# GenusTinospora: Ethnopharmacology, Phytochemistry, and Pharmacology

Authors: Sensen Chi; Gaimei She; Dan Han; Weihua Wang; Zhao Liu; Bin Liu
Journal: Evidence-Based Complementary and Alternative Medicine
 (2016)

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## Abstract

The genusTinospora includes 34 species, in which several herbs were used as traditional medicines by indigenous groups throughout the tropical and subtropical parts of Asia, Africa, and Australia. The extensive literature survey revealedTinospora species to be a group of important medicinal plants used for the ethnomedical treatment of colds, headaches, pharyngitis, fever, diarrhea, oral ulcer, diabetes, digestive disorder, and rheumatoid arthritis. Indian ethnopharmacological data points to the therapeutic potential of theT.cordifolia for the treatment of diabetic conditions. WhileTinospora species are confusing in individual ingredients and their mechanisms of action, the ethnopharmacological history of those plants indicated that they exhibit antidiabetic, antioxidation, antitumor, anti-inflammation, antimicrobial, antiosteoporosis, and immunostimulation activities. While the clinical applications in modern medicine are lacking convincing evidence and support, this review is aimed at summarizing the current knowledge of the traditional uses, phytochemistry, biological activities, and toxicities of the genusTinospora to reveal its therapeutic potentials and gaps, offering opportunities for future researches.


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## Body

## 1. Introduction

The genusTinospora (Menispermaceae), 34 species included, is a large glabrous deciduous climbing shrub [1]. The species have been widely distributed throughout the tropical and subtropical parts of Asia, Africa, and Australia. At present, 6 species and 2 varieties are found in China, in whichT.hainanensis andT.guangxiensis Lo. as endemic species are mainly distributed in Guangxi and Hainan.T.sinensis andT.capillipes are officially listed in the 2015 edition of theChinese Pharmacopoeia (中华人民共和国药典) with the Chinese names “Kuan Jin Teng” (宽筋藤) and “Jin Guo Lan” (金果榄), respectively [2]. Those herbs in genusTinospora are mostly heat-clearing and detoxifying (清热解毒). And they are commonly used as domestic folk medicine for the treatment of colds, headaches, pharyngitis, fever, diarrhea, oral ulcer, digestive disorder, and rheumatoid arthritis. For instance, Indian Ayurveda usually employsT.cordifolia in treatment for diabetes. Its obvious antidiabetic properties attract modern researches’ attention [3].T.sinensis is the major composition in a famous traditional formula “Si Wei Zang Mu Xiang” powder (四味藏木香), used for the treatment of wind-heat disease (风热病) and deficiency-heat syndrome (虚热证) [3, 4]. This family is a rich source of terpenes and alkaloids; the ongoing exploration for bioactive constituents also revealed the presence of C6-C3 derivatives, polysaccharides, and other components with exact structures. Modern pharmacological researches and clinical practices demonstrated thatTinospora species possess a wide spectrum of activities, including antidiabetic [3], antioxidation [5, 6], antitumor [7, 8], anti-inflammation [9, 10], antimicrobial [11], antiosteoporosis [12], and immunostimulation effect [13]. In clinic, genusTinospora is commonly used to protect and support the immune system, to prevent upper respiratory infections, to lower oral ulcer, to treat diabetes, to be an adjunctive therapy in cancer, and to protect the liver. However, a systematic review of these studies has not been performed to date. This paper summarized the research achievements of the medicine plants in genusTinospora ranging from clinical studies to phytochemistry and pharmacology areas. It is expected that such a paper would on one hand serve as an available database for future researches and on the other hand enable experts in the field to determine if the research on genusTinosporais on proper path.


## 2. Traditional Uses and Ethnopharmacology

Those species in this genus have traditionally been used as therapeutic remedies (Table1). On the basis of our investigations, they are clinically applied for the treatment of fever and parasitic diseases, mouth, skin, respiratory, and urinary tract infections, oral ulcer, and diabetes, in addition to being an adjunctive therapy in cancer and to protecting the liver [14–22]. The official record about species in China of genusTinospora appeared in “Bencao Gangmu Shiyi” [Zhao Xuemin, Qing dynasty, year 1765,Bencao Gangmu Shiyi (本草纲目拾遗)]. It is recorded thatT.capillipes is primarily used on the therapy of dysentery, diarrhea, and abdominal pain with heat, toxic furuncle ulcer and sore throat. It is also the traditional medicine ofMao,Dong, andYao minority of China, being applied to the treatment of acute laryngopharyngitis, tonsillitis, gastroenteritis, and bacillary dysentery [23, 24]. Earlier, “Jingzhu Bencao” [Dimaer·Danzeng PengCuo, Qing dynasty, year 1745,Jingzhu Bencao (晶珠本草)], which is an ancient Chinese Tibetan medicinal encyclopedia, recorded thatT.sinensis has been used for relieving rigidity of muscle and activating collaterals (舒筋活络) and alleviating pain (活血止痛). Its tropism of taste is Xin and Ku and in the liver channel (味辛, 苦, 主肝经). Chinese Dai Medicine (傣医药学) claimed thatT.sinensis could also regulate and invigorate the blood (调补气血) and tranquilize the mind (镇心安神). It is clinically used to treat palpitation (心悸) and improve the frail physique (体弱无力). Thailand Lanna Medicine (泰国兰纳医药学) claimed thatT.sinensis was more inclined to clear the heat and cool off the fire (清热降火) inside the body, thus usually clinically applied to relieve high fever and ameliorate the symptoms of diabetes [15–26]. In India,T.cordifolia referred to as Guduchi (plant which protects from diseases, Sanskrit) has been described in ancient text books of Ayurveda includingSushrut Samhita andCharak Samhita [27]. According to Ayurveda,T.cordifolia itself possesses a bitter, pungent, and astringent taste. The bitter taste is said to improve metabolic activity, even at a cellular level. It is documented to treat gastrointestinal diseases including dyspepsia, flatulence, gastritis, jaundice, diarrhea, splenomegaly, and hemorrhoids. It has a role in the treatment of metabolic disorders such as diabetes and kidney disorders, and it is currently more inclined to be a research focus. It is prescribed for intermittent fevers, infective conditions, urinary disorders, skin diseases, and eye diseases. In combination with other herbs, it is commonly used as an ingredient to treat gout and rheumatoid arthritis. And the well-ground whole plant is applied to fractures. Moreover,T.cordifolia shows healthy effects due to its various nutritious compounds and is considered as a general tonic [27, 28]. The diverse, significant ethnomedicinal properties possessed by this genus could be the basis for further researches to investigate the phytochemical and pharmacological aspects of this genus.Table 1
Classic prescriptions of genusTinospora in traditional and clinical usages.
Species
Preparation name/formulation
Main compositions
Medicinal uses
References
T. sinensis
Decoction
Liquidambar formosanaHance
Speranskia tuberculata(Bge.) Baill.
Angelica sinensis(Oliv.) Diels
Clematis chinensisOsbeck
Bombyx moriLinnaeus
Cinnamomum cassiaPresl
Achyranthes bidentataBl.
Spatholobus suberectusDunn
Aconitum kusnezoffiiReichb.
Atractylodes lancea(Thunb.)
Asarum heterotropoidesFr.Schmidtvar.mandshuricum(Maxim.) Kitag.
Knee joint osteoarthritis
[14]
T. sinensis
Qutan Tongluo Tang (祛痰通络汤)
Arca subcrenataLischke
Arisaema cum Bile
Drynaria fortune(Kunze) J. Sm.
Dipsacus asperWall. ex Henry
Angelica sinensis(Oliv.) Diels
Chaenomeles speciosa(Sweet) Nakai
Eucommia ulmoidesOliv.
Paeonia lactifloraPall.
Corydalis yanhusuoW. T. Wang
Angelica pubescensMaxim. F. biserrata Shan et Yuan
Akebia quinata(Thunb.) Decne.
Lumbar disc herniation
[15]
T. sinensis
Huoxue Shujin Xunxifang (活血舒筋熏洗方)
Clematis chinensisOsbeck
Sparganium stoloniferumBuch.-Ham.
Curcuma phaeocaulisVal.
Uncaria rhynchophylla(Miq.) Miq. ex Havil.
Carthamus tinctoriusL.
Salvia miltiorrhizaBge.
Ligusticum chuanxiongHort.
Achyranthes bidentataBl.
Aconitum kusnezoffiiReichb.
Aucklandia lappaDecne.
Ankylosis
[16]
T. sinensis
Sisheng Tang (四生汤)
Rheum palmatumL.
Clematis chinensisOsbeck
Speranskia tuberculata(Bge.)Baill.
Chaenomeles speciosa(Sweet) Nakai
Arisaema erubescens(Wall.) Schott
Aconitum kusnezoffiiReichb.
Aconitum carmichaeliiDebx.
Fracture
[17]
T. sinensis
Fumigation
Lycopodium japonicumThunb.
Speranskia tuberculata(Bge.) Baill.
Spatholobus suberectusDunn
Prunus persica(L.) Batsch
Carthamus tinctoriusL.
Angelica sinensis (Oliv.) Diels  Ligusticum chuanxiongHort.
Ankle fracture
[18]
T. sinensis
Decoction
—
Visceral leishmaniasis
[19]
T. capillipes
Jinguo Heji (金果合剂)
Scrophularia ningpoensisHensl.
Platycodon grandiflorum(Jacq.) A. DC.
Glycyrrhiza uralensisFisch.
Ophiopogon japonicas(L.f.) Ker Gawl.
Apis ceranaFabricius
Pharyngitis
[20]
T. capillipes
Soaking solution
—
Infusion phlebitis
[21]
T. capillipes
Powder
—
Upper respiratory tract infectionAcute tonsillitis Laryngopharyngitis Acute gastroenteritis Pneumonia Tracheitis
[22]
T. cordifolia
Decoction
Berberis aristata
Terminalia chebula
Zingiber officinale
Experimental amoebic liver abscess
[29]
T. cordifolia
Decoction
Phyllanthus niruri
Terminalia belerica
Terminalia chebula
Phyllanthus emblica
Carbon tetrachloride induced hepatotoxicity
[30]
T. cordifolia
Decoction
—
Diabetic foot ulcers
[31]
T. cordifolia
Decoction
—
Allergic rhinitis
[32]
T. cordifolia
Decoction
—
Obstructive jaundice, tuberculosis, sepsis, breast cancer
[27]
T. cordifolia
Decoction
Boerhavia diffusa
Berberis aristata
Terminalia chebula
Zingiber officinale
Entamoeba histolytica
[33]
T. cordifolia
Decoction
Zingiber officinale
Withania somnifera
Tribulus terrestris
Rheumatoid arthritis
[34]


## 3. Pharmacological Activities

Scientific studies all over the world for over 50 years have revealed much insight into the pharmacological functions ofTinospora. Crude extracts and pure compounds fromTinospora plants have shown significant antidiabetic, antioxidant, antitumor, anti-inflammatory, antimicrobial, antiosteoporosis, and immunostimulation properties. In this section, biological activities of the active compounds and mixtures on the species are highlighted.


