Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

Phytochemical and pharmacological activity of triandra (Colebr.) Diels

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2020-0256.R1 Manuscript Type:ForReview Review Article Only Date Submitted by the 03-Sep-2020 Author:

Complete List of Authors: Nutmakul, Thanutchaporn; Mahidol University Faculty of Medicine Ramathibodi Hospital, Graduate Program in Nutrition

Yanang, Bioactive compounds, Biological activity, leaves, Tiliacora Keyword: triandra

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 2 of 39

1 2 3 4 Review Article 5 6 7 Phytochemical and pharmacological activity of Tiliacora triandra (Colebr.) Diels 8 9 Thanutchaporn Nutmakul* 10 11 Graduate Program in Nutrition, Faculty of Medicine, Ramathibodi Hospital, 12 13 14 Mahidol University, Ratchatewi, Bangkok, 10400, 15 16 * Corresponding author, Email address: [email protected] 17 18 19 20 Abstract 21 For Review Only 22 23 Tiliacora triandra (Colebr.) Diels is an indigenous in Southeast Asia 24 25 belongs to the family of . It has been widely used for cuisine and 26 27 medicinal purposes since long time. Many studies have investigated and reported of its 28 29 30 chemical constituents and pharmacological activities, however these data are scattered. 31 32 Therefore, this article aims to review on its traditional uses, phytochemicals and 33 34 pharmacological properties. Traditionally, this plant has been used for treatment of 35 36 37 various diseases such as fever, diabetic, hypertension, and gastrointestinal diseases. 38 39 Many alkaloids, phenolic compounds, fatty acids, and essential oils were found in 40 41 different plant parts. Numerous pharmacological studies have been reported supporting 42 43 those traditional uses such as antioxidant, neuroprotective, antidiabetic, antiplasmodial, 44 45 46 antipyretic and anti-inflammatory, anticancer, and antimicrobial activities. Accordingly, 47 48 this valuable plant seems to have a potential to develop as a functional food and new 49 50 drugs in the future. 51 52 53 54 55 Keywords: Tiliacora triandra, Yanang, Bioactive compounds, Biological activity, 56 57 leaves 58 59 60 1

For Proof Read only Page 3 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 1. Introduction 5 6 7 Tiliacora triandra (Colebr.) Diels is a climbing plant belongs to the family of 8 9 Menispermaceae. It is commonly found in limestone hills, evergreen forest, and scrub 10 11 jungle throughout Southeast Asia (Forman, 1991). In Thailand, it is known as “ya- 12 13 14 nang”, or thao-ya-nang or thao-wan-khiao or choi-nang or yat-nang depending on the 15 16 region of Thailand (Forest Herbarium). This plant has been utilized for culinary and 17 18 medicinal purposes for a long time. The leaf is widely used as an ingredient in many 19 20 Thai dishes such as shoot soup, spiced bamboo shoots, and senna leaf curry 21 For Review Only 22 23 (Chaveerach et al., 2016) and its juice is consumed as a functional beverage 24 25 (Weerawatanakorn, Rojsuntornkitti, Pan, & Wongwaiwech, 2018). In addition, this 26 27 plant has been generally used by folk healers for treatment of many diseases and as a 28 29 30 constituent of traditional antipyretics in the national list of essential medicines of 31 32 Thailand (National Drug System Development Committee [NDSDC], 2018). Numerous 33 34 studies have been reported to evidence its health benefits and medicinal properties. 35 36 37 However, to the best of our knowledge, these valuable data are scattered and there is no 38 39 comprehensive review on this plant. Thus, the aim of this article is to review on 40 41 traditional uses, phytochemicals and pharmacological properties of different parts of 42 43 this plant. 44 45 46 2. Botanical data 47 48 Tiliacora triandra (Colebr.) Diels belongs to the Menispermaceae family. 49 50 Limacia triandra (Colebr.) Hook. f. and Cocculus triandrus Colebr are botanical 51 52 53 synonym of this plant (Forman, 1988). It is a climbing shrub whose stem is slender and 54 55 has puberulous or glabrous. Leaves are elliptic or lanceolate about 6.5-11 cm long and 56 57 2-4 cm wide. It is a dioecious plant which means male and female flowers are on 58 59 60 1

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 4 of 39

1 2 3 4 separate plant. Inflorescences are in axillary peduncle cymes with a few flowers. Male 5 6 7 flowers are yellowish, small, and have 3 or 6 petals with 3 stamens. Female flowers are 8 9 small, and have 6 petals. Fruits are drupe, obovoid 3-4 mm long, with branches 10 11 (Forman, 1991). Roots are unstable, tortuous and conical in size. The root surface is 12 13 14 grayish-yellow with longitudinal wrinkles and crossing with cracks (Singharachai, 15 16 Palanuvej, Kiyohara, Yamada, & Ruangrungsi, 2011a). 17 18 3. Local and traditional uses 19 20 Tiliacora triandra is an indigenous plant in Southeast Asia and widely utilized 21 For Review Only 22 23 in Thailand. The vine-stem is used as a cordage (Forman, 1991). The leaf is consumed 24 25 as a vegetable and popular drunk as functional beverages (Weerawatanakorn et al., 26 27 2018). It might be due to the raw leaves contain high levels of beta-carotene, vitamin A, 28 29 30 calcium, and iron (Judprasong et al., 2015). 31 32 Apart from culinary use, it has medicinal value in traditional and folk medicine. 33 34 In Cambodia, the leafy shoot mixed with other are used for treatment of 35 36 37 dysentery (Forman, 1991). In Thailand, the leaves and roots are used in the traditional 38 39 antipyretic preparations (NDSDC, 2018). Moreover, it has been widely used by folk 40 41 healers for treatment of many diseases. A decoction of the whole plant mixed with other 42 43 plants is used for healing aphthous ulcer (Neamsuvan, Tuwaemaengae, Bensulong, 44 45 46 Asae, & Mosamae, 2012). A decoction of the roots is used for lowering blood sugar 47 48 (Neamsuvan, Madeebing, Mah, & Lateh, 2015), antipyretic and antidote (Maneenoon et 49 50 al., 2015), and treating gastrointestinal diseases such as GERD, constipation, diarrhea 51 52 53 and bilharzia (Neamsuvan, Phumchareon, Bunphan, & Kaosaeng, 2016). In addition, a 54 55 decoction of fresh roots and leaves is used for treatment of hypertension by drinking 56 57 instead of plain water (Neamsuvan, Komonhiran, & Boonming, 2018). 58 59 60 2

For Proof Read only Page 5 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 4. Phytochemical constituents 5 6 7 Various chemical constituents have been reported in the roots, stems and leaves 8 9 of T. triandra as demonstrated in Figure 1. The roots and stems have many 10 11 bisbenzylisoquinoline alkaloids, of which some have been reported in both parts 12 13 14 including tiliacorine, tiliacorinine, nortiliacorinine A, tiliacorinine 2’-N-oxide and 15 16 yanangcorinine (Dechatiwongse, Chavalittumrong, & Nutakul, 1987; Nutmakul et al., 17 18 2016; Pachaly & Khosravian, 1988a; Pachaly, Tan, Khosravian, & Klein, 1986; 19 20 Wiriyachitra & Phuriyakorn, 1981). Meanwhile yanangine, dinklacorine, (Pachaly & 21 For Review Only 22 23 Tan, 1986a) tilianangine, (Pachaly & Tan, 1986b) tiliageine, protoquercitol, 24 25 tilitriandrine, (Pachaly & Khosravian, 1988b) magnoflorine, nortiliacorine A, 26 27 noryanangine, and norisoyanangine (Pachaly & Khosravian, 1988a) have been reported 28 29 30 only in the stems. In addition, many fatty acids have been reported in the stem including 31 32 5,7-dihydroxy-6-oxoheptadecanoic acid, ethyl-5,7-dihydroxy-6-oxooctadecanoate, ethyl 33 34 linolenate, ethyl linoleate, ethyl pheophorbide A, pheophorbide A (Makinde, 35 36 37 Ovatlarnporn, Sontimuang, Herbette, & Olatunji, 2020), palmitic acid, 38 39 dibenzylhydroxylamine, oleic acid and stearic acid (Makinde, Radenahmad, Adekoya, 40 41 & Olatunji, 2020). 42 43 Phytochemical studies of the leaves have been intensively investigated in the 44 45 46 past decade, and numerous bioactive compounds were explored. The alcoholic extracts 47 48 showed the presence of tannins, triterpenes, flavonoids, saponins and alkaloids 49 50 (Phadungkit, Somdee, & Kangsadalampai, 2012; Rattana, Phadungkit, & Cushnie, 51 52 53 2010). In addition, alkaloid oxoanolobine (Surapong, Benjamart, Ladachart, & Methin, 54 55 2016), and some polyphenols were identified such as santonin, minecoside, 56 57 protopseudohypericin, 3-O-methylluteolin glucoside malonylated, monoepoxy- 58 59 60 3

