Patawali Stem
Tinospora crispa (L.) Hook.f. & Thomson
Menispermaceae
Figure 1 : T. crispa. (a) Whole plant; (b)-(c) stems; (d) leaf. (Photos courtesy of Thiyagu, MARDI, 2014)
DEFINITION
Patawali stem consists of dried stem of T. crispa (L.) Hook.f. & Thomson (Menispermaceae)
SYNONYM
Tinospora rumphii Boerl, Tinospora tuberculata (Lamk) Beumee ex. K Heyne.
VERNACULAR NAMES
Heavenly elixir, heartleaf moonseed (English) [ 1 ], akar patawali, putarwali, petawali, daun akar wali, batang wali, akar seruntum (Malay), bo ye qing niu dan (Chinese) [ 2 ], kattukkodi (Tamil).
CHARACTER
| Colour | Greenish brown |
| Odour | Characteristic |
| Taste | Very bitter |
IDENTIFICATION
Plant Morphology
A woody climber up to 15 m long, older stems very prominently, tuberculate and producing very long filiform aerial roots. The stem contains an exceedingly bitter milky sap. Leaves broadly ovate to obircular, 7–14 cm x 6–12 cm. Inflorescence appearing when plant is leafless. Flowers usually come with 3 petals. Fruits are ellipsoidal, about 2 cm long and orange in colour [ 3 , 4 , 5 ].
Microscopy
Microscopic characters of T. crispa stem powder consist of cork cells in surface view, numerous starch granules with parenchyma cells, collenchymas cells; annular, spiral, reticulate, pitted and bordered pitted vessel cells, up to 160 µm in diameter; fragments of bast fibres with dentated wall and solitary crystals; fragments of lignified parenchyma and annular vessel fragments with parenchyma cells; parenchyma cell containing calcium oxalate crystals adjacent to bast fibres [ 6 ].
Figure 2 : Microscopic characters of T. crispa stem powder. (a-b) Cork cells; (c-d) starch granules; (e) collenchyma cells; (f-g) bordered pitted vessel cells; (h) fibre; (i) fibre and starch granules; (j-m) solitary crystals. [Scale bars: a,g=50 µm;b-f, h-k, m=20 µm; l=10 µm]
Colour Tests
Observed colour of solution after treatment with various reagents:
| NH4OH (25%) | Yellow |
| FeCl3 (5%) | Yellow |
Thin Layer Chromatography (TLC)
Figure 3 : TLC profiles of syringin (S) and methanol extract of T. crispa dried stem observed under (a) UV at 254 nm and (b) UV at 366 nm before spraying; (c) visible light and (d) UV at 366 nm after spraying with 10% solution of sulfuric acid in ethanol.
| Test Solutions | Extract about 0.5 g of the T. crispa dried stem with 5 mL of methanol. Sonicate the solution for 15 min and filter. |
| Standard solution | Dissolve 1 mg of syringin [CAS no.: 118-34-3] in 1 mL methanol to produce 1 mg/mL. |
| Stationary Phase | HPTLC silica gel pre-coated plate 60 F254, 10 x 10 cm. |
| Mobile phase | Chloroform : methanol : water ( 8:2:0.2 ) (v/v/v) |
| Application |
|
| Development distance | Equilibrate the chamber with the mobile phase for 15 minutes, develop vertically for 7.0 cm. |
| Drying | Air drying |
| Detection |
|
High Performance Liquid Chromatography (HPLC)
| Test solution | Extract about 0.5 g of the T. crispa dried stem with 2 mL of methanol. Sonicate the solution for 2 hr and filter the solution through a 0.45 µm PTFE syringe filter and inject the filtrate into the HPLC column. | ||||||||||||||||||
| Standard solution | Dissolve 1 mg of syringin [CAS no.: 118-34-3] in 1 mL methanol to produce 1 mg/mL. | ||||||||||||||||||
| Chromatographic system |
Detector: UV 220 nm Column: C18 (5 µm, 4.6 mm I.D. x 250 mm) (preferable Phenomenex-Luna C18) Column oven temperature: Ambient Flow rate: 1 mL/min Injection volume: 15 µL |
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| Mobile Phase (gradient mode) |
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| System suitability requirement |
Perform at least five replicate injections of the standard solutions (1 mg/mL). The requirements of the system suitability parameters are as follow:
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| Acceptance criteria |
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Figure 4 : HPLC chromatogram of syringin standard solution (1 mg/ mL) at tr = 15.542 min.
Figure 5 : HPLC full chromatogram of methanol extract of T. crispa dried stem showing peak corresponding to syringin at tr = 15.449 min.