### 3.1. Antioxidant Activity

Natural plants used in traditional Chinese medicine have antioxidant effects through different mechanisms, including DPPH radical scavenging assay, superoxide anion scavenging assay, hydroxyl radical scavenging assay, and ABTS radical scavenging method [35]. On a comparative basis,T.sinensis andT.cordifolia were tested to have equal antioxidant activities [36]. Ilaiyaraja and Khanum evaluated the antioxidant potential of different solvent extracts of leaf and stem ofT.cordifolia. Scavenging effects on DPPH, ABTS radical, hydroxyl radical, and ferric reducing antioxidant power (FRAP) were found to be highest in methanolic extract of leaf and ethyl acetate extract of stem. It is because the polarity of solvent showed a greater effect on the ordered structure of the active phenolic components [5]. The study on leaf and stem powders ofT.cordifolia followed by DPPH method found that the leaf extract powder has higher retention of antioxidant activity than the stem’s [37]. As early as 1998, Cavin et al. isolated and identified three compounds N-cis-feruloyltyramine, N-trans-feruloyltyramine, and secoisolariciresinol fromT.crispa. These ingredients proved to be more active than the synthetic antioxidant butylhydroxytoluene (BHT) [38]. Another investigation showed that the essential oil obtained from leaves ofT.cordifoliademonstrated strong DPPH radical scavenging activity with IC50 values of 25
±
0.3 μg/mL. The components identified in essential oil include alcohols (32.1%), phenols (16.6%), aldehydes (16.2%), fatty acids (15.7%), alkanes (8.3%), esters (3.2%), terpenes (1.2%), and others (4.8%) [39]. The work carried on hitherto rarely studied fruit ofT.cordifolia showed that it is also rich in antioxidant properties with the IC50 being 468 μg/g and EC50 being 1,472 μg/g, yet the effect of the fruit is not comparable to the other parts [5, 6]. These active components and mixtures could serve as valuable indications to develop them as possible antioxidant drugs for the treatment and the prevention of diseases. More importantly, oxidative stress is proved to be critical for the pathogenesis of diabetes mellitus. Clarification of antioxidant issues could help to understand the treatment of diabetes mellitus and promote the development of antidiabetic natural products [40].


### 3.2. Antidiabetic Activity

Many pharmacologic studies have clearly confirmed the antidiabetic effects of genusTinospora in vivo. Compared with other commonly seen antidiabetic drugs,T.crispa andT.cordifolia exhibit more powerful antidiabetic activity, and they have been widely used in Asia and Africa as a remedy mainly in regard to type 2 diabetes mellitus [41]. Ruan et al. explored the hypoglycemic effects of borapetoside A mediated through both the insulin-dependent and the insulin-independent pathways. They found that borapetoside A was able to increase the glucose utilization in peripheral tissues, to reduce the hepatic gluconeogenesis, and to activate the insulin signaling pathway. Comparison of the structures of three borapetosides fromT.crispa clearly suggested that the C-8 stereochemistry plays a key role in hypoglycemic effect, since the active borapetosides A and C possess 8R-chirality while the inactive borapetoside B possesses 8S-chirality. The location of glycoside at C-3 for borapetoside A (while it is C-6 for borapetoside C) and the formation of lactone between C-4 and C-6 for borapetoside A could account for the different potency in hypoglycemic action for these two compounds [42, 43]. Borapetol B is another antidiabetic active property fromT.crispa. By evaluating the blood glucose levels and stimulation of insulin secretion in normoglycemic control Wistar and diabetic Goto-Kakizaki rats, Lokman et al. provided evidence that oral administration of borapetol B has antidiabetic properties mainly due to its stimulation of insulin release [42, 44]. Antidiabetic potential ofT.cordifolia stem is also well proven [3, 45, 46]. Patel and Mishra chose sucrose and maltose as substrates to study the enzyme kinetics and the respective Michaelis-Menten constant; meanwhile maximal velocity values were estimated. Three isoquinoline alkaloids fromT.cordifolia, namely, jatrorrhizine, palmatine, and magnoflorine, showed IC50 value as sucrase inhibitor to be 36.25, 23.46, and 9.8 μg/mL and maltase inhibitor to be 22.05, 38.42, and 7.60 μg/mL. Furthermore, the increase in plasma glucose level inin vivo studies conducted on rats was significantly suppressed (P
<
0.01) by all the three alkaloids at 20 mg/kg b.wt. [47]. Kannadhasan and Venkataraman reported that the presence of antioxidant potentials in extract ofT.cordifolia was a potent healer in ameliorating diabetes like tissue damagein vivo[48]. It is perfectly consistent with the report thatT.cordifolia has a significant (P
<
0.05) effect in ameliorating the specific parameters toward normal in diabetic animals, and it has a level of efficacy that is considerably good compared to standard drug insulin [49]. These experimental results further enrich previous understanding of the antidiabetic activity of genusTinospora and provide scientific data of therapeutic efficacy against diabetes mellitus.


### 3.3. Immunostimulatory Activity

SeveralTinospora species have effects on humoral immunity, cellular immunity, and nonspecific immunity. Polysaccharide, a kind of natural macromolecule, has widespread occurrence in biological bodies and has many kinds of biological activities, which plays an important role in regulating immune system [50]. Polysaccharides inTinospora represent a structurally diverse class of macromolecules and this structural variability can profoundly affect their cell-type specificity and their biological activity in B cells. Previous studies documented that phytogenic polysaccharides could regulate animals’ immune system by combining to membrane receptors of immune cells and initiating the special signaling pathways. This includes stimulating the secretion or proliferation of macrophages, T/B lymphocytes, and natural killer (NK) cells, modulating the release of cytokines, promoting the production of antibodies, and activating the complement system [51]. (1,4)-α-D-Glucan (α-DG) is a novel immune stimulatory drug fromT.cordifolia. It exhibited macrophages activating abilities through TLR6 signaling and NF-κB activation mechanism, followed by the production of cytokines and chemokines. It can also differentially modulate cytokine production in the spleen and lung on endotoxaemic juvenile rats. In the lung, α-DG treatment reduced anti-inflammatory cytokine concentrations of IL-1β by 30%, IL-6 by 43%, IFN-γ by 46%, and IL-10 by 31% compared to endotoxaemia. In the spleen, α-DG treatment decreased the ratio of IL-1β to IL-10 by 55% [52–54]. Further data showed that α-DG led to significant tachycardia without causing hypotension. Accompanied by significant reduction in the blood hemoglobin and hematocrit concentrations, the effect did not change respiratory variable and/or plasma concentration of inflammatory cytokines in anaesthetized rats [55]. G1-4A, an arabinogalactan polysaccharide fromT.cordifolia, is a well-characterized immunomodulatory in bothin vitro andin vivo systems. Administration (12.5 mg/kg body weight) of G1-4A to mice led to splenomegaly and an increase in the numbers of T cells, B cells, and macrophages. This increase in spleen cellularity was due toin vivo proliferation of lymphocytes and upregulation of antiapoptotic genes. G1-4A induced proliferation of B cells was completely inhibited by PI3K inhibitor Ly294002, mTOR inhibitor rapamycin, and NF-κB inhibitor plumbagin. Meanwhile, Akt, ERK, and JNK were activated by G1-4A which finally resulted in the activation of IKK, degradation of IκB-α, and translocation of NF-κB to the nucleus [56–58]. In the humoral and cell-mediated immune response,T.cordifolia preparation enhanced mean hemagglutination antibody titre and reactive oxygen level against various pathogens [59–61]. Polar fractions ofT.cordifolia stem at a dose of 40 mg/kg body weight promoted the nonspecific host and defence of the immune system directly acted on peritoneal macrophages [13].T.crispa crude extract significantly stimulated RAW 264.7 cell viability (P
≤
0.05) and intracellular INF-γ, IL-6, and IL-8 expressions. Study of LC-MS showed that the immunomodulatory active compounds are cordioside, quercetin, eicosenoic acid, and boldine [62]. Other reports indicated that it is the synergistic effect of immunomodulatory active compounds in genusTinospora that contributed to the immunomodulatory activity [61, 63]. As mentioned above, polysaccharides in genusTinospora play a key role in the immunostimulatory activity. The characteristics of polysaccharides, which include backbone structure, branching degree, molecular weight, and three spiral structures, affected the activity of immunity. By activating the inherent and acquired immune system to produce antitumor immunity, the immunomodulatory activity could also contribute to the tumor immunotherapy [64].


### 3.4. Antitumor Activity

Feature chemical components ofTinospora, clerodane diterpenes, showed apparent cytotoxic activity against tumor cells [65]. Nowadays, the antitumor effect of theTinospora species has been widely studiedin vivo andin vitro. The mechanisms of the antitumor activity ofTinospora species are mainly concentrated on the cytotoxicity and cell apoptosis induced. Epoxy clerodane diterpenes were evaluated for the activity against diethylnitrosamine-induced hepatocyte carcinoma; the results concluded that they kick in blocking carcinogen metabolic activation and enhancing carcinogen detoxification [66, 67]. Aqueous ethanolic ingredients ofT.cordifolia reduced cell proliferation in dose-dependent manner and induced differentiation in C6 glioma cells. The cell proliferation inhibition was in part mediated through accumulation in S phase and G2/M arrest, with concomitant suppression of p21 expression and inhibition of cyclin-dependent kinase activity [68]. Treatment of hexane extract fraction ofT.cordifolia on Ehrlich ascites tumor bearing animals resulted in growth inhibition and induction of apoptosis in a dose-dependent mannerin vivo. The mechanism of action was caspase-3 dependent which caused a nuclear localization of apoptosis, meanwhile the formation of apoptotic bodies, nuclear condensation, typical DNA ladder, and decreased cell number and ascites volume [7]. More and Pai observed that treatment withT.cordifolia and LPS of macrophage cell line J774A.1 followed the enhanced NADH-oxidase, NADPH-oxidase, and myeloperoxidase production as compared to medium alone [69]. According to recent studies, alkaloids chemical constituents from plants have the characteristics of better antitumor activity, high effect, good tolerance, and slight side effect [70]. Alkaloid especially palmatine fromT.cordifolia significantly decreased tumor size, number and the activity of serum enzyme in DMBA induced skin cancer mice. The alkaloid could also contribute to the reduction of glutathione (GSH), superoxide dismutase (SOD), and catalase; however, it increased DNA damage [71]. The activation of macrophages is another pathway for antitumor activity. (1,4)-α-D-Glucan and G1-4A are immunological active polysaccharides fromT.cordifolia. Treatment with G1-4A on a murine lymphoma model increased T cell allostimulatory activity and secretion of IL-12 and TNFα by bone marrow derived dendritic cells, leading to the lysis of target tumor cellsin vitro. Polysaccharides can also activate macrophages, NK cells, APDs, and T and B lymphocytes to enhance antitumor immunity concerned with secretion of cytokines, activation of the complement system, induction of apoptosis of tumor cells, and intracellular signal transduction so as to achieve the tumor inhibitory effect by regulations of the immune system [56, 72]. Sonaimuthu et al. identified taxol producing endophytic fungusF.culmorum SVJM072 from medicinal plant ofT.cordifolia. Further research showed that the fungal taxol had strong activity against human cancer cell lines by MTT assay andM. tuberculosis H37Rv by Radiometric Bactec 460 assay, which could serve as an excellent alternative source and a genetically engineered species for anticancer compounds [73].