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 6 of 39

1 2 3 4 betacarotene, 3-demethoxy-9α-hydroxyligballinol-O-glucoside, p-hydroxybenzoic acid, 5 6 7 flavone glycoside cinnamic acids derivative, and flavanone glycoside (Boonsong, 8 9 Laohakunjit, & Kerdchoechuen, 2009). Quantification studies revealed high content of 10 11 phenolic compounds, vitamin E, fatty acids, and essential oils. In 100 mg of the water 12 13 14 extract contained quercetin 9,028.86 g, cyanidin 307.22 g, gallic acid 4.81 g 15 16 (Phunchago, Wattanathorn, & Chaisiwamongkol, 2015). The lyophilized leaves juice 17 18 powder contained total chlorophyll 3,551.6 mg/kg, rutin 1,762.1 mg/kg, tannic acid 19 20 21 1,213.0 mg/kg, and isoquercetinFor Review 488.1 mg/kg, catechin Only 369.9 mg/kg, quercetin 62.6 22 23 mg/kg and gallic acid 42.4 mg/kg (Weerawatanakorn et al., 2018). The GC-MS analysis 24 25 of the methanolic extract revealed relatively high levels of vitamin E (26.29%), phytol 26 27 28 (19.57%) and 1-cyclohexenylacetic acid (8.59%) and the other compounds such as 29 30 oleamide, oleic acid, neophytadiene, palmitic acid, 5-hydroxymethyl-2- 31 32 furancarboxaldehyde, and 2,6-dimethyl-3-(methoxymethyl)-benzoquinone (Chaveerach 33 34 et al., 2016). In addition, distillation of the fresh leaves yielded essential oil 0.544% and 35 36 37 the GC-MS analysis revealed that the most abundant components were isophytol 38 39 (35.27%), linoleic acid (15.71%), n-hexadecanoic acid (15.53%), and other compounds 40 41 such as linalool, α-terpineol, p-vinylguaiacol, 1-hexanol, β-damascenone, 42 43 44 neophytadiene, tetradecanoid acid, 3-hexen-1-ol, sphatulenol, benzeneacetaldehyde, and 45 46 linalool oxide (Naibaho, Laohankunjit, & Kerdchoechuen, 2012). 47 48 5. Pharmacological activity 49 50 51 5.1 Antioxidant activity 52 53 Reactive oxygen species and oxidative stress are found to be associated with 54 55 pathogenesis of several diseases such as cancer, atherosclerosis, diabetes, arthritis, and 56 57 neurodegenerative diseases. Many studies suggest evidence that antioxidants might 58 59 60 4

For Proof Read only Page 7 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 have potential to prevent and ameliorate these diseases (Rajendran et al., 2014). Several 5 6 7 parts of T. triandra have been extensively investigated and reported for their antioxidant 8 9 activities. Various in vitro assays were used for evaluation including radical scavenging 10 11 of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azinobis (3-ethylbenzothiazoline-6- 12 13 14 sulfonic acid) diammonium salt (ABTS) cation, and nitric oxide, ferric reducing 15 16 antioxidant power (FRAP), and determination of total phenolic compound (TPC) and 17 18 total flavonoid compound (TFC). 19 20 According to the results shown in Table 1, T. triandra exhibited a wide range of 21 For Review Only 22 23 antioxidant activities, which depended on various factors such as material, part used, 24 25 solvent and extraction method. Even the same ethanolic roots extract was used, one 26 27 study exhibited DPPH scavenging activity with IC value of 83.64 g/ml 28 50 29 30 (Singharachai, Palanuvej, Kiyohara, Yamada, & Ruangrungsi, 2011b), while in another 31 32 study was 15.38 g/ml (Juckmeta & Itharat, 2012). In addition, among different 33 34 35 fractions of the twigs and leaves, the ethyl acetate soluble fraction of the twigs exhibited 36 37 the highest activity for DPPH and FRAP assays, while the ethyl acetate soluble fraction 38 39 of the leaves exhibited the highest activity for ABTS assay and the n-hexane soluble 40 41 fraction of the twigs showed the best nitric oxide inhibition activity (Makinde, 42 43 44 Ovatlarnporn, Adekoya, Nwabor, & Olatunji, 2019). 45 46 For the leaves extracts, the ethanolic extract exhibited DPPH scavenging activity 47 48 with IC value of 14.51 g/ml (Phadungkit et al., 2012), while the methanolic extract 49 50 50 51 was 8.4 mg/ml (Chaveerach et al., 2016), the 80%ethanolic was 6,346.05 g extract/mg 52 53 DPPH (Nanasombat, Yansodthee &Jongjaited, 2019) and the freeze-dried leaves juice 54 55 56 was 0.29 mg Trolox equivalent/g (Chuacharoen, 2020). 57 58 59 60 5

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 8 of 39

1 2 3 4 The different solvent extraction exhibited different antioxidant activity. Among 5 6 7 petroleum ether, dichloromethane, ethyl acetate, methanol and water, the methanolic 8 9 extract displayed the best activity against DPPH and FRAP assays, and yielded the 10 11 highest TFC (Rattana et al., 2010). In another study, among water, ethanol and acetone, 12 13 14 the water extract showed the highest radical scavenging of DPPH and ABTS, reducing 15 16 power, and TPC (Singthong, Oonsivilai, Oonmetta-aree, & Ningsanond, 2014). 17 18 One study has been reported of the effect of different extraction method. Among 19 20 maceration, and batch stirring extraction at 30°C and 40°C, the ethanolic extracted by 21 For Review Only 22 23 batch stirring method at 30°C and 40°C yielded similar TPC. However, extraction at 24 25 30°C possessed the highest DPPH scavenging activity (Soradech, Kusolkumbot, & 26 27 Thubthimthed, 2018). 28 29 30 5.2 Neuroprotective activity 31 32 Tiliacora triandra has been documented as an ingredient in Thai traditional 33 34 rejuvenating and neurotonic remedies (Ingkaninan, Temkitthawon, Chuenchom, 35 36 37 Yuyaem, & Thongnoi, 2003) and possessed antioxidant activity mentioned earlier. 38 39 There are many studies reported on its neuroprotective activities (Table 2). 40 41 Alzheimer’s disease is a neurodegenerative disorder resulting in impaired 42 43 memory and behavior. One of the most promising approaches is to enhance the 44 45 46 acetylcholine level in the brain using acetylcholinesterase inhibitors. The methanolic 47 48 root extract at 0.1 mg/ml inhibited 42.29% of acetylcholinesterase activity (Ingkaninan 49 50 et al., 2003), while the 80%ethanolic extract of the leaves exhibited only 2.18% 51 52 53 inhibition (Nanasombat et al., 2019). 54 55 In animal studies, the extracts of T. triandra have been reported to improve brain 56 57 dysfunction and possess neuroprotective, neurotonic and antioxidant activities. In 58 59 60 6

For Proof Read only Page 9 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 alcoholic rat, the water extract of the aerial part at doses of 100, 200, and 400 mg/kg 5 6 7 could improve memory deficit induced by alcohol consumption, suppress 8 9 acetylcholinesterase activity, increase neuron density in hippocampus, decrease 10 11 malondialdehyde level, and increase the activities of antioxidant enzymes (Phunchago 12 13 14 et al., 2015). In cerebral ischemia/reperfusion (I/R) model, after 24 h surgery, the I/R 15 16 mice were orally administered with the ethanolic leaves extract at doses of 300 and 600 17 18 mg/kg. The extracts could enhance spatial learning, learning flexibility, spatial memory 19 20 and prevent hippocampal cell death (Thong-asa, Tumkiratiwong, Bullangpoti, 21 For Review Only 22 23 Kongnirundonsuk, & Tilokskulchai, 2017). In addition, when the mice were 24 25 administered with the extracts before I/R induction for 2 weeks, these extracts possessed 26 27 neuroprotective effects by significantly reducing calcium and malondialdehyde levels, 28 29 30 increasing the activities of antioxidant enzymes, attenuating brain infarction, and 31 32 decreasing the percentage of dead cells in the cerebral cortex and hippocampus (Thong- 33 34 asa & Bullangpoti, 2020). Moreover, these extracts possessed neurotonic effect by 35 36 37 enhancing spatial learning, memory, and learning flexibility, and increasing choline 38 39 acetyltransferase activity and hippocampal cell density in mice (Thong-asa & 40 41 Laisangunngam, 2018). 42 43 5.3 Antidiabetic activity 44 45 46 Based on the folk knowledge, the roots (Neamsuvan et al., 2015) and leaves 47 48 (Udyanin, Bumrerraj, & Nimsuntron, 2013) has been used for reducing blood sugar. 49 50 Several in vitro and in vivo antidiabetic studies, therefore, have reported supporting the 51 52 53 folk knowledge (Table 2). 54 55 α-amylase and α-glucosidase inhibition assays were used for evaluation in in 56 57 vitro studies. Inhibition of these enzymes resulted in delayed the glucose absorption in 58 59 60 7

