor activity as the doses of 200 and 400 mg/kg body weight significantly reduced the packed cell volume, tumor cell count, and restored the hematological and serum biochemical parameters towards the normal values [59].

Immunomodulatory Activity
Mallik and Nayak evaluated the immunomodulatory activity of the combination (1:1, 2:1 and 1:2 ration) of leaves of C. hirsutus and flowers of Sesbania grandiflora (L.) Pers. (Fabaceae) in mice. The 1:1 combination mixture showed potent immunostimulatory activity [60]. Rastogi et al. evaluated the immunostimulatory activity of aqueous and ethanolic extract of aerial parts of C. hirsutus in normal as well as cyclophosphamide induced immunosuppressed rats. The extracts showed an dose dependent increase in the carbon clearance, humoral antibody (HA) titre, delayed type hypersensitivity (DTH) and white blood cell (WBC) count in a dose dependent manner and authors concluded that the extract was effective to stimulate the immune system and also to protect from the immunosuppressant [61].

Anti-Diabetic Acitivty
Badole et al. reported the anti-hyperglycemic activity evaluation of aqueous extract of leaves of C. hirsutus in alloxan-induced diabetic mice. The oral administration of the extract at the dose of 250, 500, and 1000 mg/kg showed significant decrease in serum glucose level at 28th day of administration. In the oral glucose tolerance test (OGTT) in normal mice, the oral administration of extract (1000 mg/kg) increased the glucose tolerance [62]. Sangameswaran and Jayakar evaluated the anti-diabetic of C. hirsutus in streptozotocin-induced diabetic rats and the oral administration of methanolic extract (400 and 800 mg/kg) showed decrease in the blood glucose level [63].

Anti-Oxidant Activity
Srikanta and Dharmesh evaluated the anti-oxidant activity of the aqueous extract of the leaves of C. hirsutus using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and reducing power assay, and the extract showed potent DPPH free radical scavenging assay with IC 50 values of 2.75 ± 0.3 µg gallic acid equivalents (GAE)/ml and reducing power activity with the value of 65.17 ± 4.8 U/mg GAE. The extract also showed the total phenolic content of 31.83 ± 3.1 mg GAE/g [64]. Rakkimuthu et al. studied the anti-oxidant activity of C. hirsutus. The results showed potent DPPH free radical scavenging activity, ABTS free radical scavenging activity, nitric oxide scavenging activity, reducing power, inhibition of lipid peroxidation and metal chelating activity assay as ascorbic acid [65].

Hepatoprotective Activity
Thakare et al. evaluated the hepatoprotective activity of the methanolic extract of C. hirsutus in albino Wister rats with ethanol-induced hepatotoxicity and the oral administration of the extract at doses of 100, 200 and 400 mg/kg significantly lowered the levels of AST, ALT, ALP, LDH, direct and total bilirubin and cholesterol [66].   [67]. Sangameswaran and Jayakar evaluated the spermatogenic activity C. hirsutus in streptozotocin-induced diabetic rats and the increase in the sperm count was observed at a concentration of 400 mg/kg p.o (102.83 ± 1.85) and 800 mg/kg p.o. (117.83 ± 3.49) when compared to the normal group (74.83 ± 1.97) [63]. Elango and Rahuman reported the potent anti-parasitic activity of extracts of leaves of C. hirsutus against veterinary ticks and fluke [71].

Toxicological Studies
Ganapaty et al. evaluated the acute toxicity of the aqueous extract of the aerial parts of C. hirsutus in mice after oral administration of the extract at several dose ranges from of 100 to 3000 mg/kg. The extract showed sedative effect and increased urination and defecation at all doses, however no mortality was observed until 14 days after administration [67].

Patents
Some patents were also registered for the use of C. hirsutus formulations for their potential use in treatment of various diseases. For example, the patents related to the use of extract of C. hirsutus in prevention and treatment of dengue, the components of extract and the formulation were registered [72,73]. Similarly, patents related to the formulation and use of C. hirsutus alone or in combination with other medicinal plants for the treatment of tuberculosis [74] and respiratory tract diseases [75] are also registered.

Conclusions
Cocculus hirsutus is widely used in various traditional medicine systems in South Asia for the treatment of fever, skin diseases, stomach disorders, urinary diseases and also as a sedative among many other uses. The most commonly reported constituents were alkaloids such as jasminitine, hirsutine, cohirsitine and their derivatives. Some flavonoids, triterpene derivatives and volatile compounds were also reported from different plant parts. However, these chemical isolation studies were mostly carried out about 30-40 years previously, except for a few studies related to GC-MS analysis. Detailed bioassay guided isolation studies may afford pharmacologically active compounds. Extracts of different parts of C. hirsutus were evaluated for their antimicrobial, antidiabetic, immunomodulatory, hepatoprotective activities among others. However, most of these studies were based on in vitro evaluation methods and only a few studies were carried out in animal models. There are also not many studies evaluating the activity of isolated compounds. In the future, properly designed in vivo and clinical studies are necessary to evaluate the pharmacological activities of C. hirsutus along with bioassay guided chemical isolation studies to isolate and identify the active constituents. Similarly, the safety and toxicity evaluation studies are not performed in detail. The possible herb-drug interaction should also be studied in detail in the future.