### 3.5. Anti-Inflammatory and Antimicrobial Activity

Inflammation is a pathologic condition that includes a wide range of diseases such as rheumatic and immune-mediated conditions, diabetes, and cardiovascular accident. If the process of inflammatory response cannot end normally when cell debris and pathogens were cleared, the biological defence response will become causative factor. Medicinal plants and their secondary metabolites are progressively used in the treatment of diseases as a complementary medicine [74, 75]. Palmatine and jatrorrhizine, mainly separated from tubers ofT.capillipesandT.cordifolia, have widely been applied in clinic owing to their comparatively broad spectrum inhibitory activity. With the EC50 values ranged from 0.0348 to 0.8356 g/L and 0.0240 to 0.8649 g/L, respectively, palmatine and jatrorrhizine exhibited inhibition against many plant pathogens. The two alkaloids vary only in the substituents of position 3 of A ring in terms of the chemical structure. The effectiveness of protoberberine was related to the ammonium N linked at position 7 of aromatic ring in the chemical structure and the inhibitory activity of protoberberine alkaloids altered by the different substituents in A and D rings [76]. The ethanol extract ofT.smilacina showed inhibitory activities on COX-1, COX-2, 5-LO, and PA2 with the IC50 values of 63.5, 81.2, 92.1, and 30.5 μg/mL, respectively [9]. Considerable decrease in the levels of iNOS, COX-2, and ICAM-1 resulted in the reduced release of proinflammatory mediators like TNF-α, IL-4, NO, and IgE [10]. An insight into the antimicrobial activity of the ethanolic extracts ofT.cordifolia indicated that the secondary metabolites exhibited high inhibitory activity against clinically isolatedS.aureus andK.pneumoniae [11]. It is noteworthy that induction of embryogenic callus ofT.cordifoliawith the presence of additional compounds was able to inhibit both Gram-positive and Gram-negative organisms [77]. All in all, inflammation is a complex and multifaceted problem, so the treatment of inflammation is not a one-dimensional remedy. The anti-inflammation of herbal medicine belonging to genusTinospora has a favorable applied perspective; with the help of modification in structure it may assist in reaching a multidimensional therapeutic approach to inflammation in the future [78].


### 3.6. Antiosteoporosis Activity

During recent years, researches in traditional Chinese medicine for the treatment of osteoporosis are increasing, especially in the research and therapeutic application using compound medicine and external medicine, and some progress has been made [79].T.capillipesandT.cordifolia were reported to induce alkaline phosphatase activity in mesenchymal stem cells based on ecdysone compounds. At the dosage of 25 μg/mL, ethanolic extracts ofT.cordifolia stimulated the growth of osteoblasts, increased the differentiation of cells into osteoblastic lineage, and increased the mineralization of bone-like matrix on both the osteoblast model systems. Previously, Gao et al. noted that the stem extracts ofT.cordifolia in ovariectomized rats significantly prevented the bone loss in rats induced by ovariectomy without causing proliferative effects in uterus and mammary gland [80, 81]. In addition, the presence of high concentration of calcium inT.cordifolia made it a natural cure for osteoporosis [12]. Moreover, single or synergistic formulations ofT.cordifolia withZingiber officinale have been used in ancient India medicinal system to treat rheumatoid arthritis [34]. However, the morbidity of osteoporosis has been linked to several factors such as age, gender, and races. In order to get a good outcome of osteoporosis treatment, both surgical management and pharmacological treatment are necessary.


### 3.7. Other Therapeutic Activities

What is more,Tinospora has been investigated to exert other pharmacological activities. Cordifolioside A, mainly isolated inT.cordifolia, has a potentialin vivo radioprotective effect andin vitro cytoprotective activity [82]. The impressive radioprotective efficacy may be related to the attenuation of radiation induced decrease of adherence and spreading, increase of IL-1β and GM-CSF levels, and reduction of apoptosis [83]. The radiosensitization byT.cordifolia may be also due to depletion of GSH and GST, accompanied by elevated levels of LPx and DNA damage of tumor cells [84].Ethanolic extract ofT.sinensis exhibited an appreciable activity against promastigotes (IC50  37.6
±
6.2 μg/mL) and intracellular amastigotes (IC50  29.8
±
3.4 μg/mL). In hamsters, it resulted in 76.2
±
9.2% inhibition at 500 mg/kg/day × 5 oral dose levels [19–24].T.cordifolia (100 mg/kg b.wt. for 15 days daily) used in combination with cisplatin inL.donovani infected BALB/c mice selectively induced Th1 type of immune response. It was concluded by the enhanced levels of IFN-c and IL-2 with moderate decline of Th2 specific cytokines IL-4 and IL-10 [85].The least polar alkaloid fromT.crispa, columbamine, showed strong acetyl-cholinesterase inhibitory activity with IC50 48.10 μmol/L. The structure-activity relationships derived from these results suggested that the quaternary nitrogen in the skeleton has some effect but that a high degree of methoxylation is more important for acetylcholinesterase inhibition [86].T.cordifoliaethanol extract exhibited significant neuroprotection by increasing the dopamine levels (1.96
±
0.20 and 2.45 ± 0.40 ng/mg of protein) and complex I activity (77.14 ± 0.89 and 78.50 ± 0.96 nmol/min/mg of protein) at 200 and 400 mg/kg, respectively [87]. Combination of ethanolic extracts ofT.cordifolia,B.monnieri, andE.alsinoides in equal proportion provided synergistic nootropic effect on scopolamine induced amnesia in rats [88].


## 3.1. Antioxidant Activity

Natural plants used in traditional Chinese medicine have antioxidant effects through different mechanisms, including DPPH radical scavenging assay, superoxide anion scavenging assay, hydroxyl radical scavenging assay, and ABTS radical scavenging method [35]. On a comparative basis,T.sinensis andT.cordifolia were tested to have equal antioxidant activities [36]. Ilaiyaraja and Khanum evaluated the antioxidant potential of different solvent extracts of leaf and stem ofT.cordifolia. Scavenging effects on DPPH, ABTS radical, hydroxyl radical, and ferric reducing antioxidant power (FRAP) were found to be highest in methanolic extract of leaf and ethyl acetate extract of stem. It is because the polarity of solvent showed a greater effect on the ordered structure of the active phenolic components [5]. The study on leaf and stem powders ofT.cordifolia followed by DPPH method found that the leaf extract powder has higher retention of antioxidant activity than the stem’s [37]. As early as 1998, Cavin et al. isolated and identified three compounds N-cis-feruloyltyramine, N-trans-feruloyltyramine, and secoisolariciresinol fromT.crispa. These ingredients proved to be more active than the synthetic antioxidant butylhydroxytoluene (BHT) [38]. Another investigation showed that the essential oil obtained from leaves ofT.cordifoliademonstrated strong DPPH radical scavenging activity with IC50 values of 25
±
0.3 μg/mL. The components identified in essential oil include alcohols (32.1%), phenols (16.6%), aldehydes (16.2%), fatty acids (15.7%), alkanes (8.3%), esters (3.2%), terpenes (1.2%), and others (4.8%) [39]. The work carried on hitherto rarely studied fruit ofT.cordifolia showed that it is also rich in antioxidant properties with the IC50 being 468 μg/g and EC50 being 1,472 μg/g, yet the effect of the fruit is not comparable to the other parts [5, 6]. These active components and mixtures could serve as valuable indications to develop them as possible antioxidant drugs for the treatment and the prevention of diseases. More importantly, oxidative stress is proved to be critical for the pathogenesis of diabetes mellitus. Clarification of antioxidant issues could help to understand the treatment of diabetes mellitus and promote the development of antidiabetic natural products [40].


## 3.2. Antidiabetic Activity

Many pharmacologic studies have clearly confirmed the antidiabetic effects of genusTinospora in vivo. Compared with other commonly seen antidiabetic drugs,T.crispa andT.cordifolia exhibit more powerful antidiabetic activity, and they have been widely used in Asia and Africa as a remedy mainly in regard to type 2 diabetes mellitus [41]. Ruan et al. explored the hypoglycemic effects of borapetoside A mediated through both the insulin-dependent and the insulin-independent pathways. They found that borapetoside A was able to increase the glucose utilization in peripheral tissues, to reduce the hepatic gluconeogenesis, and to activate the insulin signaling pathway. Comparison of the structures of three borapetosides fromT.crispa clearly suggested that the C-8 stereochemistry plays a key role in hypoglycemic effect, since the active borapetosides A and C possess 8R-chirality while the inactive borapetoside B possesses 8S-chirality. The location of glycoside at C-3 for borapetoside A (while it is C-6 for borapetoside C) and the formation of lactone between C-4 and C-6 for borapetoside A could account for the different potency in hypoglycemic action for these two compounds [42, 43]. Borapetol B is another antidiabetic active property fromT.crispa. By evaluating the blood glucose levels and stimulation of insulin secretion in normoglycemic control Wistar and diabetic Goto-Kakizaki rats, Lokman et al. provided evidence that oral administration of borapetol B has antidiabetic properties mainly due to its stimulation of insulin release [42, 44]. Antidiabetic potential ofT.cordifolia stem is also well proven [3, 45, 46]. Patel and Mishra chose sucrose and maltose as substrates to study the enzyme kinetics and the respective Michaelis-Menten constant; meanwhile maximal velocity values were estimated. Three isoquinoline alkaloids fromT.cordifolia, namely, jatrorrhizine, palmatine, and magnoflorine, showed IC50 value as sucrase inhibitor to be 36.25, 23.46, and 9.8 μg/mL and maltase inhibitor to be 22.05, 38.42, and 7.60 μg/mL. Furthermore, the increase in plasma glucose level inin vivo studies conducted on rats was significantly suppressed (P
<
0.01) by all the three alkaloids at 20 mg/kg b.wt. [47]. Kannadhasan and Venkataraman reported that the presence of antioxidant potentials in extract ofT.cordifolia was a potent healer in ameliorating diabetes like tissue damagein vivo[48]. It is perfectly consistent with the report thatT.cordifolia has a significant (P
<
0.05) effect in ameliorating the specific parameters toward normal in diabetic animals, and it has a level of efficacy that is considerably good compared to standard drug insulin [49]. These experimental results further enrich previous understanding of the antidiabetic activity of genusTinospora and provide scientific data of therapeutic efficacy against diabetes mellitus.


## 3.3. Immunostimulatory Activity

SeveralTinospora species have effects on humoral immunity, cellular immunity, and nonspecific immunity. Polysaccharide, a kind of natural macromolecule, has widespread occurrence in biological bodies and has many kinds of biological activities, which plays an important role in regulating immune system [50]. Polysaccharides inTinospora represent a structurally diverse class of macromolecules and this structural variability can profoundly affect their cell-type specificity and their biological activity in B cells. Previous studies documented that phytogenic polysaccharides could regulate animals’ immune system by combining to membrane receptors of immune cells and initiating the special signaling pathways. This includes stimulating the secretion or proliferation of macrophages, T/B lymphocytes, and natural killer (NK) cells, modulating the release of cytokines, promoting the production of antibodies, and activating the complement system [51]. (1,4)-α-D-Glucan (α-DG) is a novel immune stimulatory drug fromT.cordifolia. It exhibited macrophages activating abilities through TLR6 signaling and NF-κB activation mechanism, followed by the production of cytokines and chemokines. It can also differentially modulate cytokine production in the spleen and lung on endotoxaemic juvenile rats. In the lung, α-DG treatment reduced anti-inflammatory cytokine concentrations of IL-1β by 30%, IL-6 by 43%, IFN-γ by 46%, and IL-10 by 31% compared to endotoxaemia. In the spleen, α-DG treatment decreased the ratio of IL-1β to IL-10 by 55% [52–54]. Further data showed that α-DG led to significant tachycardia without causing hypotension. Accompanied by significant reduction in the blood hemoglobin and hematocrit concentrations, the effect did not change respiratory variable and/or plasma concentration of inflammatory cytokines in anaesthetized rats [55]. G1-4A, an arabinogalactan polysaccharide fromT.cordifolia, is a well-characterized immunomodulatory in bothin vitro andin vivo systems. Administration (12.5 mg/kg body weight) of G1-4A to mice led to splenomegaly and an increase in the numbers of T cells, B cells, and macrophages. This increase in spleen cellularity was due toin vivo proliferation of lymphocytes and upregulation of antiapoptotic genes. G1-4A induced proliferation of B cells was completely inhibited by PI3K inhibitor Ly294002, mTOR inhibitor rapamycin, and NF-κB inhibitor plumbagin. Meanwhile, Akt, ERK, and JNK were activated by G1-4A which finally resulted in the activation of IKK, degradation of IκB-α, and translocation of NF-κB to the nucleus [56–58]. In the humoral and cell-mediated immune response,T.cordifolia preparation enhanced mean hemagglutination antibody titre and reactive oxygen level against various pathogens [59–61]. Polar fractions ofT.cordifolia stem at a dose of 40 mg/kg body weight promoted the nonspecific host and defence of the immune system directly acted on peritoneal macrophages [13].T.crispa crude extract significantly stimulated RAW 264.7 cell viability (P
≤
0.05) and intracellular INF-γ, IL-6, and IL-8 expressions. Study of LC-MS showed that the immunomodulatory active compounds are cordioside, quercetin, eicosenoic acid, and boldine [62]. Other reports indicated that it is the synergistic effect of immunomodulatory active compounds in genusTinospora that contributed to the immunomodulatory activity [61, 63]. As mentioned above, polysaccharides in genusTinospora play a key role in the immunostimulatory activity. The characteristics of polysaccharides, which include backbone structure, branching degree, molecular weight, and three spiral structures, affected the activity of immunity. By activating the inherent and acquired immune system to produce antitumor immunity, the immunomodulatory activity could also contribute to the tumor immunotherapy [64].