Figure 6 : UV spectrum of syringin (1 mg/mL) and methanol extract of T. crispa dried stem.
PURITY TESTS
| Foreign Matter |
| Not more than 2% |
| Ash Contents | |
| Total ash | Not more than 5% |
| Acid-insoluble ash | Not more than 1% |
| Loss on Drying |
| Not more than 11% |
| Extractive Values | |
| Water-soluble extracts | |
| Hot method | Not less than 18% |
| Cold method | Not less than 15% |
| Ethanol-soluble extracts | |
| Hot method | Not less than 8% |
| Cold method | Not less than 8% |
SAFETY TESTS
| Heavy Metals | |
| Arsenic | Not more than 5.0 mg/kg |
| Mercury | Not more than 0.5 mg/kg |
| Lead | Not more than 10.0 mg/kg |
| Cadmium | Not more than 0.3 mg/kg |
| Microbial Limits | |
| Total bacterial count | Not more than 105 cfu/g |
| Total yeast and mould count | Not more than 104 cfu/g |
| Bile-tolerant gram negative | Not more than 104 cfu/g |
| Specific Pathogens | |
| Salmonella spp. | Absent in 25 g |
| Escherichia coli | Absent in 1 g |
| Staphylococcus aureus | Absent in 1 g |
| Pseudomonas aeruginosa | Absent in 1 g |
CHEMICAL CONSTITUENTS
The stems contain N-trans-feruloyltyramine and N-cis-feruloyl tyramine in theethyl acetate fraction as well as a phenolicglucoside, namely, tinotuberide in thebutanol fraction [ 7 ]. Subsequently, a diterpeneglucoside, borapetoside A, and its aglycone,borapetol A were isolated from the stems, followed by the isolation ofborapetoside B, and its aglycone, borapetol B [ 8 , 9 ]. The presence of borapetoside C–H andtinotufolin A–F were also identified from thestems and leaves [ 10 , 11 , 12 , 13 ].
The cis clerodane-type furanoditerpenoids include, (2R,5R,6R,8S,9S,10S,12S)-15,16-epoxy-2-hydroxy-6-O-{β-D-glucopyranosyl-(1→6)-α-Dxylopyranosyl}-cleroda-3,13(16),14-trien-17,12-olid-18-oic acid methyl ester, (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, (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, methyl (2R,7S,8S)-8[(2S)-2-(3,4-dihydroxy-2,5-dimethoxytetrahydro-3-furanyl)-2-hydroxyethyl]-2,8-dimethyl-10-oxo-11-oxatricyclo[7.2.1.0]dodec-3-ene-3-carboxylate, (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 methylester, (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, (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, (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 and (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 [ 14 ].
The stems also contained five flavone glycosides identified as luteolin 4’-methyl ether 7-glucoside, genkwanin 7-glucoside, luteolin 4’-methyl ether 3’-glucoside, diosmetin and genkwanin [ 15 ].
The stems contained aporphine alkaloid, N-formylasimilobine 2-O–β-D-glucopyranoside, N-formylasimilobine 2-O–β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranoside (tinoscorside A), N-formylanonaine, N-formyldehydroanonaine, N-formylnomuciferine, N-demethyl-N-formyldehydronornuciferine, magnoflorine, paprazine, N-transferuloyltyramine and cytidine
[ 16 , 17 ].
The dichloromethane extract showed presence of vanillin, syringin, N formylannonain, N-formylnornuciferin, borapetosides B, C and F, N-cis-feruloyltyramine, N-trans feruloyltyramine and secoisolariciresinol [ 18 ].
The chloroform extracts of the dried stems showed presence of cycloeucalenol and cycloeucalenone [ 19 ].
The n-butanol extract of the stems was found to contain salsolinol, adenosine, uridine, tyramine, higenamine, syringing, borapetoside A, B, D and E, adenine and litcubinine [ 20 ].
The ethanolic extract showed presence of 2-O-lactoylborapetoside B, 6’-O lactoylborapetoside B, tinocrispol A, borapetosides A-F, borapetols A and B and columbin [ 21 ].
MEDICINAL USES
Uses described in folk medicine, not supported by experimental or clinical data
In folk medicine, an infusion of stem T. crispa is useful as vermifuge. A decoction of the whole plant is used for cholera [ 22 , 23 ]. This plant is useful for indigestion, stomachache, jaundice [ 22 ], sore eyes (external application), itchiness, wounds, mosquito bites [ 23 ] and fever [ 22 , 23 ]. It is also used in treatment of syphilis (external application), small pox and rheumatism [ 23 ].