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 10 of 39

1 2 3 4 digestive tract and reduced the post-prandial hyperglycemia (Tundis, Loizzo, & 5 6 7 Menichini, 2010). The 80%ethanolic extract of the leaves could inhibit the activity of 8 9 both α-amylase (78.28%) and α-glucosidase (10.30%) (Nanasombat et al., 2019). In 10 11 another study, n-hexane soluble fraction of the twigs exhibited the inhibitory activities 12 13 14 against α-amylase and α-glucosidase with the IC50 of 93.74 and 3.40 μg/mL, 15 16 respectively (Makinde et al., 2019). Six compounds, isolated from the aerial part, have 17 18 been discovered and tested for the activity. Only 5,7-dihydroxy-6-oxoheptadecanoic 19 20 21 acid possessed inhibitoryFor activity Review against both α-glucosidase Only (IC50=11.58 M) and α- 22 23 amylase (IC50=26.27 M), while the rest could inhibit only α-glucosidase with IC50 24 25 values ranging from 22.11 to 424.06 M (Makinde, Ovatlarnporn, et al., 2020). 26 27 28 In in vivo studies, after administration of the ethanolic leaves extract (300 29 30 mg/kg) for 8 weeks, the extract significantly decreased blood glucose level, increased 31 32 serum insulin level, and activated the regeneration of pancreatic Islets of Langerhans in 33 34 35 diabetic rats (Katisart & Rattana, 2017). In another study, the ethanolic leaves extract 36 37 (300 and 600 mg/kg) significantly decreased the blood glucose and serum insulin, and 38 39 increased the liver and muscle glycogen contents in mice with high sugar intake 40 41 42 (Thong-asa, Prasertsuksri, Sakamula, & Nimnuan, 2019). Furthermore, the ethanolic 43 44 extract of the aerial part significantly reduced blood glucose level, lipid profiles (except 45 46 HDL-cholesterol), and improved liver and kidney functions in high fat diet and 47 48 49 streptozotocin-induced diabetes in rats (Makinde, Radenahmad, et al., 2020). 50 51 5.4 Antiplasmodial activity 52 53 Malaria is still a global health problem, caused by Plasmodium species. 54 55 Especially, P. falciparum is the most virulent strain and could develop resistance to 56 57 58 almost all available drugs (Nutmakul et al., 2016). These stimulate efforts to 59 60 8

For Proof Read only Page 11 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 continuously discover new antimalarials. In countries where malaria is endemic, such 5 6 7 Thailand, numerous plants used to treat malaria have been investigated including T. 8 9 triandra roots which is used as antipyretics and prescribed in antimalarial preparations 10 11 in folk medicine (Dechatiwongse et al., 1987). 12 13 14 As shown in the Table 2, the methanolic roots extract inhibited clinical isolate P. 15 16 falciparum with IC50 of 17 g/ml. Five bisbenzylisoquinoline alkaloids, tiliacorinine, 17 18 tiliacorine, nor-tiliacorinine A, alkaloid G, and alkaloid H were further isolated and 19 20 21 exhibited the IC50 valuesFor ranging Review from 344 to 3533 Only ng/ml (Pavanand, Webster, 22 23 Yongvanitchit, & Dechatiwongse, 1989). In another study, the stems and roots extracts 24 25 were tested against P. falciparum chloroquine-sensitive (3D7) and chloroquine-resistant 26 27 28 (W2) strains. The chloroform extracts and methanolic extracts of both parts exhibited 29 30 potent activity against both parasite strains with IC50 values ranging from 1.22 to 5.73 31 32 g/ml. Tiliacorinine and yanangcorinine were further bioassay-guided isolated and 33 34 35 exhibited the activity against W2 strain with IC50 values of 2.14 and 1.55 g/ml, 36 37 respectively (Nutmakul et al., 2016). In addition, these compounds showed parasiticidal 38 39 effect with a slow onset of action and could potentiate the efficacy of chloroquine 40 41 42 against chloroquine-resistant strain since their structures contain many aromatic rings 43 44 and a protonated nitrogen which is a specific structure for chloroquine resistance 45 46 reversal agents (Nutmakul et al., 2020). 47 48 49 5.5 Antipyretic and anti-inflammatory activities 50 51 In Thailand, the root of T. triandra is used for treatment of fever and the leaf 52 53 juice is used for decreasing body temperature by folk healer (Maneenoon et al., 2015). 54 55 In the same way, the roots and leaves are used in traditional antipyretics listed in the 56 57 58 national list of essential medicines namely, “ha-rak” or “bencha-loga-wichian” (BLW), 59 60 9

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 12 of 39

1 2 3 4 and “mahanin-taengtong” preparations (NDSDC, 2018). Especially, BLW has been 5 6 7 widely used and confirmed its healing property by scientific approach. In yeast-induced 8 9 fever rats, oral administration of BLW powder significantly reduced rectal temperature 10 11 and the best activity was observed in T. triandra powder (Konsue, Sattayasai, Puapairoj, 12 13 14 & Picheansoonthon, 2008). In addition, the ethanolic extracts of BLW and T. triandra 15 16 showed nitric oxide inhibitory activity on LPS-induced RAW 264.7 cell lines with IC50 17 18 values of 40.36 and 54.65 μg/ml, respectively (Juckmeta & Itharat, 2012). 19 20 In addition, the lyophillized leaves juice (500 μg/ml) inhibited 56% nitrite 21 For Review Only 22 23 production in LPS-induced RAW 264.7 cell lines by down-regulation of iNOS and 24 25 COX-2 expression (Weerawatanakorn et al., 2018). In another study, oral 26 27 administration of the water leaves extract (1 g/kg) inhibited writhing reflex by 55.7% in 28 29 30 acetic acid-induced writhing model in mice (Tangsucharit, Kukongviriyapan, 31 32 Kukongviriyapan, & Airarat, 2006). The antipyretic and anti-inflammatory activities of 33 34 T. triandra extract from the roots and leaves are summarized in Table 2. 35 36 37 5.6 Anticancer activity 38 39 Tiliacora triandra is one of the plants that frequency used by folk healer in 40 41 anticancer formulation (Manosroi et al., 2015) and cancer prevention (Lumlerdkij, 42 43 Boonrak, Booranasubkajorn, Akarasereenont, & Heinrich, 2020). Several parts of this 44 45 46 plant has been investigated and reported for anticancer activity against various cancer 47 48 cell lines (Table 2). 49 50 The ethanolic root extract exhibited cytotoxicity on several lung cancer cell lines 51 52 53 including KB, Hep2, A549, COR-L23, and NCI-H226 with the IC50 values ranging 54 55 from 19.5 to 45.2 μg/ml (Juckmeta et al., 2019). The ethanolic stem extract possessed 56 57 cytotoxicity on HepG2 cell line with IC50 of 81.06 μg/ml (Lumlerdkij et al., 2020). The 58 59 60 10