## 3.4. Antitumor Activity

Feature chemical components ofTinospora, clerodane diterpenes, showed apparent cytotoxic activity against tumor cells [65]. Nowadays, the antitumor effect of theTinospora species has been widely studiedin vivo andin vitro. The mechanisms of the antitumor activity ofTinospora species are mainly concentrated on the cytotoxicity and cell apoptosis induced. Epoxy clerodane diterpenes were evaluated for the activity against diethylnitrosamine-induced hepatocyte carcinoma; the results concluded that they kick in blocking carcinogen metabolic activation and enhancing carcinogen detoxification [66, 67]. Aqueous ethanolic ingredients ofT.cordifolia reduced cell proliferation in dose-dependent manner and induced differentiation in C6 glioma cells. The cell proliferation inhibition was in part mediated through accumulation in S phase and G2/M arrest, with concomitant suppression of p21 expression and inhibition of cyclin-dependent kinase activity [68]. Treatment of hexane extract fraction ofT.cordifolia on Ehrlich ascites tumor bearing animals resulted in growth inhibition and induction of apoptosis in a dose-dependent mannerin vivo. The mechanism of action was caspase-3 dependent which caused a nuclear localization of apoptosis, meanwhile the formation of apoptotic bodies, nuclear condensation, typical DNA ladder, and decreased cell number and ascites volume [7]. More and Pai observed that treatment withT.cordifolia and LPS of macrophage cell line J774A.1 followed the enhanced NADH-oxidase, NADPH-oxidase, and myeloperoxidase production as compared to medium alone [69]. According to recent studies, alkaloids chemical constituents from plants have the characteristics of better antitumor activity, high effect, good tolerance, and slight side effect [70]. Alkaloid especially palmatine fromT.cordifolia significantly decreased tumor size, number and the activity of serum enzyme in DMBA induced skin cancer mice. The alkaloid could also contribute to the reduction of glutathione (GSH), superoxide dismutase (SOD), and catalase; however, it increased DNA damage [71]. The activation of macrophages is another pathway for antitumor activity. (1,4)-α-D-Glucan and G1-4A are immunological active polysaccharides fromT.cordifolia. Treatment with G1-4A on a murine lymphoma model increased T cell allostimulatory activity and secretion of IL-12 and TNFα by bone marrow derived dendritic cells, leading to the lysis of target tumor cellsin vitro. Polysaccharides can also activate macrophages, NK cells, APDs, and T and B lymphocytes to enhance antitumor immunity concerned with secretion of cytokines, activation of the complement system, induction of apoptosis of tumor cells, and intracellular signal transduction so as to achieve the tumor inhibitory effect by regulations of the immune system [56, 72]. Sonaimuthu et al. identified taxol producing endophytic fungusF.culmorum SVJM072 from medicinal plant ofT.cordifolia. Further research showed that the fungal taxol had strong activity against human cancer cell lines by MTT assay andM. tuberculosis H37Rv by Radiometric Bactec 460 assay, which could serve as an excellent alternative source and a genetically engineered species for anticancer compounds [73].


## 3.5. Anti-Inflammatory and Antimicrobial Activity

Inflammation is a pathologic condition that includes a wide range of diseases such as rheumatic and immune-mediated conditions, diabetes, and cardiovascular accident. If the process of inflammatory response cannot end normally when cell debris and pathogens were cleared, the biological defence response will become causative factor. Medicinal plants and their secondary metabolites are progressively used in the treatment of diseases as a complementary medicine [74, 75]. Palmatine and jatrorrhizine, mainly separated from tubers ofT.capillipesandT.cordifolia, have widely been applied in clinic owing to their comparatively broad spectrum inhibitory activity. With the EC50 values ranged from 0.0348 to 0.8356 g/L and 0.0240 to 0.8649 g/L, respectively, palmatine and jatrorrhizine exhibited inhibition against many plant pathogens. The two alkaloids vary only in the substituents of position 3 of A ring in terms of the chemical structure. The effectiveness of protoberberine was related to the ammonium N linked at position 7 of aromatic ring in the chemical structure and the inhibitory activity of protoberberine alkaloids altered by the different substituents in A and D rings [76]. The ethanol extract ofT.smilacina showed inhibitory activities on COX-1, COX-2, 5-LO, and PA2 with the IC50 values of 63.5, 81.2, 92.1, and 30.5 μg/mL, respectively [9]. Considerable decrease in the levels of iNOS, COX-2, and ICAM-1 resulted in the reduced release of proinflammatory mediators like TNF-α, IL-4, NO, and IgE [10]. An insight into the antimicrobial activity of the ethanolic extracts ofT.cordifolia indicated that the secondary metabolites exhibited high inhibitory activity against clinically isolatedS.aureus andK.pneumoniae [11]. It is noteworthy that induction of embryogenic callus ofT.cordifoliawith the presence of additional compounds was able to inhibit both Gram-positive and Gram-negative organisms [77]. All in all, inflammation is a complex and multifaceted problem, so the treatment of inflammation is not a one-dimensional remedy. The anti-inflammation of herbal medicine belonging to genusTinospora has a favorable applied perspective; with the help of modification in structure it may assist in reaching a multidimensional therapeutic approach to inflammation in the future [78].


## 3.6. Antiosteoporosis Activity

During recent years, researches in traditional Chinese medicine for the treatment of osteoporosis are increasing, especially in the research and therapeutic application using compound medicine and external medicine, and some progress has been made [79].T.capillipesandT.cordifolia were reported to induce alkaline phosphatase activity in mesenchymal stem cells based on ecdysone compounds. At the dosage of 25 μg/mL, ethanolic extracts ofT.cordifolia stimulated the growth of osteoblasts, increased the differentiation of cells into osteoblastic lineage, and increased the mineralization of bone-like matrix on both the osteoblast model systems. Previously, Gao et al. noted that the stem extracts ofT.cordifolia in ovariectomized rats significantly prevented the bone loss in rats induced by ovariectomy without causing proliferative effects in uterus and mammary gland [80, 81]. In addition, the presence of high concentration of calcium inT.cordifolia made it a natural cure for osteoporosis [12]. Moreover, single or synergistic formulations ofT.cordifolia withZingiber officinale have been used in ancient India medicinal system to treat rheumatoid arthritis [34]. However, the morbidity of osteoporosis has been linked to several factors such as age, gender, and races. In order to get a good outcome of osteoporosis treatment, both surgical management and pharmacological treatment are necessary.


## 3.7. Other Therapeutic Activities

What is more,Tinospora has been investigated to exert other pharmacological activities. Cordifolioside A, mainly isolated inT.cordifolia, has a potentialin vivo radioprotective effect andin vitro cytoprotective activity [82]. The impressive radioprotective efficacy may be related to the attenuation of radiation induced decrease of adherence and spreading, increase of IL-1β and GM-CSF levels, and reduction of apoptosis [83]. The radiosensitization byT.cordifolia may be also due to depletion of GSH and GST, accompanied by elevated levels of LPx and DNA damage of tumor cells [84].Ethanolic extract ofT.sinensis exhibited an appreciable activity against promastigotes (IC50  37.6
±
6.2 μg/mL) and intracellular amastigotes (IC50  29.8
±
3.4 μg/mL). In hamsters, it resulted in 76.2
±
9.2% inhibition at 500 mg/kg/day × 5 oral dose levels [19–24].T.cordifolia (100 mg/kg b.wt. for 15 days daily) used in combination with cisplatin inL.donovani infected BALB/c mice selectively induced Th1 type of immune response. It was concluded by the enhanced levels of IFN-c and IL-2 with moderate decline of Th2 specific cytokines IL-4 and IL-10 [85].The least polar alkaloid fromT.crispa, columbamine, showed strong acetyl-cholinesterase inhibitory activity with IC50 48.10 μmol/L. The structure-activity relationships derived from these results suggested that the quaternary nitrogen in the skeleton has some effect but that a high degree of methoxylation is more important for acetylcholinesterase inhibition [86].T.cordifoliaethanol extract exhibited significant neuroprotection by increasing the dopamine levels (1.96
±
0.20 and 2.45 ± 0.40 ng/mg of protein) and complex I activity (77.14 ± 0.89 and 78.50 ± 0.96 nmol/min/mg of protein) at 200 and 400 mg/kg, respectively [87]. Combination of ethanolic extracts ofT.cordifolia,B.monnieri, andE.alsinoides in equal proportion provided synergistic nootropic effect on scopolamine induced amnesia in rats [88].