Biological and pharmacological activities supported by experimental data
Antibacterial activity
Ethanol (95%v/v) extract of T. crispa dried stem inhibited the growth of eight methicilin-resistant Staphylococcus aureus isolated strains with minimum inhibitory concentrations (MIC) of 0.40-0.78 mg/mL and minimum bacterial counts (MBC) of 0.78-1.56 mg/mL by using checkerboard assay [ 24 ].
Aqueous, ethanol, and chloroform extracts of T. crispa dried stem (25, 50, 75, and 100% concentration/strength) inhibited the growth of Clostridium diphtheria, Streptococcus pneumonia and Shigella flexneri in a dose-dependent manner by using in vitro disc diffusion assay. From the same assay, the growth of Escherichia coli was only inhibited by aqueous and chloroform extracts at concentrations of 50-100% while the growth of Staphylococcus aureus was inhibited by ethanol extract at concentrations of 75% and 100%
[ 25 ].
Antiplasmodial activity
Ethanol (95% v/v) extract of T. crispa stem administered intraperitoneally (20, 40, and 80 mg/kg/day) to female ICR mice (pre-inoculated with Plasmodium yoelii, aged 6 weeks) for 20 days exhibited antiplasmodial activity in a dose-dependent manner with lower percentage of P. yoelii (18.12 ±2.24%) compared to ethanol (30%v/v) control (45.45 ±3.67%) on day 8. A pre-inoculated mouse given 80 mg/kg/day extract was alive without P. yoelii anymore in its blood stream on day 20 [ 26 ].
Antifilarial activity
Aqueous extract of T. crispa (10 mg/mL) against Dirofilaria immitis with mean relative movability (RM) value of 30.15% after 24 hours treatment (p < 0.05) compared to DMSO control (100%) [ 27 ].
Aqueous extract of T. crispa stem (0.5, 1.0, 5.0, and 10.0 mg/mL) exhibited antifilarial activity against adults worms of Brugia malayi (harvested from 4-6 weeks old male cat infected with L3-stage larvae from Aedes togoi) with the mean relative movability (RM) values of adults worms ranging between 0-100, in a dose-dependent manner, after 24 hours incubation compared to RPMI1640 culture medium as control (mean RM value of 100) [ 28 ].
Antinociceptive activity
Ethanol extract of T. crispa dried stem administered intraperitoneally in a single dose (30, 100, and 300 mg/kg body weight) to male Balb-C mice (aged 5-7 weeks) significantly exhibited antinociceptive activity in a dose-dependent manner with writhing response values of 3-15 (p ≤ 0.05) compared to saline control (36 ±1.45) by using acetic acid-induced writhing test and latency of discomfort of 10.84-13.16 seconds (p ≤ 0.05) compared to saline control (8.12 ±0.71) at 240 minutes by using hot plate test [ 29 ].
Anti-inflammatory activity
Ethanol extract of T. crispa dried stem administered intraperitoneally in a single dose (30, 100, and 300 mg/kg body weight) to Sprague Dawley rats (aged 8-10 weeks) significantly exhibited anti-inflammatory activity in a dose-dependent manner with the means of paw edema ranging between 0.11-0.41 mL/h (p ≤ 0.05) compared to saline control (0.57 ±0.05) at 1 hour by using carrageenan-induced paw edema test [ 30 ].
Antihyperglycemic activity
Water-ethanol extract of T. crispa stem (400 µg/mL) significantly increased 2-deoxy-D-glucose uptake (214.49 ±9.00% above basal, p < 0.01) in isolated rat’s L6 skeletal muscle cells after 24 hours incubation compared to metformin (205.80 ±6.28% above basal). However, this extract-stimulated glucose uptake may adversely be inhibited by 3.5 µM cycloheximide (glucose uptake < 100%) compared to partially inhibited metformin-stimulated glucose uptake (< 150%). This extract also significantly increased AMPKα1 (2.10 ±0.38 fold above basal, p < 0.05) and PPARγ (6.73 ±0.88 fold above basal, p < 0.05) mRNA levels after 15 minutes treatment while significantly increased GLUT1 mRNA level (1.29 ±0.38, p < 0.05) after 4 hours compared to control [ 31 ].