For Proof Read only Page 13 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 methanolic leaves extract showed cytotoxicity against human colon cancer cell line 5 6 7 (HT-29) with IC50 value of 203.52 μg/ml (Manosroi et al., 2015). In another study, the 8 9 ethyl acetate, methanolic and water extracts exhibited cytotoxicity against oral cavity 10 11 cancer (KB) and lung cancer (NCI-H187) cell lines with IC50 values ranging from 11.93 12 13 14 to 32.15 μg/ml, and oxonanolobine, a main active compound isolated from the 15 16 methanolic extract, possessed the activity against NCI-H187 with IC50 of 27.6 μg/ml 17 18 (Surapong et al., 2016). 19 20 Moreover, different extracts of the leaves were tested against sensitive (A549) 21 For Review Only 22 23 and multidrug resistant (A549RT-eto) lung cancer cells. The dichloromethane extract 24 25 exhibited the most cytotoxic effect against both sensitive and resistant cells with IC50 of 26 27 22.0 and 48.5 μg/ml, respectively. However, although the hexane extract was inactive, a 28 29 30 mixture of fatty acids isolated from the hexane extract possessed a multidrug resistance 31 32 reversing activity by enhancing P-glycoprotein function in A549RT-eto cell line 33 34 (Kaewpiboon, Winayanuwattikun, Yongvanich, Phuwapraisirisan, & Assavalapsakul, 35 36 37 2014). 38 39 Tiliacorinine, an alkaloid isolated from the roots and stems, displayed 40 41 antiproliferative effect on cholangiocarcinoma cell lines and antitumor activity in 42 43 animal model. It inhibited growth of four human cholangiocarcinoma cell lines with 44 45 46 IC50 values ranging from 4.5-7.0 μM by inducing apoptosis via caspase-activation 47 48 pathways and rapidly reduced tumor growth in cholangiocarcinoma xenografted mice 49 50 (Janeklang et al., 2014). 51 52 53 5.7 Antimicrobial activity 54 55 The ethanolic roots extract exhibited wide spectrum antimicrobial activity 56 57 against Gram positive bacteria (Stahylococcus aureus, methicillin-resistant S. aureus, 58 59 60 11

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 14 of 39

1 2 3 4 and Streptococcus pyrogenes), Gram negative bacteria (Escherichia coli, Shigella spp, 5 6 7 Salmonella typhimurium, Acinetobacter buamannii, and Bacillus subtilis) and Candida 8 9 albicans with the inhibition zone ranging from 6.8 to 16.3 mm (Nuaeissara, Kondo, & 10 11 Itharat, 2011). In another study, the n-hexane and ethyl acetate fractions of the aerial 12 13 14 part exhibited the activity against B. cereus, E. coli, S. aureus, and Listeria 15 16 monocytogenes with the MBC values ranging from 1.5 to 12 mg/ml (Makinde et al., 17 18 2019). The essential oil extracted from the leaves possessed the activity against S. 19 20 aureus, B. cereus, E. coli, and Salmonella spp. with inhibition zone of 16, 14, 13 and 10 21 For Review Only 22 23 mm, respectively and the MIC values of 6.25 μl/ml. (Naibaho et al., 2012). Moreover, 24 25 Tiliacorinine, 2’-nortiliacorinine, and tiliacorine, isolated from the roots, were tested 26 27 against 59 clinical isolates of multidrug-resistant Mycobacterium tuberculosis and 28 29 30 exhibited the MIC values ranging from 0.7 to 6.2 μg/ml. (Sureram et al., 2012). The 31 32 antimicrobial activity of this plant is summarized in Table 2. 33 34 5.8 Other activities 35 36 37 The ethanolic leaves extract (30 mg/plate) inhibited mutagenicity of nitrite 38 39 treated 1-aminopyrene on S. typhimurium TA 98 and TA 100 by 44.84% and 58.84%, 40 41 respectively (Phadungkit et al., 2012). In another study, the ethanolic and water extracts 42 43 of the roots at concentrations of 5-15 mg/ml showed strong mutagenic inhibition 44 45 46 ranging from 88.42 to 123.12%. These extracts did not exhibit directly mutagenic 47 48 towards on both strains but exhibited indirect mutagenicity induced by nitrosation 49 50 (Singharachai et al., 2011b). 51 52 53 Since BLW has long been used for treatment of fever and skin rash, BLW and 54 55 its constituent plants were investigated for anti-allergic activity. The ethanolic extract of 56 57 BLW showed inhibitory effect on the release of -hexosaminidase from RBL-2H3 cells 58 59 60 12

For Proof Read only Page 15 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 with IC value of 39.8 μg/ml, whereas T. triandra was inactive (IC >100 μg/ml) 5 50 50 6 7 (Juckmeta, Thongdeeying, & Itharat, 2014). 8 9 In anti-grey hair activity, the ethanolic leaves extract not only possessed 10 11 antioxidant activity, it stimulated tyrosinase activity with SC50 values ranging from 3.46 12 13 14 to 3.97 mg/ml without cytotoxicity to human dermal skin fibroblast and melanoma 15 16 (B16F10) cells. In addition, at concentration of 0.05–1.0 mg/ml, the extract exhibited 17 18 the potential of stimulating melanin ranging from 128.69 to 131.46% (Soradech et al., 19 20 2018). 21 For Review Only 22 23 6. Toxicity 24 25 Based on the folk and traditional knowledge, the root and aerial parts of T. 26 27 triandra are often prepared by boiling with water and no side effects reported. Several 28 29 30 toxicity studies are scientific evidence supporting this knowledge. The water extract of 31 32 the whole plant did not produce acute or subchronic toxicity in rat, in terms of mortality, 33 34 changes in internal organ weight, gross appearance and histopathology of internal 35 36 37 organs, hematological profiles, and animal behaviors (Sireeratawong et al., 2008). In 38 39 another studies, the water extract of the stems and roots did not exhibit cytotoxicity on 40 41 peripheral blood mononuclear cells (IC50>100 μg/ml) while the dichloromethane and 42 43 methanolic extracts exhibited cytotoxicity with IC values ranging from 8.17 to 26.26 44 50 45 46 μg/ml (Nutmakul et al., 2016). In the same way, the water extract of the roots was less 47 48 toxic on Artemia salina than the ethanolic extract with LC50 values of 200 and 44 49 50 g/ml, respectively (Singharachai et al., 2011b). 51 52 53 7. Future perspectives 54 55 The aerial and root parts of T. triandra have been used as a culinary ingredient 56 57 and a medicinal plant for a long time without any side effect reported. The aerial part, 58 59 60 13

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 16 of 39

1 2 3 4 especially leaves, contain rich of nutrients and many phytochemicals such as flavonoids 5 6 7 and other phenolic compounds. In addition, it has been reported to have antioxidant, 8 9 neuroprotective, antidiabetic, anti-inflammatory, and anticancer activities. From a 10 11 commercial perspective, the aerial part of this plant should be further studied in details 12 13 14 for development as functional foods or nutraceuticals. For the root part, it is generally 15 16 used in traditional medicine to treat fever including malaria fever and has scientific 17 18 studies supporting the traditional uses. Moreover, the root extracts and its 19 20 bisbenzylisoquinoline alkaloids possessed antimicrobial and anticancer activities which 21 For Review Only 22 23 should be further studied on mechanisms of action and efficacy in human in order to 24 25 develop as new drugs. 26 27 8. Conclusion 28 29 30 Tiliacora triandra (Colebr.) Diels is an indigenous plant in Southeast Asia 31 32 which has been long time used for cuisine and medicinal purposes. Numerous studies 33 34 have been investigated and reported particularly in Thailand. However, these interesting 35 36 37 data are scattered. This article is the first review of the traditional uses, phytochemicals 38 39 and pharmacological properties of this plant. Apart from high nutritional value, many 40 41 ethnomedicinal survey studies revealed its medicinal properties for treatment of several 42 43 diseases such as fever, diabetic, hypertension, and gastrointestinal diseases. These 44 45 46 properties might be attributed to the presence of a variety of bioactive compounds such 47 48 as bisbenzylisoquinoline alkaloids, flavonoids, phenolic compounds, fatty acids, and 49 50 essential oils which have been found in different parts of this plant. Various 51 52 53 pharmacological activities such as antioxidant, neuroprotective, antidiabetic, 54 55 antiplasmodial, antipyretic and anti-inflammatory, anticancer, and antimicrobial 56 57 activities, have been reported as evidence supporting the traditional uses. In addition, 58 59 60 14

For Proof Read only Page 17 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 based on the history of consumption and use in medicinal remedies without any 5 6 7 reported side effect and proving by several toxicity studies, this valuable plant seems to 8 9 be safe and has potential to develop as functional foods and new drugs in the future. 10 11 However, further studies on mechanisms of action and efficacy in human are still 12 13 14 needed to be explored. 15 16 Conflict of interest 17 18 The author declares that there is no conflict of interest. 19 20 References 21 For Review Only 22 23 Boonsong, P., Laohakunjit, N., & Kerdchoechuen, O. (2009). Identification of 24 25 polyphenolic compounds and colorants from Tiliacora triandra (Diels) leaves. 26 27 Agricultural Science Journal, 40(Suppl.)(3), 13-16. 28 29 30 Chaveerach, A., Lertsatitthanakorn, P., Tanee, T., Puangjit, N., Patarapadungkit, N., & 31 32 Sudmoon, R. (2016). Chemical constituents, antioxidant property, cytotoxicity 33 34 and genotoxicity of Tiliacora triandra. International Journal of Pharmacognosy 35 36 37 and Phytochemical Research, 8(5), 722-729. 38 39 Chuacharoen, T. (2020). Development of yanang (Tiliacora triandra) powder using 40 41 freeze drying in use for sherbet. Paper presented at the International Conference 42 43 Business Education Social Sciences Tourism And Technology. 44 45 46 Dechatiwongse, T., Chavalittumrong, P., & Nutakul, W. (1987). Isolation of the in vitro 47 48 antimalarial principles from Tiliacora triandra Diels. Bulletin of the Department 49 50 of Medical Sciences, 29(1), 33-38. 51 52 53 Forest Herbarium. Thai Plant Name. Available from Forest Botany Division Tem 54 55 Smitinand's Thai Plant Names Retrieved 26 May 2020 56 57 https://www.dnp.go.th/botany/mplant/index.html 58 59 60 15