## 4. Chemical Constituents

The chemical constituents ofTinosporahave long been the pursued objectives of the researchers, and multiple classes of chemical constituents have been isolated and reported. Within these compounds, diterpenoids are the most dominant constituent found in the currently studied species in this genus. Up to now, 223 compounds have been isolated from genusTinospora, including terpenoids, alkaloids, steroids, C6-C3 derivatives, and polysaccharides as well as other components. The compounds list and their structures are shown in Table 2 and Figure 1.Table 2
The compound list of genusTinospora.
Number
Compounds
Species (part of plant)
References
Terpenoids
1
Tinocapillins C
T. capillipes(R)
[123]
2
Tinocallone A
T. capillipes(R)
[107]
3
Tinocallone B
T. capillipes(R)
[107]
4
Tinocordin
T. cordifolia
[124]
5
Tinocapillins A
T. capillipes(R)
[123]
6
Fibaruretin H
T. sagittata(R)
[125]
7
Tinocapillins B
T. capillipes(R)
[123]
8
Fibaruretin G
T. sagittata(R)
[125]
9
Sagitone
T. sagittatavar. yunnanensis(R)
[93]
10
Tinosponone
T. cordifolia(S)
[113]
11
Tinosporaside
T. cordifolia(S)
[100, 113]
12
Tinosporaside tetraacetate
T. cordifolia(S)
[113]
13
Amritoside D
T. cordifolia(S)
[109]
14
Tinocordioside
T. cordifolia(S)
[109]
15
Tinocordioside tetraacetate
T. cordifolia(S)
[109]
16
Boropetol B
T. crispa(S)
[126]
17
Tinocrisposide
T. crispa(S)
[127]
18
Boropetoside F
T. tuberculata(S)
[128]
19
(2R,5R,6S,9S,10S,12S)-15,16-Epoxy-2-hydroxy-6-O-(β-D-glucopyranosyl)-cleroda-3,7,13(16),14-tetraen-17,12-olid-18-oic acid methyl ester
T. crispa
[114]
20
(5R,6S,9S,10S,12S)-15,16-Epoxy-2-oxo-6-O-(β-D-glucopyranosyl)-cleroda-3,7,13(16),14-tetraen-17,12-olid-18-oic acid methyl ester
T. crispa
[91]
21
(5R,6R,8S,9R,10S,12S)-15,16-Epoxy-2-oxo-6-O-(β-D-glucopyranosyl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester
T. crispa
[91]
22
(2R,5R,6R,8S,9S,10S,12S)-15,16-Epoxy-2-hydroxy-6-O-
β-D-glucopyranosyl-(1→6)-α-D-xylopyranosyl-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester
T. crispa
[91]
23
(2R,5R,6R,8R,9S,10S,12S)-15,16-Epoxy-2-hydroxy-6-O-(β-D-glucopyranosyl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester
T. crispa
[91]
24
(5R,6R,8S,9R,10R,12S)-15,16-Epoxy-2-oxo-6-O-(β-D-glucopyranosyl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester
T. crispa
[91]
25
Borapetoside B
T. crispa
[38, 91]
26
Borapetoside C
T. crispa
[38, 91]
27
Tinophylloloside
T. sagittatavar. yunnanensis(R)
[92]
28
Epitinophylloloside
T. sagittatavar. yunnanensis(R)
T. capillipes(R)
[107]
29
Tinoscorside C
T. cordifolia
[94]
30
Borapetoside F
T. cordifolia
[94]
31
Borapetoside B
T. cordifolia
[94]
32
Tinocrispol A
T. crispa(S)
[129]
33
Amritoside C
T. cordifolia(S)
[109]
34
2-O-Lactoylborapetoside B
T. crispa(S)
[129]
35
6′-O-Lactoylborapetoside B
T. crispa(S)
[129]
36
Borapetoside H
T. tuberculata(S)
[95]
37
Octa-O-acetylborapetoside H
T. tuberculata(S)
[95]
38
Borapetoside H
T. crispa(S)
[96]
39
Tinospinoside C
T. sagittatavar. yunnanensis(R)
[97]
40
Tinospinoside A
T. sagittata(Oliv.)Gagnep.(R)
[98]
41
Tinospinoside B
T. sagittata(Oliv.)Gagnep.(R)
[98]
42
Rumphioside A
T. rumphii(S)
[117]
43
Rumphioside B
T. rumphii(S)
[117]
44
Rumphioside C
T. rumphii(S)
[117]
45
Rumphioside D
T. rumphii(S)
[117]
46
Furanoid diterpene glycoside
T. cordifolia(S)
[130]
47
Cordioside
T. cordifolia(S)
[99]
48
Cordioside tetraacetate
T. cordifolia(S)
[99]
49
Cordifoliside D
T. cordifolia(S)
[100]
50
Cordifoliside E
T. cordifolia(S)
[100]
51
Boropetoside G
T. tuberculata(S)
[128]
52
Malabarolide
T. malabarica(S)
[131]
53
Tinosposinenside A
T. sinensis(S)
[101]
54
Tinosposinenside B
T. sinensis(S)
[101]
55
Tinosposinenside C
T. sinensis(S)
[101]
56
1-Deacetyltinosposide A
T. sinensis(S)
[103]
57
Tinosineside A
T. sinensis(S)
[103, 104]
58
Tinosineside B
T. sinensis(S)
[104]
59
Penta-O-acetyl-tinosineside A
T. sinensis(S)
[104]
60
Hexa-O-acetyl-tinosineside A
T. sinensis(S)
[104]
61
(3R,4R,5R,6S,8R,9S,10S,12S)-15,16-Epoxy-3,4-epoxy-6-O-(β-D-glucopyranosyl)-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester
T. crispa
[91]
62
Tinospinoside D
T. sagittata(R)
[105, 132]
63
Tinosporide
T. cordifolia(S)
[113]
64
Furanolactone clerodane diterpene
T. cordifolia(S)
[113]
65
Furanolactone clerodane diterpene
T. cordifolia(S)
[113]
66
Tinosporicide
T. malabarica(S)
[113]
67
Jateorin
T. smilacina(S)
[114]
68
Palmatoside F
T. smilacina(S)
[114]
69
(5R,10R)-4R,8R-Dihydroxy-2S,3R:15,16-diepoxycleroda-13(16),17,12S,18,1S-dilactone
T. cordifolia(S)
[110]
70
Menispermacide
T. malabarica(S)
[127]
71
Tinosporaside
T. cordifolia(S)
[113]
72
Ioa-hydroxy columbin
T. malabarica(S)
[131]
73
Tinoside
T. capillipes(Rh)
[107]
74
Isocolumbin
T. capillipes(Rh)
[107]
75
Columbin
T. craveniana(R)
T. sagittatavar. yunnanensis(R)
T. capillipes(R)
[92, 93, 106, 107, 132, 133]
76
Palmatoside C
T. sagittatavar. yunnanensis(R)
T. capillipes(R)
[93, 107, 115]
77
Fibleucin
T. sagittatavar. yunnanensis(R)
T. capillipes(R)
[93]
78
Isocolumbin
T. capillipes(R)
T. sagittata(R)
T. craveniana(R)
[92, 106, 107, 133]
79
Tinoside
T. sagittata(R)
T. capillipes(R)
[106]
80
6-Hydroxy columbin
T. sagittata(R)
[92]
81
(1R,4S,5R,8S,9R,10S,12S)-15,16-Epoxy-4-O-(β-D-glucopyranosyl)-cleroda-2,13(16),14-triene-17(12),18(1)-diolide
T. crispa
[91]
82
Tinospinoside E
T. sagittatavar. yunnanensis(R)
[98]
83
8-Hydroxy columbin
T. cordifolia
[116, 128]
84
Tinospin E
T. sagittata(R)
[105, 112]
85
Cordifolide B
T. cordifolia(S)
[108]
86
Cordifolide C
T. cordifolia(S)
[108]
87
Furanolactone diterpene
T. cordifolia(S)
[113]
88
Boropetol A
T. crispa(S)
[134]
89
Boropetoside A
T. crispa(S)
[134]
90
Cordifolide A
T. cordifolia(S)
[108]
91
Menispermacide
T. smilacina(S)
[114]
92
Borapetoside A
T. crispa
[91]
93
Tinosporaclerodanoid
T. cordifolia(S)
[102]
94
Tinotufolin A
T. tuberculata(L)
[116, 128]
95
Tinotufolin B
T. tuberculata(L)
[116, 128]
96
Amritoside B
T. cordifolia(S)
[109]
97
Tinotufolin C
T. crispa(S)
[96]
98
Methyl(1α,4αa,5α,6β,8aα)-5-2-(3-furan-3-ene-2-one)ethyl-1,2,3,4,4a,5,6,7,8,8a-decahydro-1,2-dihydroxy-1-naphthalenecarboxylate
T. rumphii(L)
[115]
99
Tinotufolin F
T. crispa(S)
[96]
100
Tinotufolin E
T. crispa(S)
[96]
101
Sagittatayunnanoside B
T. sagittatavar. yunnanensis(R)
[97]
102
Sagittatayunnanoside A
T. sagittatavar. yunnanensis(R)
[97]
103
Sagittatayunnanoside C
T. sagittatavar. yunnanensis(R)
[97]
104
Sagittatayunnanoside D
T. sagittatavar. yunnanensis(R)
[97]
105
Amritoside A
T. cordifolia(S)
[109]
106
Tinosagittone A
T. sagittata(R)
[92]
107
Tinosagittone B
T. sagittata(R)
[92]
108
Tinocapilactone A
T. capillipes
[107]
109
Tinocapilactone B
T. capillipes
[107]
110
Tinosporafuranol
T. cordifolia(S)
[102]
111
Tinosporaclerodanol
T. cordifolia(S)
[102]
112
Tinosporafurandiol
T. cordifolia(S)
[100]
113
(2aβ,3α,5aβ,6β,7α,8aα)-6-2-(3-Furanyl)ethyl-2a,3,4,5,5a,6,7,8,8a,8b-decahydro-2a,3-dihydroxy-6,7,8b-trimethyl-2H-naphtho1,8-bcfuran-2-one
T. rumphii(L)
[104–115]
114
Boropetoside D
T. tuberculata(S)
[116, 128]
115
Boropetoside E
T. tuberculata(S)
[116, 128]
116
Tinotufolin D
T. crispa(S)
[96]
117
Borapetoside E
T. rumphii(S)
[118]
118
Rumphioside I
T. rumphii(S)
[118]
119
Rumphioside F
T. rumphii(S)
[117]
120
Rumphioside E
T. rumphii(S)
[117]
121
(2R,7S,8S)-8-[(2S)-2-(3,4-Dihydroxy-2,5-dimethoxytetrahydro-3-furanyl)-2-hydroxyethyl]-2,8-dimethyl-10-oxo-11-oxatricyclo[7.2.1.02,7]dodec-3-ene-3-carboxylate
T. crispa
[91]
122
Borapetoside D
T. crispa
[91]
123
Baenzigeride B
T. baenzigeri(S)
[120]
124
Baenzigeroside B
T. baenzigeri(S)
[120]
125
Baenzigeride A
T. baezigeri(S)
[120]
126
Baenzigeroside A
T. baezigeri(S)
[120]
127
Cycloeuphordenol
T. malabarica(S)
[113]
128
Cycloeucalenol
T. crispa(S)
[120]
129
Cycloeucalenone
T. crispa(S)
[120]
130
Tinosinenside
T. sinensis(S)
[101]
131
11-Hydroxymustakone
T. capillipes
[61, 121, 135]
132
Tinocordiside
T. capillipes
[61, 121, 135]
133
Tinocordifolioside
T. sinensis(S)
T. cordifolia(S)
[103, 122, 136]
134
Tinocordifolin
T. cordifolia(S)
[136]
135
Tinocordifolioside tetraacetate
T. cordifolia(S)
[136]
136
Angelicoidenol-2-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside
T. cordifolia
[94]
Alkaloids
137
Berberine
T. cordifolia(S)
[113]
138
Palmatine
T. cordifolia(S)
T. malabarica(S)
[127, 137]
139
Jatrorrhizine
T. cordifolia(S)
T. capillipes(R)
[137, 138]
140
13-Hydroxycolumbamine
T. sagittata(R)
T. capillipes(R)
[14, 139]
141
13-Hydroxyjatrorrhizine
T. sagittata(R)
T. capillipes(R)
[14, 139]
142
Demethyleneberberine
T. sagittata(R)
T. capillipes(R)
[14, 139]
143
13-Hydroxypalmatine
T. sagittata(R)
T. capillipes(R)
[14, 139]
144
Columbamine
T. sagittata(R)
T. capillipes(R)
[14, 139]
145
Jatrorrhizine
T. sagittata(R)
T. capillipes(R)
T. sinensis(S)
[14, 106, 139, 140]
146
Isocolumbamine
T. hainanensis(S)
T. capillipes(R)
T. sagittata(R)
[106]
147
Stepharanine
T. sagittata(R)
T. capillipes(R)
[14, 139]
148
Dehydrodiscretamine
T. sagittata(R)
T. capillipes(R)
[14, 139]
149
Dehydrocorydalmine
T. sagittata(R)
T. capillipes(R)
[14, 106, 139]
150
Palmaturbine
T. sinensis
T. craveniana(R)
[14, 133, 139, 140]
151
Palmatine
T. sinensis(S)
T. capillipes(R)
T. craveniana(R)
[103, 106, 133, 140]
152
S-trans-N-Methyltetrahydrocolumbamine
T. hainanensis(S)
[141]
153
S-trans-Cyclanoline
T. hainanensis(S)
[141]
154
3-Hydroxy-2,9,11-trimethoxy-5,6-dihydro-isoquino3,2-a-isoquinolinylium
T. sinensis
[140]
155
4,13-Dihydroxy-2,8,9-trimethoxydibenzo [a,g] quinolizinium
T. crispa
[86]
156
Berberine
T. smilacina(S)
[92]
157
Magnoflorine
T. cordifolia(S)
T. malabarica(S)
T. capillipes(R)
[113, 138]
158
Menisperine
T. capillipes(R)
T. cordifolia(S)
[113, 138]
159
N-Formyl anonaine
T. crispa(S)
T. malabarica(S)
[113]
160
N-Formyl nornuciferine
T. crispa(S)
[113]
161
N-Acetyl nornuciferine
T. crispa(S)
[38]
162
N-Formylasimilobine-2-O-β-D-glucopyranoside
T. crispa(S)
[96]
163
N-Formylannonain
T. capillipes
[61, 135]
164
Magnoflorine
T. sagittata(R)
T. capillipes(R)
[14, 106, 139]
165
Menisperine
T. sagittata(R)
T. capillipes(R)
[14, 139]
166
Tembetarine
T. sagittata(R)
T. capillipes(R)
[14, 139]
167
Reticuline
T. sagittata(R)
T. capillipes(R)
[14, 139]
168
Tembetarine
T. cordifolia(S)
[113]
169
Higenamine
T. crispa
[142]
170
Adenine
T. crispa
[142]
171
Adenosine
T. crispa
[143]
172
Uridine
T. crispa
[142]
173
Salsolinol
T. crispa
[142]
174
(−)-Litcubinine
T. crispa
[142]
175
Tyramine
T. crispa
[142]
176
N-trans-Feruloyl tyramine
T. tuberdata(S)
[38]
177
N-cis-Feruloyl tyramine
T. tuberculata(S)
[36–38]
178
N-trans-Feruloyl tyramine diacetate
T. cordifolia(S)
[38, 136]
179
Kokusaginine
T. malabarica(S)
[113]
180
N-Methyl-2-pyrrolidone
T. capillipes
[61, 135]
181
Choline
T. cordifolia(S)
[137]
182
2,3-Dihydroxy-N-(2S,3S,4R)-1,3,4-trihydroxyicosan-2-yltetracosanamide
T. oblongifolia
[144]
C6-C3-C6
183
Tinosporinone
T. malabarica(HW)
[113]
184
5-Alloxy-6,7,4′-trimethyl flavone
T. malabarica(HW)
[113]
185
Kaempferol
T. malabarica(S)
[113]
186
Astragalin
T. malabarica(S)
[113]
187
Quercitroside
T. malabarica(S)
[113]
188
(−)-Epicatechin
T. cordifolia(S)
[145]
189
Ecoisolariciresinol
T. crispa
[38]
190
Secoisolariciresinol-9′-O-β-D-glucopyranoside
T. capillipes
[38, 94]
191
Sagitiside A
T. sagittatavar. yunnanensis(R)
[105, 112]
192
(+)-Lyoniresinol-2α-O-β-D-glucopyranoside
T. sagittatavar. yunnanensis(R)
[105, 112]
193
(+)-5′-Methoxyisolariciresinol-3α-O-β-D-glucopyranoside
T. sagittatavar. yunnanensis(R)
[105, 112]
194
3(a,4-dihydroxy-3-methoxybenzyl)-4-(4-hydroxy-3-methoxybenzyl) tetrahydrofuran
T. cordifolia(S)
[113]
195
Lirioresino-β-dimethyl ether
T. sinensis
[140]
196
Pinoresinol-di-O-glucoside
T. capillipes
[94]
197
(−)-Pinoresinol-4-O-β-D-glucopyranoside
T. sinensis(S)
[103]
198
8′-Epitanegool
T. sinensis(S)
[103]
199
Syringin
T. crispa
[142]
200
Tinoscorside D
T. capillipes
[94]
201
Cordifolioside A
T. capillipes
[38, 61, 135]
202
Docosyl-3,4-dihydroxy-trans-cinnamate
T. oblongifolia
[144]
203
2-(40-Hydroxyphenyl)-ethyl lignocerate
T. oblongifolia
[144]
204
Tinotuberide
T. crispa(S)
[113]
205
Isoorientin 2′′-O-(E)-sinapate
T. crispa(L)
[146]
206
2′′-(E)-p-coumarate
T. crispa(L)
[146]
207
Cosmosiin 6′′-(E)-ferulate
T. crispa(L)
[146]
208
Cosmosiin 6′′-(E)-cinnamate
T. crispa(L)
[146]
Steroids
209
β-Sitosterol
T. oblongifolia
[144]
210
β-Sitosterol glycoside
T. oblongifolia
[144]
211
β-Sitosterol
T. cordifolia(S)
T. sinensis(S)
T. craveniana(R)
[102, 140]
212
2-Deoxy crustecdysone
T. capillipes(R)
[113]
213
2-Deoxy-3-epicrustecdysone
T. capillipes(R)
[113]
214
2-Deoxy-3-epicrustecdysone-3-O-D-glucopyranoside
T. capillipes(R)
[113]
215
20α-Hydroxy ecdysone
T. cordifolia(S)
[113]
216
20β-Hydroxy-ecdysone-2-O-β-glucopyranoside
T. craveniana(R)
[147]
217
20β-Hydroxyecdysone
T. sagittata(R)
T. capillipes(R)
T. craveniana(R)
[14, 106, 133, 139]
218
2-Deoxy-20β-hydroxyecdysone
T. sagittata(R)
T. capillipes(R)
[14, 106, 139]
219
2-Deoxy-3-epi-20β-hydroxyecdysone
T. sagittata(R)
T. capillipes(R)
[14, 139]
220
2-Deoxy-20β-hydroxyecdysone-3-O-glucopyranoside
T. sagittata(R)
T. capillipes(R)
[14, 106, 139]
221
Polypodine B 20,22-acetonide
T. cordifolia
[94]
222
Makisterone A
T. hainanensis(S)
[148]
223
24-Epi-makisterone A
T. hainanensis(S)
[148]Figure 1
The structures of compounds isolated fromTinospora.