Anti-oxidant activity
Aqueous extract of T. crispa stem (dose) scavenged DPPH free radicals (86.51 ±0.07%) and inhibited oxidation of ferric ions (0.89 ±0.07mmol/L, p < 0.05) compared to vitamin C (96.36 ±0.90%, 1.05 ±0.00mmol/L) and BHT (96.51 ±0.95%, 1.03 ±0.03mmol/L) controls. This extract also inhibited lipid peroxidation (39.2 ±5.14%) by using thiobarbituric acid assay compared to vitamin C (73.2 ±5.14%) and BHT (75.8 ±6.08%) controls. The extract contained flavonoids (1.58 µg/mg of catechin, 0.85 µg/mg of luteolin, 1.44 µg/mg of morin and 1.38 µg/mg rutin) and phenolic (0.29 ±0.01 mg gallic acid equivalent/100 g of fresh sample) [ 31 ].
Methanol, chloroform, and water extracts of T. crispa stem showed significant total phenolic content (79.00-255.33 mg gallic acid equivalents/g extract, p < 0.05) by using Folin-Ciocalteu method and flavonoid content (2.67-9.53 mg quercetin equivalents/g extract, p < 0.05) by using Dowd method. Only methanol extract inhibited the scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals with IC50 value of 12 µg/mL compared to vitamin C control by using DPPH free radical scavenging assay [ 32 ].
Aqueous extract of T. crispa dried stem was prepared through different temperatures with respective time length, i.e. 20°C/24h, 40°C/12h, 60°C/6h, 80°C/3h, and 100°C/15 min. The extract at 60°C/6h significantly inhibited DPPH free radicals with 85.95 ±0.52% inhibition (p<0.05) compared to 20°C/24h (66.86 ±0.55%) and also inhibited thiobarbituric acid (TBA) (39.20 ±2.97%) [ 32 ].
Antihypercholesterolemic activity
Aqueous extract of T. crispa stem (450 mg/kg/day body weight) given to male New Zealand white rabbits fed with high cholesterol diet for 10 weeks significantly increased the levels of glutathione peroxidase (494.32 ±96.72U/L) and high density lipoprotein (HDL) (9.23 ±1.36mmol/L) (p <0.05) compared to untreated high cholesterol diet rabbits (220.41 ±19.69U/L , 0.86 ±0.11mmol/L). This extract also significantly decreased the levels of triglycerides and low density lipoprotein (LDL) (8.38 ±0.99mmol/L) (p < 0.05) compared to untreated high cholesterol diet rabbits (16.46 ±1.60 mmol/L). No foam cell formation was observed in the aorta of rabbits [ 34 ].
Cardiotonic activity
Triterpenes (cycloeucalenol and cycloeucalenone) isolated from crude chloroform extract of T. crispa dried stem (5.6×10-5 M for each triterpene) exhibited mild cardiotonic effects on isolated rat’s right and left atria (male Wistar rats weighed 250-300 g) compared to ethanol (80%v/v) control [ 35 ].
Cytochrome inhibitory activity
Methanol and ethanol-soluble fractions from aqueous extract of T. crispa stem (1.65 mg/mL) respectively inhibited activity of drug metabolizing enzymes from human liver microsomes, i.e. CYP3A4 (>70% inhibition; <30% inhibition) compared to ketoconazole control and CYP2D6 (>70% inhibition; <30% inhibition) compared to quinidine control
[ 36 ].
Methanol extract of T. crispa stem (0.5 mg/mL) inhibited the activity of drug metabolizing enzymes from human liver microsomes, i.e. CYP3A4 (>30% inhibition) compared to troleandomycin control (37.9% inhibition) and CYP2D6 (<30% inhibition) compared to paroxetine control (60.7% inhibition) [ 37 ].
Cytotoxic activity
Methanol and water crude extract of T. crispa dried stem (5-500 µg/mL) showed less cytotoxicity effect (IC50 value > 500 µg/mL) on HL-60, MCF-7 and HepG2 cancer cell lines by using MTT assay [ 38 ].
Antiproliferative activity
Methanol, chloroform, and water extracts of T. crispa stem inhibited the growth of MCF-7 (IC50 = 33.75-42.75 µg/mL), MDA-MB-231 (IC50 = 44.83-51.25 µg/mL), HeLa (IC50 = 46.13-53.83 µg/mL), and 3T3 (IC50 = 52.58-65.50 µg/mL) cancer cell lines, in a dose-dependent manner compared to tamoxifen control by using MTT assay [ 32 ].
Aqueous extract of T. crispa dried stem inhibited the growth of Caov-3 (IC50 = 80 µg/mL compared to cisplatin control), HepG2 (IC50 = 60 µg/mL compared to cisplatin control), MCF-7 (IC50 = 60 µg/mL compared to tamoxifen control), and HeLa (IC50 = 79 µg/mL compared to tamoxifen control) cancer cell lines on day 3 by using MTT assay [ 33 ].