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 18 of 39

1 2 3 4 Forman, L. L. (1988). A synopsis of Thai Menispermaceae. Kew Bulletin, 43(3), 369- 5 6 7 407. 8 9 Forman, L. L. (1991). MENISPERMACEAE. In T. Smitinand & K. Larsen (Eds.), 10 11 Flora of Thailand (Vol.5(3)). Bangkok: The Chutima Press. 12 13 14 Ingkaninan, K., Temkitthawon, P., Chuenchom, K., Yuyaem, T., & Thongnoi, W. 15 16 (2003). Screening for acetylcholinesterase inhibitory activity in plants used in 17 18 Thai traditional rejuvenating and neurotonic remedies. Journal of 19 20 Ethnopharmacology, 89(2), 261-264. 21 For Review Only 22 23 Janeklang, S., Nakaew, A., Vaeteewoottacharn, K., Seubwai, W., Boonsiri, P., Kismali, 24 25 G., Suksamrarn, A., Okada, S., & Wongkham, S. (2014). In vitro and in vivo 26 27 antitumor activity of tiliacorinine in human cholangiocarcinoma. Asian Pacific 28 29 30 Journal of Cancer Prevention, 15(17), 7473-7478. 31 32 Juckmeta, T., & Itharat, A. (2012). Anti-inflammatory and antioxidant activities of Thai 33 34 traditional remedy called “Ya-Ha-Rak”. Journal of Health Research, 26(4), 205- 35 36 37 210. 38 39 Juckmeta, T., Pipatrattanaseree, W., Jaidee, W., Dechayont, B., Chunthorng-orn, J., 40 41 Andersen, R. J., & Itharat, A. (2019). Cytotoxicity to five cancer cell lines of the 42 43 respiratory tract system and anti-inflammatory activity of Thai traditional remedy. 44 45 46 Natural Product Communications, 14(5). 47 48 Juckmeta, T., Thongdeeying, P., & Itharat, A. (2014). Inhibitory effect on β- 49 50 hexosaminidase release from RBL-2H3 cells of extracts and some pure 51 52 53 constituents of Benchalokawichian, a Thai herbal remedy, used for allergic 54 55 disorders. Evidence-based complementary and alternative medicine, 2014. 56 57 58 59 60 16

For Proof Read only Page 19 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Judprasong, K., Puwastien, P., Rojroongwasinkul, N., Nitithamyong, A., Sridonpai, P., 5 6 7 & Somjai, A. (2015). Ya-nang, leaves, raw. Available from Institute of Nutrition, 8 9 Mahidol University Thai Food Composition Database Retrieved 26 May 2020 10 11 http://www.inmu.mahidol.ac.th/thaifcd 12 13 14 Kaewpiboon, C., Winayanuwattikun, P., Yongvanich, T., Phuwapraisirisan, P., & 15 16 Assavalapsakul, W. (2014). Effect of three fatty acids from the leaf extract of 17 18 Tiliacora triandra on P-glycoprotein function in multidrug-resistant A549RT-eto 19 20 cell line. Pharmacognosy magazine, 10(Suppl 3), S549-S556. 21 For Review Only 22 23 Katisart, T., & Rattana, S. (2017). Hypoglycemic activity of leaf extracts from Tiliacora 24 25 triandra in normal and streptozotocin-induced diabetic rats. Pharmacognosy 26 27 Journal, 9(5). 28 29 30 Konsue, A., Sattayasai, J., Puapairoj, P., & Picheansoonthon, C. (2008). Antipyretic 31 32 effects of Bencha-Loga-Wichien herbal drug in rats. Thai Journal of 33 34 Pharmacology, 29(1), 79-82. 35 36 37 Lumlerdkij, N., Boonrak, R., Booranasubkajorn, S., Akarasereenont, P., & Heinrich, M. 38 39 (2020). In vitro protective effects of plants frequently used traditionally in cancer 40 41 prevention in Thai traditional medicine: An ethnopharmacological study. Journal 42 43 of Ethnopharmacology, 250, 112409. 44 45 46 Makinde, E. A., Ovatlarnporn, C., Adekoya, A. E., Nwabor, O. F., & Olatunji, O. J. 47 48 (2019). Antidiabetic, antioxidant and antimicrobial activity of the aerial part of 49 50 Tiliacora triandra. South African Journal of Botany, 125, 337-343. 51 52 53 Makinde, E. A., Ovatlarnporn, C., Sontimuang, C., Herbette, G., & Olatunji, O. J. 54 55 (2020). Chemical constituents from the aerial part of Tiliacora triandra (Colebr.) 56 57 58 59 60 17

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 20 of 39

1 2 3 4 Diels and their α-glucosidase and α-amylase inhibitory activity. Natural Product 5 6 7 Communications, 15(1), 1-4. 8 9 Makinde, E. A., Radenahmad, N., Adekoya, A. E., & Olatunji, O. J. (2020). Tiliacora 10 11 triandra extract possesses antidiabetic effects in high fat diet/streptozotocin- 12 13 14 induced diabetes in rats. Journal of Food Biochemistry, e13239. 15 16 Maneenoon, K., Khuniad, C., Teanuan, Y., Saedan, N., Prom-in, S., Rukleng, N., 17 18 Kongpool, W., Pinsook, P., & Wongwiwat, W. (2015). Ethnomedicinal plants 19 20 used by traditional healers in Phatthalung province, Peninsular Thailand. Journal 21 For Review Only 22 23 of Ethnobiology and Ethnomedicine, 11(1), 43. 24 25 Manosroi, A., Akazawa, H., Akihisa, T., Jantrawut, P., Kitdamrongtham, W., Manosroi, 26 27 W., & Manosroi, J. (2015). In vitro anti-proliferative activity on colon cancer cell 28 29 30 line (HT-29) of Thai medicinal plants selected from Thai/Lanna medicinal plant 31 32 recipe database “MANOSROI III”. Journal of Ethnopharmacology, 161, 11-17. 33 34 Naibaho, N. M., Laohankunjit, N., & Kerdchoechuen, O. (2012). Volatile composition 35 36 37 and antibacterial activity of essential oil from yanang (Tiliacora triandra) leaves. 38 39 Agricultural Science Journal, 43(2(Suppl.)), 529-532. 40 41 Nanasombat, S., Yansodthee, K., & Jongjaited, I. (2019). Evaluation of antidiabetic, 42 43 antioxidant and other phytochemical properties of Thai fruits, vegetables and 44 45 46 some local food plants. Walailak Journal of Science and Technology, 16(11), 851- 47 48 866. 49 50 National Drug System Development Committee. (2018). National list of essential 51 52 53 medicines 2018. Thai Government Gazette Volume 135. Retrieved from 54 55 http://www.fda.moph.go.th/sites/drug/Shared%20Documents/New/nlem2561.PDF 56 57 58 59 60 18