### 4.1. Terpenoids

#### 4.1.1. Diterpenes

Diterpenoids with clerodane-type skeleton represent the second most prevalent chemical class of the genus with over one hundred. They are the characteristic-hot constituents containing 20-C atom forming a 6/6 bicyclic skeleton. According to different stereochemistry of the decalin ring junction, this type of structure is grouped intoneo-clerodane,ent-clerodane, andnor-neo-clerodane. The skeleton ofneo-clerodane andent-clerodane-type diterpene is the same, with some differences in the configuration. The spatial locations of angular methyl group linked at C-5, C-8, and C-9 are differed. Specifically, it is C-5(S), C-8(R), and C-9(S) forneo-clerodane and C-5(R), C-8(S), and C-9(R) forent-clerodane.Nor-neo-clerodane is an oxidation product ofneo-clerodane, and one more five-membered unsaturated lactone ring is added compared withneo-clerodane [89]. We prefer to divide the diterpenoids into another two major types. A naphthalene ring fused to a lactone ring forms the main basic structure of the diterpenoids. The C-9 position often has a six-membered side chain and usually forms a furan-ring moiety. Lactone ring commonly exists in this type of compounds. Most of the lactone rings connect between C-8 and C-12 constituting the three six-membered rings with the similar arrangement of phenanthrene. Meanwhile, those ones without a lactone ring linked at C-8 and C-12 account for another small part of terpenoids. Apart from what is mentioned above, the few ones with isomerization in ring C then are in another group.(1) Diterpenes with Type 1 Skeleton. Compounds with type 1 skeleton are abundant in this genus. Due to the decalin core motif, Diels-Alder cycloaddition proved to be a valuable tool in their construction [90]. However, different synthetic routes result in the different positions of double bond and distinct substituents of the parallelized 6/6 bicyclic skeleton. These compounds are mostly reported fromT.cordifolia,T.crispa, andT.sagittata, while other plant species possess a few. Compounds1–60 are typical clerodane-type diterpenes characterized by the presence of one or more double bond or methyl metabolites attached to the clerodane core [91–105]. Compounds61–86 have distinct connecting mode of lactone ring linked at C-1/C-18 with different spatial configurations of substituent groups [91–93, 106–112]. Compounds87–92, as a tiny type, caused more attention to be paid to the ethane thiolate group [101, 108, 113, 114]. Tinosporaclerodanoid (93) is a particular clerodane-type diterpene without a furan-ring at C-12 superseded by a butane terminated glycol, never isolated from a natural or synthetic source before. Location of the carbonyl group is determined to be at C-1 [102].(2) Diterpenes with Type 2 Skeleton. This is another type of clerodane diterpene without the C-8/C-12 linking lactone ring, yielding a variant genre of diterpenes featured by the decalin ring junction of different stereochemistry. In these compounds, the C-8 position always contains a methyl or methoxycarbonyl group, which obstructs the formation of the lactone bridge between C-8 and C-12; see compounds94 to112. They are mainly identified fromT.cordifolia,T. tuberculate,T. sagittatavar. yunnanensis, andT.sagittata. There is an ester bond connected between C-1 and C-4 (106–108). One more sugar glucose unit in sagittatayunnanoside C (103) indicates that it may be the derivative of sagittatayunnanoside A (102) via the glycosylation of hydroxyl at C-18 [97, 115].(3) Diterpenes with Other Structures. Compounds113–116 are structurally similar, and the differences between the basic structures are the oxidation degree of ring A. The diversity among their lactone ring may be an influencing factor causing these compounds to exhibit varying degrees of biological activities against the same cancer cell line [96, 115, 116]. Compounds117–120 were isolated from the stem ofT.rumphii, and the lactone ring linking between C-6 and C-8 is quite remarkable [117, 118]. Compounds121-122 fromT.crispa have the same nucleus structure as compound117’s. Compounds123–126 are rearranged clerodane diterpene, obtained from the stems ofT.baenzigeri, of which baenzigeride (123) and baenzigeroside (124), as the novel skeleton, arising from thecis-ent-neo-clerodane epoxide are considered to be the precursor of compounds125 and126[119].


#### 4.1.2. Other Terpenes

There are ten compounds in this category, involving three triterpenoids (127–129), six sesquiterpenoids (130–135), and one monoterpenoid (136). Cycloeuphordenol is a novel 31-nortriterpene inT.malabarica. The other two new triterpenes are cycloeucalenol and cycloeucalenone producing mild cardiotonic effects [120]. A new rearranged cadinane sesquiterpene glycoside, tinosinenside, is tested to have inhibitory activities againstR-glucosidase [101]. Tinocordiside, consisting of a tricyclic skeleton with a cyclobutane ring, is found in the aqueous fraction ofT.cordifolia [61, 121]. Compounds133–135 possessing the core structure of the parallelized five-membered and seven-membered rings are new daucane-type sesquiterpenes [122]. Compound136 is the only monoterpenoid isolated from the methanol extract ofT.cordifolia [94].


### 4.2. Alkaloids

#### 4.2.1. Berberine Alkaloids

Twenty berberine alkaloids from genusTinospora have been reported (137–156). Commonly, the exact positions of methoxy groups are linked at C-2, C-3, C-9, and C-10. Rings C and D are coplanar with each other and in good conjugation, yet rings A and B are not in the same plane because of the saturated carbons at C-5 and C-6. If the methyl linked at C-2 or C-3 is lost, it cannot be well stabilized by the vicinal unsaturated ring C. When the methyl is lost from C-9, a stable intermediate could be formed. After the loss of two H atoms at C-5 and C-6 (rings A and B are in a plane at this time), the two methoxy groups at C-2 and C-3 can also turn into a methylenedioxy group [139]. The polarity of the overall compound interrelates with the inhibitory of cholinesterases [86].