Clinical studies
A randomized double-blind placebo-controlled crossover trial to study the hypoglycemic effect of T. crispa was conducted involving 36 patients who met the metabolic syndrome criteria by National Cholesterol Education Program Adult Treatment Panel III and did not consume any oral hypoglycemic drug. These patients were divided into two groups, i.e. a group given 250 mg T. crispa dry extract powder capsule (consists of 0.98% dry weight marker substance A) and another given placebo, at 30 minutes before meal twice daily for two months period. After two months, each group of patients received the other treatment. The fasting blood glucose (112.06 ± 13.98mg/dL) and triglyceride (135.78 ± 65.59mg/dL) levels in treatment group were significantly decreased (p < 0.05) and the HDL level (48.03 ±9.95mg/dL) was significantly increased (p < 0.05) while total cholesterol (189.31 ±33.91mg/dL) and LDL (114.11 ±30.82mg/dL) levels were not significantly changed (p>0.05), respectively compared to baseline. No significant difference in above measured parameters, calorie intakes, and body weights between treatmentand placebo groups during the treatment period. 91.6% of total patients adhered to T. crispa (assessed by capsule count method). The extract increased level of liver enzymes (AST and ALT) more than three times the baseline levels [ 41 ].
A randomized double blind placebo controlled trial to study the hypoglycemic effect of T. crispa was conducted involving 40 patients (included dropouts of six patients) with type 2 diabetes mellitus and glycosylated hemoglobin more than 8.5%, aged above 35 years old, who consumed adequate dose of oral hypoglycemic drug for at least two months, and refused insulin injection. These patients were divided evenly into two groups, i.e. a group given 1 g of T. crispa powder capsule and another given placebo, thrice daily for six months period. Fasting plasma glucose, glycosylated hemoglobin, and insulin levels in treatment group were not significantly different (p ≥ 0.0.5) compared to baseline and placebo. Body weight of treatment group decreased significantly but cholesterol increased significantly (p ≤ 0.05). Changes of liver enzymes profile were not observed [ 42 ].
SAFETY INFORMATION
Preclinical studies (Toxicology studies)
Acute toxicity
Oral single dose acute toxicity study using aqueous mixture of dried powder of T. crispa stem on female Sprague Dawley rats (aged between 8 and 12 weeks old) showed no toxic effect on the parameters observed which includes behaviors, body weight, food and water intakes. All rats were observed for 14 days prior to necropsy. No death was found throughout the study period. Necropsy revealed no significant abnormality. No-observed-adverse-effect level (NOAEL) is more than 2,000 mg/kg body weight[ 39 ].
Ethanol (95%v/v) extract of T. crispa stem (1, 2, and 4 g/kg body weight) given single dose orally to ICR mice (25±2 g body weight) showed no sign of toxicity after seven days observation [ 40 ].
Chronic toxicity
Ethanol (95%v/v) extract of T. crispa stem (0.02, 0.16, and 1.28 g/kg/day) given orally to Wistar rats (200-230 g body weight) for 180 days showed no effect on hematological parameters (hematocrit, white blood cells, platelet, neutrophil, eosinophil, lymphocyte, monocyte). The extract at the concentration of 1.28 g/kg/day significantly decreased creatinine and potassium levels, reduced rats’ body weight (p<0.05, both genders) and showed morphological changes (bile duct proliferation and focal liver cell hyperplasia) on male rats’ liver compared to control [ 41 ].
Others (Adverse reaction, contraindication, side effect, warning, precaution)
Adverse Reaction
Diabetic patient may experience increased appetite, flatulence and dizziness while receiving 250 mg T. crispa dry extract powder capsule (consists of 0.98% dry weight marker substance A) [ 41 ].
Precaution
Extract-thioacetamide treated rats showed significant increase in the levels of liver enzymes and liver body weight ratios. Histological examination revealed hepatocytes degeneration, centrilobular necrosis of hepatocytes, and inflammatory cell infiltration containing lymphocytes and mononuclear cells on liver of the rats [ 43 ].
250 mg T. crispa dry extract powder capsule (consists of 0.98% dry weight marker substance A) given to diabetic patients, at 30 minutes before meal twice daily for two months period, increased the level of liver enzymes (AST and ALT) more than three times the baseline levels. This extract may be a risk factor to elevated liver enzymes [ 41 ].
DOSAGE
Information and data have not been established
STORAGE
Store below 30°C. Protect from light and moisture.
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