For Proof Read only Page 21 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Neamsuvan, O., Komonhiran, P., & Boonming, K. (2018). Medicinal plants used for 5 6 7 hypertension treatment by folk healers in Songkhla province, Thailand. Journal of 8 9 Ethnopharmacology, 214, 58-70. 10 11 Neamsuvan, O., Madeebing, N., Mah, L., & Lateh, W. (2015). A survey of medicinal 12 13 14 plants for diabetes treating from Chana and Nathawee district, Songkhla province, 15 16 Thailand. Journal of Ethnopharmacology, 174, 82-90. 17 18 Neamsuvan, O., Phumchareon, T., Bunphan, W., & Kaosaeng, W. (2016). Plant 19 20 materials for gastrointestinal diseases used in Chawang district, Nakhon Si 21 For Review Only 22 23 Thammarat province, Thailand. Journal of Ethnopharmacology, 194, 179-187. 24 25 Neamsuvan, O., Tuwaemaengae, T., Bensulong, F., Asae, A., & Mosamae, K. (2012). A 26 27 survey of folk remedies for gastrointestinal tract diseases from Thailand's three 28 29 30 Southern border provinces. Journal of Ethnopharmacology, 144(1), 11-21. 31 32 Nuaeissara, S., Kondo, S., & Itharat, A. (2011). Antimicrobial activity of the extracts 33 34 from Benchalokawichian remedy and its components. Journal of the Medical 35 36 37 Association of Thailand, 94, S172-S177. 38 39 Nutmakul, T., Pattanapanyasat, K., Soonthornchareonnon, N., Shiomi, K., Mori, M., & 40 41 Prathanturarug, S. (2016). Antiplasmodial activities of a Thai traditional 42 43 antipyretic formulation, Bencha-Loga-Wichian: A comparative study between the 44 45 46 roots and their substitutes, the stems. Journal of Ethnopharmacology, 193, 125- 47 48 132. 49 50 Nutmakul, T., Pattanapanyasat, K., Soonthornchareonnon, N., Shiomi, K., Mori, M., & 51 52 53 Prathanturarug, S. (2020). Speed of action and stage specificity of Bencha-loga- 54 55 wichian, a Thai traditional antipyretic formulation, against Plasmodium 56 57 58 59 60 19

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 22 of 39

1 2 3 4 falciparum and the chloroquine-potentiating activity of its active compounds, 5 6 7 tiliacorinine and yanangcorinine. Journal of Ethnopharmacology, 258, 112909. 8 9 Pachaly, P., & Khosravian, H. (1988a). New bisbenzylisoquinoline alkaloids from 10 11 Tiliacora triandra. Planta Medica, 54(5), 433-437. 12 13 14 Pachaly, P., & Khosravian, H. (1988b). Tilitriandrin: a new bisbenzylisoquinoline 15 16 alkaloid from Tiliacora triandra. Planta Medica, 54(6), 516-519. 17 18 Pachaly, P., & Tan, T. J. (1986a). Alkaloide aus Tiliacora triandra Diels 19 20 (Menispermaceae), 2. Mitt.1) Yanangin, ein neues Bisbenzylisochinolin-Alkaloid. 21 For Review Only 22 23 Archiv der Pharmazie, 319(9), 841-849. 24 25 Pachaly, P., & Tan, T. J. (1986b). Alkaloide aus Tiliacora triandra Diels 26 27 (Menispermaceae), 3. Mitt.1). Die Struktur von Tilianangin, einem neuen 28 29 30 Bisbenzylisochinolin-Alkaloid. Archiv der Pharmazie, 319(10), 872-877. 31 32 Pachaly, P., Tan, T. J., Khosravian, H., & Klein, M. (1986). Alkaloide aus Tiliacora 33 34 triandra Diels (Menispermaceae), 1. Mitt. Die Struktur von Yanangcorinin, einem 35 36 37 neuen Bisbenzylisochinolin-Alkaloid. Archiv der Pharmazie, 319(2), 126-133. 38 39 Pavanand, K., Webster, H. K., Yongvanitchit, K., & Dechatiwongse, T. (1989). 40 41 Antimalarial activity of Tiliacora triandra Diels against Plasmodium falciparum 42 43 in vitro. Phytotherapy Research, 3(5), 215-217. 44 45 46 Phadungkit, M., Somdee, T., & Kangsadalampai, K. (2012). Phytochemical screening, 47 48 antioxidant and antimutagenic activities of selected Thai edible plant extracts. 49 50 Journal of Medicinal Plants Research, 6(5), 662-666. 51 52 53 Phunchago, N., Wattanathorn, J., & Chaisiwamongkol, K. (2015). Tiliacora triandra, 54 55 an anti-intoxication plant, improves memory impairment, neurodegeneration, 56 57 58 59 60 20

For Proof Read only Page 23 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 cholinergic function, and oxidative stress in hippocampus of ethanol dependence 5 6 7 rats. Oxidative medicine and cellular longevity, 2015, 918426. 8 9 Rajendran, P., Nandakumar, N., Rengarajan, T., Palaniswami, R., Gnanadhas, E. N., 10 11 Lakshminarasaiah, U., Gopas, J., & Nishigaki, I. (2014). Antioxidants and human 12 13 14 diseases. Clinica Chimica Acta, 436, 332-347. 15 16 Rattana, S., Phadungkit, M., & Cushnie, B. (2010). Phytochemical screening, flavonoid 17 18 content and antioxidant activity of Tiliacora triandra leaf extracts. Paper 19 20 presented at the The 2nd Annual International Conference of Northeast Pharmacy 21 For Review Only 22 23 Research. 24 25 Singharachai, C., Palanuvej, C., Kiyohara, H., Yamada, H., & Ruangrungsi, N. (2011a). 26 27 Pharmacognostic specification of five root species in Thai traditional medicine 28 29 30 remedy: Ben-Cha-Lo-Ka-Wi-Chian. Pharmacognosy Journal, 3(21), 1-11. 31 32 Singharachai, C., Palanuvej, C., Kiyohara, H., Yamada, H., & Ruangrungsi, N. (2011b). 33 34 Safety evaluation of Thai traditional medicine remedy: Ben-Cha-Lo-Ka-Wi- 35 36 37 Chian. Journal of Health Research, 25(2), 83-90. 38 39 Singthong, J., Oonsivilai, R., Oonmetta-aree, J., & Ningsanond, S. (2014). Bioactive 40 41 compounds and encapsulation of Yanang (Tiliacora triandra) leaves. African 42 43 journal of traditional, complementary, and alternative medicines, 11(3), 76-84. 44 45 46 Sireeratawong, S., Lertprasertsuke, N., Srisawat, U., Thuppia, A., Ngamjariyawat, A., 47 48 Suwanlikhid, N., & Jaijoy, K. (2008). Acute and subchronic toxicity study of the 49 50 water extract from Tiliacora triandra (Colebr.) Diels in rats. Songklanakarin 51 52 53 Journal of Science and Technology, 30(5), 611-619. 54 55 Soradech, S., Kusolkumbot, P., & Thubthimthed, S. (2018). Development and 56 57 characterization of microemulsions containing Tiliacora triandra Diels as an 58 59 60 21

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 24 of 39

1 2 3 4 active ingredient for antioxidant and melanogenesis stimulating activities. Journal 5 6 7 of Applied Pharmaceutical Science, 8(3), 46-54. 8 9 Surapong, R., Benjamart, C., Ladachart, T., & Methin, P. (2016). Chemical constituents 10 11 and in vitro anticancer activity of Tiliacora triandra leaves. Pharmacognosy 12 13 14 Journal, 8(1). 15 16 Sureram, S., Senadeera, S. P. D., Hongmanee, P., Mahidol, C., Ruchirawat, S., & 17 18 Kittakoop, P. (2012). Antimycobacterial activity of bisbenzylisoquinoline 19 20 alkaloids from Tiliacora triandra against multidrug-resistant isolates of 21 For Review Only 22 23 Mycobacterium tuberculosis. Bioorganic & Medicinal Chemistry Letters, 22(8), 24 25 2902-2905. 26 27 Tangsucharit, P., Kukongviriyapan, V., Kukongviriyapan, U., & Airarat, W. (2006). 28 29 30 Screening for analgesic and anti-inflammatory activities of extracts from local 31 32 vegetables in Northeast Thailand. Srinagarind Medical Journal, 21(4), 305-310. 33 34 Thong-asa, W., & Bullangpoti, V. (2020). Neuroprotective effects of Tiliacora triandra 35 36 37 leaf extract in a mice model of cerebral ischemia reperfusion. Avicenna journal of 38 39 phytomedicine, 10(2), 202-212. 40 41 Thong-asa, W., & Laisangunngam, H. (2018). Enhancing effect of Tiliacora triandra 42 43 leaves extract on spatial learning, memory and learning flexibility as well as 44 45 46 hippocampal choline acetyltransferase activity in mice. Avicenna journal of 47 48 phytomedicine, 8(4), 380-388. 49 50 Thong-asa, W., Prasertsuksri, P., Sakamula, R., & Nimnuan, T. (2019). Effect of 51 52 53 Tiliacora triandra leaf extract on glycemic control in mice with high sugar intake. 54 55 Sains Malaysiana, 48(9), 1989-1995. 56 57 58 59 60 22