#### 4.2.2. Aporphine Alkaloids

Nine (157–165) aporphine alkaloids are isolated and characterized in this genus. When the hydroxyl is at C-1, it seems more favorable that the H2O-group tends to be lost in successive fragmentation. Possessing two pairs of vicinal hydroxyl and methoxy groups (usually between C-1 and C-2 and C-10 and C-11), two molecules of methanol moieties tend to be lost consecutively. In mass spectrometry, the losses of methyls and the second molecule of methanol may as well be considered to be minor fragmentation. Such changes may be related to the steric interactions between the hydroxyl at C-1 and the epoxy group at C-10 and C-11. Meanwhile, the stable aromatic ring prevents diversifications in the core structure [139].


#### 4.2.3. Other Alkaloids

Compounds166–169 are benzylisoquinoline alkaloids which play a significant role as a minute part. Compounds170–174 fromT.crispa acted in concert on the cardiovascular system of anesthetized rats [142, 143]. Compounds175–178 are homologous provided with identical basic skeleton [149, 150].


### 4.3. C6-C3 and C6-C3-C6 Constituents

Other components isolated from the genus include six flavonoids, ten lignans, and several others [94, 103, 140]. Compounds183–188 belonging to flavonoid are the antioxidant profile of genusTinospora. It is noteworthy that (−) epicatechin was reported for the first time in the stem extract ofT.cordifolia[145]. Compounds189–198 are lignan in which192–194 were tested having obvious antioxidant activity [112]. Syringin (199) isolated from the crude butanol extract ofT.crispa acted in concert with other active ingredients on the cardiovascular system [142]. Tinoscorside D (200) was isolated and elucidated based on spectroscopic data from the methanol extract ofT.cordifolia aerial parts [94].


### 4.4. Steroids

Tinospora plants also contain steroids that are usually encountered in natural sources, and those presented in genusTinospora are mostly derivatives of β-sitosterol. Up to now, fifteen of this kind of ingredients have been isolated and identified. Among them, β-sitosterol and β-sitosteryl glucoside were frequently found inTinospora species [110, 113, 139, 144].


### 4.5. Polysaccharides

In the last decades polysaccharides from genusTinospora have caught a great deal of attention due to their broad spectrum of therapeutic properties and relatively low toxicity, particularly being immune-enhancing agents. The composition of polysaccharides fromT.cordifolia was reported as follows: glucose 98.0%, arabinose 0.5%, xylose 0.8%, galactose 0.3%, rhamnose 0.2%, and mannose 0.2% [151, 152]. Through subjecting to GC-MS analysis, the types of monosaccharides’ linkages were investigated to be terminal-glucose, 4-xylose, 4-glucose, 4,6-glucose, and 2,3,4,6-glucose [153]. Sharma et al. reported that the active polysaccharide fractions mainly constitute glucose, fructose, and arabinose as monomer units. The polymorphonuclear leukocyte function test concluded that they were responsible for immune enhancement [135]. Administration of the polysaccharide fraction fromT.cordifolia with tumor challenge inhibited (72.0%) the metastases formation in the lungs of syngeneic C57BL/6 mice [154]. Bioassay guided fractionation and separation of the ethanolic extract ofT.crispa leaf led to four acylated glycosylflavonoids. Among the isolated compounds, isovitexin 2′′-(E)-p-coumarate (206) showed the best activity against α-glucosidase with an IC50 value of 4.3
±
1.4 μM [146].


## 4.1. Terpenoids

### 4.1.1. Diterpenes

Diterpenoids with clerodane-type skeleton represent the second most prevalent chemical class of the genus with over one hundred. They are the characteristic-hot constituents containing 20-C atom forming a 6/6 bicyclic skeleton. According to different stereochemistry of the decalin ring junction, this type of structure is grouped intoneo-clerodane,ent-clerodane, andnor-neo-clerodane. The skeleton ofneo-clerodane andent-clerodane-type diterpene is the same, with some differences in the configuration. The spatial locations of angular methyl group linked at C-5, C-8, and C-9 are differed. Specifically, it is C-5(S), C-8(R), and C-9(S) forneo-clerodane and C-5(R), C-8(S), and C-9(R) forent-clerodane.Nor-neo-clerodane is an oxidation product ofneo-clerodane, and one more five-membered unsaturated lactone ring is added compared withneo-clerodane [89]. We prefer to divide the diterpenoids into another two major types. A naphthalene ring fused to a lactone ring forms the main basic structure of the diterpenoids. The C-9 position often has a six-membered side chain and usually forms a furan-ring moiety. Lactone ring commonly exists in this type of compounds. Most of the lactone rings connect between C-8 and C-12 constituting the three six-membered rings with the similar arrangement of phenanthrene. Meanwhile, those ones without a lactone ring linked at C-8 and C-12 account for another small part of terpenoids. Apart from what is mentioned above, the few ones with isomerization in ring C then are in another group.(1) Diterpenes with Type 1 Skeleton. Compounds with type 1 skeleton are abundant in this genus. Due to the decalin core motif, Diels-Alder cycloaddition proved to be a valuable tool in their construction [90]. However, different synthetic routes result in the different positions of double bond and distinct substituents of the parallelized 6/6 bicyclic skeleton. These compounds are mostly reported fromT.cordifolia,T.crispa, andT.sagittata, while other plant species possess a few. Compounds1–60 are typical clerodane-type diterpenes characterized by the presence of one or more double bond or methyl metabolites attached to the clerodane core [91–105]. Compounds61–86 have distinct connecting mode of lactone ring linked at C-1/C-18 with different spatial configurations of substituent groups [91–93, 106–112]. Compounds87–92, as a tiny type, caused more attention to be paid to the ethane thiolate group [101, 108, 113, 114]. Tinosporaclerodanoid (93) is a particular clerodane-type diterpene without a furan-ring at C-12 superseded by a butane terminated glycol, never isolated from a natural or synthetic source before. Location of the carbonyl group is determined to be at C-1 [102].(2) Diterpenes with Type 2 Skeleton. This is another type of clerodane diterpene without the C-8/C-12 linking lactone ring, yielding a variant genre of diterpenes featured by the decalin ring junction of different stereochemistry. In these compounds, the C-8 position always contains a methyl or methoxycarbonyl group, which obstructs the formation of the lactone bridge between C-8 and C-12; see compounds94 to112. They are mainly identified fromT.cordifolia,T. tuberculate,T. sagittatavar. yunnanensis, andT.sagittata. There is an ester bond connected between C-1 and C-4 (106–108). One more sugar glucose unit in sagittatayunnanoside C (103) indicates that it may be the derivative of sagittatayunnanoside A (102) via the glycosylation of hydroxyl at C-18 [97, 115].(3) Diterpenes with Other Structures. Compounds113–116 are structurally similar, and the differences between the basic structures are the oxidation degree of ring A. The diversity among their lactone ring may be an influencing factor causing these compounds to exhibit varying degrees of biological activities against the same cancer cell line [96, 115, 116]. Compounds117–120 were isolated from the stem ofT.rumphii, and the lactone ring linking between C-6 and C-8 is quite remarkable [117, 118]. Compounds121-122 fromT.crispa have the same nucleus structure as compound117’s. Compounds123–126 are rearranged clerodane diterpene, obtained from the stems ofT.baenzigeri, of which baenzigeride (123) and baenzigeroside (124), as the novel skeleton, arising from thecis-ent-neo-clerodane epoxide are considered to be the precursor of compounds125 and126[119].


### 4.1.2. Other Terpenes

There are ten compounds in this category, involving three triterpenoids (127–129), six sesquiterpenoids (130–135), and one monoterpenoid (136). Cycloeuphordenol is a novel 31-nortriterpene inT.malabarica. The other two new triterpenes are cycloeucalenol and cycloeucalenone producing mild cardiotonic effects [120]. A new rearranged cadinane sesquiterpene glycoside, tinosinenside, is tested to have inhibitory activities againstR-glucosidase [101]. Tinocordiside, consisting of a tricyclic skeleton with a cyclobutane ring, is found in the aqueous fraction ofT.cordifolia [61, 121]. Compounds133–135 possessing the core structure of the parallelized five-membered and seven-membered rings are new daucane-type sesquiterpenes [122]. Compound136 is the only monoterpenoid isolated from the methanol extract ofT.cordifolia [94].


## 4.1.1. Diterpenes

Diterpenoids with clerodane-type skeleton represent the second most prevalent chemical class of the genus with over one hundred. They are the characteristic-hot constituents containing 20-C atom forming a 6/6 bicyclic skeleton. According to different stereochemistry of the decalin ring junction, this type of structure is grouped intoneo-clerodane,ent-clerodane, andnor-neo-clerodane. The skeleton ofneo-clerodane andent-clerodane-type diterpene is the same, with some differences in the configuration. The spatial locations of angular methyl group linked at C-5, C-8, and C-9 are differed. Specifically, it is C-5(S), C-8(R), and C-9(S) forneo-clerodane and C-5(R), C-8(S), and C-9(R) forent-clerodane.Nor-neo-clerodane is an oxidation product ofneo-clerodane, and one more five-membered unsaturated lactone ring is added compared withneo-clerodane [89]. We prefer to divide the diterpenoids into another two major types. A naphthalene ring fused to a lactone ring forms the main basic structure of the diterpenoids. The C-9 position often has a six-membered side chain and usually forms a furan-ring moiety. Lactone ring commonly exists in this type of compounds. Most of the lactone rings connect between C-8 and C-12 constituting the three six-membered rings with the similar arrangement of phenanthrene. Meanwhile, those ones without a lactone ring linked at C-8 and C-12 account for another small part of terpenoids. Apart from what is mentioned above, the few ones with isomerization in ring C then are in another group.(1) Diterpenes with Type 1 Skeleton. Compounds with type 1 skeleton are abundant in this genus. Due to the decalin core motif, Diels-Alder cycloaddition proved to be a valuable tool in their construction [90]. However, different synthetic routes result in the different positions of double bond and distinct substituents of the parallelized 6/6 bicyclic skeleton. These compounds are mostly reported fromT.cordifolia,T.crispa, andT.sagittata, while other plant species possess a few. Compounds1–60 are typical clerodane-type diterpenes characterized by the presence of one or more double bond or methyl metabolites attached to the clerodane core [91–105]. Compounds61–86 have distinct connecting mode of lactone ring linked at C-1/C-18 with different spatial configurations of substituent groups [91–93, 106–112]. Compounds87–92, as a tiny type, caused more attention to be paid to the ethane thiolate group [101, 108, 113, 114]. Tinosporaclerodanoid (93) is a particular clerodane-type diterpene without a furan-ring at C-12 superseded by a butane terminated glycol, never isolated from a natural or synthetic source before. Location of the carbonyl group is determined to be at C-1 [102].(2) Diterpenes with Type 2 Skeleton. This is another type of clerodane diterpene without the C-8/C-12 linking lactone ring, yielding a variant genre of diterpenes featured by the decalin ring junction of different stereochemistry. In these compounds, the C-8 position always contains a methyl or methoxycarbonyl group, which obstructs the formation of the lactone bridge between C-8 and C-12; see compounds94 to112. They are mainly identified fromT.cordifolia,T. tuberculate,T. sagittatavar. yunnanensis, andT.sagittata. There is an ester bond connected between C-1 and C-4 (106–108). One more sugar glucose unit in sagittatayunnanoside C (103) indicates that it may be the derivative of sagittatayunnanoside A (102) via the glycosylation of hydroxyl at C-18 [97, 115].(3) Diterpenes with Other Structures. Compounds113–116 are structurally similar, and the differences between the basic structures are the oxidation degree of ring A. The diversity among their lactone ring may be an influencing factor causing these compounds to exhibit varying degrees of biological activities against the same cancer cell line [96, 115, 116]. Compounds117–120 were isolated from the stem ofT.rumphii, and the lactone ring linking between C-6 and C-8 is quite remarkable [117, 118]. Compounds121-122 fromT.crispa have the same nucleus structure as compound117’s. Compounds123–126 are rearranged clerodane diterpene, obtained from the stems ofT.baenzigeri, of which baenzigeride (123) and baenzigeroside (124), as the novel skeleton, arising from thecis-ent-neo-clerodane epoxide are considered to be the precursor of compounds125 and126[119].