For Proof Read only Page 25 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Thong-asa, W., Tumkiratiwong, P., Bullangpoti, V., Kongnirundonsuk, K., & 5 6 7 Tilokskulchai, K. (2017). Tiliacora triandra (Colebr.) Diels leaf extract enhances 8 9 spatial learning and learning flexibility, and prevents dentate gyrus neuronal 10 11 damage induced by cerebral ischemia/reperfusion injury in mice. Avicenna 12 13 14 journal of phytomedicine, 7(5), 389-400. 15 16 Tundis, R., Loizzo, M. R., & Menichini, F. (2010). Natural products as alpha-amylase 17 18 and alpha-glucosidase inhibitors and their hypoglycaemic potential in the 19 20 treatment of diabetes: an update. Mini-Reviews in Medicinal Chemistry, 10(4), 21 For Review Only 22 23 315-331. 24 25 Udyanin, W., Bumrerraj, S., & Nimsuntron, K. (2013). Herbal-usage behavior for 26 27 reducing blood sugar in diabetics: basic information for developing service system 28 29 30 of diabetics in Huai Thalaeng district. Community Health Development Quarterly 31 32 Khon Kaen University, 1(1), 11-24. 33 34 Weerawatanakorn, M., Rojsuntornkitti, K., Pan, M.H., & Wongwaiwech, D. (2018). 35 36 37 Some phytochemicals and anti-inflammation effect of juice from Tiliacora 38 39 triandra leaves. Journal of Food Nutrition Research, 6(1), 32-38. 40 41 Wiriyachitra, P., & Phuriyakorn, B. (1981). Alkaloids of Tiliacora triandra. Australian 42 43 Journal of Chemistry, 34(9), 2001-2004. 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 23

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 26 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Figure 1 Chemical structures of some compounds identified in Tiliacora triandra 42 43 (Colebr.) Diels. (A) alkaloids (B) miscellaneous (C) chlorophyll and its breakdown 44 45 46 products (D) fatty acids (E) essential oils (F) vitamin E (G) flavonoids and (H) other 47 48 phenolic compounds. 49 50 51 52 53 54 55 56 57 58 59 60 1

For Proof Read only Page 27 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Review Only 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Figure 1 (cont.) Chemical structures of some compounds identified in Tiliacora 57 58 triandra (Colebr.) Diels. 59 60 2

For Proof Read only Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 28 of 39

1 2 3 4 Table 1. In vitro antioxidant activities, total phenolic content (TPC) and total flavonoid content (TFC) of different parts and extracts of 5 6 7 Tiliacora triandra (Colebr.) Diels. 8 9 Plant parts and DPPH assay ABTS assay FRAP assay 10 Other assays References 11 extracts (IC50) (IC50) Fe(II) equivalent 12 13 Roots extracts 14 For Review Only 15 Ethanol 83.64 μg/ml Singharachai et 16 17 al. (2011b) 18 19 20 Ethanol 15.38±0.25 μg/ml Juckmeta et al. 21 22 (2012) 23 24 Aerial extracts 25 26 Nitric Oxide assay (IC50) 27 28 n-hexane fraction 2,436.83±22.49 μg/ml 190.04±8.59 μg/ml 402.21±13.62 μM/mg 30.50±0.47 μg/ml Makinde et al. 29 30 of twigs (2019) 31 32 Ethyl acetate 424.16±2.69 μg/ml 21.62±0.03 μg/ml 1,116.54±3.9 μM/mg 87.51±8.72 μg/ml 33 34 fraction of twigs 35 36 37 38 39 40 41 42 1 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 29 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Plant parts and DPPH assay ABTS assay FRAP assay 5 Other assays References 6 extracts (IC ) (IC ) Fe(II) equivalent 7 50 50 8 9 Ethyl acetate 2,412.83±34.39 μg/ml 18.70 ± 3.68 μg/ml 276.59 ± 11.22 μM/mg 1,760.09±11.81 μg/ml 10 11 fraction of leaves 12 13 Leaves extracts 14 For Review Only 15 Ethanol 14.51±0.67 μg/ml Phadungkit et 16 17 al. (2012) 18 19 Methanol 8.4 mg/ml Chaveerach et 20 21 al. (2016) 22 23 Total Flavonoid Content 24 25 Petroleum ether 113.81±0.85 ppm 0.49±0.00 mmol/mg 7.22±0.08 mmolQE/mg Rattana et al. 26 27 28 Dichloromethane 75.57±1.68 ppm 0.37±0.00 mmol/mg 17.04±0.06 mmolQE/mg (2010) 29 30 Ethyl acetate 15.02±0.47 ppm 0.58±0.00 mmol/mg 14.71±0.08 mmolQE/mg 31 32 Methanol 9.63±0.53 ppm 0.734±0.13 mmol/mg 18.68±0.28 mmolQE/mg 33 34 Water 16.19±0.45 ppm 0.151±0.00 mmol/mg 2.01±0.07 mmolQE/mg 35 36 37 38 39 40 41 42 2 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 30 of 39

1 2 3 4 Plant parts and DPPH assay ABTS assay FRAP assay 5 Other assays References 6 extracts (IC ) (IC ) Fe(II) equivalent 7 50 50 8 9 80%Ethanol 6,346.05±1.17 μg 0.27±0.02 mmol/g 22.63±1.53 mgCE/g Nanasombat et 10 11 extract/mg DPPH Total Phenolic Content al. (2019) 12 13 101.25±1.81 mgGAE/g 14 For Review Only 15 Water 0.197±0.018 mg/g 0.077±0.011 mg/g 0.054±0.002 mmol/g 97.899±1.735 mgGAE/g Singthong et al. 16 17 Ethanol 0.333±0.024 mg/g 0.191±0.059 mg/g 0.034±0.001 mmol/g 26.703±1.642 mgGAE/g (2014) 18 19 Acetone 0.419±0.091 mg/g 0.312±0.056 mg/g 0.014±0.001 mmol/g 16.456±2.968 mgGAE/g 20 21 Ethanol 0.1007±0.01 mg/ml 50.15±3.82 mgGAE/g Soradech et al. 22 23 (maceration) (2018) 24 25 Ethanol (batch 0.0968±0.01 mg/ml 58.02±4.11 mgGAE/g 26 27 28 stirring at 30°C) 29 30 Ethanol (batch 0.1131±0.01 mg/ml 59.13±4.81 mgGAE/g 31 32 stirring at 40°C) 33 34 freeze-dried leaves 0.29±0.02 mgTE/g 0.34±0.03 mgGAE/g 0.94±0.04 mgGAE/g Chuacharoen 35 36 juice (2020) 37 38 39 40 41 42 3 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 31 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 IC =50% inhibitory concentration; DPPH=1,1-diphenyl-2-picryl hydrazyl; ABTS=2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) 5 50 6 7 diammonium salt; FRAP=Ferric Reducing Antioxidant Power; QE=Quercetin equivalent; CE=Catechin equivalent; GAE=Gallic acid 8 9 equivalent; TE=Trolox equivalent. 10 11 12 13 14 For Review Only 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 4 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 32 of 39

1 2 3 4 Table 2. Other pharmacological activities of Tiliacora triandra (Colebr.) Diels. 5 6 7 Plant Extracts/ 8 Activity Model used Major findings References 9 part compounds 10 11 Neuroprotective In vitro. AChE Root MeOH extract 42.29% inhibition at 0.1 mg/ml. Ingkaninan et al. 12 13 inhibition assay (2003) 14 For Review Only 15 16 Leaf 80%EtOH extract 2.18% inhibition at 0.1 mg/ml. Nanasombat et al. 17 18 (2019) 19 20 In vivo. Aerial Water extract memory deficit, AChE activity, Phunchago et al. 21 22 Alcoholic rat part (100, 200, and 400 neuron density, MDA level, and (2015) 23 24 mg/kg, p.o.) SOD, CAT, and GSH-Px activities. 25 26 27 In vivo. Cerebral Leaf EtOH extract spatial learning, spatial memory, Thong-asa et al. 28 29 ischemia/reperfusion (300 and 600 learning flexibility and prevent (2017) 30 31 mice mg/kg, p.o.) hippocampal cell death. 32 33 34 35 36 37 38 39 40 41 42 5 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 33 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Plant Extracts/ 5 Activity Model used Major findings References 6 7 part compounds 8 9 Pretreatment: calcium level and MDA Thong-asa and 10 11 level, GSH, SOD and CAT activities, Bullangpoti 12 13 attenuate brain infarction, and dead cells (2020) 14 For Review Only 15 in the cerebral cortex and hippocampus. 16 17 18 In vivo. Male mice Leaf EtOH extract spatial learning, spatial memory, Thong-asa and 19 20 (300 and 600 learning flexibility, choline Laisangunngam 21 22 mg/kg, p.o.) acetyltransferase activity and (2018) 23 24 hippocampal cell density. 25 26 Antidiabetic In vitro. α-amylase and Leaf 80%EtOH extract 78.28% α-amylase inhibition and 10.30%α- Nanasombat et al. 27 28 29 α-glucosidase inhibition glucosidase inhibition at 1 mg/ml. (2019) 30 31 assays Twig n-hexane fraction IC50α-amylase=93.74 μg/ml. Makinde et al. 32 33 IC50α-glucosidase=3.40 μg/ml. (2019) 34 35 36 37 38 39 40 41 42 6 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 34 of 39

1 2 3 4 Plant Extracts/ 5 Activity Model used Major findings References 6 7 part compounds 8 9 Aerial 5,7-dihydroxy-6- IC50α-amylase=26.27 M. Makinde, 10 11 part oxoheptadecanoic IC50α-glucosidase=11.58 M. Ovatlarnporn et al. 12 13 acid (2020) 14 For Review Only 15 In vivo. STZ-induced Leaf EtOH extract blood glucose level, serum insulin Katisart and 16 17 18 diabetic rats (300 mg/kg, p.o. level, and activated the regeneration of Rattana (2017) 19 20 for 8 weeks) pancreatic Islets of Langerhans. 21 22 In vivo. Mice with high Leaf EtOH extract blood glucose, serum insulin, and Thong-asa et al. 23 24 sugar intake (300 and 600 liver and muscle glycogen contents. (2019) 25 26 mg/kg, p.o.) 27 28 29 In vivo. High-fat diet/ Aerial EtOH extract blood glucose level, lipid profiles, and Makinde, 30 31 STZ induced diabetic part (100 and 400 liver and kidney functions. Radenahmad, et al. 32 33 rats mg/kg, p.o. for (2020) 34 35 30 days) 36 37 38 39 40 41 42 7 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Plant Extracts/ 5 Activity Model used Major findings References 6 7 part compounds 8 9 Antiplasmodial In vitro. Clinical isolate Root MeOH extract MeOH extract: IC50=17 g/ml, Pavanand et al. 10 11 Plasmodium falciparum and isolated Tiliacorinine: IC50=3533±281 ng/ml, (1989) 12 13 compounds Tiliacorine: IC =675±96 ng/ml, 14 For Review Only50 15 16 Nor-tiliacorinine A: IC50=558±41 ng/ml, 17 18 alkaloid G: IC50=344±44 ng/ml, 19 20 alkaloid H: IC50=916±122 ng/ml. 21 22 In vitro. Stem, Different extracts The CH2Cl2 and MeOH extracts of the stem Nutmakul et al. 23 24 P. falciparum CQ root and isolated and root: IC =1.22 to 5.73 g/ml against (2016) 25 50 26 27 sensitive (3D7) and compounds both strains of parasites. 28 29 resistance (W2) strains Tiliacorinine: IC50W2=2.14g/ml. 30 31 Yanangcorinine: IC W2=1.55 g/ml. 32 50 33 34 35 36 37 38 39 40 41 42 8 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 36 of 39

1 2 3 4 Plant Extracts/ 5 Activity Model used Major findings References 6 7 part compounds 8 9 Tiliacorinine and Yanangcorinine showed Nutmakul et al. 10 11 parasiticidal effect and potentiated the (2020) 12 13 efficacy of CQ against CQ-resistant strain. 14 For Review Only 15 Antipyretic In vivo. Yeast-induced Root Powder Significantly reduced rectal temperature Konsue et al. 16 17 18 fever rats (40 mg/kg, p.o.) from the first hour after yeast injection. (2008) 19 20 Anti- In vitro. LPS induced Root EtOH extract NO inhibitory activity: IC50=54.65±5.34 Juckmeta and 21 22 inflammatory NO production in μg/ml. Itharat (2012) 23 24 RAW264.7 Leaf Lyophillized 56% nitrite production inhibition at 500 Weerawatanakorn 25 26 leaves juice μg/ml. et al. (2018) 27 28 29 In vivo. Writhing test in Leaf Water extract 55.7% inhibition of writhing reflex. Tangsucharit et al. 30 31 mice (1 g/kg, p.o.) (2006) 32 33 Anticancer In vitro. Various lung Root EtOH extract IC50KB=42.1±4.5 μg/ml, Juckmeta et al. 34 35 cancer cell lines IC50Hep2= 45.2±7.1 μg/ml, (2019) 36 37 IC A549=33.6±0.6 μg/ml, 38 50 39 40 41 42 9 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 37 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Plant Extracts/ 5 Activity Model used Major findings References 6 7 part compounds 8 9 IC50COR-L23=25.7±7.9 μg/ml, 10 11 IC50NCI-H226=19.5±1.4 μg/ml. 12 13 In vitro. Hepatoma cell Stem EtOH extract IC50HepG2=81.06 μg/ml. Lumlerdkij et al. 14 For Review Only 15 line (2020) 16 17 18 In vitro. Colon cancer Leaf MeOH extract IC50HT-29=203.52 μg/ml. Manosroi et al. 19 20 cell line (2015) 21 22 In vitro. Oral cavity Leaf Different extracts EtOAc extract: IC50KB=15.81 μg/ml, Surapong et al. 23 24 cancer (KB) and lung IC50NCI-H187=33.38 μg/ml. (2016) 25 26 cancer (NCI-H187) cell MeOH extract: IC KB=32.15 μg/ml, 27 50 28 29 lines IC50NCI-H187=11.93 μg/ml. 30 31 Water extract: IC50KB=12.06 μg/ml, 32 33 IC50NCI-H187=12.27 μg/ml. 34 35 Oxonanolobine (isolated from MeOH 36 37 extract): IC NCI-H187=27.6 μg/ml. 38 50 39 40 41 42 10 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul Page 38 of 39

1 2 3 4 Plant Extracts/ 5 Activity Model used Major findings References 6 7 part compounds 8 9 In vitro. Sensitive Leaf Different extracts CH2Cl2 extract: IC50A549=22.0 μg/ml, Kaewpiboon et al. 10 11 (A549) and multidrug IC50A549RT-eto=48.5 μg/ml (2014) 12 13 resistant (A549RT-eto) EtOH extract: IC50A549=67.3 μg/ml, 14 For Review Only 15 lung cancer cells IC A549RT-eto=73.0 μg/ml. 16 50 17 18 Human CCA in vitro Stem, Tiliacorinine IC50KKU-M055=4.5 μM, Janeklang et al. 19 20 and in vivo root IC50 KKU-M213=5.7 μM, (2014) 21 22 IC50KKU-M214=6.1 μM, 23 24 IC50KKU-100=7.0 μM. 25 26 Tiliacorinine rapidly reduced tumor growth 27 28 29 in CCA xenografted mice. 30 31 Antimicrobial In vitro. Disc diffusion Root EtOH extract The extract exhibited antibacterial and Nuaeissara et al. 32 33 antifungal activities with the inhibition (2011) 34 35 zone 6.8 to 16.3 mm. 36 37 38 39 40 41 42 11 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 39 of 39 Songklanakarin Journal of Science and Technology SJST-2020-0256.R1 Nutmakul

1 2 3 4 Plant Extracts/ 5 Activity Model used Major findings References 6 7 part compounds 8 9 In vitro. Broth Aerial n-hexane and The extracts exhibited antibacterial activity Makinde et al. 10 11 microdilution part EtOAC fractions with MIC=0.39 to 6.25 mg/ml and (2019) 12 13 MBC=1.5 to 12 mg/ml. 14 For Review Only 15 In vitro. Disc diffusion Leaf Essential oil The essential oil exhibited antibacterial Naibaho et al. 16 17 18 and broth microdilution activity with the inhibition zone 10 to 16 (2012) 19 20 mm, and MIC=6.25 μl/ml. 21 22 In vitro. Root Tiliacorine, MIC=0.7 to 6.2 μg/ml against clinical Sureram et al. (20 23 24 Antimycobacterial Tiliacorinine, and isolates of multidrug-resistant 25 26 activity 2′-nortiliacorinine Mycobacterium tuberculosis. 27 28 29 30 31 =increase; =decrease; AChE=Acetylcholinesterase; MDA=malondialdehyde; SOD= Superoxide dismutase; CAT= Catalase; GSH- 32 33 Px=Glutathione Peroxidase; STZ=Streptozotocin; CQ=chloroquine; LPS=lipopolysaccharide; NO=nitric oxide; CCA=Cholangiocarcinoma 34 35 36 37 38 39 40 41 42 12 43 For Proof Read only 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60