## 4.1.2. Other Terpenes

There are ten compounds in this category, involving three triterpenoids (127–129), six sesquiterpenoids (130–135), and one monoterpenoid (136). Cycloeuphordenol is a novel 31-nortriterpene inT.malabarica. The other two new triterpenes are cycloeucalenol and cycloeucalenone producing mild cardiotonic effects [120]. A new rearranged cadinane sesquiterpene glycoside, tinosinenside, is tested to have inhibitory activities againstR-glucosidase [101]. Tinocordiside, consisting of a tricyclic skeleton with a cyclobutane ring, is found in the aqueous fraction ofT.cordifolia [61, 121]. Compounds133–135 possessing the core structure of the parallelized five-membered and seven-membered rings are new daucane-type sesquiterpenes [122]. Compound136 is the only monoterpenoid isolated from the methanol extract ofT.cordifolia [94].


## 4.2. Alkaloids

### 4.2.1. Berberine Alkaloids

Twenty berberine alkaloids from genusTinospora have been reported (137–156). Commonly, the exact positions of methoxy groups are linked at C-2, C-3, C-9, and C-10. Rings C and D are coplanar with each other and in good conjugation, yet rings A and B are not in the same plane because of the saturated carbons at C-5 and C-6. If the methyl linked at C-2 or C-3 is lost, it cannot be well stabilized by the vicinal unsaturated ring C. When the methyl is lost from C-9, a stable intermediate could be formed. After the loss of two H atoms at C-5 and C-6 (rings A and B are in a plane at this time), the two methoxy groups at C-2 and C-3 can also turn into a methylenedioxy group [139]. The polarity of the overall compound interrelates with the inhibitory of cholinesterases [86].


### 4.2.2. Aporphine Alkaloids

Nine (157–165) aporphine alkaloids are isolated and characterized in this genus. When the hydroxyl is at C-1, it seems more favorable that the H2O-group tends to be lost in successive fragmentation. Possessing two pairs of vicinal hydroxyl and methoxy groups (usually between C-1 and C-2 and C-10 and C-11), two molecules of methanol moieties tend to be lost consecutively. In mass spectrometry, the losses of methyls and the second molecule of methanol may as well be considered to be minor fragmentation. Such changes may be related to the steric interactions between the hydroxyl at C-1 and the epoxy group at C-10 and C-11. Meanwhile, the stable aromatic ring prevents diversifications in the core structure [139].


### 4.2.3. Other Alkaloids

Compounds166–169 are benzylisoquinoline alkaloids which play a significant role as a minute part. Compounds170–174 fromT.crispa acted in concert on the cardiovascular system of anesthetized rats [142, 143]. Compounds175–178 are homologous provided with identical basic skeleton [149, 150].


## 4.2.1. Berberine Alkaloids

Twenty berberine alkaloids from genusTinospora have been reported (137–156). Commonly, the exact positions of methoxy groups are linked at C-2, C-3, C-9, and C-10. Rings C and D are coplanar with each other and in good conjugation, yet rings A and B are not in the same plane because of the saturated carbons at C-5 and C-6. If the methyl linked at C-2 or C-3 is lost, it cannot be well stabilized by the vicinal unsaturated ring C. When the methyl is lost from C-9, a stable intermediate could be formed. After the loss of two H atoms at C-5 and C-6 (rings A and B are in a plane at this time), the two methoxy groups at C-2 and C-3 can also turn into a methylenedioxy group [139]. The polarity of the overall compound interrelates with the inhibitory of cholinesterases [86].


## 4.2.2. Aporphine Alkaloids

Nine (157–165) aporphine alkaloids are isolated and characterized in this genus. When the hydroxyl is at C-1, it seems more favorable that the H2O-group tends to be lost in successive fragmentation. Possessing two pairs of vicinal hydroxyl and methoxy groups (usually between C-1 and C-2 and C-10 and C-11), two molecules of methanol moieties tend to be lost consecutively. In mass spectrometry, the losses of methyls and the second molecule of methanol may as well be considered to be minor fragmentation. Such changes may be related to the steric interactions between the hydroxyl at C-1 and the epoxy group at C-10 and C-11. Meanwhile, the stable aromatic ring prevents diversifications in the core structure [139].


## 4.2.3. Other Alkaloids

Compounds166–169 are benzylisoquinoline alkaloids which play a significant role as a minute part. Compounds170–174 fromT.crispa acted in concert on the cardiovascular system of anesthetized rats [142, 143]. Compounds175–178 are homologous provided with identical basic skeleton [149, 150].


## 4.3. C6-C3 and C6-C3-C6 Constituents

Other components isolated from the genus include six flavonoids, ten lignans, and several others [94, 103, 140]. Compounds183–188 belonging to flavonoid are the antioxidant profile of genusTinospora. It is noteworthy that (−) epicatechin was reported for the first time in the stem extract ofT.cordifolia[145]. Compounds189–198 are lignan in which192–194 were tested having obvious antioxidant activity [112]. Syringin (199) isolated from the crude butanol extract ofT.crispa acted in concert with other active ingredients on the cardiovascular system [142]. Tinoscorside D (200) was isolated and elucidated based on spectroscopic data from the methanol extract ofT.cordifolia aerial parts [94].


## 4.4. Steroids

Tinospora plants also contain steroids that are usually encountered in natural sources, and those presented in genusTinospora are mostly derivatives of β-sitosterol. Up to now, fifteen of this kind of ingredients have been isolated and identified. Among them, β-sitosterol and β-sitosteryl glucoside were frequently found inTinospora species [110, 113, 139, 144].


## 4.5. Polysaccharides

In the last decades polysaccharides from genusTinospora have caught a great deal of attention due to their broad spectrum of therapeutic properties and relatively low toxicity, particularly being immune-enhancing agents. The composition of polysaccharides fromT.cordifolia was reported as follows: glucose 98.0%, arabinose 0.5%, xylose 0.8%, galactose 0.3%, rhamnose 0.2%, and mannose 0.2% [151, 152]. Through subjecting to GC-MS analysis, the types of monosaccharides’ linkages were investigated to be terminal-glucose, 4-xylose, 4-glucose, 4,6-glucose, and 2,3,4,6-glucose [153]. Sharma et al. reported that the active polysaccharide fractions mainly constitute glucose, fructose, and arabinose as monomer units. The polymorphonuclear leukocyte function test concluded that they were responsible for immune enhancement [135]. Administration of the polysaccharide fraction fromT.cordifolia with tumor challenge inhibited (72.0%) the metastases formation in the lungs of syngeneic C57BL/6 mice [154]. Bioassay guided fractionation and separation of the ethanolic extract ofT.crispa leaf led to four acylated glycosylflavonoids. Among the isolated compounds, isovitexin 2′′-(E)-p-coumarate (206) showed the best activity against α-glucosidase with an IC50 value of 4.3
±
1.4 μM [146].


## 5. Metabolism and Pharmacokinetics

To date, there are few pharmacokinetics studies of the extracts of this genus. Previous pharmacokinetics studies ofTinospora mainly focused on its diterpenes and its derived compounds that fitted to a one-compartment model. Shi et al. reported that columbin (75) and its derivatives (72,74,80, and83) are commonly present in the root ofT.capillipesandT.sagittata. Columbin is the major urinary metabolites in rats to which these compounds were orally given. These components could be absorbed and metabolized by the gastrointestinal tract. But pharmacokinetic studies showed that absorption of diterpene components was poorly absorbed through the gastrointestinal tract and they were quickly eliminated. Owing to the similar structures, these compounds have parallel pharmacokinetic parametersin vivo. Columbin is eliminated with systemic clearance of 5.04
±
2.92 L/h/kg after i.v. administration demonstrating a little higher blood flow rate (3.3 L/h/kg in rats) than the hepar. It could be concluded that columbin was rapidly cleared via hepatic clearance and extrahepatic clearance. The volume of distribution at terminal phase was 8.73
±
3.11 L/kg demonstrating a much higher total body water of 0.67 L/kg. The above researches suggested that columbin has extensive distribution into extravascular systems. Because columbin is almost insoluble in water, the absolute oral bioavailability of columbin is only 3.18
±
2.22%. The time to reach the maximum plasma drug concentration is less than 5 mins. The aforementioned indicate that the absorption of columbin is limited and quickly eliminated [107, 155, 156]. So it is necessary to improve its absorptionin vivo for clinical application in further research on pharmaceutical preparation. Since few pharmacokinetics studies of the extracts ofTinospora species have been reported, the needs for mechanism of actions on them are necessary in view of the wide bioactivities and extensive uses, and metabolism and pharmacokinetics studies are warranted to find out its most perspective activities.


## 6. Safety Evaluation

The clinical existence for herbs of genusTinospora over centuries are in a way themselves evidences of therapeutic utility. No adverse effects have been reported in this genus prescribed under reasonable doses application.T.cordifolia is advocated as a tonic in infants and children to facilitate growth. The LD50 value forT.cordifolia is higher than 1 g/kg for oral administration [157]. Acute toxicity study with the dose of 3 g/kg demonstrated thatT.cordifolia does not have any side effects and reported no death of the experimental rats [158]. When administered in doses of 0.1 g/kg for 12 weeks,T.cordifolia does not trigger any unfavorable factors on liver and renal function parameters in rats. It precipitated the increment of leukocytosis with neutrophilia in rats while no such effect was observed in healthy humans [27, 159].T.cordifolia treatment does not display clastogenicity and DNA damaging effect in bone marrow erythrocytes and peripheral blood lymphocytes [160]. And no neurological impairment or marked central nervous system depressant activities were shown [161]. Administration ofT.cordifolia to healthy volunteers has been found to be safe in phase I study, and the drug was well tolerated [157, 162]. It has also been shown not to exert any conspicuous adverse reactions on the gastrointestinal system, renal system, cardiovascular system, and central nervous system [163]. However, systematic toxicological tests and safety evaluations have not been carried out extensively within theTinospora species. Generally speaking, it could be used safely at low and middle dosage. Since test for toxicology of chemical constituents in this genus are rare until now, more toxicity researches remain to be done.


## 7. Conclusions

The review paper summarized a total of 223 compounds and abundant clerodane-type diterpene components that were reported from the genusTinospora, with 152 references cited. We noted thatTinospora has an extensive distribution, targeted clinical application, and diverse biological and pharmacological activities of pure compounds and extracts. Previous phytochemical researches on the genus revealed the extensive presence of terpenoid, alkaloid, C6-C3 derivative, polysaccharide, and other compound types. The pharmacological activities were mainly regarded on antidiabetic, antioxidant, antitumor, anti-inflammatory, antimicrobial, antiosteoporosis, and immunostimulation activities. From the review, it can be seen that phytochemical investigations, pharmacological researches, and traditional clinical application mainly focus on 4Tinospora species,T.sagittata,T.capillipes,T.cordifolia, andT.crispa. The related chemical and biological activities toward otherTinospora species are still blank. So, plenty of further studies are necessary in order to illustrate the chemodiversity and to make full use of the biological significance of the compounds and extracts ofTinospora, especially the antidiabetic and antioxidant activities. Future works will be focused on the following aspects: to useTinospora species as chemopreventive and therapeutic agent for various diseases, or to use them as adjuncts to exiting drugs to the treatment of diabetes, or to discover important and interesting biologically active compounds from these herbs by bioactivity-guided isolation. The authors wish the review can provide valuable data for explorations and advanced researches ofTinospora species.



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*Source: 9232593-2016-08-28.xml*